reactions of immature stages of noctuid moths to juvenoids

Oxygen consumption b y larvae and pupae of Spodoptera littoralis 85

Inderte sich im Verlauf der Entwicklung erheblich. Dabei hatte die Art der Nahrung deut- lichen Einflui3 auf das Gewicht und die Entwicklungsdauer der Raupen und somit auf den Sauerstoffverbrauch. Der Verbrauch an Sauerstoff nahm mit dem Wachstum der Larve und mit der Temperatur zu. Wihrend des Puppenstadiums folgte der Verbrauch einer U-formi- gen Kurve. Die Abhangigkeit des Sauerstoffverbraudx von verschiedenen Nahrungspflan- Zen wird naher betrachtet.

References

ABDEL-SALAM, F. A.; HASSAN, S., 1962: Laboratory rearing procedure for the Egyptian cotton leafworm. Prodenia litura (F.), for toxicological studies. 3rd Cotton Confr. . . Cairo, pp. 1869-1877 (in Arabic).

ABOUL-NASR, A. E.; EL-IBRASHY, M. T., 1969: O n the respiratory metabolism in the pupae of Spodoptera littoralis (Boisd.), (Lepidoptera, Noctuidae). Bull. SOC. ent. Egypte 43, 3 1 1-3 17.

ABOUL-NASR, A. E.; MAHER ALI, A.; ESAAC, E. G.; EL-GOGARY, S., 1975: Effect of rearing Spodoptera littoralis (Boisd.) on different host plants in relation to life span, body weight and fecundity. Z. ang. Ent. (in press).

BADR, N. A., 1967: The development and reproduction of the cotton leafworm Prodenia litura on different host plants. M. Sc. Thesis, Univ. of Alexandria.

ELDEFRAWI, M. E.; TOPPOZADA, A.; MANSOUR, N.; ZEID, M., 1964: Toxicological studies on the Egyptian cotton leafworm, Prodenia litura. I. Susceptability of different larval instars of Prodenia to insecticides. J. Econ. Entomol. 57, 591-593.

KEISTER, M.; BUCK, J., 1964: Respiration: some exogenous and endogenous effects on rate of remiration. In: ROCKSTEIN, M., (ed.), The Physiology of Insecta. Vol. 3, pp. 617-658. .. . . ~ ,. -. New kork : Academic Press.

junius. J. Insect Physiol. 16, 449-459. PETITPREN, M. F.; KNIGHT, A. W., 1970: Oxygen consumption of the dragon fly, Anax

UMBREIT, W. W.; BURRIS, R. H.; STAUFFER, J. F., 1964: Manometric Techniques. Minnea- polis: Burgess.

Reactions of immature stages of noctuid moths to juvenoids

By F. SEHNAL', M. M. METWALLY~, I. GELBI?

With 2 figures

Abstract

Acetone solutions containing 0.01 O / o or more of juvenoids inhibit hatching when administered to freshly laid eggs of Spodoptera littoralis and Mamestra brassicae. Application of 1-100 y g of juvenoids to penultimate instar larvae of these species induces light body coloration in the last instar larvae. The effect on body coloration, however, is ambiguous in Autograph gamma. Metamorphosis is most readily inhibited by administering juvenoids within the second half of the feeding period in the last larval instar and at the start of the pupal instar. The isopropyl 1 ~-methoxy-3,7,1~-trimethyl-2,4-dodecadienoate was the most active compound out of 32 juvenoids examined. It affected A . Ramma at 0.05 pg,

Entomological Institute of the Czechoslovak Academy of Sciences, Praha. ' Present address : Department of Economic Entomology, Al-Azhar University, Nasr City, Cairo, Egypt.

2. ang. Enr. 81 (1976), 85-102 @ 1976 Verlag Paul Parey , Hamburg und Berlin ISSN 0044-2240 I ASTM-Coden: ZANEAL

86 F. Sehnal, M . M . Metwally , I . GelbiC

S. littoralis at 0.8 pg, M . brassicae at 6 pg, and Scotia ipsilon at 50 ,ug per specimen. The treated larvae either grow much bigger than the controls, rarely undergo an extra larval moult, and eventually they produce giant pupae (often inviable); or they develop into intermediate forms showing species specific distribution of larval and pupal features. These observations complemented with other evidence suggest that the juvenile hormone normally is involved in induction of the solitary phase in the development of certain lepidopterans as well as in the control of the length of the last larval instar and of the body size which the larvae attain before pupation.

