TRIBOLIUM INFORMATION BULLETIN

NUMBER 10 - March, 1967

Foreword v

Editor’s Random Notes vi

Announcements x

Stock Lists 1

New Mutants 65

Notes - Research

Quantitative differences between the “aureate” and normalPhenotypes in T. castaneum, Mary Ackermann 86

Acarophenax tribolii – a parasitoid of Tribolium, William W. Allen 87

Further simplification of a bioassay method with adultsOf T. confusum Duv., B. Berck 88

Sex differences in recombination values for linkage Group V of T. castaneum, A. A. Dewees 89

Population performance of single – and mixed – species of flour beetles, H. E. Erdman 91

Ontogeny and X-radiation sensitivity of the flour beetle,Tribolium castaneum, mutant – sooty, H. E. Erdman 91

Metamorphic and adult life span modifications of flourBeetles Tribolium confusum, X-irradiated as pupae, H.E.Erdan 92

Comparative studies with Tribolium. III. The productivityof T. castaneum and T. confusum on synthetic diets,I.R. Franklin, J. Chandra and A. Sokoloff 93

Egg-laying rate of virgin females of Tribolium castaneum at different temperatures, Ma. C. Fuentes and R.G. Ruano 96

Algae flour as nutrition for Tribolium confusum, P. Geisert 101

The use of vital dyes for marking Tribolium eggs in freshAnd aged flour, Frank K. Ho 103

Identification of eye mutants in Tribolium larvae,Frank K. Ho 105

Susceptibility of Sitophilus granaries to moderate cold,R. W. Howe and B. D. Hole 107

Quinoid secretions in Tribolium confusum, R. K. Ladisch and S. K. Ladisch 108

Sexual dimorphism in the pupal setae of Tribolium,E. L. Lange 111

Regression of the sex ratio on maternal grandfather’s age,I.M. Lerner and N. Inouye 113

Evaluation of an anti-feeding compound as a protectant Against stored-products insects, S. R. Loschiavo 115

The distribution of Oryzaephilus Mercator Fauvel in Canada, S. R. Loschiavo and L.B. Smith 115

Factors involved in the survival of Tribolium confusumPopulations, D. J. McDonald and Mrs. L. Stoner 116

Cytochemical studies of Sitophilus granarius mycetomes,A.J. Musgrave and S. B. Singh 119

Mycetomes as possible obligate symbiotes of Sitophilus,A.J. Musgrave and I. Grinyer 119

A quantitative approach to the feeding of HymenolepisDiminuta eggs to the flour beetle, Tribolium confusum, D.M. O’Brian, A.Levine and F.F. Katz 120

The behavior and biology of Flour beetles, genus Tribolium, as studied in laboratory gradients of Temperature and humidity, A.K. Onyearu 121

The scanning electron microscope, R. F. W. Pease, T.L. Hayes and A. Sokoloff 122

The response of larvae of Trogoderma granarium Everts to-100 C, Elisabeth M. Reynolds and Barbara M. Rundle 131

Allelism of “mottled” (mt) and “melanotic stink glands” (msg)In Tribolium castaneum, T.H. Schmitz and D.C. Englert 132

The chromosome numbers of some stored productColeoptera, D. D. Shaw 133

Sex chromosome variation in D. maculates and D. frischii, D. D. Shaw 134

The effects of synchrony of egg batches on fitness Characters and competition in Tribolium castaneum,R. R. Sokal 135

A comparison of fitness characters and their responses to Density in stock and selected cultures of wild type and blackTribolium castaneum, R. R. Sokal 142

Preliminary population studies with mutants of TriboliumCastaneum Herbst. I. The paddle gene, A. Sokoloff 147

Preliminary population studies with mutants of TriboliumCastaneum Herbst. II. The black gene, A. Sokoloff 149

Preliminary population studies with mutats of TriboliumCastaneum Herbst. III. The jet gene, A. Sokoloff 152

Preliminary population studies with mutants of Tribolium castaneum Herbst. IV. The pearl gene,A.Sokoloff 153

Preliminary population studies with mutants of Latheticus Oryzae Waterh. I. The pearl gene, A. Sokoloff 161

Additions to established linkage groups, A. Sokoloff,M. Ackermann and B. Heinze 169

Additional sex-linked lethals in Tribolium castaneum Herbst,A.Sokolof, N. Inouye and R.S. St. Hilaire 169

Further studies of productivity of Tribolium cstaneum andTribolium confusum in homo- and heterospecific matings,A.Sokoloff and J. Lanier 173

Influence of collecting frequency on egg-laying rate of fecundatedAnd virgin females in Tribolium castaneum,

Ma. P. Tagarro and F. Orozco 175

Sex pheromones and defensive secretions from TenebrionidBeetles, W. Tschinkel 179

Some observations on mating frequencies in TriboliumCastaneum strains, D. Wool 182

Selection for 13-day larval size in Tribolium under twoNutritional levels, Y. Yamada and A. E. Bell 186

Relative fitness of selected strains under differentEnvironments, Y. Yamada and A. E. Bell 189

A method for isolating C. turcicus to obtain virgin females,M. Ackermann 191

A method for enhancing propagation of poorly viable flourbeetles, J. Lauck 191

Technique to determine the contents of cocoons of CryptolestesTurcicus, J. T. Robertson and L.B. Smith 192

New method for weighing Tribolium pupae, W. Rumball 192

A method for rearing Eleodes (longicollis?) (Coleoptera:Tenebrionidae) in the laboratory, A. Sokoloff 194

Directory – Geographical 196

Directory – Alphabetical 219

NEW MUTANTS

A. Dermestes maculates (Dermestidae)

*REPORT OF D. D. SHAW

1. Deformed antennae (def). A mutant reported in TIB 9:58, now proved to be controlled by an autosomal recessive gene of-variable penetrance and giving apparently normal viability. The mutant is similar in appearance to the phenoldeviant “branched” in Tribolium castaneum. Linkage tests with “rufous” and “fuscous” are being carried out.

Deformed antenna Normal

2. Fuscous (fu) (Mali). This mutant was isolated from a culture from Mali and is phenotypically similar to fuscous already described in TIB 8:42. Tests of allelism are under way.

3. Muddy. An eye-color mutant isolated from a culture of Indian origin. The eyes

appear grey-brown compared with the normal brown black. Genetics not yet studied.

4. Sexpitles. Originated in a single male offspring from a “rufous” x “white tip” cross. The male has no sex-pit. Genetics not yet studied.

5. White tip (Wt). This mutant was described in TIB 9 and has proved to be controlled by a dominant gene giving normal viability. The mutant is phenotypically similar to that described by Philip (1940) which was controlled by

an autosomal recessive gene. Linkage tests with rufous and fuscous are being carried out.

B. Tribolium castaneum (Tenebrionidae)

*REPORT OF CONSTANTINO, DITTMAR, HANCOCK, LANGE AND SHIDELER.

1. Corn oil sensitive (cos) – Costantino, 1964. An autosomal recessive gene identified during an investigation of the genetic basis of selection response for small 13-day larval weight in a population of Tribolium castaneum. A preliminary report on this gene was given by Constantino, Bell, and Rogler (Nature 210:221, 1966. The properties of the cos gene are as follows:

a. Larval growth of the cos homozygote, as measured by 13-day larval weight, varies inversely to the concentration of corn oil in the diet.

b. Viability also varies inversely to the concentration of corn oil in the diet (83% viable, at 13 days, in the diet with no supplemental corn oil decreasing to 51% in the diet with 5% corn oil).

c. The degree of dominance, with respect to 13-day larval weight when grown in a diet with 5% corn oil, is essentially complete.

d. .Gas-liquid chromatographic analyses of normal (+/+) and sensitive (cos/cos) individuals grown on diets with and without supplemental corn oil suggest that excessive quantities of linoleic acid in the cos homozygote may be the basis for the observed sensitivity to corn oil.

Linkage studies and further biochemical analyses are presently underway.

2. Grossly deformed (gdf) – Lange, 1965. Autosomal recessive of good expression isolated from Purdue Foundation. The pupae have the following appearance:

a. Elytra shortened and barely extending to the abdomen.b. Tibia is narrower in width and curved in a manner similar to bowleg.c. Urogomphi are reduced in size.d. Genital lobes are absent.e. Eyes are similar to squint or bar eye.f. Club of the antennae is fused.g. Gin traps are reduced in size.h. Setae are reduced to a large but variable amount.

The adults have the following appearance:

a. Elytra shortened and cover about half of the abdomen.b. Flight wings normal.c. Tibia has a form similar to bowleg.d. Eyes appear similar to squint but some facets are present.e. Antennal club is fused with a variable number of stalk segments, with generally the

first five segments of the stalk unfused.

The homozygous recessive is lethal in the adult stage probably because of desiccation brought about by the reduced elytra. It is not allelic to squint and is not linked to Bar eye. Further linkage studies are in progress.

3. Bumpy (by) – Hancock. Autosomal recessive found in sub-population of Purdue + Foundation, has variable expression and incomplete penetrance, similar in phenotype to Sokoloff’s ble, yields 3% recombination with Sa. Bumpy is being forwarded to Sokoloff’s laboratory for allelism tests with ble.

4. Miniature appendaged (maD) – Dittmar. Spontaneous sex-linked recessive found in a multiple recombination study, allelic to Sokoloff’s ma and similar in expression.

5. Multi-urogomphi (Mu) – Dittmar. Autosomal dominant found in Purdue b Foundation, incomplete penetrance with expression varied giving one or two additional urogomphi in larvae and pupae. Preliminary recombination studies for Mu suggest independence of linkage groups 2, 3, 4, 5, 7 and 8. Similar in expression to eu described by Lasley and Sokoloff in TIB 3 and has been forwarded to that laboratory for further comparisons.

6. Reduced elytra (re) – Shideler. Autosomal recessive of reduced viability found in sub-population of the Purdue black Foundation, incomplete penetrance and variable expression, elytra are severely reduced and sometimes missing.

7. split-back (sb) – Shideler. Autosomal recessive found in a sub=population of the Purdue perarl Foundation, has expression varying from shortened elytra to severe splitting or diverging of elytra. Similar in phenotype to the description of short elytra (Ho and Dawson, shH & D, Sokoloff, shS, TIB 1962) and has been forwarded to them for tests of allelism.

*REPORT OF R.W. LEMON AND D. G. BLACKMAN

1. mahogany (my). Autosomal recessive (TIB 9:59), not allelic with black or sooty. Linked with sooty and Bar eye in linkage Group IV (cross over values of 43% and 21.5% respectively). Results of three point crosses not yet known.

2. Pearl-like (TIB 9:59). Linkage tests for groups III, IV, V, VII and X proved negative. Crosses with ptl in group IX were inconclusive due to incomplete penetrance of ptl. The tests with Mo in group VI showed possible linkage, but difficulty in classifying Mo beetles made it necessary to repeat the tests which are not yet concluded. However, a very recent cross between “pearl-like” and “ivory” produced an F1 all mutant beetles; it is therefore possible that “pearl-like” is a reoccurrence of “ivory.”.

*REPORT OF A. SOKOLOFF

1. Charcoal (Chr) – Ackermann, 1966. Found during the course of determining whether “prothoracic margin irregular,” an abnormality involving the anterior margin of the prothorax was heritable (it was probably a phenodeviant). Autosomal

dominant with recessive lethal effects. Charcoal beetles resemble tawny and sooty. No synthetic lethal effect has been observed with Spa, Be, Fta and Bamp.

2. diminutive appendages (dim) – Heinze, 1966. Spontaneous in a stock of Bamp. An autosomal recessive with good penetrance and fairly good viability. The elytra are very short, exposing two or three abdominal segmens. The membranous wings fail to fold and protrude beyond the elytral tips. Many adults display a split of the abbreviated elytra, beginning about one-third of the length of the elytra from the scutellum. The legs are also much shorter than normal but maintain proper proportions as in miniature appendaged (see figure below). The mutant can readily be identified in the pupa stage by the short elytra which do not completely cover the membranous wings (as in the normal) and the legs are noticeably shorter. A more careful examination reveals fusions of antenna- and tarsomeres. These are summarized in Tables 1 and 2. This mutant is not allelic with ov or aa..

Table 1.Antennal fusions in dim. (15 beetles of each sex scored)

Males Females_____

Right Left Right Left

0 11 4 5 74-5 4 3 4 26-7 -- -- 1 --6-7-8 -- 1 -- --7-8 -- -- 2 17-8-9 -- -- -- 18-9 1 1 4 38-11 -- -- -- 110-11 -- -- -- 1

Table 2.Number of tarsal segments involved in fusions or missing in Dim.

(15 beetles of each sex scored)

Pair of Legs

1 st pair 2 nd pair 3 rd pair

0 1 2 3 4 0 1 2 3 4 0 1 2 3

Females Right 5 1 7 1 1 7 4 2 3 - 5 3 7 - Left 5 3 4 2 1 5 4 4 1 - 5 6 3 1

Males Right 8 0 6 2 - 6 1 7 1 - 8 2 5 - Left 8 2 3 1 1 11 3 2 - - 8 1 5 1

3. fused antennal segments-3b (fas-3b) – Heinze, 1966. Spontaneous, in a population of black and pearl scored for these genes for many generations in studies of natural selection. (Originally called “aggregate antennae” but found to be allelic with fas-3, therefore renamed.) Incompletely recessive, with fusions of antennameres as shown in the accompanying table. It may be noted that male antennae exhibit more fusions of the club. Female antennae show more fusions involving both funicular and club segments. Also, male antennae exhibit more fusions which involve fewer segments, while the fusions in female antennae are more extensive (involving more segments).

4. Knobby prothorax (knp) – Sokoloff, 1966. Spontaneous in a stock of Mo mas. Possibly a phenodeviant, but more likely autosomal recessive of incomplete penetrance. Penetrance is greater when knp females rather than males are used in backcrosses, and greater in backcrosses in general than in F1 x F1 crosses (expression in the latter being very weak) characterized by the fact that the prothorax has more or less symmetrical “knobs” extending lateroposteriorly. These are as large as ¼ - 1/3 the length of the prothorax. The prothoracic margin continues around the knobs, but many of these have a “suture” more or less continuous with the posterior like the prothorax but the ventral part may be sclerotized. This mutation is similar to knp in T. confusum. (TIB 9:62, see illustration on front cover, TIB 9).

5. Overhung split (ohs) – Heinze, 1966. Common in au stocks, but does not appear to be linked to au. Its appearance is like that shown in the figure below, with the elytra longer than the abdomen and exhibiting a pronounced split and sometimes tips curling down. Some may extend beyond the abdomen without splitting. Not allelic with we, spl, te, dve, bal, or cye. Probably a recessive with poor penetrance.

Table 3.Segments involved in fusions in the fas-3b mutant

(50 males and 50 females scored)

Males Females

Right Left Right Left

0 9 7 7 64-5 -- -- 1 14-6 -- -- 1 14-8 -- 1 -- --4-10 -- -- -- 14-11 -- 1 1 35-6 3 4 3 25-7 -- -- 1 15-8 1 1 -- --5-9 1 -- -- --5-11 2 3 3 16-7 1 5 1 56-8 3 -- 3 26-9 1 2 1 --6-10 -- -- -- 16-11 5 4 6 87-8 3 3 2 27-9 1 -- 1 --7-11 2 3 5 38-9 5 2 4 48-11 -- 1 2 29-10 1 3 1 39-11 4 7 4 610-11 13 13 11 5

6. Prothoracic margin irregular (pmi) – Hoy, 1965. Spontaneous in a population started two years previously with 12 Kr irradiated male mated with anon-irradiated

female. The anterior ventral margin of the prothorax appears as in the figure on page 77. A phenodeviant. From a cross of two pmi beetles only 2/578 were pmi.

7. Red-2 (r-2) – Heinze, 1966. One adult was found in a test cross between au s and Be which had chestnut-colored eyes; this male was mated to produce offspring with pale eyes as young beetles, and darker red eyes as older adults. An allelism test with red showed the mutation to be its allele; mode of inheritance tests showed it to also be an x-linked recessive gene, as is red.

8. Reduced tarsi and antennae (rta) – Heinze, 1966. In a “scalloped prothorax” stock (initially derived from an irradiation experiment which had been performed several years before) were found 10 adults and two pupae with antennae and legs reduced in segment number.

Table 4 shows the antennameres, Table 5 the tarsomeres involved in fusions and Table 6 the total number of tarsomeres seen in those legs with no apparent fusions but with 0-5 tarsomeres.

It is evident that the modal number of fusions is in antennameres 4-5, but also there appears to be a sex-difference, males having the club segments affected while that in females does not exhibit fusions. The tarsomeres were reduced to 1-2 per leg in most cases, some legs having no tarsomeres at all. The mutation is readily identifiable in the pupa stage, the legs appearing noticeably shorter than normal, as in Fta (see Plate 5, H, I, J in Sokoloff, 1966). Preliminary crosses suggest an autosomal recessive mode of inheritance.

Table 4.Antennal fusions found in a sample of “reduced tarsi and antennae” (rta) in T.

castaneum (N = 21 females and 21 males)

Males Females Right Left Right Left

0 11 12 11 132-3 -- -- -- 13-5 -- -- -- 14-5 8 6 9 55-6 -- 2 1 --

6-7 1 -- -- --7-8 -- 1 -- 19-10 4 2 -- --10-11 1 1 -- --

Table 5.Tarsomeres involved in fusions in “reduced tarsi and antennae” (rta)

In T. castaneum (N = 21 females and 21 males)

Pair of Legs

1 st Pair 2 nd pair 3 rd pair

0 1 0 1 0 1

Males Right 20 1 19 2 21 -- Left 16 5 21 -- 20 1

Females Right 20 1 20 1 21 --Left 21 -- 21 -- 20 1

Table 6.Total number of segments observed in the tarsi of “reduced tarsiAnd antennae” (rta) in T. castaneum (N = 21 beetles of each sex)

Leg and Tarsus Number___________________________

1 2 3 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4

Male Right 3 4 1 0 1 12 - 2 3 - - 16 1 1 4 - 15 Left 4 3 0 1 0 3 - 6 1 - - 14 - 4 3 - 14

Female Right 2 4 - - 3 12 - 2 1 - - 18 1 7 2 1 10 Left 3 3 1 - - 14 - 3 1 - 1 16 2 8 2 - 9

9. Rugose elytra (rue) – Heinze, 1966. A spontaneous mutation commonly seen in populations derived from aureate. The adult has very finely corrugated elytra, giving them an almost granular appearance. Mode of inheritance tests show that rue can be separated from au but the expression is a little less remarkable in non-aureate adults. The rue mutation is an autosomal recessive with good penetrance and viability, and not allelic to rough.

10. Short antenna-3 (sa-3) – Heinze and Hoy, 1966. In a cye fas stock were found several adults and pupae with very short and stubby legs, as well as short antennae and elytra. In allelism tests with aa-1 47/102 adults were seen to have weakly affected legs; with stock aa ov, 15/233 adults had very weakly affected

legs; with stock sa/sa all adults had strongly shortened appendages. The new mutant is, therefore, considered to be allelic with sa and will be called sa-3. A mode of inheritance test shows sa-3 to be an autosomal, incompletely recessive with 100% penetrance and high viability.

11. Short median abdominal projection (smp) – Ackermann, 1966. Autosomal recessive of apparently uniform expression and complete penetrance. A mutation somewhat resembling but not as pronounced as Bamp (Blunt abdominal and metathoracic projection, Hoy, 1965). (See TIB 9:60 for illustration of Bamp). It may be most readily identified by a shorter median abdominal projection of the apparent first abdominal segment, with the result that the tip does not extend between the hind coxae and is not accommodated by the inverted-V-shaped metasternellum of the metathorax (see figure on p.77). The mesosternellum and the intercoxal process of the metathorax barely join each other between themesothoracic legs. The mesosternellum appears somewhat ventrally convex. In crosses of smp x the F1 x F1 gave 187+:63 smp F2 and the backcross 196+:123 smp. In the reciprocal cross the ratios were 257+:58 smp for F2 and the backcross 109+:59 smp. Also noted was a large number of matings which failed and failed again, indicating a possible sterility factor.

12. Truncated elytra-2 (te-2) – Heinze, 1966. In a cross between r Mr x sy. Wild type several male adults and pupae were seen with short, folded te-like elytra. A good stock was built up and an allelism test with te showed the new mutant to be allelic. Since the new te has very strong expression and seems to be healthier than the old, a new stock shall be maintained.

Sex-linked Lethals

All the sex-linked lethal described below were found in the Berkeley synthetic strain in experiments designed to test the Cavalli-Sforza’s model of mutation frequency as a regression on the age of the maternal grandfather (see note by Lerner and Inouye elsewhere in this issue of TIB).

1. Lethal-5 (1-5_ -- Sokoloff and Inouye, 1964. Located about 25 units to the left of py (away from r) (see note by Sokoloff, Inouye and St. Hilaire). Allelic with 12 and 14.

2. Lethal-6 (1-6) – Sokoloff and Inouye, 1964. Located about 12 units to the left of py (away from r).

3. Lethal-7 (1-7) – Sokoloff and Inouye, 1964. Located about 20 units to the left of py (away from r).

Figure Legend

Upper Row, Center. – The “prothoracic margin irregular” phenodeviant in T.castaneum. Note that the probasisternum is indented on both sides between the Midline and the tergosternal suture.