1 Introduction

Many moths of the family Noctuidae (Lepidoptera) are serious pests in agri- culture and forestry. Some noctuids are also interesting from a theoretical point of view. For example, caterpillars of some migratory species vary in colour in dependence on their population density; it was suggested by FAURE (1943a, b) that these species may form gregarious and solitary phases as do the locusts. The parallelism with locusts would indicate hormonal control of the occurrence of phases in noctuids but few relevant experiments were done. The available data on the r8le of endocrines in the control of growth and development of noctuids (EL-IBRASHY 1971, 1973) indicates that this group differs from species like Galleria mellonella or Bombyx mori that are usually thought to be fairly representative of all Lepidoptera. Treatments of noctuids with juvenoids were either ineffective (EL-IBRASHY and MANSOUR 1970; BRANSHBY-WILLIAMS 1972) or affected the insects only at relatively high doses (BENSKIN and VINSON 1973; GUERRA et al. 1973; ABDALLAH et al. 1974; GAWAAD et al. 1974).

Both the economic significance of noctuids and our ignorance of the r81e of hormones in their development called for a detailed examination of the effects of juvenoids on these insects. We have chosen for our study represent- atives of four major subfamilies of noctuids. The results, which are described below, permit conclusions on the r81e of juvenile hormone in the development of noctuids and on the possible use of juvenoids in their control.

2 Material and methods

The following species were used in this study: the Y-silver moth, Autograph (Plusia) gamma L. (Plusiinae); the greasy (blaek) cutworm, Scotia ipsilon Hufn. (= Agrotis ypsilon Rott.) (Noctuinae); the cabbage armyworm, Mamestra (Barathra) brassicae L. (Hadeninae); and the Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.) (= Prodenia litura [F.]) (Amphipyrinae). For comparative reasons, we have also included data on the effectiveness of juvenoids on the waxmoth Galleria mellonella L. (Galleriidae); some of the figures reported here were taken from the paper by J A R O L ~ M et al. (1969).

All four species of noctuids wcre reared in the laboratory at 20-25°C and 18 hrs photophase. Cultures of A. gamma and S. ipsilon were established from the adults caught in the vicinity of Prague by Dr. I. Novdk of the Institute for Plant Protection, Praha- Ruzyh. The larvae of A . gamma were fed with fresh dandelion leaves (Taraxacum offici- nale) and those of S. ipsilon either the leaves of burdock (Arctiurn sp.) or an artificial diet which was developed by WEISYANN and SVATARAKOVA (1973). The composition of this medium is given in table 1 . The breeding of S. littoralis on the same diet was started from pupae obtained from Prof. M. Hafez of the Faculty of Science, Cairo University, Egypt. The initial culture of M . brassicae was provided by Dr. L. Varjas of the Research Institut for Plant Protection in Budapest, Hungary. The recipe for the food for M. brassi-

Reactions of immature stages of noctuid moths t o juvenoids 87

cue larvae (table 1) was made available to us by Dr. 2. HOSTOUNSK~ of the Entomological Institute CSAV, Praha.

The larvae of noctuids were reared in colonies of up to 50 specimens in 3 liter containers. Cultures were checked daily and freshly ecdysed penultimate or last instar larvae were removed. They were then usually kept in groups of 1 to 5 specimens in Petri dishes (diameter 5-10cm). In certain experiments, the larvae were reared in larger groups in bigger containers. Daily supply of food and regular cleaning of the dishes kept mortality at a negligible level. Dishes with fully grown caterpillars of S. ipsilon, M . brassicae, and

Table 1. Composition of larval diets

Diet for Autographa and Ingredients Spodoptera‘

Beans Brewer’s yeast “Pangamin” Agar Ascorbic acid Sorbic acid Vitamin mixture3 Preserving solution4 Natrium benzoate Formaldehyde (40 O i o ) Nipagin (natrium p-hydroxybenzoate) Linseed oil Destilled water

Diet for Mamertra‘

160 g 51.2 g 20.5 g

5.1 g 1.6 g - - -

3.2 ml 3.2 g 3.2 ml

1024 ml

1 Beans are soaked overnight in 600 ml water, boiled, mashed, and mixed with hot solution of agar in the other 600 ml water. Remaining ingredients are added to the diet under continuous mixing. * Beans are ground and added, along with the other ingredients, into the hot solution of agar in water. 3 Composed from 1497 g glucose, 0.3 g vitamin A, 0.18 g vitamin D, 1.5 g ascorbic acid, 3.0 g choline chloride, 0.075 g menadione, 0.15 g p-aminobenzene, 0.15 g niacin, 0.075 g riboflaflavin, 0.045 g pyridoxine, 0.045 g thiamine, 0.105 g calcium pantotheate, 0.003 g folic acid, 0.015 g vitamin BE, 0.33 g inositol, and 0.66 g biotin. 4 15 g Nipagin (natrium 1-hydroxybenzoate), 20 g sorbic acid, and 170 ml 96 O h ethanol.

S. littoralis were half-filled with saw dust in which the insects pupated. A . gamma pupated in cocoons attached to the walls of Petri dishes and to dandelion leaves. The adults of all species were kept in 5 liter jars and fed on a 10 O / o solution of honey in water. They deposited eggs on strips of folded paper. Under these conditions, the culture of A . gamma was maintained for three generations, that of S. ipsilon for ten generations, and those of M . brassicae and S . littoralis for about 30 generations.

The juvenoids (fig. 1) were obtained from the Institute of Organic Chemistry and Biochemistry, CSAV, Praha, (compounds I, IV, V, VIII-XI, XIII, XVII-XXI, X X V till XXVIII) and from the Zoecon Corporation, Palo Alto, California (the remaining substances). They were dissolved in acetone and applied by means of calibrated capillaries to the eggs, larvae, and pupae. Three batches of eggs were “flooded” with an acetone solution in each test. Since the amount absorbed by one egg could not be determined, the ovicidal activities of juvenoids were expressed in concentrations inhibiting hatching of 50 O/O of the treated eggs (EC-50 - cf. SLAMA et al. 1974). Eggs were treated within 12 hrs after deposition and their hatchability was recorded two days after the eclosion of controls. Each t a t was repeated at least twice.

Larvae and pupae were treated with measured amounts of juvenoids. The penultimatc instar larvae received regularly 1 pl and the last instar larvae and pupae 2 ,ul of juvenoid solution per specimen. At least two groups of six insects were taken for each assay. Activities of juvenoids were expressed as doses in microgrammes per specimen which affected the larval-pupal or pupal-adult transformation in 50 O / o of the treated insects (ED-50 - cf. SLAMA e t al. 1974).

88 F. Sehnal, M . M . Metwally, I . Gelbir

Fig. 1. Chemical structures of tested juvenoids

3 Results

3.1 Effects of juvenoids on larval coloration

The coloration of last instar larvae of A. gamma, M. brassicae, and S. littoralis in our stock culture varied from very light to nearly black. The caterpillars were accordingly divided into four colour categories (table 2) . Most caterpillars of A . gamma were green with greyish longitudinal stripes and were scored as category 2 . The caterpillars of both M . bvassicae and S. littoralis were mostly brown or dark brown with a clear colour pattern and belonged to categories 3 and 4, respectively.

Application of juvenoids to the penultimate instar larvae of M . bvassicae and S. littoralis caused a lighter body colour in the last instar larvae. The effect was obtained with 100 pg of compounds 111, XIV, XVI, and XXIX. Substance XVI seemed most active and was thus used in a closer examination of the pigmentation changes (table 3). It was found that the degree of colour change depended on the dose applied and on the time of application. An effect was produced only after treatments performed within the first three quarters of the penultimate instar; larvae in the middle of that instar seemed to be most sensitive.