Upper Right. -- The “incomplete metathoracic projections” mutant in T.confusum. Note the posterior projections of the metathorax are short, failing to form an inverted V to accommodate the medial abdominal projection of the first apparent abdominal segment.

Lower Left – The prosternumless (psl) mutation in T.confusum. Both the probasis- Ternum and mesobasisternum sternella are reduced. The prosternellum Forms a small ridge and as a result of its reduction in size the coxae of the Forelegs are freer, and not as widely separated. The mesosternellum fails

to fuse with the intercoxal process of the metabasisternum, again freeing the coxae of the middle pair of legs and the mesobasisternum acquired a suture-like appearance (represented by stipples).

Lower Middle. – incomplete meso (basi) sternum (ims). Illustration of the mutant in T. castaneum described in TIB 6:25 and in T. confusum in TIB 9:68.

Lower Right. – “short median abdominal projection” mutant in T. castaneum. Note that the “point” of the first apparent abdominal segment is blunt.

C. Tribolium confusum

*REPORT OF A. SOKOLOFF

Autosomal

1. Black (b) – Ho and Ackermann, 1966. Spontaneous in a mixed population XXIII-12 of T. anaphe and T. confusum. Not allelic with ebony nor with ebony-2. A recessive allele of black.

2. Engraved meta (basi) sternum dots (ems dt) – Heinze, 1966. Spontaneous. Found in a “frosted” (allele of dpe) stock. In adults, characterized by a very small indentation or hole just above the middle of one or both antecoxal pieces (see sketch) but not connected to the latter as in jac, jagged antecoxal piece (see Plate 7F in Sokoloff, 1966). There may be 1-3 dots per basisternum, and not necessarily the same number of dots on each side. Allelic to ems, and like ems autosomal recessive with incomplete penetrance.

3. Legless (lgl) – Hoy and Sokoloff, 1966. Spontaneous in a Reduced eye stock, five males and three females were found with a body color resembling mahogany (much lighter than Park’s ebony), and both forelegs missing down to the coxa. Outcrossed to wild type and scoring F2 no or few beetles have legs missing or partly so. But, mated to ech other, they produce progeny without one or both prothoracic legs. The elytra in all beetles are rugose, they may be a little shorter than the abdomen in a few beetles, and the proximal third of the elytra may fail to meet at the midline. Those beetles with both legs missing, taken out of the flour, are often seen moving with the lower part of the head on the glass container, being pushed as the beetles attempt to walk. Others can hold the body off the container as they walk. Tentatively this condition has been designated as a phenodeviant because beetles with the legs absent may not be seen at all in F2. However, since there is some evidence that the legs may develop and turn white, it is possible that this may be an aging effect young beetles exhibiting normal legs which, a few weeks later, fall off. Attempts are therefore being made to work out the mode of inheritance by examining body color and/or the wrinkled condition of the elytra.

4. Pointed elytra-like (pel) – Sokoloff, 1963. Elytra mostly divergent and pointed, somewhat shorter than normal resembling dve and/or pointed elytra of T. castaneum. (See Plate 9, i, j in Sokoloff, 1966). Occasionally, there may be beetles with elytra only short (not divergent) resembling sh in T. castaneum. Another form of expression: elytra longer than abdomen and drooping at the sides. Autosomal recessive, good viability.

5. Prosternumless (psl) – Sokoloff, Ackermann and Daly, 1966. Autosomal, incompletely recessive (in reciprocal crosses of psl x + about 10 per cent of the F1 beetles showed the character). (Found during tests of allelism between sep and dep.) This mutation is characterized by a reduction of the sternellum of the prothorax and mestothorax to a vestigial, keel-like structure (see figure on p.77). As a result, the globose coxae of the fore- and middle-legs lie ventral to the prosternum and mesosternum (“above” as one examines the ventral aspect of the beetle as it lies on its back). Since the mesobasisternum fails to reach the intercoxal process of the metasternum, the mesobasisternum appears to “sink in” above the coxae (i.e., it is directed postero-dorsally). The mesobasisternum acquires a shiny ridge along the midline resembling a suture. The probasisternum also may develop a less pronounced (and not shiny) suture along the midline. In some beetles the tergosternal suture becomes less pronounced.

6. Stunted (stt) – Sokoloff and Lauck, 1966. Spontaneous. Autosomal recessive mutation found in some crosses involving pointed-elytra-like (pel, q.v.). A gene with pleiotropic effects producing, like the sex-linked ma, miniature appendaged, individuals with the whole body shorter than normal, but the effect being most pronounced on the appendages as follows:

a. Antennael Thesre is considerabke, often asymmetrical, fustion of the antennameres. The antennae appear elbowed, and because of fusion and reduction in size of segments 7-8 the antennae may appear clubbed resembling that in T. castaneum. The distribution of fusions in the two sexes is shown in Table 1 which also indicates that the antennameres most often fused are 7 and 8. It is also evident that the gene often fails to express itself on the side of the body, producing one antenna normal and another abnormal. In the affected antennae there may be as few as two and as many as eight antennameres in fustions, some of which may involve more than two antennameres. When four consecutive segments are fused, usually they will form a kidney-shaped structure pointing the

other way. Summarized in Table 2 are the numbers of antennameres which may be affected in one antenna. Table 3 shows how funicular and club segments or both may be affected.

b. Legs. All the podomeres are reduced (the femur, in particular, becoming globase), resulting in a beetle with all legs considerably shorter (in a manner similar to that illustrated for ma in T. castaneum in Sokoloff, 1960, Can. J. Genet. Cytol 2:28-33). The tarsomeres are affected, the distribution of fusions being as shown in Table 4. Table 5, summarizing the fusions in the antennae and the tarsi of this mutant, appears to indicate that the chief difference between the two sexes is in the tarsi, the males exhibiting a greater number of fusions than females.

c. Mouth parts. Although this point was not investigated thoroughly there seems to be a partial fusion at least of some maxillary palpomeres.

d. Elytra and membranous wings. These are considerably reduced: the elytra are about half as long as the abdomen, often divergent, and often they exhibit a blister on the lateral margin. The veins and striae on the caudal half of the elytron become less pronounced and often hardly visible. The membranous wings trail (the tips of the wings fail to fold under the proximal portion), but they extend beyond the elytra, almost as far as the tip of the abdomen. All the veins appear to be present, but the cells are considerably reduced.

e. Other parts of the body. The prosternal process and the post-coxal bridges are reduced, with the result that a fairly extensive area of the prosternum in front of the mesobasisternum appears unsclerotized. The lateral parts of the coxae sockets appear to be more open than in the normal, so that at least the coxae of the fore- and hind-legs have a greater freedom to rotate within them. The intercoxal process of the apparent first abdominal segment appears somewhat shorter so it does not fit as well between the metasternellal. The tergal aspect of the abdomen is, of course, exposed. This circumstance, as well as the fact that the hind-legs are too short to enable the beetle to free its hind-end and the fore-legs are too short to free

Table 1.Distribution of fused antennameres in the T. confusum mutant

“stunted” (N = 20 males and 20 females)

Segments Males FemalesInvolved Right Left Right Left

1-4 -- -- -- 1 3-4 3 1 2 -- 3-5 1 1 2 1 3-8 -- -- 1 1 4-5 2 1 2 1

4-8 2 1 -- -- 4-11 1 -- 1 -- 5-6 4 5 3 -- 5-7 3 -- 1 1 5-8 1 -- 1 -- 5-11 1 -- 1 -- 6-7 2 1 2 2 6-8 1 3 1 2 6-11 -- -- 1 -- 7-8 11 9 8 10 7-9 -- 2 -- 1 7-11 -- -- -- 1 8-9 -- -- 2 2 8-11 -- -- -- 1 9-10 5 3 2 4 9-11 1 1 -- -- 10-11 2 1 1 1

Table 2.Numbers of segments involved in antennal fusions of “stunted”

(N = 20 males and 20 females) NSegments Males FemalesInvolved Right Left Right Left

2 32 21 20 22 3 4 8 4 4

4 1 1 2 1 5 -- 1 2 1 6 2 1 -- -- 7 1 -- 1 -- 8 -- 1 -- --

Table 3.Part of antennae where fusions were found in “stunted”

(N = 20 males and 20 Females)

Males Females Right Left Right Left

Funicle 30 25 20 21Club 8 6 3 4Both 1 2 5 5

Table 4.Distribution of fused (or missing) tarsomeres in the T. confusum

Mutant “stunted” (N = 20 males and 20 females)

NTarsomeres

Leg in Fusion Males FemalesPair or Missing Right Left Right Left

0 1 -- 1 4 1 2 5 11 - 6

1 2 15 12 3 6 3 3 2 3 3 4 -- 1 2 1 5 -- -- -- --

0 1 2 6 9 1 5 6 6 5

2 2 10 13 7 5 3 3 1 -- 1 4 -- -- 1 -- 5 -- -- 1 --

0 -- -- 4 2 1 7 7 4 1

3 2 13 18 13 18 3 2 1 -- -- 4 -- -- 1 --

Table 5.Number of fused segments in antennae and tarsi of male and female

stunted T. confusum (N = 20 males and 20 females)

Antennae Tarsi

Total Ave. No. Segments Ave. of Segments Ave. of Fusions of involved Segments/ Fused Segments/Sex Side N Fusions N Fusions N Fusions

Males R 36 1.8 97 4.8 114 5.7 L 33 1.6 75 3.7 120 6.0

Females R 42 1.4 79 3.9 97 4.8 L 28 2.1 79 3.9 86 4.3

the head from the pupal skin, results in a great mortality in the imago attempts to eclose from the pupa. It is quite probable that because of such major changes in the appendages and the overall size reduction of the body that this mutant might be recognized in the larval stage.

It is clear that both in the case of antennamere and tarsomere fusions (or deletions) occur more frequently in females than in males. In both sexes the first three segments appear to escape fusion. In males the modal group appears to be in the fusion of segments 4-5; in females these segments as well as segments 8-9 appear equally susceptible to fusion. Also, if club segments are involved, they appear to fuse more often in females than in males. The females showed somewhat more fusions of tarsomeres than did the males.

7. Wide split-1 (wspl-1) – Lauck, 1966. A mutation involving the leytra was found in the legless mutation. It appears as the figure on Plate 11 of Sokoloff, 1966, and is allelic with a similar mutation found, but apparently not reported, by P.S. Dawson.

Sex-linked

1. Alate prothorax (apt) – Strong, 1966. Originally found in a T. confusum synthetic strain maintained in soy plus yeast for many generations. This recessive, sex-linked, homeotic mutant is apparent in both pupae and adults. Both show large elytra-like growths on the dorso-lateral aspect of the prothorax. Although somewhat distorted, these growths show many of the characteristics of elytra (see illustration on front cover). The normal expression is similar to the strongest expression found in T. castaneum, apt, and rarely is as weak as the normal expression of T. castaneum, apt (i.e., small knobs on the dorso-lateral aspect of the prothorax). The expression is fairly constant and the penetrance appears to be complete. The viability, about 75%, is surprisingly high for such a drastic mutation. Most deaths take place during the molt from the pupa to the adult stage. Linkage data with eslt and lp are available (see note elsewhere inTIB) and experiments with red eye are in progress.

Tests of Allelism

1. Black (b) – Ho and Ackermann, 1966. Black (b) in T. confusum has proved to be a recessive allele of the semidominant black. It has been redesignated as b-4, Ho and Ackermann, 1966.

2. Frosted, reported in T. confusum (TIB 8:62) is allelic with dirty pearl eye (dpe) and it is therefore renamed dpe-1, Hoy and Heinze, 1966.

Addenda and CorrigendaIncomplete metathoracic projections (imp) (TIB 8:52) in T. castaneum is an

autosomal recessive with slightly variable expression and incomplete penetrance (about 15% of individuals fail to show the character).

ACKERMANN, MARYDepartment of GeneticsUniversity of CaliforniaBerkeley, California

*Quantitative differences between the “aureate” and Normal phenotypes in T. castaneum

The “aureate” mutation produces beetles whose bodies appear more pubescent than the wild type. Counts of body hairs were made on typical adult beetles shosen at random from the au and normal stocks. Counts were made through a compound microscope at 150X magnification for sternites and 660X magnification for wings, using an ocular reticle calibrated for a square approximately 0.1 mm to a side for the sternite, and 0.024 mm for the wing counts. One of the membranous wings and the abdomen of ten beetles of each type had been temporarily mounted in paraffin oil. One square area from the central portion of each of the five visible sternites, and five contiguous areas along the main axis of the wing (near the distal tip to avoid veins) were chosen, and in each the number of bristles or hairs was determined.

The results are shown in Table 1. From the data in the table it can be seen that, although the difference is not significant for the membranous wings, it is significant for each of the abdominal sternites, the number of bristles being increased two- or threefold in the mutant.

Table 1.Numbers of setae in normal and aureate abdominal

Sternites and wings in T. castaneum (N = 10)

Apparent abdominal sternite Normal aureate_____

1 9.4 + 0.2 30.6 + 1.42 10.7 + 0.4 27.4 + 1.73 9.7 + 0.4 28.3 + 1.54 10.6 + 0.3 27.4 + 1.1

5 9.9 + 0.4 23.7 + 2.1

Total 10.6 + 0.17 27.25 + .69

Membranous wings 10.84 + 2.36 11.10 + 1.66

Thus, from our observations, the aureate gene seems to affect only the sclerotized portions of the exoskeleton. Scanning electron micrographs (see research note elsewhere in this bulletin) show that there is a definite increase of cervical setae (at the anterior margin of the prothorax, particularly at the gular border) and on the antennae.

Most remarkable is the fact that the interommatidial bristles, which are single in normal beetles, are doubled under the influence of au.

This work was supported by USPHS grant GM-08942.

ALLEN, WILLIAM W.Division of Entomology and AcarologyUniversity of CaliforniaBerkeley, California

*Acarophenax tribolii – a parasitoid of Tribolium

In 1964 small mites were found infesting some of the Tribolium cultures at Berkeley. Since a general decline in the vigor of the cultures was associated with these infestations, it was necessary that some remedial action be taken. A review of theliterature revealed that these mites, belonging to the family Pymotidae, had been described by Newstead and Duvall (1918) as Acarophenax tribolii. In addition to the taxonomic description they gave a generalized account of the life history and habits of these mites. More recently Cross (1965) reviewed the family Pymotidae and summarized what was known about the life history of Acarophenax tribolii by referring back to the paper by Newstead and Duvall (1918).

The adult mites are found attached to the bodies of both adult and larval beetles. Commonly 10 to 12 adult mites may be found on the bodies of the beetles. There is some question whether the mites feed on the adults and larvae of Tribolium, or whether the relationship is merely phoretic.

After several days the adult mites drop from the beetles and seek out a Tribolium egg. The mites attach themselves to the eggs, and in a day or two suck out the contents. During feeding there is marked enlargement of the body of the female (physogastry), and all power of locomotion is lost. Complete development of the immature mites takes place within the body of their mother. There is good evidence that mating takes place before the adult females leave the body of the mother. Since development takes only three or four days and commonly there may be 12 to 14 progeny per mother, it is understandable why infestations can build up rapidly.

Since establishment of new Tribolium cultures from small numbers of adults observed to be free of mites was unsuccessful in eliminating infestations, it was obvious that more drastic action would be necessary to clean up the cultures. A number of tests were conducted to determine if certain chemicals could be found that would control the mites without having an adverse effect on the beetles. These tests revealed that there was selectivity in the insecticide endosulfan (Thiodan) and the acaricide Morestan when used in the flour at a rate of 300 ppm of the actual material. Because Morestan generally

is regarded to be of lower toxicity to insects it was selected for use in cleaning up the cultures.

In actual practice it was found that Morestan did have a detrimental effect on the beetles particularly when they were held at higher temperatures. This difference can be explained by the fact that the initial tests were run with adults and mature larvae rather than the early larval instars. Nevertheless, it was possible to use Morestan to help free the cultures of the mites. This was done by placing beetles in flour treated with 300 ppm Morestan and holding them at a temperature of 200C. After 10 days the beetles were removed from the treated flour and used to establish new cultures in clean flour. Through use of this treatment and greater care with the cultures it has been possible to hold the mites to a very low level.

Literature Cited

Cross, A. E. 1965. The generic relationships of the family Pymotidae (Acarina: Trombidiformes). University of Kansas Science Bull. 45 (2):275 pp.

Newstead, R. and H. M. Duvall. 1918. Bionomic, morphological, and economic report on the acarids of stored grain and flour. Royal Soc. Rep. of Grain Pests (War) Committee, 2:19-25.

BERCK, B.Canada Department of AgricultureWinnipeg 19, Manitoba, Canada

Further simplification of a bioassay method with Adults of T. confusum Duv.

A simplified bioassay method used by the author for screening and validation tests with fumigant gases was described in TIB 9 (2). The method employed 100 cc. all-glass syringes (1) as combined test cages and micro fumigation chambers.

In the current work with phosphine (3), it was found that not all the syringes that had been tested were uniformly gas-tight at all positions of the syringe piston. In a search for alternative leak-proof test chambers, 125 ml. Erlenmeyer flasks and 4 oz. medicine bottles fitted with gas-tight stoppers and septums were much cheaper, more durable and easier to clean and use than the syringes. They were used in a simplified bioassay method for the simultaneous determination of LD50 and LT50 values (4). Changes in CO2-02-N2 relationships in these miniaturized test chambers are readily monitored. The main advantages are that a wise range of gas concentrations and temperatures can be tested in a relatively small space, and insect mortalities can be readily assessed over a wide time span. Thus, it was possible to determine the LT50 of 0.002 mg. phosphine per litre air during a 14-day test with adults of T. confusum Duv.

Literature Cited

1. Berck, B. 1965. Sorption of ethylene dbromide, ethylene dichloride and carbon tetrachloride by cereal products. J. Agr. Food Chem. 13:248-254.

2. Berck, B. 1966. Simplified bioassay method with adults of T. confusum Duv. Tribolium Information Bull. 9:74-75.

3. Berck, B. 1967. Paper in preparation.

4. Berck, B. 1967. Paper in preparation.

DEWEES, A. A.Population Genetics InstitutePurdue UniversityLafayette, Indiana

*Sex differences in recombination values for Linkage group V of T. castaneum

In a study to establish the linkage relationships among the eye color mutants, ruby (rb) and maroon (m), and the jet (j) body color mutant of linkage group V in T. castaneum, it was noted that the recombination values were not equal for the two sexes. However, Lasley (1960, TIB, p. 14) reported evidence for equality of recombination in both sexes between jet and split in the same linkage group.

The phenomenon of unequal crossing over in reciprocal crosses was first pointed out by Sokoloff (1963, TIB 6; 1964) for markers of the seventh linkage group of T. castaneum (male rates higher than female rates) and was later studied by Johnson (1966) with reference to linkage groups IV and VII.

Preliminary tests (Dewees, 1965, TIB 8:72) indicated that rb was located about 27 units to the left of j near m. Due to the difficulties in classifying adult maroon beetles, a regular three-point linkage test of m, rb, and j ws not undertaken. However, a modified two-point linkage test of m and rb in the trans phase was set up with the jet gene included as a marker on the ruby chromosome. The progeny were scored in the late pupal stage as either wild type or mutant with regard to eye color and the recombinants (wild type) were then test mated to rb j/ rb j beetles to determine the most probable sequence of the three loci. From 87 single pair matings of m rb +/m rb + x + rb j/m + + , 60 wild type recombinants were recovered from a total of 8,085 progeny. In the reciprocal cross from 96 matings only eight out of 13,246 progeny had wild type eyes. The calculated recombination rates and their corresponding 95% fiducial limits are shown below.

Heterozygous Recombination rate Parent between m and rb 95% fiducial limit

Male .0148 ( .0113, . 0191) Female .0012 ( .0005, .0024)

Clearly, these rates are statistically different. The gene sequence was determined to be m – rb – j.

To further investigate this sex difference, regula two-point linkage tests of rb and j were conducted. Both cis and trans phases were examined in reciprocal crosses. Within each sex the recombination rates observed for the cis and trans phases were not significantly different at the 1% probability level, therefore the results presented below represent pooled estimates.

Heterozygous No. of Total No. of Recombination rates Parent matings progeny between rb and j + s.e.

Male 19 1974 .3283 + .0212Female 20 3196 .2118 + .0145

Here also it can be seen that the recombination rate in the males is significantly greater than the rate observed in females. Whether or not these two instances represent a general phenomenon for the fifth linkage group is not known, however further tests involving markers of this linkage group are being planned.

As for the possible mechanism responsible for sex differences in recombination rates, Sokoloff (1964) discusses evidence from other organisms that seems to indicate that such a phenomenon could result when the two sexes differ in the distribution of a single chiasma. However, an equally plausible explanation might be one in which there is a differential segregation of chromosomes in one sex, probably the female, such that the recombinant chromosome is more likely to be incorporated in the polar body during second division segregation thus lowering the number of recombinant progeny produced. This type of mechanism could very well account for the several cases in which male recombination rates are observed to be higher than female rates.

ERDMAN, H. E.Biology DepartmentBattelle-NorthwestRichland, Washington 99352

Population performance of single—and mixed—species of flour beetles

Knowledge on the interaction(s) among environment, population structure and inter-species competition is beeded to understand better species survival and population performance. Single—andmixed—species population of flour beetles were cultured at

250C, 290C and 350C and censused monthly for eight months prior to chronic gamma irradiation. Results of the radiation will be reported later.

Tribolium confusum Duval (“Chicago Standard”) and Tribolium castaneum Herbst (sooty) were sexed as pupae and held until sexually mature. Fifteen replicates of single species populations (begun with 20 pairs) and of mixed-species populations (begun with 10 pairs of both species) were censused as to numbers of larvae, pupae and adults. All live forms and eggs were returned to new food. Eggs were not counted and adults were counted as to species because of their color difference.

Population stability was achieved only at the high temperature. Adults’ forms were most abundant and showed least variability, probably due to their longer life relative to the other life cycle stages.

Fitness, measured on total number of life forms, indicated that Tribolium confusum was better adapted when not in competition at 290C; whereas Tribolium castaneum was better adapted at the temperature extremes. In mixed-species population, Tribolium castaneum was progressively less fit than Tribolium confusum. Numbers of total life forms per population replicate after eight months were greatest at 250C and least at 320C, indicating the temperature effect on population performance.

ERDMAN, H. E.Biology DepartmentBattelle-NorthwestRichland, Washington 99352

Ontogeny and X-radiation sensitivity of the flour beetle, Tribolium castaneum, mutant : sooty

Nonlinearity of radiation sensitivity during the life cycle reflects the differential sensitivities of dividing cells compared to nondividing cells and the degree of differentiation. This report defines the radiation sensitivities of the flour beetle, Tribolium castaneum (mutant: sooty) throughout its life cycle for specific stages which show developmental changes in somatic and gonadic tissues.

Approximately 100 individuals at each of nine different stages during the life cycle were X-rayed. X-rays were delivered at 1 kR/min by a generator operated at 30 ma, 250 kvp, 0.25 mm cooper + 1.0 mm aluminum filtration, and a 2.5 inch target-subject distance. Culture conditions were 30 + 10C and 70 + 10 per cent relative humidity.

Virgin adults were crossed after radiation. An exposure was considered as sterilizing if no progeny developed with 2 – 3 months and as lethal if individuals failed to mature or died within 3 – 4 weeks post eclosion. Exposures were performed at 1000 R increments, or in some cases, at 500 R increments.

The weakest link in the life cyle was the 1 – 3 hour old egg in which nuclei are rapidly dividing and undifferentiated.

The differential radiosensitivity between somatic cells and gonad anlagen diverged markedly during post-larval stages because:

(1) Germ cells remained at a radiosensitive stage in cell division, and(2) The increased differentiation of somatic tissue required greater exposures for

lethality.

ERDMAN, H. E.Biology DepartmentBattelle-NorthwestRichland, Washington 99352

Metamorphic and adult life span modifications of flour beetles. Tribolium confusum, X-irradiated as pupae

Adult life span in mammals and insects was decreased when immature stages were irradiated supposedly by induced mutations in somatic cells. When pupae are radiated one would expect any lethal effect on genetic material would be eliminated during metamorphosis into the adult, instead of occurring after the adult stage successfully developed. This investigation determined whether metamorphosis and length of life could be altered when flour beetle pupae were X-irradiated.

T. confusum Duval (“Chicago Standard” stock) pupae, age 25-26 days, were sexed and irradiated. The X-ray unit operated at 250 kv, 0.86 mm copper half value layer, 0.25 mm copper and 1.0 mm aluminum filtration. The anode was 2.5 in. from the subject and the apparatus delivered approximately 1 kr/min. Doses were 0, 1, 2.5, 5, 7.5, 10, 20, 30, 40, 50, 60 and 70 kr. Each of the five replicates at eah dose contained ten pupae of each sex in 10 g food. Deaths were noted twice weekly. Culture conditions were 29 + 10 C and 60-75 per cent relative humidity.

Lethality data for males and females were combined, because sex made no difference in flour beetles.

Emergence of adults from pupae given 1 or 2.5 kR was comparable to that of control. Metamorphic incompletion increased progressively for doses from 5 to 70 kR, but metamorphosis was not delayed. Partial metamorphosis occurred in some individuals given 5 kR and greater doses—that is, thorax and head were adult but the elytra, although pigmented, was pupal in size and position and the abdomen retained pupal characteristics. Some individuals given 30 kR and greater were unable to cast off their

meconium. Death probably resulted from intestinal obstruction and not from starvation as heavily radiated adults ingest.

Reduction in length of life of adults radiated as pupae was a sensitive indication of radiation damage. Mortality increased progressively with increased exposure.

Exact cause(s) of death remain unknown because nuclear effects were difficult to differentiate from cytoplasmic effects. Death was probably due to chemical alteration of the cytoplasm because pupae developed adult structures. Antimetabolites could result from radiation-induced alterations in the chemistry of pigmentation. Other biochemical processes continue from juvenile stages to the adult and interruption of such processes could result in adverse effects on metamorphosis and adult life span when pupae are irradiated.

FRANKLIN, I. R., J. CHANDRA AND A. SOKOLOFFDepartment of GeneticsUniversity of CaliforniaBerkeley, California 94720

Comparative studies with Tribolium. III. The productivity of T. castaneum and T. confusum on synthetic diet.

A synthetic medium described by Naylor (1964) will allow development of T.castaneum and T. confusum, but has been shown to be a poor diet, in terms of productivity, compared to “natural” diets, such as wheat and corn flours (Sokoloff, et al., 1966b). However, the productivity of both species is raised approximately tenfold in Naylor”s medium to which brewer’s yeast has been added, and is comparable to productivity in commonly used diets, such as wheat + yeast and corn+ yeast. Since yeast supplements are generally considered to be an important source of vitamins, it was considered desirable to examine the effect on productivity of varying the vitamin content of the Naylor medium. The vitamin mixture described by Naylor contains varying amounts of micotinic acid, thiamine, pantothenate, riboflavin, pyridoxine, carnitine, folic acid and biotin, and the level of this mixture is 134 ug/fram of medium. Five media were tested containing different concentrations of vitamin mixture, and for comparison a sixth medium containing 5% brewer’s yeast was used. Fifteen females and 10 males of each species were transferred eah day for four days into five grams of the test diets, and the adult progeny were counted during the following two months. Each species was replicated three times, and the average productivity replicate for the two species and the six diets as shown in Table 1.

Table 1.Supplement to Naylor’s medium vitamin mixture ug/gram diet

5%Species 0 50 100 200 400 Yeast

T. castaneum 1 39 70 47 40 605T. confusum - 86 91 102 79 391

While it is apparent that the vitamin mixture is necessary for development in each species, there is little difference between the four diets in which the vitamin supplement was added. The differences in productivity between the two species is comparable with the conclusions of Sokoloff, et al. (1966b) that T. confusum performs better than T. castaneum on “poor” diets, and worse on “good” diets.

It was suggested by Sokoloff, et al. (1966a) that the two species T. castaneum and T.confusum may differ in their requirements for certain amino acids. Various combinations of eight amino acids, including those suggested by the above authors, were added to the Naylor medium and the productivity of both species was measured. The following concentrations of finely ground crystalline amino acies were used (Table 2).

Table 2.Amino acid concentrations used in enriching the Naylor medium

Amino acid Concentration

alanine 2.5 mg/gram medium

aspartic acid 3.5 “ “ “

cystine 1.5 “ “ “

glycine 4.0 “ “ “

leucine 4.5 “ “ “

methionine 1.0 “ “ “

threonine 2.0 “ “ “

tyrosine 3.0 “ “ “

As before, 15 females and 10 males were transferred four times into five grams of fresh medium, and the total number of adults emerging in the subsequent two months were counted. Thirty-two diets were used, representing a 1l8 replicate of a 28 factorial

design. None of the main effects were significant for either species, as illustrated by the analysis of variance table (Table 3).

Table 3.Analysis of variance

Mean Square Source d.f. CS CF

Main effects 8 197.8 n.s. 586.0 n.s.Two factor interactions 20 203.9 n.s. 1,096.1 n.s.Residual 3 61.8 218.2

31

These experiments show that the vitamins and amino acids which were tested are not limiting the productivity of either species in Naylor’s medium, and the poor performance of T. castaeum and T. confusum is due to absence of other factors which are present in brewer’s yeast.

This investigation was supported by USPHS grant GM-08942.

Literature Cited

Naylor, A.F. 1964. Possible value of casein, gluten, egg albumin or fibrin as whole proteins in the diet of two strains of the flour beetle Tribolium confusum (Tenebrionidae). Can. J. Zool. 42:1-9.

Sokoloff, A., I. R. Franklin, L. F. Overton and F. K. Ho. 1966a. Comparative studies with Tribolium I. Productivity of T. castaneum and T. confusum in several commercially available diets. J. Stored Prod. Res. 1:295-311.

Sokoloff, A., I. R. Franklin and R. K. Lakhanpal. 1966b. Comparative studies with Tribolium II. Productivity of T. castaneum and T. confusum in natural, semisynthetic and synthetic diets. J. Stored Prod. Res. 1:313-324.

FUENTES, Ma. C. AND R.G. RUANOLaboratorio de Genetica de Poblaciones Instituto Nacional de Investigaciones AgronomicasMadrid, Spain

*Egg-laying rate of virgin females of Tribolium castaneum at different temperatures

To start quantitative genetics studies in connection with the genotype x environment interation, some tests were run to determine the variation of the egg-laying rate in virgin females of Tribolium castaneum when they are exposed to different temperatures.From some previous studies testing the egg-laying at 280 C, 320 C and 360 C during a four day period comprised between the seventh and the eleventh days after adult emergence, we observed that both 280 C and 360 C were not as favorable environments as 320 C when egg-laying is the character studied.

We ran two experiments testing egg-laying during consecutive periods of 48 hours. The first test started at 240 C going two by two degrees up to 420C and afterwards going in the same way down to 240C again. The second one started at 420C going down to 240C and upto 420C, also increasing or decreasing by two degrees in each 48 hour period. Temperatures greater than 420C begin to be lethal.

At the same time some controls were maintained at 330 C in both tests to evaluate changes due to microenvironment ariations through the testing periods, other uncontrolled effects and to take into account the decreasing effect of the females age after the first days of a greater lay. Such controls were females of the same age as the experimental ones.

Enough pupae from the “Consejo” strain of Tribolium castaneum were sexed to have 200 females emerging on the same day, at which time they were placed in individual vials. During the larval and pupal stage and until day 7 after adult emergence, they were maintained at a temperature of 330C and a humidity of 70% RH; humidity was not changed in any case during the experiments. At day 7 after emergence fresh medium was put in each vial and at that time 100 females were shifted to 240C conditions and 100 remained at 330 C. At day 9 eggs were collected by suction-sifting and counted, and the experimental females were changed to 260 C, leaving the controls at 330 C. The same procedure was followed every two days going up with the experimental temperatures until day 25, when experimental females were subjected to 420 C conditions. At day 27 and after egg collection, they were shifted to 400 C again and so on going down arriving at day 43 when they were placed in the 240 C conditions. The test finished at day 45 after egg collection and count.

The second test was run in the same way but starting with the 100 experimental females at 420 C and going down to 240 C and up to 420 C.

Table 1 gives for both tests the average egg-laying figures obtained.from the experimental and ontrol females at specified temperatures. Deviations from controls and corrected figures with the average for all the controls in both tests are also given. Table 2 includes the figures averaged for every temperature disregarding any possible influence or effects of the previous temperatures.

Table 1Mean Number of eggs produced by T. castaneum at different

temperatures (oC) in consecutive period of 48 hours

Table 2.Figures for each temperature, averaging data in Table 1

Testing Control Deviation Data Temperature at 330 C from adjusted to 0 C m m control controls*_______

24 5.71 13.21 - 7,50 5.1326 7.84 12.79 - 4.95 7.6828 8.68 11.50 - 2.82 9.8130 10.85 13.57 - 2.72 9.9132 12.80 12.73 + 0.07 12.7034 14.73 12.34 + 2.39 15.0236 12.91 13.06 - 0.15 12.48

38 10.17 12.53 - 2.36 10.2740 5.52 12.98 - 7,46 5.1742 1.58 11.43 - 9.85 2.78

*Standard error for corrected figures: + 0.92

In order to see the results more clearly, the corrected figures for both tests are plotted in gaph 1. Graph 2 presents four second order para-bolas adjusted to the points of graph 1 by the least squares method. Equations of such parabolas are:

Test 1, increasing temperatures: y = - 0.18 + 0.25 x -- 0.36 x2Test 1, decreasing temperatures: y = 0.60 + 0.16 x – 0.51 x2Test 2, increasing temperatures: y = 1.45 + 0.36 x – 0.62 x2 Test 2, decreasing temperatures: y = 0.70 + 0.58 x – 0.44 x2

Using the corrected figures it is concluded:

(a) In our strain, temperatures around 33 or 340 C are the optimum for egg-laying. Temperatures above or below those seem to be worse; the greater the distance from the optimum the smaller is the average egg-laying rate.

(b) Response to the same temperature does not seem to be the same when progressive temperatures are increasing or decreasing, having gotten an average of 8.90 and 9.29 eggs respectively. Test 1 (starting at 240C) has an average figure of 9.04 and test 2 (starting at 420 C) 9.15 eggs. In any case these differences do not appear to be significant, nor is the interaction of those possible effects. We reach the tentative conclusion when we see both data and graphics, that in general laying is greater with decreasing temperatures.

(c) It is interesting to note that even though the differences are not significant we are tempted to conclude that after the females have been submitted to a very high temperature the laying rate afterwards is larger, because the combination of test 2 (starting at 420 C) and decreasing temperatures (coming from 420 C) has the best effect. This could be explained by assuming that at 420 C the females experience greater stress retaining eggs which, after some days, they lay in excess.

GEISERT, P.Oak Park River Forest High SchoolOak Park, Illinois

Algae flour as nutrition for Tribolium confusum

Algae can be cultivated in heavy water resulting in fully deuterated species. In an attempt to determine the effect of deuteration on higher organisms I planned to use deuterated algae as nutrition for Tribolium confusum. As a preliminary study Scenedesmu obliquus grown in water culture was processed into a flour and fed to Tribolium to determine the feasibility of using this algae as deuterated nutrition for the beetle.

The preliminary survey used 10 adults/vial containing two grams of algae flour prepared by desiccating and grinding water grown algae. The amount of materials used was limited by the ability to produce the algae. Various concentrations of the algae flour were prepared using flour media to dilute the algae.

The populations were surveyed with the following results (see Table 1).

Algae as presently processed would not make a good “carrier” of deuterium for studies with Tribolium. The flour beetle should be able to support itself in a high concentration of algae before an evaluation of the effect of deuterium is attempted. A more carefully controlled evaluation of the effect of algae on Tribolium is presently being planned, and modifications are being made in the preparation of the algae flour.

I feel bacterial toxins were probably present in the previous algal preparations and future techniques will attempt to separate the algae from other microorganisms and their metabolic products.

Table 1

Algal concentration_______________________ Date

100% 60% 50% 40% 30% 20% 15% 10% 0%

ADULTS 5-4 10 10 10 10 10 10 10 10 105-12 8 9 10 10 10 10 10 10 95-19 1 8 8 9 9 10 9 10 86-2 0 7 6 8 8 10 9 10 76-14 0 6 5 8 8 9 7 10 87-5 0 4 5 5 7 13 11 11 9

LARVAE 5-4 0 0 0 0 0 0 0 0 05-12 0 0 0 0 0 0 0 0 05-19 0 0 0 1 0 0 0 0 26-2 0 0 0 4 3 5 6 6 156-14 0 0 1 1 2 11 7 8 67-5 0 5 0 10 12 8 6 14 10

EGGS 5-4 0 0 0 0 0 0 0 0 05-12 0 0 0 8 - 37 83 45 935-19 0 0 3 20 16 30 52 36 806-2 0 0 0 25 4 32 56 21 13

PUPAE Total count of pupae from 5-4 to 7-5

0 0 0 0 1 2 5 6 12

HO, FRANK K.Department of GeneticsUniversity of CaliforniaBerkeley, California 94720

*The use of vital dyes for marking Tribolium eggs In fresh and aged flour

The development of a technique for marking eggs with dyed flour (Rich 1956) has facilitated the measurement of oviposition and cannibalism rates in a population. This technique has been used advantageously by Sonleitner (1961), and Ho and Dawson (1966). In the studies of Rich, and Ho and Dawson, 0.5 per cent neutral red dye concentration was used. Sonleitner preferred to use only0.2 to 0.3 per cent dye concentration. For coloring the eggs, commercial lour is considered the most ready-to-use choice. If whole wheat flour is used, however, it should be sifted through a number 5 bolting cloth sieve. This insures the eggs to be thoroughly coated with the fine-colored

flour particles. To prepare the flour, dye should be mixed and ground into the flour, then sifted through the number 5 bolting cloth sieve and stored in a high humidity condition for at least a week before using. Marked eggs are obtained by allowing beetles to oviposit in the dyed medium. Eggs are removed from such egg farms using the number 2 or number 5 bolting cloth sieves.

In some population studies, more than two or three strains or species of eggs may be used in an experiment. The dyes for marking egg, other than the neutral red mentioned may be needed. For this purpose, the safety range of Bismarck brown y, methylene blue and methyl green in Tribolium studies has been observed. All these dyes are well-known stains which have been used in histology and bacteriology for many years.

In addition, the aged-colored flour and the fresh-colored flour were tested for a nutritive comparison. The fresh-colored flour in this study was prepared and stored in the incubator at 290 C, 60% R.H. with a cheese cloth cover over the bottles one week before using; the aged-colored flour was prepared and stored in the same manner, after which the cheese cloth was replaced with an air tight cover and stored in room condition for more than three years. T. cstaneum and T. confusum synthetic strain beetles, two weeks old, were used as the parents of the egg farms. In the colored flour testing series, five jars included one control and four colored flour for each species. After three days, 500 eggs were collected from each jar; 100 eggs were placed in each vial containing eight grams of colored or white flour. A total of 25 vials for each species were used. The cultures were introduced into an incubator maintained at 290 C and 60% relative humidity. The adults were censused 45 days later. These experiments were carried out in the same manner for both fresh and aged flour. The results are summarized in Table 1.

It is evident that no appreciable effect is produced when Tribolium are reared in the colored flour from egg through adult stages. The beetle emerging rate from five replicates of each colored flour was satisfactory. It is very interesting to know that flour aged more than three years can be used almost as effectively as the fresh-colored flour for both species.

Table 1Per cent of Tribolium adults emerged from 500 eggs in fresh colored,aged colored and commercial white flour (five replicates for each test)

T. castaneum T. confusumFresh Aged Fresh Aged

Flour flour flour flour

Neutral red 81.00 62.20 85.00 82.00Bismarck brown y 86.60 85.00 83.60 80.00Methyl green 93.20 81.00 82.00 67.50Methylene blue 74.40 80.50 84.00 76.00Control 79.00 82.10 78.60 80.50

It should be pointed out that the emerging rate of beetles in vials containing neutral red in the aged flour for T. castaneum, methyl green in the aged flour for T. confusum, methylene blue in the fresh flour for T. castaneum, and methylene blue in the aged flour for T. confusum, was slightly lower than the emerging rate of the controls.

The results indicate that the vital dyes used are not poisonous to Tribolium when being used for egg coloring at a 0.5 per cent or less concentration in the flour.

The observation also suggests that all the dyes used in this study give brightly colored, easily distinguishable eggs.

This work was supported in part by USPHS grant GM-08942.

Literature Cited

Ho, F. K. and P. S. Dawson. 1966. Egg cannibalism by Tribolium larvae.Ecology 47:318-322.

Rich, E. R. 1956. Egg cannibalism and fecundity in Tribolium. Ecology37:109-120.

Sonleitner, F. J. 1961. Factors affecting egg cannibalism and fecundity in populations of adult Tribolium castaneum. Herbst. Physiol. Zool. 34:233-255.

HO, FRANK K.Department of GeneticsUniversity of CaliforniaBerkeley, California 94720

*Identification of eye mutants in Tribolium larvae

The previous studies (Good, 1936 and Ho, 1960) indicated that the arrangement of the ommatidia (facets), the shape and size of the compound eyes, and the distance between eyes in the pupal and the adult stages can be used to identify the species of Tribolium castaneum and T. confusum. In the larval stage no compound eyes have developed. However, the setal map of the larva has been used advantageously for their species identification (Ho, 1967 in press).

Tribolium has been used for genetic studies only recently. At present, over 150 mutants are described, including 11 eye mutants in T. castaneum and nine in T. confusum (Sokoloff, 1966). To distinguish the wild type and eye mutant larvae, examination of their stemmatal characteristics has been considered a practical technique. In general, three types of stemmata (eyespot) are present in Tribolium larvae. In wild type and in some mutants with deforemed eyes (microcephalic, and Bar eye) the

stemmata appear black; in the eye mutants red, pink, eyespot, light eyespot, ruby spot and dirty pearl eye of T. confusum, they are brown. There are no visible stemmata in pearl, platinum eye, Microphthalmic, frosted, and chestnut eye mutants, probably because the lack of pigment renders these stemmatal structures invisible or because the stemmata are too small or absent. In the squint (sq) beetle no ommatidia form, with the result that the ocular diaphragm, unaffected by sq, forms in the pupa and lies exposed in its normal position (Sokoloff and Dawson, 1963: Sokolof, 1966). The sq gene apparently suppresses the formation of the stemmata as well, since these structures are not visible in the larva. In microcephalic adults, the eyes are variably reduced and they bear very few facets. In some cases, the eye on the side may be absent. If these beetles are selected, they produce produce progeny with both eyes missing (Sokoloff, 1966). Therefore sometimes, in a microcephalic stock, three types of larvae may be found: those with stemmata missing on either side, those with stemmata missing on both sides, and those with stemmata present on both sides. If the stemma is present, it is always black. The color of the stemmata in larvae usually corresponds with the color of the compound eyes of the resulting pupae and adults. The data are summarized in Table 1.

The stemmata are located near the antennae from the first instar through the early part of the last larval instar. They are paired and more or less fused, and are located on each side of the head. In the later part of the last larval instar, the stemmata migrate along the head margin to a position near the vertex, and gradually become lighter, finally disappearing when the larva nears pupation (Ho, 1961). In these last larval stages the mutant may not be distinguishable from wild type.

This work was supported in part by USPHS grant GM-08942.

Table 1.The type of stemmata of Tribolium larvae

Wild type or Type ofSpecies eye mutants Symbol stemmata

T. castaneum wild type + blackBar Be blackRed r brownPink pPK InvisiblePearl P invisibleIvory i invisibleSquint sq invisible

Microphthalmic Mo invisibleChestnut c invisibleplatinum eye pte invisiblemicrocephalic mc usually invisible;

if present, blackglas gl undetermined

(specimen not available)

T. confusum wild type + blackred r brownpink pK browneyespot es brownlight eyespot es lt brownruby spot rus browndirty pearl eye dpe brownpearl P invisiblefrosted fro invisibleruby rby undetermined

(specimen not available)

Literature Cited

Good, N. E. 1936. The flour beetles of the genus Tribolium. U. S. Dept. Agric. Tech. Bull. 498.

Ho, F. K. 1960. Discrimination between the pupae of Tribolium confusum.

Duv. And T. castaneum (Hbst.). Ann. Entomol. Soc. Amer. 53:280-281.

Ho, F. K. 1961. Optic organs of Tribolium confusum and T. castaneum and Their usefulness in age determination. Ann. Entomol. Soc. Amer. 54: 921-925.

Ho, F. K. 1967. Identification of Tribolium larvae by their setal characteristics.Ann. Entomol. Soc. Amer. (in press).

Sokoloff, A. 1966. The genetics of Tribolium and related species. Academic Press In., New York, 212p.

Sokoloff, A. and P. S. Dawson. 1963. Linkage studies in Tribolium castaneum Herbst. IX. The map position of antennapedia, squint, short elytra and elbowed antenna. Can. J. Genet. Cytol. 5:450-458.

HOWE, R. W. AND B. D. HOLEPest Infestation LaboratorySlough, England

*Susceptibility of Sitophilus granaries to moderate cold.

This experiment was performed following the failure in several successive years to establish populations of granary weevil in freshly-harvested wheat and barley stored in farm bins. Cultures were prepared by groups of 800 adult weevils on successive lots of 320 g of Manitoba wheat for one day at 250 C and 70% R. H. Each culture was split into 32 samples and stored at 250 C and at a predetermined age each sample was moved

into a room controlled at 150 C and left there for eight weeks. In all, 24 samples of every age from 1 to 39 days old at 250 C were exposed at 150 C and a similar number kept as controls. On return to 250 C the samples were sieved regularly and the fecundity of a sample of the emerged adults was measured for one week. Very young eggs and the pre-pupal stage seem to be susceptible to exposure at 150 C, a temperature close to the developmental threshold.

Age at % alive Age at % aliveExposure No. after Eggs per exposure No. after Eggs per Days emerged 6 weeks beetle days emerged 6 week beetle

1 119 98 8.1 11 687 98 6.8 2 187 99 9.9 12 754 98 8.3 3 360 99 8.1 13 691 99 8.7 4 463 98 9.4 14 607 98 7.5 5 651 94 7.8 15 622 91 6.3 6 669 99 9.0 16 592 89 6.8 7 717 100 9.4 17 568 70 3.6 8 662 99 8.1 18 512 63 3.9 9 608 99 8.3 19 462 64 2.4 10 625 99 7.9 20 404 44 1.5

Age at % alive Age at % aliveExposure No. after Eggs per exposure No. after Eggs per Days emerged 6 weeks beetle days emerged 6 week beetle

21 316 37 0.7 31 551 56 4.322 272 45 1.4 32 640 72 5.623 244 27 1.0 33 728 84 4.824 221 33 0.4 34 707 84 6.825 238 26 0.8 35 728 90 5.826 297 20 1.9 36 751 -- --27 353 30 3.1 37 731 -- --28 404 35 3.0 38 708 -- --29 499 50 4.3 39 723 -- --30 497 58 5.6 Control 804 100 8.4

Predominant stage in each group

1 -- 6 eggs 26 – 28 prepupa7 – 10 larvae 1 29 – 34 pupa

11 -- 14 2 35 – 37 adults in grain 15 -- 19 3 38 – 39 adults emerged

20 -- 25 4

LADISCH, ROLF K.Cancer Research UnitImmaculata CollegeImmaculata, Pennsylvania

LADISCH, STEPHAN K.Chemistry DepartmentThe Johns Hopkins UniversityBaltimore, Maryland

Quinoid secretions in Tribolium confusum

We have most recently perfected a micro-polarographic method for the quantitative determination of simple alkyl-parabenzo-quinones in the odorous secretion of the flour beetle T. confusum. The method is capable of measuring quantities of quinine a small as 1 x 10-4 mgs, making it an excellent tool for the study of quinines in individual specimens of insects—even in melanotic stink gland mutants msg (Sokoloff, 1964) which bear the chemicals at as low as the submicrogram level. The procedure also furnishes conclusive information on the minor amounts of hydroquinone that invariably appear to accompany the quinine in the insect secretion. Furthermore, data of electrochemical significance concerning the reversibility of the redox process and the extent of electron transfer can readily be obtained by evaluating the analytical evidence from these extremely small amounts of test substance. These special aspects, and details of our new method will be discussed elsewhere.

In essence, the beetle to be studied is crushed in 500 microliters phosphate buffer solution, pH 7, and the mixture is subjected to conventional direct current polarographic electrolysis. Light is carefully excluded from the sample to minimize possible photo-reduction. The resultant polarogram shows the amount of quinine (cathodic wave height), the amount of hydroquinone (anodic wave height), and the half-wave potential. The latter provides evidence on the identity of the insect chemicals in comparison with synthetic alkyl-parabenzoquinones, i.e., in this instance with 2-ethyl-1, 4-benzoquinone, and 2-methyl-1, 4-benzoquinone. We have used a Leeds and Northrup Polarotron P-40 and a Sargent Model III Polarograph. The sample holder for the Polarotron was redesigned with an electrolysis chamber of 250 microliters minimum capacity, and with a bridge containing de-aerated electrolyte.

Typical polarograms of the very small amounts of quinine and hydroquinone determined in mutants msg are depicted in Fig. 1. Computed quinone and hydroquinone values for both the wild-type and the mutant insects are listed in Table 1, for ten individual beetles in each case. Am average amount of 52.6 micrograms quinine per beetle was thus found for the wild-type culture which was several months old. This result is somewhat higher than that previously arrived at with a more indirect analytical procedure. The latter has disclosed progressive biosynthesis of quinine in T. confusum as the beetles grow to maturity, from zero in newborn adults to approximately 35 micrograms per beetle at the age of four weeks (Ladisch, 1965).

Fig. 1. Polarograms of quinoid secretions in melanotic stink gland mutantsT. confusum msg. Each curve refers to an individual insect fromThe culture. Data obtained in supporting electrolyte .05M alkali phosphate buffer pH 7,0, at 250 C. Drop time, 3.58 sec; mass flow Hg, 2.33 mgs/sec.Corrected for residual current.

Table 1.Quinoid compounds in T. confusum, micrograms/insect.

Tenspecimens each of wild-type and mutant cultures.(Q, Quinone; HQ, Hydroquinone) Wild-type culture mutant msg

Q HQ Total Q HQ Total

57.9 1.2 59.1 None 1.1 1.169.0 2.4 71.4 1.1 1.9 3.053.1 1.5 54.6 None 1.8 1.836.6 2.4 39.0 2.2 2.3 4.562.1 3.0 65.1 None 1.3 1.331.2 2.1 33.3 0.3 1.4 1.735.1 2.7 37.8 None 0.5 0.540.2 6,0 46.2 None 0.1 0.164.8 5.4 70.2 1.8 2.9 4.775.6 3.3 78.9 2.8 2.9 5.7

Average Q: 52.6 Average Q: 0.8Average HQ: 3.0 Average HQ: 1.6

The present data for mutants msg confirm the findings of Engelhardt, Rapoport, and Sokoloff (19650 with respect to the greatly reduced amount of quinine in these beetles. In contrast, hydroquinone was not detected in the mutants by these authors, who extracted the volatile compounds by passing a stream of air over the insects and condensing the sublimed matter in cold traps. This method would appear unsuitable to demonstrate hydroquinone in insects as this compound will not sublime under these conditions due to its low vapor pressure of a few microns Hg at the temperatures employed. Information on the vapor pressure of insect quinines may be found in a recent article by Ladisch and Suter (1965). Evidently, as is now shown, there is more hydroquinone than quinine in the glands of these beetles (Fig. 1, Table I).

Hydroquinone has not been previously detected in wild-type insects by Loconti and Roth (1953), Ladisch and McQue (1953), and Ladisch (1963; 1965), all of whom have used sublimation methods in the isolation of the beetle quinines. Sixteen hundredths of a microgram of hydroquinone per beetle was reported by Engelhardt et al. (1965) for wild-type insects. Yet, this compound was most likely a degradation product formed in vitro from the quinine after collection, and is probably not related to hydroquinone existing in the glands of the beetles. We have found inect quinines from T. confusum to convert photochemically with ease to hydroquinone. In aqueous solution at pH 7, and exposed to light of 750 foot-candles intensity for one hour, these quinines undergo complete reduction. The solution is stable when stored in darkness. We shall discuss this phenomenon in detail elsewhere. Employing the described direct method of analysis, we have now found an average of 3.0 micrograms of glandular hydroquinone per insect of the wild-type strain (Table 1).

The presently described analytical procedure is easy to perform and most reliable. It has proved to be of special benefit in our current study of quinoid agents which we have been able to detect in several genera and numerous species of the family Tenebrionidae.

Literature Cited

Engelhardt, M., H. Rapoport and A. Sokoloff. 1965. Science 150:632/Ladisch, R. K. 1963. Penna. Acad. Sci. Proc. 37:127; ibid. 1965. 39:3Ladisch, R. K. and B. McQue. 1953. Science 118:324.Ladisch, R. K. and Sister St. A. Suter. 1965. Penna. Acad. Sci. Proc. 39:42.Loconti, D. J. and L. M. Roth. 1953. Ann. Entom. Soc. Am. 46:281.Sokoloff, A. 1964. Can. J. Genet. Cytol. 6:259.

We thank Dr. Alexander Sokoloff, University of California, for a culture of mutants T. confusum msg. This study was supported in part by a grant from the Damon Runyon Memorial Ffund for Cancer Research. We shall preset a more comprehensive treatment of this subject, including potential environmental health aspects of quinoid toxicants from stored-food insects at the April 1967 annual meeting of the Pennsylvania Academy of Sciences.

LANGE, EUGENE L.Population Genetics Institute Purdue UniversityLafayette, Indiana

*Sexual dimorphism in the pupal setae of Tribolium

The setae of Tribolium larvae and pupae have been examined by several people. The variation that has been noted has dealt with specific differences (Mertz, 1961; Ho, 1960, 1964). No known intraspecific difference has been reported previously. This is a bit surprising considering the mutants affecting seta number and shape in Drosophila. The following is a description of a pupal sex difference in the position and number of setae in Tribolium castaneum Herbst.

On the posterior margin of the eighth sternite of the pupae are a series of setae, the pregenital setae (Fig. 1). The most lateral pairs are the long first lateral and the short second lateral. They are slightly lateral and cephalad of the genital lobes and are present in both sexes in this position. The second lateral is sometimes absent. Medially to these pairs of setae are the first and second medial setae. The second medial is the smaller and more sephalad of them. In males (left half of Fig. 1) both of the medials may be present although usually only the first medial is found. They are approximately equally spaced between the laterals. In females only the first medial is present, and it is about as far from the first lateral as the second lateral is.

Fig .1.Composite Male-female tribolium Castaneum showing pupal sex differences in setae

LERNER, I. MICHAEL AND NOBUO INOUYEDepartment of GeneticsUniversity of CaliforniaBerkeley, California 94720

*Regression of the sex ratio on maternal grandfather’s age

1961 Cavalli-Sforza proposed a method of estimating the spontaneous mutation rate for sex-linked lethal by the regression of the sex-ratio of still (a) and live-born (b) children on the age of the maternal grandfather at the time of the mother’s birth. The rationale of these methods was given by him in the original paper as well as in a 1962 publication. Both he and Krebhiel (1966) found significant positive regressions on samples of humans by method (a). For method (b) Cavalli-Sforza (1961) obtained, on material involving some 67,000 women, a negative regression which was not significantly different from zero. He demonstrated that (b) would be a less sensitive measure of the effect, and proposed a massive study on a scale sufficiently large to obtain significant results, The results have so far not been published.

Meanwhile, we have undertaken a similar study of our synthetic stock Tribolium castaneum and T. confusum with method (b). Two separate experiments with the first of the species, and a single with the second, containing ten replicates each (five males mated to 10 stock females per replicate) were performed. In them, daughters were obtained from males at monthly intervals (with some minor exceptions), and ten daughter per replicate were mated to ten stock males and permitted to produce pupae, which were then sexed. The age of the grandfathers spanned the period of 2 to 24 months, and any of those that died were replaced by males of the same age. All the stocks were maintained at 290C and 70% relative humidity. The total number of pupae examined was over 150,000.

The pooled data for the different experiments are presented in Table 1 separately for the two species. It is apparent from mere inspection that no linear regression is detectible in T. confusum. The data for T. castaneum are not as unequivocal, and hence a chi-square test was carried out:

Regression chi-sq. 0.70 for 1 d.f.Residual chi-sq. 20.23 for 20 d.f. Total chi-sq. 20.91 for 21 d.f.

The discrepancy in the total is likely due to rounding off figures in the calculation. There does not seem to be a significant negative regression, although the possibility of deviations from chance in some other manner is not excluded.

Thus it seems that the extent of our material bears out Cavalli-Sforza’s suggestion that method (b) is not very sensitive, so that on the basis of our data, no estimate of mutation rate is possible, They are, nevertheless, presented here, since if other attempts

at studying the same problem are made it may be possible to use them in combination with other material.

Table 1.Sex ratio and age of grandfather

Age of maternal T. castaneum T. confusumGrandfather at the No. of No. ofTime of mother’s pupae % pupae % Eclosion sexed males sexed males

2 3341 51.393 3883 49.704 5663 49.87 4750 50.575 5808 49.69 5658 48.306 5839 48.36 3899 49.817 2628 50.02 4067 49.798 6049 50.24 5252 49.959 6305 48.96 5371 50.1210 6761 50.39 4718 50.5911 5961 50.39 4876 51.3512 5659 49.48 4983 50.4113 6946 49.55 4587 49.8614 7803 49.97 2204 49.0915 6423 49.7616 5238 49.7717 5619 49.4718 2152 50.6519 4100 49.0220 1750 48.3421 1710 49.5322 1488 47.5123 666 48.65

24 1307 48.13______________________________ Total 99216 49.61 54248 49.99

Literature Cited

Cavalli-Sforza, L. L. 1961. Un metodo per la stima della frequenza di mutazioneNell’uomo: Risultati preliminari. Atti Assoc. Genet. Ital., 6:151-162.

Cavalli-Sforza, L.L. 1962. Demographic attack on genetic problems. The use ofVital and Health Stat. for Genet. And Rad. Studies (UN-WHO, N.Y.): 221-233.

Krehbiel, E.L. 1966. An estimation of the cumulative mutation rate for sex-linked Lethal in man which produce fetal deaths. Am. J. Human Genet. 18:127-143.

This work was supported in part by USPHS grant GM-08942.

LOSCHIAVO, S. R.Canada Department of Agriculture Research Station25 Dafoe RoadWinnipeg 19, Manitoba, Canada

Evaluation of an anti-feeding compound as a protectant against stored-products insects

An anti-feeding compound, 24055 discovered by American Cyanamid Company was initially reported to inhibit feeding by certain phytophagous chewing insects. This interesting property suggested that the compound might be exploited as a protectant against stored-products insects. Laboratory experiments were conducted to determine whether 24055 applied to the outer surface of cotton sacks prevented penetration and oviposition by flour beetles and grain beetles. The compound was used as a 25% wet-table powder in water and applied to empty sacks at different rates of application.

Adults of Tribolium confusum, Cryptolestes turcicus and Oryzaephilus surinamensis did not penetrate or lay eggs through the mesh of treated sacks filled with flour. They died or became moribund within one week of exposure. This compound was shown to cause mortality of flour beetles by volatile action and by contact action, the former having the greater effect. Its ability to protect packaged cereal products is currently being investigated. Its anti-feeding and insecticidal properties are being examined in relation to reproduction and mortality of treated adults.

LOSCHIAVO, S. R. AND L. B. SMITHCanada Department of Agriculture Research Station25 Dafoe RoadWinnipeg 19, Manitoba, Canada

The distribution of Oryzaephilus Mercator Fauvel in Canada

Since late summer 1966, we have received an unusually large number of reports of the merchant grain beetle, Oryzaephilus Mercator Fauvel occurring in packaged cereal products. This species had not been reported in Canada before 1952. It was recorded from Ottawa in 1952 and Vancouver in 1954.

This beetle superficially resembles the saw-toothed grain beetle, O. surinamensis and was considered by many workers to be a variant of the latter species. However, Howe (1953, 1956) and Slow (1956) confirmed Fauvel’s earlier finding (1889) that O. Mercator was a separate species. We know that the merchant grain beetle has frequently been erroneously identified as the saw-toothed grain beetle. Consequently, we suspect that the former species is distributed in Canada far more widely and for a longer time than indicated by the two reported records mentioned above.

The increasing frequency of reports of the merchant grain beetle suggests that it is becoming firmly established as a pest of stored products in households, retail stores, and storage warehouses in Canada. It has been found in flour, rolled oats, biscuit products, peanuts, walnuts, puffed rice, cake mix, chocolate bars, dried fruits and vegetables, flax seed, Oriental foods, and copra.

A survey is being conducted to determine whether this species enters the country on certin imported products from which it may spread to other food commodities via normal handling, shipping, and storing operations.

MCONALD, D. J. AND L. STONER (MRS.)Department of BiologyDickinson CollegeCarlisle, Pennsylvania

*Factors involved in the survival of Tribolium confusum populations

Flour beetle populations can persist for long periods of time in small volumes of unrenewed flour. One factor that may influence the longevity of a population under these conditions is the effectiveness of the self-limiting mechanisms that restrict population size, for it seems likely that a smaller population would be able to persist longer than a larger one. However, there may be other factors influencing longevity and these might be revealed by a close study of populations from genetically distinct strains that differ in several population characteristics. In these first experiments two strains were used, McGill black and a wild type (red) strain. One hundred single pair populations of each strain and another hundred populations of mixed pairs, one red adult and one black, were started in 5 gms of flour medium. These three population types are referred to hereafter as the R. B. AND H. (heterozygous) populations. The populations were censused at two-week intervals and since the medium was never renewed they eventually became extinct. Table 1 shows some of the data obtained during weeks 10 through 28 inclusive. Since the adult and egg numbers of the H populations fall between the R and B populations it is possible that these characteristics are determined by the body color genes which exhibit no dominance.

Table 1.Mean characteristics of red, black, and heterozygous populations

Population type Tests of Significance between

Red Heterozygous Black R and B R and H H and B Live 97.6 + 19.5 81.2 + 17.2 71.1 + 13.8 t = 11.0 t = 6.3 t = 4.5Adults P .001 P .001 P .001

Egg 20.5 + 7.3 28.7 + 10.0 42.6 + 13.6 t = 14.2 t = 6.6 t = 8.2

Numbers P .001 P .001 P .001

Weeks 37.4 + 6.5 45.1 + 5.4 39.9 + 5.6 t = 2.9 t = 9.1 t = 6.7Duration P .005 P .001 P .001

Since the H populations, although bigger than the B populations, survive several weeks longer, it is clear that factors other than the adult population size can influence survival in a depleted environment. However, within each population type there is a negative correlation between adult population size and duration. Table 2 shows this as well as the

Table 2.Correlations between adult numbers (A), egg numbers

(E), and weeks duration (D)

Population type Red Heterozygous Black TotalCorrelationcoefficient

rAE -.70 P .001 -.41 P .001 -.49 P .001rAD -.72 P .001 -.88 P .001 -.67 P .001rED .49 P .001 .37 P .001 .32 P .001

PartialCorrelationCoefficient

rAD.E -.97 P .001 -1.01 P .001 -.76 P .001rED.A -.02 P .10 .06 P .10 -.02 P .10rAE.D -.94 P .001 -.42 P .001 -1.46 P .001

fact that the egg number is apparently not a factor in determining duration. When the effects of the negative correlations between eggs and duration are not significant. Since the level of adult numbers is established early in the population’s life, it is possible that this level can influence the population’s chance of surviving a period of stress, generated by depleted resources. Since, in the present instance, a smaller population has a greater chance of surviving, the results suggest a way in which natural selection acting on the whole population as a unit might establish self-limiting population mechanisms. In another experiment, using the same strain, when the population reaches its peak number of adults at eight weeks, all the adults are discarded then and every fourth week thereafter. Table 3 shows some of the data. Again, for reasons not yet known, the H populations survive longest although not significantly longer than the B populations. Within each population type the total number of adults produced is positively correlated with duration. This can be interpreted to mean that populations differ in their efficiency in converting flour into beetles,

Table 3.Correlation between the total number of live adults produced (T),The duration of the population in weeks (D), ad the mean number

of adults at the peak period, weeks six and eight (P)

Means T tests Red Heterozygous Black R and B R and H H and B

Total liveAdults produced 166 157 148 .0001 .01 .005

Weeks duration 41 51 47 .005 .0001 P = .11

Peak adults 92 76 74 .0001 .0001 P = .38x 6-8 weeks

Total Correlation coefficient Red Heterozygous Black

rTD . 43 P .01 .31 P .01 .54 P .01

rPD - .043 P .1 -.24 P .02 .05 P .1

rTP .39 P .01 .06 P .1 .34 P .01

PartialCorrelationCoefficient

rTD.P .528 P .01 .345 P .01 .593 P .01

rTP.D .519 P .01 .157 P .1 .443 P .01

rPD.T -.305 P .01 -.287 P .01 -.214 P .05

and the more efficient populations have an increased chance of surviving. However, the rate at which this conversion takes place should have some effect on duration. Those populations producing more beetles early in the population’s life should exhaust the environment sooner. Consequently, a negative correlation might be expected between the peak number produced at weeks 6 ad 8 and duration. However, since the peak number is part of the total number, and the total is positively correlated to duration, the negative relationship between peak number and duration only emerges in the partial correlation coefficients. Other characteristics, such as the mean weight of the individuals in the population, measured as the weight of pupae, appeared to have no effect on population survival.

MUSGRAVE, A. J. AND S. B. SINGH

Department of ZoologyUniversity of GuelphGuelph, Canada

*Cytochemical studies of Sitophilus granaries mycetomes

Cytochemical studies of the outer membranes and chromatinic material in the inherited, supposedly symbiotic mycetomal micro-organisms of Sitophilus granaries have revealed the presence of considerable amounts of DNA occurring as either consolidated dots or a spongy network. There is also evidence that, at times, the organisms are surrounded by cell walls.

MUSGRAVE, A. J. AND I. GRINYERDepartment of SoologyUniversity of GuelphGuelph, Canada

*Mycetomes as possible obligate symbiotes of Sitophilus

The peripheral membranes of the micro-organisms of the mycetocytes of adult midgut caecae and of larval mycetomes of Sitophilus granaries (L.). GG strain, have been examined with an electron microscope. The majority of the mycetocytes were depleted of intracellular organelles but contained large numbers of mycetomal micro-organisms, most of which exhibited only one peripheral membrane. Some mycetocytes, however, had well-developed ultrastructure and harbored mycetomal micro-organisms which showed two peripheral membranes, namely a cell wall and plasma membrane. Intermediate conditions also occurred.

It is suggested that the absence of host-provided membranes around the micro-organisms categorizes them as obligate symbiotes.

O’BRIAN, DENNIS M., ANDREA LEVINE ANDFRANK F. KATZDepartment of BiologySeton Hall UniversitySouth Orange, New Jersey 07079

A quantitative approach to the feeding of Hymenolepis diminuta Eggs to the flour beetle, Tribolium confusum

All stock cultures were maintained at 300 C, 92 to 96% relative humidity and 12L:12D light conditions. The medium consisted of 95% bleached Gold Medal Wondra® flour and 5% National Active Dry ® yeast and was changed at least every two weeks. Eggs

collected from apopulation of beetles four to six weeks following their eclosion were used to set up the experimental groups which consisted of virgin adults maintained in individual vials throughout the duration of the experiment.

At six to eight weeks of age, the experimental beetles were starved for a period of five to six days prior to being allowed to feed for a 48-hour period on a known number of eggs. The eggs were obtained from five to ten freshly obtained terminal gravid segments of H. diminuta and embedded in a 17% gelatin, 2% sucrose medium. The solvent for preparing the gelatin and sucrose consisted of three parts distilled water: one part supernatant of a mammalian saline extract of freshly-obtained homogenized gravid segments. This extract was centrifuged for a period of five minutes at 1800 rpm and the supernatant was removed. The eggs and diluted supernatant was added to the gelatin sucrose mixture just prior to gellation. After examination of the surrounding feeding area and feculae for eggs or egg shells, it appeared that no eggs or embryos were passed through the digestive tract during the 48- hour feeding period, and the 24-hour period after replacement on the regular diet. Thus, it was possible to determine the number of eggs consumed per beetle by subtracting the number remaining at the end of the 48 hour feeding period from the initial number. Selective staining with Trypan Blue (0.02 to 0.04% in mammalian saline) in addition to observational studies indicated over 90% viability of the eggs freshly obtained from gravid segments and those kept for the 48-hour period in the gelatin medium. In addition, eggs maintained for 48 hours in the gelatin medium did not take up the stain at a faster rate than freshly obtained eggs, indicating no decrease in the relative viability of the eggs. The majority of beetles that fed consumed between one and 60 eggs, 21 to 29% becoming infected, with a mean of 1.5 to 3.0 cysticercoids per infected beetle.

ONYEARU, A. K.University of St. AndrewsDundee, Scotland

The behavior and biology of flour beetles, genus Tribolium as studied in laboratory gradients of temperature and humidity

Under the supervision of Dr. F. L. Waterhouse, Natural History Department of Queens College, University of St. Andrews, Dundee, Scotland, Dr. A. K. Onyearu has recently completed a three-year research program on the behavior and biology of Tribolium spp. including some genetic strains and geographical populations of Tribolium castaneum, Hbst.

The laboratory studies were mainly conducted in a gradient environment. The equipment for this has been described earlier (Graham, Onyearu and Waterhouse, 1965).

Tribolium populations employed in these investigations include:

1. Seven geographical stocks of Tribolium castaneum obtained from:

Umuahia - (Eastern Nigeria)Onadam - (Western Nigeria)Kano - (Northern Nigeria)Kenya - (East Africa)Kingston - (Jamaica, West Indies)Rangoon - (Burma)Tokyo - (Japan)

2. Three genetic strains of Tribolium castaneum namely:

Black, Pearl and Mahogany. The last is a recent find described elsewhere. (Onyearu and Graham, in press).

3. Laboratory strains of :Tribolium confusum duValTribolium anaphe Hint.Tribolium madens Charp.Tribolium destructor Uytt.

The following aspects were covered in these investigations:

1. Adult distributions in the gradient environments.2. Oviposition as a functional aspect of adult orientation on the gradients of

temperature and humidity.3. Developmental periods in gradient environments as well as under the uniformly

controlled conditions of the C. T. Room.

Results of these investigations have served to emphasize the need for relating experimental conditions and experimental animals to those occurring in the field to which the original ecological problems relate. Details of these studies formed the theme of a Doctoral thesis recently submitted to the University of St. Andrews (Onyearu, 1966). Further publications now under preparation are to be made elsewhere.

Literature Cited

Graham, W. M., A. K. ONYEARU AND f. l. Waterhouse. 1965. Temperature and moisture gradient equipment. Can. Ent. 97 (8):880-996.

Onyearu, A. K. 1966. Laboratory studies on the behavior and biology of flourBeetles (Genus Tribolium), in gradient environments, with specialReference to species and strain comparisons. Ph. D. Thesis, UniversityOf St. Andrews, Scotland.

Onyearu, A. K. and W. M. Graham. 1967. Life history studies of two body colorMutants of Tribolium castaneum Hbst. (Coleoptera Tenebrionidae). (In press).

PEASE, R. F. W., T. L. HAYES AND A. SOKOLOFF

Electron Research Lab., Donner Lab.,Lawrence Radiation Lab., Department of GeneticsUniversity of California, Berkeley

The scanning electron microscope

The scanning electron microscope utilizes a fine, moving beam of electron (diameter as small as 50 A) to probe the specimen for a variety of kinds of physical, chemical and electrical information. As the scanning beam is swept across the specimen, the electron beam of a standard cathode ray tube is driven in synchrony with it and the brightness of the cathode ray beam is modulated by the signal from one of several detectors. The secondary radiation produced by the interaction of the scanning electron beam is not imaged but is only used to convey information about the particular point being bombarded. The image is a result of the synchrony between the scanning beam in the microscope column and the beam of the cathode ray tube. The resulting 1:1 correspondence of points on the specimen with points on the face of the cathode ray tube serves to identify the location of the information seen by the detectors. The separation of information and localization allows many different kinds of information to be gained while utilizing the localization possible with an electron beam. Different kinds of secondary radiation are produced by the interaction of the electron beam with the specimen and each carries a particular kind of information. For example, visible light may be produced and give information about the molecular structure of the biological material itself or the location of light producing stains within the specimen. Operated in this mode, the scanning electron microscope could be described as a high resolution fluorescence microscope (Pease et al. 1966a). Secondary electrons are another form of radiation produced by the interaction of the scanning beam with the specimen. Since production of these secondary electrons is a function of the angle between the scanning beam and the surface of the specimen, stereoscopic information can be obtained. The scanning electron microscope operated in this mode can be described as a high resolution stereoscopic microscope (Hayes et al. 1966). In addition, ultraviolet radiation, characteristic X-rays, backscattered electrons, energy loss electrons and specimen current are all produced in the specimen and might be used to build up the scanning electron microscope image. The principles of the instrument and several of its modes of operation have been recently reviewed (Oatley et al. 1965).

The present paper deals with an attempt to visualize living Tribolium by using the scanning electron microscope in the secondary electron mode of operation. There are several advantages both physiologically and morphologically if the specimen can be viewed while it is living. Morphologically, the reduction in the possibility of artifacts and the simplicity of sample preparation are important. Physiologically, the possibility of observing “on-going” processes would be most valuable, as well as utilizing the tiny electron beam as a micro-radiation source in radiobiological studies (Pease et al. 1966b).

Literature Cited

Pease, R. F. W. and T. L. Hayes. 1966a. Nature 210:1049.Hayes, T. L., R. F. W. Pease and L. W. McDonald. 1966.

Lab. Invest. 15:1320-1326.Oatley, C. W., W. C. Nixon and R. F. W. Pease. 1965. In Advances in Electronics and Electron Physics, 21:181-247, Academic Press Inc., New York.Pease, R. F. W., T. L. Hayes, A. S. Camp and N. M. Amer. 1966. Science 154:1185-1186.

REYNOLDS, ELISABETH M. AND BARBARA M. RUNDLEPest Infestation LaboratorySlough, Bucks, England

The response of larvae of Trogoderma granarium Everts to – 100 C

Some preliminary observations of the response of fully grown larvae of Trogoderma granarium Everts to low temperatures have been made by placing samples of approximately 50 larvae in a deep freeze cabinet at – 100 C for various periods of time. The larae were taken directly to the cabinet from cultures maintained at 300 C and after exposure were placed on food and returned to 300 C. The larvae were then examined, initially at weekly intervals and then fortnightly or three weekly, for six months.

Periods of exposure to -100 C of from two hours to 14 days were tested. Exposures of up to 3.5 hours appeared to have no lasting effect on the larvae. From 7.5 to 72 hours larval mortality increased to 96%. No larvae survived exposures of five days or longer.

All the exposures tested immobilized the larvae. On their return to 300 C some larvae remained immobile for a long time. Such larvae displayed no sign of damage and it was impossible to tell from their appearance whether they would eventually die or resume development. Dead larvae became desiccated and dark in color.

After short exposures some larvae were still immobile at the end of the observation period. Increase in exposure not only increased mortality but also appeared to decrease the period of immobility before the larvae could be seen to be dead.

These findings are demonstrated in Table 1 below. In this summary which combines the results of three separate tests some slight approximations in dates have been made in order to divide the observation period into the five intervals shown. Results for exposures giving less than 100% kill have been combined into groups.

Table 1.Numbers of survivors and dead larvae found during the observation

Period at 300 C following exposure to – 100 C.

Observation period, days from start Total insects found

Length of of exposureExposure Stages atTo -100 C found 0-30 31-60 61-90 91-125 126-166 Adults end dead

0, 1.5 adults 129 3 2 0 0 3.5 h dead Larvae 2 1 0 1 0 134 1 4

7.5-41 h adults 33 5 0 1 8 dead larvae 91 65 13 5 4 47 7 178

48-72 h adults 8 3 1 4 1 dead larvae 170 30 20 2 0 17 4 222

5 days dead larvae 47 2 0 1 0 0 0 50

7 days “ 36 8 2 1 0 0 0 4710 days “ 37 17 1 0 0 0 0 5512 days “ 38 10 1 0 0 0 0 4914 days “ 43 7 0 0 0 0 0 50

SCHMITZ, THOMAS H. 1 AND DUWAYNE C. ENGLERTDepartment of ZoologySouthern Illinois UniversityCarbondale, Illinois

Allelism of “mottled” (mt) and “melanotic stink glands” (msg) in Tribolium castaneum

Two mutations of similar phenotype have been reported, “melanotic stink glands, “msg (Sokoloff and Hoy, TIB 8:55-56), and “mottled”. Mt (Englert, TIB 9:59-60). Both of these mutations are affected by apparent deposition of polymerized darkly pigmented secretions composed of ethylquinones in the region of the stink glands.

1 Present address: Population Genetics Institute, Purdue University, Lafayette, Indiana.

Both mutations are inherited as autosomal recessives exhibiting incomplete penetrance and variable expressivity. Under ordinary laboratory conditios “mottled” exhibits an average penetrance of 63 per cent. In the stock received from the Berkeley Stock Center, msg was found to exhibit a penetrance of approximately 60-70 per cent under our laboratory conditions. However, the viability of the stock ws less than good. Linkage tests of

the two had indicated a relationship with linkage group III (includes “black”, b). Therefore, a test for allelism utilizing single pair matings between the two stocks was conducted. From 29 single pair matings, only 16 of 178 beetles (8.9 per cent) exhibited what ould be called the mutant phenotype.

A second test was conducted, this time utilizing mass matings in an attempt to increase productivity. A total of 464 progeny were examined, 17 of which exhibited the mutant phenotype (3.7 per cent). Neither of these tests could be considered conclusive, so to further establish allelism, the msg stock was crossed to the Purdue wild foundation stock to give a similar genetic background to that of mt. Mutant progeny from F2 segregants were used to start the “new” msg stock.

Beetles from the reconstituted msg stock were crossed with mt beetles to again check for allelism. The results revealed only 3 of 132 beetles which were of the “mutant” phenotype (0.8 per cent), indicating that similar genetic background did not increase the penetrance of the “mutant” phenotype. Thus, in the classical sense of allelism the two mutants cannot be considered to be allelic.

SHAW, DAVID D.Pest Infestation LaboratorySlough, Bucks, England

The chromosome numbers of some stored product Coleoptera

Species Karyotype

DERMESTIDAE

Dernestes nacykatys 8 II + Xyp“ “ 8 II + Xy1y2“ “ 8 II + Xy1y2y3 JOHN 7 SHAW 1966

D frischii 8 II + XypD frischii 8 II + Xy1y2D ater 8 II + XypD haemorrhoidalis 8 II + XYD lardarius 8 II + XypD peruvianus 8 II + Xyp

Trogoderma parabile 9 II + XypT. glabrum 9 II + XypAnthrenus verbasci 8 II + XypA. Flavipes 8 II + Xyp

OSTOMATIDAE

Tenebroides mauritanicus 11 II + Xyp SHAW (Unpublished)

TENEBRIONIDAE Alphitobius diaperinus 9 II + XOTenebrio molitor 9 II + XypT.obscurus 9 II + XypGnathocerus cornutus 9 II + Xyp

SHAW, DAVID D.Pest Infestation LaboratorySlough, Bucks, England

Sex chromosome variation in D. maculates and D. frischii

Five cultures of Dermestes maculates were available for cytological examination. The individuals from stocks originating in Australia, South Africa, Nigeria and Sudan all showed a standard Xy parachute sex bivalent but all the individuals examined from a Indian culture were characterized by multiple y’s. In 32 out of 33 individuals examined, two y chromosomes were present leading to the formation of a sex multiple of three at meiosis. This multiple shows regular meiotic behavior giving a consistent X-2y segregation with no observed deviation.

In a single individual of the Indian strain, three y chromosomes were found and here the meiotic behavior was less regular. At first metaphase, two principal patterns of orientation were observed.

First, and most frequently (35 out of 40 cells scored), the three y’s co-orientated with the single X giving a regular X-3y segregation. In five other cells one of the y’s was orientated to the same pole as the X giving an Xy-2y pattern of segregation. The consequences of this irregular behavior were seen at second division for in addition to (8 + X) and (8 : 3y) cell types, (8 + X + y) and (8 + 2y) cells were also seen.

Five of the 25 males of D. frischii examined also turned out to be the Xy1y2 type.

Two possible explanations can be offered to account for these multiple y variants. Either they represent a polymorphism which exists in nature or, alternatively, we are dealing with a system of supernumerary y-chromosomes which have arisen in culture presumably as a consequence of inbreeding coupled with reduced competition.

A 3y strain of D. maculates has now been selected and it is hoped to isolate females which may possess a y-chromosome due to the irregular behavior of the 3 y’s at

meiosis. (In conjunction with Dr. B. John, Department of Genetics, Birmingham University, England,)

SOKAL, ROBERT R.Department of Entomology The University of KansasLawrence, Kansas

*The effects of synchrony of egg batches on fitness characters and competition in Tribolium castaneum.

In a previous study (Sokal and Karte, 1964) the effects of density on three characters affecting fitness (survival to adulthood, dry weight of adults, and length of developmental period) were described for ++, +b and bb individuals in pure culture and in mixed cultures at varying proportions. It was believed that these findings would help explain the results of a selection experiment (Sokal and Sonleitner, 1965; a more extensive manuscript is in preparation) in which by 10 generations the frequency of b increased from 0.25 to 0.53, other cultures started at gene frequency 0.50 had increased to 0.58, while yet othes started at 0,75 had maintained themselves approximately at this value. Analysis of the data revealed, however, that the results of the earlier study differed in two important details from those of the selection experiment. In the study of Sokal and Karten (1964) black had consistently higher or at least equal survival to adulthood when compared to ++, while in the selection experiment the wild type strain invariably had appreciably higher survival than bb. Also developmental period of bb was less than that of the wild type in the experiment of Sokal and Karten (at least at the lower densities), while in the selection experiment, the black strain developed considerably slower than the wild type strain (bY as much as 19 days).

Study of these results and another experiment reported later in this issue (Sokal, 1967) ruled out genetic differences between the original stocks and the selected strains as responsible for the differences in survival and developmental period. Another possible cause of the divergent results of the two sets of experiments is that Sokal and Karten used egg batches obtained from 4-hr egg collections while the selection study was based on 3-day egg yields. It is assumed that the latter egg batches are les synchronous and may therefore be responsible for differences in survival and developmental period. The present paper tests this hypothesis.

The materials and techniques employed are identical to those reported in Sokal and Karten (1964). The design differed in that only two densities, 20/g and 100/g were set up with pure cultures of ++, +b and bb, and with mixed cultures representing Hardy-Weinberg proportions for gene frequencies 0.1, 0.5 and 0.9 of black. For each combination of conditions 4-hr as well as 3-day eggs were tested. Replication for each experimental condition was four vials for the pure cultures and 0.5qb, and eight vials for

the other gene frequencies at density 20/g, while at density 100/g the respective replications were three and six vials. The entire experiment was repeated four times.

Survival to adulthood: The overall survival not broken down by genotype is shown in Table 1. The 4-hr data compare well with previous results by Sokal and Karten (1964) and Sokal and Huber (1963). Percentages of survival at densities 20/g are higher than at 100/g, even for the ++ cultures which previously did not show this trend. At density 20/g the 3-day egg batches did not differ from the 4-hr vials in per cent survival, but at 100/g they are significantly lower than the same eggs at density 20/g and also the 4=hr cultures at density 100/g. The lowest single per cent survival is in the 3-day cultures of ++ which dip to 49.61%.

Table 1. Overall adult survival at different gene frequencies, and two densities For 4-hr and 3-day egg batches

Gene frequency

Egg batch Density 0.00 0.10 0.50 +b 0.90 1.00

4 hrs 20/g 81.98 79.84 85.78 87.97 83.20 85.31 100/g 71.01 64.39 76.65 60.24 77.68 71.21

3 days 20/g 83.90 83.33 80.26 86.72 78.25 86.25 100/g 49.61 55.43 60.75 62.85 63.76 66.79

Figure 1 illustrates the survivorships as percentages of input (averaged over the four experiments) for pure and mixed cultures at the three gene frequencies, two densities and for the two types of egg batches. Although the details of this figure differ somewhat from corresponding Figures 1 and 4 in Sokal and Karten (1964), the general trends are identical, showing heterozygous superiority under most conditions and a trend toward higher survival of wild type with increasing gene frequency of black at the highest density. Some instances of genetic facilitation are again demonstrated. However, with respect to the main feature of the experiment, the effect of synchrony of the egg batches on survival, no apparent difference can be illustrated. This is borne out by a factorial analysis of variance which shows only density and replication as significant main effects. Most significant interactions involve replication as one of the factors, the outcomes of the several experiments having fluctuated considerably.

Dry weight of adults: These results (not illustrated here) are quite comparable to the findings of Sokal and Karten (1964). Again, weights are in the relation +b ++ bb. This relation is maintained at both densities and for both types of egg batches. Weight is not affected by synchrony of egg batches. Beetles at density 20/g are considerably heavier than those at 100/g.

Length of developmental period: This variable is affected by density and differs considerably among genotypes. Table 2 shows the relations among developmental periods of the three genotypes in this experiment and in that of Sokal and Karten. Note the reversal in relationship between ++ and bb for the two densities of the 4-hr cultures. However, in the 3-day egg batches, patterned after the selection experiment, this reversal does not occur. Thus, the 4-hr cultures actually come closer to the bb ++ pattern of the selection experiment (based on 3-day egg yields at high densities) than do the 3-day cultures. Mean developmental period was apparently affected markedly by the synchrony of the egg hatch, yet when mean hatching period, the time from the start of the experiment until the hatching of each individual egg, was calculated it was seen that the increase in developmental period in the 3-day eggs could be accounted for by their greater mean hatch time.

Table 2.Relation among developmental periods of the three genotypes.

Egg batchDensity 4 hours 3 days

20/g This study ++ bb = +b ++ = bb = +bSokal & Karten “64 ++ bb +b

100/g This study +b bb ++ ++ +b bb Sokal & Karten “64 bb +b ++

More informative than means are cumulative frequency distributions of emerging adults of the three genotypes shown in Figure 2. Figures 2a and 2b are representative of the general findings. They illustrate density 100/g of the 3-day batches in pure culture and at gene frequency 0.9, respectively. It is clear from these graphs that the ++ strain lags consistently behind the other two, which for most of the experimental conditions produced coincident curves, or if they did separate, showed the bb to be slightly ahead of the +b. This relationship (of ++ lagging behind bb) was also found in separate, unpublished experiments carried out by F. J. Sonleitner in our laboratory. Notice how in the pure strains (Figure 2a) the emergence patterns are much more diffuse and development takes longer than in the mixed strins at gene frequency 0.9. This culture exhibits genetic facilitation, with all three strains developing faster and closer together as shown by the steeper slope of the curves. Under only one condition (100/g of the 4-hr egg batches in pure culture; see Figure 2c) was ++ not the slowest strain.

Conclusion: While the relations described above have considerable interest, they do not demonstrate any major effects due to greater or lesser synchronization in egg batches and are thus not able to explain the differences between the early results of Sokal and Karten (and now also the present results) and those observed in the selection experiment of Sokal and Sonleitner.

Contribution No.1351 from the Department of Entomology, The University of Kansas. This is paper No. 5 in a series on the ecological genetics of Tribolium. Numbers 1 through 4 are listed at the end of paper No. 6 (Sokal, 1967, in this issue of TIB). This research was supported by the National Science Foundation under grant GB-2170 and by a Public Health Research Career Program Award (No.3-K3-GM-22, 021-O1S1) from the National Institute of General Medical Sciences. The technical assistance of Miss Yu-Jen Chen, Mrs. Maxine L. Howe and Mrs. Cornella B. Tollefson are very much appreciated.

Literature Cited

Sokal, R. R. 1967. A comparison of fitness characters and their response to density inStock and selected cultures of wild type and black Tribolium castaneum, TIB 10: 142-147.

Sokal, R. R. and I. Huber. 1963. Competition among genotypes in Tribolium castaneumAt varying densities and gene frequencies (the black locus). Genetics 49:195-211

Sokal, R. R. and F. J. Sonleitner. 1965. Components of selection in Tribolium (Coleoptera) AND HOUSEFLIES. Proceedings of the XII International Congress of Entomology. 274-275.

Figure 1. Adult survival expressed as percentages of egg input averaged over the replicates and experiments of the study and shown for the two types of egg batches, two densities and three gene frequencies employed. Leftmost column with noncontiguous bars represents the results of rearing the beetles in pure culture. Hollow bars represent the ++ genotype, hatched bars +b and black bb.

SOKAL, ROBERT R.Department of EntomologyThe University of KansasLawrence, Kansas

*A comparison of fitness characters and their responses to density in stock and selected cultures of wild type and black Tribolium castaneum.

The wild type and black control strains of a selection experiment (Sokal and Sonleitner, 1965; a more extensive manuscript is in preparation) differed in two important respects from the stock cultures from which they were derived. Invariably the wild type strain had appreciably higher adult survival than the bb cultures, this difference increasing at the higher densities. By contrast, experiments with the stock cultures carried out by Sokal and Karten (1964) and Sokal (1967) had shown that black had consistently higher, or at least equal, survival to adulthood when compared with ++. However, these experiments were carried out in 8 g of flour in 6-dram shell vials, while the observations in the selection experiment had been made in 40g of flour contained in half-pint Mason jars.

A second discrepancy between the two types of experiments is that the developmental period of bb was less than that of the ++ strains under all but one set of conditions in the stock cultures, while in the selection experiment bb took consistently longer to develop than ++.

The purpose of the present experiments is to delineate more sharply the nature of the differences in survival to adulthood, dry weight of adults, and length of developmental period between the controls of the selection experiment and the stock cultures from which they were derived. These comparisons were carried out under conditions of the earlier experiments by Sokal and Karten (1964) and Sokal (1967), i.e., in 8 g of flour in shell vials. While an analysis of these differences is of primary importance for an under-standing of the results of the selection experiment, the findings are here interpreted in terms of the treatment to which the controls in the selection experiment had been subjected and are of some general interest from this point of view.

The materials and techniques are identical to those reported in Sokal and Karten (1964). However, only pure strains ++ and bb were tested at four densities, 5/g, 20/g, 50/g, and 100/g. The two strains were the standard UPF wild type and black stock cultures employed in work in our laboratory (see stock list) and the ++ and bb controls from the RSE selection experiment (Sokal and Sonleitner, 1965). The controls were taken from generations 25 and 26, respectively, of the second replicate for these two strains. The eggs for these experiments were obtained during a 4-hour period. Replication for each experimental condition was 10 vials at density 5/g, 4 vials at density 20/g, and 3 vials each at densities 50/g and 100/g. The entire experiment was repeated two times.

Survival to adulthood: Survival as percentage of egg input is graphed in Figure 1, which shows that survival decreases with an increase in density for all tested cultures. In both the stock and selection controls, the black strain ad a consistently higher survival, quite in contrast with our findings under the conditions of the selection experiment. Thus, the genetically determined differences in survival between the black and wild type stocks have not changed in the selection controls, or if they have, these differences are not expressed under the conditions of these experiments (8 g of flour in 6-dram shell vials). I may therefore be that the reverse relationship observed in the selection experiment is due to the environment peculiar thereto (40 g of flour in half-pint jars) or is only expressed in that environment. An overall decrease in survival of the selection controls is noticeable in Figure 1. The survival is expressed in degrees (because of the angular transformation); on the average the selection controls have five degrees lower survival than the corresponding stock cultures. Investigation of per cent hatchability of eggs revealed that these differences in survival are due to differences in larval or pupal survival.

Dry weight of adults: These relations (not illustrated) are generally consistent with previous findings in various experiments (e. g., Sokal and Karten, 1964). The ++ strain is heavier than the bb strain and there are no differences between the stock cultures and the selection controls.

Length of developmental period: Here, relations in the stock cultures are as described in earlier studies, with the ++ strain having a longer developmental period than bb at the low densities. While even at density 100/g the average developmental period of the ++ is half a day longer than that of the bb, the steeper increase of the developmental period of bb in response to density is evident. Studies by Sokal ad Karten (1964) and Sokal (1967) have shown that under these conditions of high density bb has a longer developmental period than ++. This relationship is illustrated in the selection controls in Figure 2. Since the actual selection experiment was run at asymptotic densities, between 82/g and 90/g, it may simply be that differences in developmental period observed in that experiment are reactions to the density in the cultures. Notice, however, that the selected strains hve on the average a developmental period two days longer than their counterparts from stock cultures. These findings can be corroborated by unpublished data by F. J. Sonleither who, using the earlier generations 12 and 13 of the selection controls at density 12.5/g, found that the ++ control had a mean of 30.5 days while the bb controls had a mean of 29.3 days. Comparable figures obtained by him for stock cultures were 29.8 days versus 28.5 days.

Conclusions: This experiment is unable to explain the reversion of survival of ++ and bb in the selection experiment. This may be due to the difference in environmental conditions in the jars as contrasted with the vials of this experiment. This point is now being investigated. As for length of developmental period, the genotype-density interation evident for the selection controls in Figure 2 and matching experiences in stock cultures by Sokal and Karten (1964) and Sokal (1967) may be able to explain the longer developmental period of bb in the selection experiment, although the differences observed there are far greater than the difference of about two days noted in this experiment.

Of general interest are the overall differences in survival and developmental period between the stock cultures and the selection controls. The selection controls had been subjected to a pattern of stock-keeping in which adults from a culture were permitted to oviposit for three days, then removed from the culture and the eggs reared until almost all of the adults had emerged. This has apparently resulted in inadvertent selection for long developmental periods. No effort was made to use only the earliest emerging beetles as progenitors, such as might be done in a Drosophila experiment where the investigator is eager to carry out as many generations as possible. Such selection might have led to short developmental period (see Hunter, 1959, for a striking example). In our selection experiment, the most successful beetles were those which remained as relatively small larvae for a considerable period of time, not pupating until most other pupae had already done so. Thus, their chances of being cannibalized were minimized. Selection for such slow-developing larvae would, of course, retard the mean developmental period of the entire strain, which appears to have taken place both in the wild type ad bb selection controls (also in hybrid strains not reported on here). The lower overall survival of the selection controls vis0a0vis the stock cultures may simply be due to their longer developmental period, during which they are exposed to more vicissitudes of the environment or may reflect that more of the earlier pupae are cannibalized by the larger numbers of remaining larvae in the selection controls.

Contribution No.1352 from the Department of Entomology, The University of Kansas. This is paper No. 6 in a series on the ecological genetics of Tribolium. Nos. 1 through 5 are Schlager (1963), Sokal and Huber (1963), Sokal and Karten (1964), Karten (1965), and Sokal (1967), respectively. This research was supported by the National Science Foundation under grant GB-2170 and by a Public Health Research Career Program Award (No. 3-K3-GM-22, 021-O1S1) of the National Institute of General Medical Sciences. The technical assistance of M. Young-chen Chang, Mrs. Maxine L. Howe, and Mrs. Cornella B. Tollefson are very much appreciated.

Literature Cited

Hunter, P. E. 1959. Selection of Drosophila melanogaster for length of larval period.Zeitschrift fur Vererbungslehre 90:7028.

Karten, I. 1965. Genetic differences and conditioning in Tribolium castaneum.Physiological Zoology 38:69-7S.

Schlager, G. 1963. The ecological genetics of the mutant sooty in populations of Tribolium castaneum. Evolution 17:254-273.

Sokal, R.R. 1967. The effects of synchrony of egg batches on fitness characters andCompetition in Tribolium castaneum TIB 10:135-141.

Sokal, R. R. ad I. Huber. 1963. Competition among genotypes in Tribolium castaneum At varying densities and gene frequencies (the sooty locus). American Naturalist 97:169-184.

Figure 1. Adult survival expressed as degrees (angular transformation of percentage of Egg input) averaged over the replicates and experiments of the study. Results are shown for the four strains tested at the four densities.

Figure 2. Mean developmental period in days averaged over the replicates andExperiments of the study. Results are shown for the four strains tested

At the four densities.

Sokal, R. R. and I. Karten. 1964. Competition among genotypes in Tribolium castaneum at varying densities and gene frequencies (the black locus). Genetics 49:195-211.

Sokal, R. R. and F. J. Sonleitner. 1965. Components of selection in Tribolium (Coleoptera) And houseflies. Proceedings of the XII International Congress of Entomology, 274-275.

SOKOLOFF, ADepartment of GeneticsUniversity of California, BerkeleyAnd Natural Sciences Division,California State College,San Bernardino

*Preliminary population studies with mutants of Tribolium Castaneum Herbst. I. The paddle gene.

Note: This and the following experiments in this series were performed a few years back. They had to be discontinued either because the incubator became an incinerator; the populations became infected with protozoan parasites; or because while moving from one part of the country to another constant conditions could not be maintained. In all the series of experiments reported below the beetles were introduced into four ounce glass jars containing 50 gras of standard medium (whole wheat flour enriched with brewer’s yeast in a proportion 19:1). The lids had punched out centers replaced with silk bolting cloth to allow free transpiration and thus prevent accumulation of moisture and development of mold. All jars were kept in a 290 C, 70 per cent R. H. incubator. The medium was renewed every two weeks. Censuses were taken several months apart. Live beetles of all stages were returned to their jars after census. Both dead and live beetles were scored, the latter after being etherized for 5-10 minutes. The live beetles were returned to their respective jars and the dead ones discarded after counting. Thus, it is possible that adults represented cohorts of several overlapping generations.

It may be pointed out that where populations are large, the computations of gene frequency give essentially the same results when taking into consideration only the live beetles as when both live and dead beetles are summed to produce a larger sample. This conclusion is particularly valid for the pearl gene, which is peculiar in that its adaptive value is about equal (or sometimes higher) than its wild type allele.

Since these experiments were carried out some of these mutants have been used in populations where artificially discrete generations of a month have been created. (See, for example, Sokoloff, 1964, Proc. Ent. Congress, London, 1964). This is a much more satisfactory method permitting the accumulation of 12 generations per year, which is about what would be obtained in the case of Drosophila melanogaster populations. The data are presented with little comment, in the hopes that they may help others planning similar experiments.

The founding population of each of the six replicates involving pd consisted of 25 + , 25 + , 25 pd , 25 pd . The data are given in Table 1, where wild type and paddle progeny, whether living or dead, are given according to sex. The gene frequencies, obtained directly from the males, and estimated for the females are also given. It is clear that for the short period of observation there is no consistent trend, male’s frequencies sometimes exceed those of females and vice versa. It is clear, however, that the gene

frequencies obtained for the two sexes are clearly different.

The pd gene is not very useful if one wants to identify the genotype or phenotype of dead males and females, because the antennae and/or tarsi often break off.

This work was supported in part by USPHS grant GM 08942.

SOKOLOFF, A.Department of GeneticsUniversity of California, BerkeleyAnd Natural Sciences DivisionCalifornia State College, San Bernardino

Preliminary population studies with mutants of Tribolium castaneum Herbst. II. The black gene

Two allelic mutations were available: the black mutant derived from the Chicago wild type strain, and the black mutant derived from the McGill wild type strain. Eight replicate populations were set up with 25 +/+ ; 25 +/+ (Chicago wild) and 25 b/b ; 25 b/b (derived from Chicago +/+). Eight replicate populations were set up with 25 +/+ , 25 +/+ (Chicago wild) and 25 b/b ; 25 b/b derived from the much more productive McGill wild type strain (Sokoloff, Shrode and Bywaters, 1965? Phys. Zool). The data as well as estimated and real frequencies are given in Tables 1 and 2, respectively. Half of the replicates in each had to be discontinued at the end of three months because of incubator failure, and the last two observations 17 and 23 months after the experiments were begun, were made after the populations had been taken out of the incubator and subjected to room and lower temperatures while they were being transported across the country. The gene frequencies (estimated and real) of the two populations are, however,

initially different, the McGill black being greater than the Chicago black. At the end of two years, however, the Chicago black populations consist of black at a gene frequency between 0.40 and 0.50 while the McGill black populations ended up with a gene frequency between 0.10 and 0.40.

Aside from these differences it is clear that the two populations differ in:

(1) Density (i.e. number of adults observed at census).(2) Mortality.

This work was supported in part by USPHS grant GM 08942.

SOKOLOF, A.Department of GeneticsUniversity of California, BerkeleyAnd Natural Sciences DivisionCalifornia State College, San Bernardino

Preliminary population studies with mutants of Tribolium castaneum Herbst. III. The jet gene.

The autosomal recessive body color gene jet, and the Chicago wild type (from which it was originally derived) were introduced in equal numbers in regard to sex and genotype (25 + ; 25 + ; 25 j ; 25 j ). Half of the cultures were continued for 23 months in the same manner as the other populations. The data and gene frequencies are summarized in Table 1.

This work was supported in part by USPHS grant GM 08942.

Table 1.Phenotype and changes in gene frequency of jet. (The founding population

Consisted of 25 + , 25 + : 25 j , 25 j .)

Adults found

Months +/+ j / j EstimatedReplicate later live dead live dead gene frequency

1 3 923 95 164 12 .3883 9 699 383 143 58 .4121

17 565 741 121 153 .4200 23 321 359 34 37 .3952

2 2 1411 98 333 28 .4369 10 1071 1242 258 238 .4406

3 3 928 87 187 12 .4095 9 754 505 166 89 .4247

17 576 840 107 165 .3959

23 589 815 72 75 .3300

4 2 1341 180 321 83 .48934 repeat 2 1042 43 388 12 .4209

8 1234 429 384 90 .4871

5 3 754 80 215 27 .47119 547 380 166 85 .4825

17 520 658 136 165 .4553 23 376 448 45 71 .3270

6 3 1277 120 367 15 .4724

7 3 871 79 247 15 .47009 676 471 215 134 .4912

17 544 696 106 211 .4039 23 380 948 33 85 .2826

8 2 1642 72 475 22 .4703 10 1316 1393 291 360 .4702

SOKOLOFF, A.Department of GeneticsUniversity of California, BerkeleyAnd Natural Sciences DivisionCalifornia State College, San Bernardino

Preliminary population studies with mutants of Tribolium castaneum Herbst.IV. The pearl gene.

In these experiments the gene ws introduced as homozygotes and as heterozygotes according to the following scheme:

Set 1 Set 2___________

+ /+ p / p + / p + / + p / pM F M F M F M F M F

50 50 -- -- -- 1 50 49 -- --50 49 -- 1 -- 10 50 40 -- --

50 45 -- 5 -- 50 50 -- -- -- 50 25 -- 25 50 50 -- -- -- --50 --- -- 50 25 -- -- 50 25 --

-- 50 50 -- -- 25 50 -- -- 25

-- 25 50 25 -- 10 50 -- -- 40-- 5 50 45 -- 1 -- -- 50 49

-- 1 50 49 -- -- 50 50

The results are summarized in Tables 1 and 2. Essentially the results show that in most of the replicates set up the frequency of the pearl gene remains as that introduced almost ad infinitum.

Table 1.Phenotype and gene grequency of pearl in populations with varying

Initial gene frequencies of pearl introduced as homozygotes.

Adults found

Months + / + p / p GeneFounders elapsed live dead live dead frequency

A 50 + ; 50 +1 3 849 136 --- ---

2 3 1367 47 --- ---2 repeat 3 1343 78 --- ---

3 3 844 161 --- ---

4 3 1665 71 --- ---4 repeat 3 1051 70 --- ---

B 49 +/+ ; 1 p/p ; 50 +/+

1 3 807 165 --- ---

2 3 1653 56 1 1 . .02408 4 1419 354 --- 1 .02366

6 1255 643 --- 4 .04584

3 3 883 142 1 --- .03122

4 3 1425 40 --- --- 6 941 81 1 --- .03125

C 45 +/+ ; 5 p/p ; 50 +/+

1 3 942 101 3 --- .053552 3 1508 55 3 1 .05053

4 1262 124 2 2 .05364

3 3 847 148 4 --- .06328

4 3 1565 47 5 --- .05561 4 1386 205 3 3 .06129

D 25 +/+ ; 25 p/p; 50 +/+

1 3 981 138 24 --- .1449

2 2 1229 50 24 1 .1385 4 1615 146 49 13 .1844

3 3 997 124 25 .1477

4 2 1271 43 25 .1366 5 1512 189 40 13 .1738

E 25 + ; 25 p ; 25 + ; 25 p

1 3 954 79 135 16 .35712 3 1016 76 298 35 .4834

5 1194 135 346 43 .4758 6 1365 345 391 76 .4631 14 1058 1832 297 326 .4211

3 3 893 107 109 12 .32853 repeat 3 846 --- 208 --- .4442

7 1177 318 214 85 .4095

4 3 1122 62 367 16 .4917 4 1164 97 378 44 .5007

4 repeat 3 946 128 255 53 .4730 9 796 855 173 191 .4250

5 3 747 102 135 12 .3841

6 2 1111 43 304 15 .4653 4 1151 128 310 40 .4636 6 1348 507 361 124 .4553 9 1139 774 340 177 .4613 14 760 872 220 194 .4498

7 3 766 73 124 11 .3723 9 740 428 121 49 .3723

17 632 814 105 175 .3955 22 240 --- 40 --- .3793

7 repeat 3 736 --- 206 --- .4677 7 1352 294 288 54 .4147

8 2 961 66 329 21 .5041 4 915 106 279 43 .4897

8 repeat 2 884 57 266 19 .4822 5 1184 456 277 130 .4459

10 1032 858 238 178 ,4247

F 25 +/+ ; 25 p/p ; 50 p/p

1 3 652 53 244 25 .5255

2 2 642 35 489 29 .6584 4 736 59 553 59 .6595 6 675 520 495 509 .6767

3 3 653 54 257 29 .5367

4 3 819 51 551 32 .7080 4 360 457 114 380 .6139 6 1252 89 543 35 .5488

G 5 +/+ ; 45 p/p ; 50 +/+

1 3 208 26 558 82 .8557

2 2 129 27 978 53 .93204 139 13 1015 132 .9397

3 3 124 11 438 84 .8913

4 2 106 3 709 52 .93534 151 30 929 184 .9274

H 1 +/+ ; 49 p/p ; 50 p/p

1 3 11 4 694 96 .9814

2 2 17 --- 1049 39 .98214 24 8 1083 148 .9873

3 3 28 7 674 128 .9789

4 2 20 --- 989 25 .99034 29 3 1122 227 .9883

10 41 17 972 916 .9850

I 50 p/p ; 50 p/p Pearl frequency

1 3 --- --- 610 148 1.0

2 2 --- --- 1034 44 1.04 --- --- 1122 219 1.0

3 3 --- --- 469 105 1.0

4 2 --- --- 1072 544 --- --- 869 262

Table 2.Phenotype and gene frequency of pearl in populations with arious

Initial gene frequencies of pearl introduced as heterozygotes.

Adults foundMonths +/+ p/p Pearl

Founders later live dead Live dead frequency

J 1 +/p ; 49 +/+ ; 50 +/+

1 3 926 150 0 0

2 3 1342 45 0 04 1285 228 0 06 1321 485 0 0

3 3 989 173 0 0

4 2 1111 38 0 04 1562 248 0 0

K 1 +/p ; 49 +/+ : 1 +/p ; 49 +/+

1 3 988 136 0 0 ?

2 3 1170 33 2 .040745 1568 245 3 1 .046917 1531 93 1 0 ,02481

3 3 921 140 0 0 ?

4 3 1148 64 0 0 ?4 1553 191 0 0 ?6 1296 673 1 0 .02254

L 5 +/p ; 45 +/+ ; 5 +/p ; 45 +/+ mutant Gene Live dead frequency

1 3 1038 117 0 0 ?

2 2 1082 48 2 0 .042044 1331 298 4 5 .074126 1358 425 5 3 .06684

3 3 903 132 0 0 ?

4 3 1398 41 8 0 .074393 1736 218 13 2 .087286 1488 659 12 4 .08801

M. 25 +/p ; 25 +/+ ; 25 +/p ; 25 +/+

1 3 1000 85 69 4 .2511

2 3 1406 69 90 8 .25164 1543 186 104 10 .2487

3 3 976 114 81 5 .2708

4 2 1060 28 70 2 .26265 1431 215 103 5 .24816 986 547 78 29 .2554

N 25 +/p ; 25 p/p ; 25 +/p ; 25 p/p

1 3 741 77 360 39 .5719

2 3 599 37 493 33 .67285 590 146 414 131 .6522

7 858 426 517 238 .6085

3 3 531 82 364 36 .6284

4 3 708 29 637 29 .68895 599 117 532 150 .69846 632 266 522 212 .6780

O 5 +/p ; 45 p/p ; 5 +/p ; 45 p/p

1 3 112 13 427 85 .8965

2 2 177 6 1033 43 .92444 153 23 1025 188 .9345

3 3 181 22 497 86 .8612

4 3 181 11 994 72 ,92055 195 31 1071 147 .9184

P 1 +/p ; 49 p/p ; 1 +/p ; 49 p/p

1 3 16 3 555 146 .9867

2 2 24 2 979 49 .98764 24 8 1119 343 .9892

3 3 65 6 562 106 .9507

4 2 10 2 872 43 .99354 18 3 1028 208 .99166 25 6 1062 508 .9903

Q 1 +/p ; 49 p/p ; 50 p/p

1 3 8 0 631 150 .9949

2 2 12 0 841 38 .99324 22 6 996 378 .9899

3 3 5 2 656 168 .9958

4 2 8 1 1077 47 .99604 7 1 1062 198 .9968

SOKOLOFF, A.Department of GeneticsUniversity of California, BerkeleyAnd Natural Sciences DivisionCalifornia State College, San Bernardino

Preliminary population studies with mutants of Latheticus Oryzae Watern. I. The pearl gene.

These experiments were designed to test, in a comparative way, the performance of pearl in populations. For this reason the founders in the various sets were introduced in a manner similar to the experiment reported above for Tribolium castaneum. The first notable difference between the twospecies is that the number of adults produced by Latheticus is far lower than that obtained in Tribolium cultures. Furthermore, the developmental period of Latheticus in these cultures is astonishingly slow. Cultures set up six to eight months before may contain only the original adults, the progeny being in the late larva or in the pupa stage. The cultures appear as if the larvae are all of the same age, and they remain thus for a long period of time. Eggs and small larvae apparently are eaten by the older larvae as they are produced. At the other end, the first few pupae forming are destroyed by the younger larvae. The small size of the populations as well as the long developmental period makes it impractical to pursue population studies with this organism.

The data, summarized in Tables 1 and 2, insofar as they go, appear to indicate that when pearl is frequent and wild type infrequent the results in the two species are comparable.

Table 1.Frequency changes of pearl in Latheticus oryzae introduced initially

At frequencies from 0-1.0 into each of six replicates as homozygotes.

Adults found

Months +/+ p/p GeneFounders later live dead live dead frequency

A 50 + ; 50 :

1 8 114 29 --- --- 0

2 6 124 44 --- --- 0 11 218 176 --- --- 0

3 8 93 7 --- --- 0 4 7 188 92 --- --- 0

11 241 179 --- --- 0

5 8 93 7 --- --- 0

6 7 136 91 --- --- 0 11 190 178 --- --- 0

B 49 + ; 1 p/p ; 50 +

1 8 111 40 1 ---

2 5 74 31 1 --- 7 231 39 --- ---

3 8 102 18 1 ---

4 7 186 66 1 --- 11 166 162 0 2

5 8 96 4 2 ---

6 7 124 55 1 --- 11 256 105 --- 1

C 45 +/+ ; 5 p/p ; 50 +

1 8 99 11 6 ---

2 5 72 23 5 --- 7 204 68 9 2 .1972

3 8 101 18 4 1

4 7 112 79 4 --- 11 209 152 2 3 .1189

5 8 89 7 5 ---

6 7 142 92 4 1 11 174 177 3 2 ..1185

D 25 +/+ ; 25 p/p ; 50 +/+

1 8 85 32 25 ---

2 5 67 6 25 ---

7 131 131 21 12 .3345

3 8 70 20 26 ---

4 7 66 43 21 411 175 95 22 8 .3162

5 8 73 9 26 ---

6 7 200 96 21 211 178 97 21 13 .3317

E 25 +/+ ; 25 p/p ; 25 +/+ ; 25 p/p

1 8 55 17 49 --- .636420 47 19 34 5 .609426 85 56 33 36 .5732

2 5 32 18 41 9 7 194 61 37 5 .3761

3 8 55 7 50 --- .668120 19 33 23 7 .698426 0 15 13 27 .8528

4 7 34 16 47 211 74 76 37 25 .5408

5 8 59 11 48 2 .645520 23 18 20 5 .615526 54 37 32 19 .5993

6 7 113 98 41 5 .423111 163 74 29 20 .4139

F 50 +/+ ; 50 p/p

1 8 61 20 47 1

2 5 46 4 44 6 7 145 65 35 9

3 8 68 21 48 2

4 7 87 43 35 1111 222 105 11 33 .3444

5 8 56 10 45 4

6 7 94 79 41 1011 221 111 31 25 .3799

G 25 +/+ ; 25 p/p ; 50 p/p

1 8 30 5 82 10

2 6 21 4 63 12 8 102 14 164 32 .7926

3 8 29 4 71 7

4 7 49 42 71 5111 102 65 144 62 .7432

5 8 25 4 75 7

6 7 54 27 102 3711 46 56 75 80 .7766

H 5 +/+ ; 45 p/p ; 50 p/p

1 8 4 4 97 7

2 5 3 2 75 20 7 10 2 194 60 ,9772

3 8 10 4 92 8

4 7 13 6 121 5211 15 10 204 85 .9594

5 8 5 --- 90 6

6 7 6 2 84 2711 6 5 145 86 .9770

I 1 +/+ ; 49 p/p ; 50 p/p

1 8 1 --- 96 15

2 5 1 --- 86 13

7 3 2 150 76 .9891

3 8 1 --- 95 11

4 7 --- 1 88 1511 --- --- 282 133

5 8 2 --- 98 10

6 7 1 --- 79 2011 2 1 108 85 .9923

J 50 p/p ; 50 p/p

1 8 --- --- 101 18

2 7 --- --- 131 4011 --- --- 229 116

3 contaminated with wild type

4 7 --- --- 85 2311 --- --- 202 82

5 8 --- --- 96 5

6 7 --- --- 92 4811 --- --- 216 128

Table 2.Phenotype and gene frequency of pearl in populations with various

Gene frequencies of pearl introduced as heterozygotes

Adults found

Months +/+ p/p GeneFounders later live dead live dead frequency

K 1 +/p ; 49 +/+ ; 50 +/+

1 8 101 10 --- ---

2 7 173 92 2 ---

11 257 130 --- 1 .05075

3 8 100 9 --- ---

4 5 104 19 --- --- 9 278 101 --- ---

5 8 101 5 --- ---

6 5 116 24 --- --- 9 207 152 --- ---

L 10 +/p ; 40 +/+ ; 50 +/+

1 8 93 7 1 ---

2 7 94 11 --- --- 11 206 164 2 --- .07332

3 8 102 15 --- ---

4 5 87 13 --- --- 9 181 97 --- ---

5 8 97 8 --- ---

6 5 90 9 1 --- 9 207 130 1 --- .05439

M 50 +/p ; 50 +/+

1 4 97 4 1 ---

2 4 94 5 1 --- 8 152 90 6 1 .1676

3 5 97 2 1 ---

Q 25 +/p ; 25 p/p ; 50 +/+

1 6 75 6 26

2 5 64 9 25 9 91 55 19 6 .3824

3 6 80 8 25

4 5 69 4 23 1 9 129 108 14 8 ,2914

5 6 66 6 25

6 5 73 2 20 5 9 178 91 19 9 .3071

S 10 +/p ; 40 p/p ; 50 p/p

1 6 10 --- 85 7

2 5 9 1 82 7 9 12 8 96 62 .9421

3 6 10 --- 90 5

4 5 9 --- 83 7 9 17 10 83 130 .9421

5 6 10 2 86 6

6 5 10 --- 86 4 9 18 6 118 89 .9466

T 1 +/p ; 49 p/p : 50 p/p

1 6 1 3 97 ---

2 5 1 --- 97 22 9 2 1 151 109 .9943

3 6 2 --- 97 3

4 5 1 --- 90 8 9 2 --- 176 75 .9960

5 6 2 --- 96 3

6 5 1 --- 87 66 .9967

M 50 +/p ; 50 +/+

4 5 97 3 --- --- 9 59 39 --- ---

5 5 98 3 --- ---

6 4 95 3 2 --- 8 172 106 9 --- .1771

N 50 +/p ; 50 +/p

1 5 98 2 --- ---

2 4 95 5 --- --- 8 181 90 47 9 .4139

3 5 98 2 --- ---

4 4 95 3 1 --- 8 114 69 16 6 .3491

5 5 99 3 --- 1

6 4 99 --- 1 --- 8 71 1 28 ---

P 50 +/+ ; 25 +/p ; 25 p/p

1 6 74 8 24 1

2 5 72 4 23 1 9 184 123 10 15 .2744

3 6 76 4 25 1

4 5 59 16 19 6 9 150 113 5 14 .2858

5 6 77 5 24 1

6 5 70 5 22 3 9 163 137 6 23 .2967

SOKOLOFF, A., M. ACKERMANN AND B. HEINZEDepartment of GeneticsUniversity of California,Berkeley, California

*Additions to established linkage groups

I. T. castaneum

A. X-chromosome

1. Lethal-5 (1-5), located about 25 units to the left of py (away from r ). Allelic with 12 and 14.

2. Letal-6 (1-6), located about 12 units to the left of py (away from r ).

3. Lethal-7 (1-7), located about 20 units to the left of py (away from r ).

B. Autosomes

Aureate is located about 42 units away from black. Three-point crosses To locate it in respect to other gees are now in progress.

II. T. confusum

A. X-chromosome

1. Alate prothorax, apt, is between es lt and lp, about four units to the left of lp.

This work was supported by USPHS grant GM 08942.

SOKOLOFF, A., N. INOUYE AND R. S. ST. HITAIREDepartment of GeneticsUniversity of California, BerkeleyAnd Natural Sciences Division,California State College , San Bernardino

*Additional sex-linked lethal in Tribolium castaneum Herbst.

In an experiment designed to determine the frequency of lethal as a regression on the age of the maternal grandfather (see Lerner and Inouye in the present issue of TIB) several females gave aberrant sex-ratios and two of these, on retesting, proved to be heterozygous for lethal. The material was originally derived from the Berkeley synthetic

strain marked with sooty (for details on its construction and maintenance see Lerner and Ho. 1961, Am. Nat. 95:329).

In order to locate these lethal and determine possible allelism, four virgin females from stock 29c and eight from stock 68a were mated with py r males. Because of lack of time it was not possible to set up the sequential matings immediately with a consequent over lap in generations. Female virgins were tan mated with py r males, four from 29c and eight from 68a. In the former, one female proved to be heterozygous for the lethal producing 1+ and 4 py r males, and 6 + and 4 py r females. The “+” virgin females (py r +/+ + 1x) were placed in individual creamers and remated with py r males, allowed to lay eggs for a week and transferred to fresh medium four times at intervals of a week to increase the number of progeny. From 68a two females designated as 68a-1 and 68a-6 and producing 9 py r : 14+, 1 py, 6 py r , and 3+, 1 py, 13 py r : 18 +, 1 r, 1 py, 10 py r , respectively, were the source of the carriers of the other lethal. In 68a-1 eight “+” females and in 68a-6 twelve “+” females were heterozygous for the lethal. These females were separated in individual creamers and allowed to remain with their progeny until the latter emerged as adults.

Results and Conclusion

Experiment 29c

The various broods in experiment 29 c have been tested and found homogeneous. Therefore, the data have been pooled and they are shown in Table 1. They make it possible to determine that the crossover frequency for the three genes involved is :

Males Females

r - py 44/425 = 10.35 78/917 = 8.51

r – 1 29c 136/425 = 32.00

py – 1 29c 104/425 = 24.47

Therefore, the order is 1 s9c – py – r. Previous studies have located 1s at 22.78 units from py (from 90/395 crossovers detected between py and 12) and 14 at 25.45 units from the same gene (from 70/275 crossovers between py- and 14). 12 and 14 have been found allelic (Sokoloff and Dawson, 1963, Can. J. Genet. Cytol. 5:138). Chi square tests of homogeneity between 1s, 14 and 129c indicate the data are homogeneous. Therefore, 129c must be considered as a recurrence of 12 and it is designated as 1-5. (cf. section on New Mutants.)

Tables 2 and 3 summarize the data for experiment 68.

The data for 68a-1 indicate the crossover frequencies are:

Males Females

py - r 47/398 = 11.81 92/613 = 15.01

r – lethal 87/398 = 21.86

py – lethal 46/398 = 11.56

so the order is : lethal – py – r.

The data for 68a-6 give the following crossover values:

Males Females

py - r 30/416 = 7.21 84/822 = 10.22

r - lethal 99/416 = 23.80

py - lethal 81/416 = 19.47

and the order of the three genes is lethal - py - r.

68a-1 and 68a-6 have been tested for allelism by the Chi square that for homogeneity with 12, 14 and 15. The statistical tests suggest these lethal are not allelic with them nor with each other. Neither are they allelic with 13. Therefore, 68a-1 is redesignated 16 and 68a-6 17.

We do not know whether the original female whose progeny indicated the presence of a sex-linked lethal carried both lethal or whether a second lethal occurred during the course of these experiments.

This work was supported by USPHS grant GM 08942.

Table 1. Progeny of py r +/++ 29c x py r +/.

Phenotype Male Female

py r 283 266

+ + 98 573

py 38 38

r 6 40

Total 425 917

Table 2. Progeny of py r +/++ 68a-1 x py r + (nine successful creamers)

Phenotype Male Female

py r 308 216

++ 43 305

py 44 56

r 3 36

Total 398 613

Table 3. Progeny of py r +/++ 68a-6 x py r +/. (12 successful creamers)

Phenotype Male Female

py r 311 268

+ + 75 470py 24 36r 6 48

Total 416 822

SOKOLOFF, A AND J. LANTERDepartment of GeneticsUniversity of California, BerkeleyAnd Natural Sciences DivisionCalifornia State College, San Bernardino

Further studies of productivity of Tribolium castaneum and Tribolium confusum in homo- and heterospecific matings

In a previous paper (Sokoloff, Shrode and Bywaters, 1965, Phys. Zool. 38:165), it was shown that productivity (defined as the number of adult progeny per female within a specific interval or the number of fertile eggs per female surviving to the adult stage) is a reliable genetic character. It was used in a previous study by Sokoloff and Inouye (TIB 6:61) to determine whether females of T. castaneum (CS) or T. confusum (CF) are affected in their reproductive capacities by the presence of males of the other species. It was found that when CF females are in association with CS males their productivity drops

somewhat, but not as much as when CS females their productivity drops somewhat, but not as much as when CS females are in creamers together with CF males. This previous experiment left unanswered the question whether this drop in productivity, observed particularly for CS, was not the result of depriving the females from their mating partners. Tagarro and Rico (TIB 9:12) showed that when males are removed from cultures where females are already fecundated, they continue to lay fertile eggs at a slightly lower rate than when males were present, but that this difference is not significant. The period they tested was between days 7 and 11 after the females had eclosed. Since in the Sokoloff and Inouye experiments the females were left without males for a much longer period, the present experiments were performed.

For each of the species or species combination the experimental set up was as follows: into each of 10 creamers were introduced four pairs of beetles 10 days old (females and males had been isolated as pupae) on a Thursday. At the same time on Monday the imagoes were transferred into new containers, the old medium with eggs being returned to the original creamers after counting the eggs. The adults were moved to new quarters every twenty-four hours for three days, and the eggs were counted before returning them to their respective creamers. Every week the beetles were manipulated in the following way: the first week the females in all 10 creamers had males of the same species as partners. The second week the set was broken into two subsets. From subset A the males were removed altogether, and these females are referred as “widows”. From subset B half of the males were removed and replaced by males of the other species. We refer to these females as “bigamists”. On the third week the females of subset A are supplied with males of the other species, and refer to them as “miscegynists, type I”. From subset B the remaining two males of the same species are removed and replaced by males of the other species, and the females referred to as

“miscegynists, type II”. Finally, on the fourth week, the foreign males are removed from both subsets and males of the same species re-introduced. (The two subsets will be referred to as “repurified I” and “repurified II”, respectively.)

If any dead beetles were found, they were sexed and replaced by beetles of the same age, sex and species.

Fertility of the eggs was determined by counting larvae when they were three weeks old.

The data have been analyzed to determine any significant difference between the three successive 24-hour periods. Since no significant difference has been found, the values of the three successive days have been pooled providing 30 observations for the pure species (10 replicates x 3 days) and 15 for the subsets (5 replicates x 3 days). The results are summarized in Table 1.

Comparisons of the means by t-test indicates that none of the differences observed for CF are significant. For CS significant differences can be shown at the .02 level for the means of egs produced by “pure species” and “widows” and at the .01 level for “pure species” vs. “miscegynists-I”, “miscegynists-II”, and “repurified”. Significant differences are obtained at the .01 level for fertility of eggs of “pure species” vs. “bigamists”, “miscegynists-I” and “miscegynists-II”. The values obtained for “pure species” and “re-purified II” are significantly different at the .02 level.

This experiment appears to indicate that when inseminated CF females are introduced with CS males either they reject them, or if copulation takes place between them, the foreign sperm play no role in fertilization of CF eggs. On the other hand, if CS females are confined with CF males (and no CS males are present) they inseminate them , and the foreign sperm may fertilize a fairly large number or all of CS eggs. The result is that females exhibit partial or complete sterility. These sterile or semisterile CS females recover almost immediately from any “damage” the foreign males may have produced if CS males are reintroduced.

Therefore, it is concluded that in mixed species cultures heterospecific matings should not influence greatly the outcome of competition if the numbers of the two species are large. Where the numbers of CS are small (and CF large), the absence of sufficient CS males to service the CS females present might result in the loss of CS from those cultures.

This work was supported by USPHS grant GM-08942.

TAGARRO, MA. P. AD F. OROZCOLaboratorio de Genetica de PoblacionesInstituto Nacional de Investigaciones AgronomicasMadrid, Spain

Influence of collecting frequency on egg-laying rate of fecundated and virgin females in Tribolium castaneum

Egg-laying rate is the quantitative character we are using more in our genetic research with Tribolium castaneum. We have defined a period comprised between the 7 th

and the 11th days after adult emergence to measure that character.

Egg collecting and counting is a time-consuming task, so we were interested to reduce as much as possible the number of collections in such testing period. At 330 C and 70% RH larvae hatch 66 + 6 hours after the eggs are laid. So with fecundated females it is necessary to collect no later than every other day (two collections in the four-day period). With virgin females this problem does not exist, so it is possible to collect once at the end of the testing period.

However, we wondered whether collecting every day, every other day, or at the end of the four-day period could have some influence in the total number of eggs laid or counted. Favorable effect of fresh medium after a collection, disturbing effect of the suction sifting, cannibalism, etc., could contribute to find different figures in the total lay of the four days.

Four trials were run comparing 24 vs 48 hours collecting frequencies both with fecundated and with virgin females. Two more trials were run to compare 48 vs. 96 hours collecting frequencies only with virgin females. We used our “Consejo” strain of Tribolium castaneum. Fecundated females were mated at adult emergence and the males (one per female) were maintained during the testing period. At day 7th after emergence the medium in each vial was changed. Egg collecting was made by suction sifting every day, every other day or at day 11, according to the corresponding treatment, and afterwards fresh medium was added. The figure analyzed was the total number of eggs laid per female during the four-day testing period.

Table 1 includes the analysis of each of the four experiments run with fecundated females comparing 24 vs. 48 hours collecting frequencies, together with a pooled analysis of the four as a whole. We can see that in no case there exist differences between treatments.

Table 2 includes similar information as in Table 1 but with the data obtained with virgin females. The results in this case are not as clear as with fecundated females because in the A trial we find significant differences to the .05 level in favor of 48 h. and in the B one slightly significant (only to .10 level) but in favor of the 24 h. frequency. However, when considering the pooled analysis we do not find significant differences to any level between treatments and the significance for the interaction reflects the

difference of results obtained in A and B. The greater influence of uncontrolled effects in egg laying of virgin females as compared with the lay of fecundated ones, widely observed by us in many experiments, could explain the anomalous results of A. In a practical sense we can consider the non-significant differences found for treatments in the pooled analysis as favorable.

Table 3 contains the analysis of the two experiments comparing 48 vs. 96 hours collecting frequencies with virgin females and the pooled analysis of both.

Table 1. Means, standard errors, df, MS and F values of the analysis of Four experiments studying the influence of 24 vs. 48 hours Collecting frequencies on the laying rate of fecundated females.

Experiment Means and S. E. Sources df MS F

A 24 h. 63.5 + 2.7 Treatments 1 46.04 0.2748 h. 65.5 + 2.7 Error 45 173.50

B 24 h. 76,9 + 3.6 Treatments 1 218.11 0.6948 h. 72.5 + 4.0 Error 43 317.11

C 24 h. 34.4 + 2.9 Treatments 1 154.47 0.9648 h. 38.3 + 2.8 Error 37 161.00

D 24 h. 61.0 + 3.3 Treatments 1 108.15 0.4048 h. 58.0 + 3.5 Error 45 273.09

Pool 24 h. 60,4 + 1.6 Experiments 3 10,881.46 46.61*48 h. 58.9 + 1.6 Treatments 1 110.06 0.47

Exp. X Treat. 3 138.90 0.59Error 170 233.47

*Significant to .005 level.

Table 2. Means, standard errors, df, MS and F values of the analysis of Four experiment studying the influence of 24 vs. 48 hours Collecting frequencies on the laying rate of virgin females.

Experiment Means and S. E. Sources df MS F

A’ 24 h. 9.8 + 1.1 Treatments 1 252.01 6.6248 h. 13.7 + 1.1 Error 62 38,04

B’ 24 h. 19.4 + 1.7 Treatments 1 243.95 3.77*

48 h. 14.9 + 1.6 Error 46 64.65

C’ 24 h.. 9.3 + 1.5 Treatments 1 21.03 0.4848 h. 7.9 + 1.5 Error 38 44.17

D’ 24 h. 14.1 + 1.9 Treatments 1 89.78 1.0148 h. 16.8 + 1.9 Error 48 89.05

Pool 24 h. 12.9 + 0.8 Experiments 3 635.57 10.9348 h. 13.6 + 0.8 Treatments 1 25.59 0.44

Exp. X Treat. 3 193.73 3.33Error 194 58.17

*Significant to .10 level; to 0.04 level; to .005 level.

Table 3. Means, standard errors, df, MS and F values of the analysis of Two experiments studying the influence of 48 vs. 96 hours Collecting frequencies on the laying rate of virgin females.

Experiments Means and S. E. Sources df MS F

E 48 h. 12.8 + 1.2 Treatments 1 0.66 0.0196 h. 13.0 + 1.2 Error 96 69.09

F 48 h. 28.4 + 2.3 Treatments 1 0.66 0.0096 h. 28.3 + 2.3 Error 96 250.33

Pool 48 h. 20.6 + 1.3 Experiments 1 11.694.88 73.23*96 h. 20.6 + 1.3 Treatments 1 0.00 0.00

Exp. X Treat. 1 1.30 0.01Error 192 159.71

*Significant to .005 level.

No differences at all are found between treatments in any experiment nor in the pooled analysis. This result also helps to interpret the figures in Table 2 in the sense that the significant differences observed in A and B must be due to chance effects.

In all sets of trials differences between experiments are always significant. This is not surprising because many uncontrolled effects influence the number of eggs laid by females of Tribolium castaneum. Pooling the analysis of different trials is quite fair because we have proportional or nearly proportional subclass numbers; analysis with means of treatments x experiments cells give the same results reported here.

Therefore, we can conclude tht under our experimental conditions, when we are interested to measure the egg-laying rate from day 7 to day 11 after adult emergence, it does not make any difference whether we collect eggs every day or every other day in both fecundated and virgin females and even in doing only one collection at the end of the four days with virgin females. The longest “between collections” period with fecundated females is 48 hours because larvae may hatch before three days.

This is a partial and preliminary study included in Grant No. FG-Sp-137 of P.L. 480, contract with the USDA.

TSCHINKEL, W.Department of BiochemistryUniversity of CaliforniaBerkeley, California

*Sex pheromones and defensive secretions from Tenebrionid beetles

Studies on the sex pheromones and defensive secretions of Tenerbrionid beetles are under way in this laboratory. Sex pheromones have been demonstrated in Tenebrio molitor and Zophobas rugipes (a large Central American species) and a biological assay for the pheromone of Tenebrio molitor has been devised (Tschinkel, Willson and Bern,1967). Attempts to isolate the sex pheromone of Tenebrio molitor are in progress.

The secretion of the prothoracic defensive glands of Zophobas rugipes is being studied by various chromatographic and spectral techniques. These glands secrete phenols, but are otherwise homologous to the quinine-secreting prothoracic stink glands of Tribolium (Roth, 1943).

Disturbed larvae of Zophobas rugipes frequently squirt hemolymph along with an acrid odor. This phenomenon and an effect of larval crowding on pupation are under study.

Attempts are being made to establish a number of Tenebrionid beetles (especially subfamily Tenebrioninae) in laboratory cultures for comparative studies on their sex pheromones and defensive secretions. Success has been limited. The results are summarized in Table 1.

Literature Cited

Roth, L. M. 1943. Studies on the gaseous secretion of Tribolium confusum Duval. II. The odoriferous glands of Tribolium confusum. Ann. Ent. Soc. Am. 36:397-424.

Tschinkel, W. R., C.D. Willson and H. A. Bern. 1967. Sex pheromone of the mealworm beetle (Tenebrio molitor). J. Exp. Zool. 164:81-85.

WOOL, DAVIDDepartment of EntomologyThe University of KansasLawrence, Kansas

*Some observations on mating frequencies in TriboliumCastaneum strains

Introduction. In a series of papers, Ehrman and associates (Ehrman, 1965, 1966; Ehrman et al., 1965) described a phenomenon observed earlier by Petit (1958) with important evolutionary implications. When two strains of Drosophila are mated in unequal proportions, in some cases the rarer strain, regardless of genotype, will mate more often than expected on the basis of its frequency in the observation chamber. The series of experiments reported here was designed to look for a similar phenomenon in two strains of Tribolium. In the course of the experiment, some interesting observations on the mating habits of these strains were made, which are also reported here.

Materials and Methods. The standard UPF wild type and black strains of T. castaneum employed in work in this laboratory (see stock list) served as experimental material. Large numbers of pupae were recovered from multiple cultures of the two stocks, sexed and then put, in unisexual groups of 10, in 6-dram holding vials containing about 4g of flour. The bb beetles emerged two days later than the ++ adults and therefore were two days younger throughout the experiment. The first experiment was set up four days after the emergence of bb adults, and the last one two weeks later.

Mating was observed in two-inch Syracuse dishes with 20 and 20 in varying genotypic proportions. All the males were marked by paint at least three days before the experiment to distinguish true mating pairs from the frequent x mounts (see results). The genotype of each sex and time of mating was recorded. Each observation dish was watched constantly for two consecutive 30-minute periods, during which a considerable number of copulations was observed. Two series of observations were made. In the first, after the end of the fixed observation period, all the males were removed, and the females were transferred individually to vials with about 4g of flour, where they were left to oviposit for three days, after which they were discarded. The genotype of the was recorded on the vial, and the resulting brood was classified by genotype to infer the type or types of males that had sired it.

In the second series, the FF and MM were separated after the first 30 minutes’ observation period. The “old” MM were given a new group of virgin FF and the “old” FF, a fresh group of (the previous genotypic proportions being maintained). The two dishes were observed simultaneously for another 30 minutes, after which all the beetles were discarded. No brood analysis was carried out in this series.

The first series was set up at proportions 10:90 and 25:75, with both wild type and black being rare in turn. The second series was run at 10:90 only. Two 50:50 observations were carried out as controls.

Results. Previous to the main experiment, couples (in copula) were recovered from large wild type and black populations and the sex of the participants determined by microscopic examination. It appeared that wild type or black will mate with any Tribolium available, regardless of sex. Mounting of MM on other MM occurred in 30.5% of the cases among the wild type and 34.5% among the blacks (based on 127 pairs); a test of independence yielded X2 = 0.277 and fails to show any difference in behavior between the strains in this respect. In all subsequent experiments the males were marked, and only true ( M x F) matings are considered in the following sections.

The observations in the Syracuse dishes may be summarized in the following form; the asterisks refer to total X2 for all replicates.

The symbols (++) or (bb) following asterisks indicate the genotype that mated more often. Significant heterogeneity among the replicates is shown by the superscript h.

All new x new cases were observed in the first 30-minute periods. All other combinations were in the second 30-minute periods.

The mean number of copulations in 30 minutes for 10:90 proportions differed greatly among the four combinations: new FF x new MM (21.7), new FF x old MM (12.5), old FF x new MM (15.0), and old FF x old MM (6.75). Analyzing the variance of the 26 replicates, the following table shows a highly significant mean square among combinations.

Source of variation df MS

Genotype of rare strains 1 1.78Combinations 3 248.26***Interaction 3 28.74Error 18 17.35

Single degree of freedom comparisons showed a highly significant difference between the mean number of copulations between new FF x new MM and the mean of the other three taken as a whole (P << .001), and a significant difference between old FF x old MM and the two (old FF x new MM and new FF x old MM) combinations taken together (P < .01). No significant difference was found between the mean of new FF x old MM versus old FF x new MM

Similar results were obtained in the eight 25:75 replicates, which contained only old FF x old MM and new FF x new MM combinations.

Surprisingly, the results of the brood analysis of the 10:90 proportions showed that very few of the in the observation dish were fertilized although, based on the average mating frequency, every one of the 20 could have been fertilized. (Similar results were obtained in the 25:75 series.)

Number of matings Number of fertilizedRare strain observed females

++ 25 629 7

bb 24 1227 13

Control (50:50) 23 5

In a few cases, a F was mated in sequence to a heterotypic M and then a homotypic M. Only 5 of 10 FF were fertilized by any of the males: 3 bb FF and 2 ++ FF. The brood of the bb FF contained only the offspring of the first (++) MM. The brood of the ++FF contained mainly the first (bb) male’s offspring, although 3.5 – 8.5% of the offspring were from the second mating.

Discussion. As can be seen from the results presented above, the Petit-Ehrman phenomenon does not occur in these two strains of Tribolium under our test conditions. However, it was found that black FF, when rare, consistently mated much more frequently than expected, mostly with wild type males. In all cases where a significant deviation from expected mating frequencies for MM or FF was present, the frequency of ++ MM x bb FF matings was very much higher than expected (P << .001 by the G-test). This trend in the bb FF was consistent for all replicates. The only case in which heterogeneity was significant for the FF was caused by an exceptionally large deviation from expectation in one of the six replicates. No clear trend could be shown for the MM.

Such a behavior, if reflected in the offspring, could result in an increasing gene frequency of black.

Virgin pairs showed the highest mating frequency per half hour. When both and had mated in the half hour preceding the experiment, they showed the lowest number of mating. In cases where one sex was virgin, while the other had mated in the half hour preceding the experiment, the mating frequency ws in-between, with no significant difference between the reciprocal crosses. Possibly mated may resist a subsequent mating shortly after the first one, and the may need a period of rest between matings.

It is interesting to note that even in the old x new and new x old combinations, the black females, when rare, mated significantly more often than expected. Presumably they are unable to resist the males as effectively as the wild-type females.

Why the number of fertilized females should be so much lower than the number of observed copulations is obscure. When b was rare (wild-type common) more were fertilized tan when wild type was rare. Some of the matings could have been infertile because of the unusual conditions of light and substrate in the observation dish. By contrast, when in another experiment 20 and 20 at proportions 10:90 and 50:50 were left in flour for 12 hours, 18, 18 and 20 females were fertilized in the cases where + was rare, the two alleles equally frequent, and b rare, respectively.

The brood analysis data are not helpful in testing the Petit-Ehrman phenomenon because the evidence shows a considerable number of double matings (mating of one female with more than one male). Regrettably, this can be demonstrated only if the female mated with both types of males. Ignoring all evident double matings, no significant higher mating frequency of the rare form can be shown.

Contribution No.1350 from the Department of Entomology, the University of Kansas. This is paper No. 7 in a series on the ecological genetics of Tribolium. Numbers 1 through 5 are listed at the end of paper No.6 (Sokal, 1967) in this issue of TIB. This research was supported by a National Science Foundation Grant GB-2170 (Robert R. Sokal, principal investigator).

Literature Cited

Ehrman, L. 1965. Direct observation of sexual isolation between allopatric andSympatric strains of the different Drosophila paulistorum races.Evolution 19:459-464.

Ehrman, L. 1966. Mating success and genotype frequency in Drosophila. Animal Behavior 14:332-339.

Ehrman, L., B. Spassky, O. Pavlovsky, T. Dobzhansky. 1965. Sexual selection, geotaxis, and chromosomal polymorphism in experimental populations of Drosophila pseudoobscura. Evolution 19:337-346.

Petit, C. 1958. Le determinisme genetique et psycho-physiologique de la

Competition sexuelle chez Drosophila melanogaster. Bull. Biol.France Belgique 92:248-329.

Sokal, R. R. 1967. A comparison of fitness characters and their response to density in stock and selected cultures of wild-type and black Tribolium castaneum.Tribolium Information Bulletin 10:142-147.

YAMADA, YUKIO AND A. E. BELLPopulation Genetics InstitutePurdue UniversityLafayette, Indiana

*Selection for 13-day larval size in TriboliumUnder two mutritional levels

Selection experiments for larval growth under two nutritional levels were conducted for sixteen generations so as to evaluate the effectiveness of various selection methods in the presence of genetic-environmental inter-action.

A strain of Tribolium castaneum, Purdue “+” Foundation stock was used as the experimental organism. The two levels of nutrition, so-called Good and Poor rations, which were originally formulated by R. H. Hardin, were used as the environments. The primary difference between the two rations depends on the content of brewer’s dry yeast and corn oil. The Good contains 10% of dry yeast and 5% corn oil but none at all in the Poor.

The character for selection is the 13-day larval weight in two directions. Genetic parameters of the initial population for the character were: 0.40 in heritability under both environments and the genetic correlation between Good and Poor performance was 0.80.

The experimental populations which were originated from the base population by random sampling are listed in Table 1.

Table 1. Symbols of experimental populations

GL = Selected for large under the Good level every generation.

PL = Selected for large under the Poor level every generation.

GPL = Selected for large on average performance under both levels every generation.

GPL = Selected for large under Good and Poor in alternating generation.

GS = Selected for small under Good every generation.

PS = Selected for small under Poor every generation.

GPS = Selected for small on average performance under both levels every generation.

GPS = Selected for small under Good and Poor in alternating generations.

C = Unselected controls consisted of 20 pair matings, each contributes one male and one female to the next generation.

The experiment was repeated twice but one week apart. Each set of replicated experimental populations was sampled from two different sublines derived from the base population a few generations prior to the initiation of selection.

The mating and selection were made in such a way that each pair produces eggs in the creamer which contains standard wheat medium for 48 hours, and then the parents are transferred to Good medium for 24 hours, Poor ration for 24 hours and additional Good or Poor ration for 24 hours, so as to have two creamers of the same ration for selection purpose depending on the population, until the 7th generation. For instance, GL had two Goods and one Poor, while PL had towo Poors and one Good. The GPL and GPS had only one Good and One Poor. Each mating randomly sampled five larvae for each creamer weighed and the sum of two creamers was used as the selection criterion. The measurement taken under the opposite environment rather than for selection was used as the measure of correlated trait. Since the 8th generation inclusively, two Goods and two Poors were measured for all populations in the same manner. Once the families for selection were decided, the full sibs raised under the standard medium were picked up randomly and sexed for mating to produce the next generation. Therefore, the parental individuals in all lines were never exposed to either Good or Poor but standard medium. The individuals measured were discarded after weighing. This technique eliminates any carry-over environmental effect from parental generation.

Collect the desired pupae for selection. The chips are then collected, their wights recorded, and returned to the pool.

The pupae show no tendency to roll about on the pan provided the weighing room is kept warm but, if necessary, the pan grid can be constructed of a wire mesh to prevent such movement. The number of pupae which can be weighed together is then limited only by the size of pan or size of scale.

SOKOLOFF, A.Department of GeneticsUniversity of California, BerkeleyAnd Natural Sciences DivisionCalifornia State College, San Bernardino

A method for rearing Eleodes (longicollis?) (Coleoptera:Tenebrionidae) in the laboratory

The stink beetle Eleodes (longicollis?), an inhabitant of the desert areas of the Southwestern United States and Mexico, when irritated does a “headstand” (lowering its head and raising the posterior end of the abdomen) and discharges quinines (see illustrations in Eisner, T. and J. Meinwald, 1966. Defensive secretions of Arthropods. Science 153:1341-1350). Because of repeated encounters with this beetle outdoors as well as indoors (janitors often place specimens on my desk should I want to keep them), attempts have been made to maintain the adults in captivity and to find a medium in which these beetles can be maintained in stock in the laboratory.

The adult specimens were initially kept in baby food jars provided with grapes as sources of food and moisture. This food appeared to be adequate since the beetles lived on it for about a month and the female began to lay eggs in clusters. The latter were transferred to another jar containing standard flour beetle medium (whole wheat flour + brewer’s yeast in a proportion of 19:1). The larvae were seen eating this medium, but a few days later they had died, still in the first larvae instar.

In order to stimulate oviposition, the adult female was placed in flour beetle medium, and this proved to be a highly satisfactory food for Eleodes, provided that a source of moisture was available. This was supplied by adding a piece of apple or some other fruit. The increase in moisture induces the growth of mold, so adult beetles have to be transferred rather frequently to fresh medium. Nonetheless, the effort pays off, for at this writing, the beetles have lived in this medium for about five moths, and the female continues to lay eggs. The larvae also require transferring to fresh medium every so often because of the tendency of the medium to cake and eventually become moldy when pieces of fruit are added as a source of moisture. The eggs, somewhat larger than those in Tribolium but alike in this genus of a white color, are laid in clutches of about 20-30. The larvae were only about 1-1/2 to 2 mm long in the first instar. At about 240C, the largest larvae have reached a size of almost three centimeters in three months, and they resemble the larvae of Tenebrio molitor. Out of 110 small larvae introduced in this jar, only about 10 per cent survive because the immature stages of Eleodes are highly cannibalistic. It was possible to observe the cannibalistic activities of these organisms when the larvae were about 1 cm long. At least two tunnels within which the larvae travelled were evident. They were against the glass wall of the container, and a larva from the supper gallery had made its way to the lower gallery where it had encountered a larva of about the same size; it apparently had dealt the latter a bite on the tergite of the mesothorax which largely immobilized -- at least there was no apparent effort on the part of the larva preyed upon to escape--and the predator larva had half of its head within the

body of the larva it was eating. When the larvae are removed from the medium one does not find any pieces of dead larvae, unless the larvae was recently killed.

The medium, insofar as it has been possible to observe, provides larvae the necessary nutritional requirements for good health and development. It is possible, therefore, that the tendency toward cannibalism is a trait characteristic of the family.

It is evident from the above that beetles in this family other than Tribolium and other flour beetles have potentialities of being raised in the laboratory and thus become potentially useful organisms in research population genetics and population ecology.

The writer has not tried it, but it comes to mind that Tenebrio molitor, the mealworm, and Zophobus, another Tenebrionid which normally lives on bat guano, can be reared on bran provided a source of water such as a slice of potato, is provided to the beetles every so often. The survival of Eleodes could also be enhanced by rearing this species in bran medium distributed in a container in layers between paper toweling and to which wood shavings are provided so the smaller larvae can escape the predatory activities of larger larvae. The lack of an incubator at San Bernardino for this particular purpose has prevented the exploration of the question of the optimal conditions of temperature and R.H. for the development of this beetle from egg to adult.

P.S. A visit to the laboratory of Dr. Clyde Willson, Biochemistry Department, University of California, Berkeley, after the above note was written, revealed that Mr. Walter Tschinkel is extremely interested in developing techniques for rearing a wide variety of Tenebrionidae (see Research Note elsewhere in this issue). Mr. Tschinkel informs me that according to Dr. Eisner, Eleodes can be kept on bran for as long as six or seven years in the laboratory (if supplied with water). During this time they produce numerous progeny, but they fail to reach the adult stage. Whether the present diet will prove to be more suitable so Eleodes will undergo metamorphosis remains to be seen.