Reactions of immature stages of noctuid moths to juvenoids

Table 2. Classification of the dorsal body coloration of caterpillars

89

~~~~~~~~~~

Degree of Species coloration Autographa Mamestra Spodoptera

1 Uniformly light green or slightly greyish.

2 Grey-green with grey longitudinal stripes. Head yellow with two small darker sports.

3 The stripes and head are dark grey, head with two large black spots.

bladr. 4 Stripes dark, head

Leight beige with a darker spot behind head and with white- yellow longitudinal stripes on body sides. Light brown with darker spots on some segments Longitudinal stripes are yellow-brown. Brown with 4 darker and 4 lighter points on every segment.

Nearly uniformly very dark or black.

Uniformly very light brown or reddish.

Light brown with darker lateral longitudinal stripes.

Dark brown with yet darker stripes.

Nearly uniformly very dark or black.

Table 3. Effect of compound XVI on larval coloration'

Applied dose

Average degree of colorarion2 Mamestra Spodoptera Autographa

single grouped population

1.45 2.5 3.7 1.3 1.4 1.4 2.3 3.5 1.4 2.1

1000 pl aceton (control) 1.6 2.5 2.2 2.9 3.2

The compound was applied on caterpillars in the middle of the penultimate instar and the effects were evaluated about three days after the ecdysis into the last larval instar. Caterpillars of Autographa were kept either solitarily (single) or in groups of 5-10 specimens (grouped), andlor in synchronized colonies of 50 caterpillars (population). Cater- pillars of Mamestra and Spodoptera were always reared in groups.

The indicated degrees of coloration represent averages of the classification of 4C100 caterpillars in each experimental group with the aid of Table 2 . Caterpillars of Auto- g r a p h and Spodoptera were kept either on white or on black background but because the background did not influence their response to juvenoids the results of these experimental series were cumulated.

100 PCg 10 Pg

The effect of juvenoids on the coloration of A . gamma was variable. In one series of experiments, neither the solitarily nor the gregariously kept larvae were affected (table 3). On the other hand, larvae treated in the 4th instar with 100 pg/specimen of XVI were not affected in the 5th instar but were lighter than the controls in the 6th (last) larval instar. In another series of experiments performed with caterpillars recently collected in the field, three groups of fifty larvae accommodated in half-liter containers produced after a treatment with XVI darker last instar larvae than the controls kept under the same conditions.

90 F . Sehnal, M . iM. Metwally , I . Gelbii

3.2 Inhibition of metamorphosis

Further development of penultimate instar larvae treated with juvenoids was examined in S. littoralis. All 80 larvae which had received 100 pg/ specimen of XVI ecdysed into last instar larvae and half of them sub- sequently pupated. The pupal ecdysis occurred 9 days after the last larval ecdysis, two days later than in controls, and the pupae weighed an average of 485 mg (320 mg in controls). The other half of experimental larvae produced superlarvae aiid ecdysed again as pupae 9 days later. These pupae weiched an average of 534 mg.

Similar effects were produced with treatments to the last instar larvae. In A. gamma, M . brassicae, and S . littoralis the instar lasted as long as 12 days instead the normal 7-9 days. In S. ipsilon, the interecdysial period was sometimes prolonged from the normal 12 days to 25 days and exceptionally it was even longer. Due to prolonged feeding, the caterpillars attained giant body sizes. For example, S. littoralis larvae treated with 20 pg of XVI weighed up to 2630 mg whereas the body weight of controls did not surpass 900 mg. Similar giants were occasionally seen also in S. ipsilon and M . brus- ricae. The giant larvae eventually either perished or pupated but adults emerged only exceptionally.

The increase in body size without intervening ecdysis occurred mostly in solitary cultures of caterpillars which were treated with moderate amounts of effective juvenoids within the first half of the last larval instar. In other cases, the affected insects frequently did not differ from the controls by their body size but moulted into superlarvae and larval-pupal intermediates instead of pupae. These morphologically affected insects were divided into five categories (tablc 4, fig. 2). A few perfect supcr!zrvae (effect 5) were obtained in S. ipsilon and S . littoralis. Typically the most affected individ-

-. Iab le 4 Clacdication of the cffectc of luvenoida on the larval-pupal transformation

C Normal pupae. 1 Punae with remnant t ot larval orolezs o r with shorter aooendaees ifies. 2i.

I ~I &. \ ., ,

m d 2j). The $core also includes insects which remain in the larval exuvia a n d possess large intersegmental menibraries (fig. 2cj. Insects with prepupal body shape, often incapable of leaving the larval cxuvi'i. P u p i l cuticle prevails but large patches of larval cuticle appear o n the dorsal !Spodopt~i- ,z) or on the 1:iteral (rcniaininji species) parts of the body (figs. 2d,

3 Larval-pi!pnl interniediates with considerably metamorphosed head a n d appcndages, partl). degzncrated larval prolegs, and a i n g disks ubually everted i n i l i~to,qr .zph~ and Scoria, rarely in Mamestra, and esceptionally i n Spodo- p t c r ~ . The pupal cuticle occur\ on prunotum and iiiesonotLiin (rarely also on thi. l a c t abdoininal tergittss) i n A i 4 t o g r ~ p h ~ . on pronotuni aiid lair threc aldominal rergitci i n Scotia, :inJ i n Iatcr.il regions o f most abdominai seg- m c n t s i n Spodoptcra; i n M a n i e s t r a i t foriris irrcgular spots on all bod,-

2

211).

sc$ments (fig\. 2c, 2gj. 4 [mpcrfcct s i iper la rv~ic w i r h long antrnn. ie ; append

i i i f i c t e n r i a t ~ d i n A:rtvgr,zph,r ,uid d i f fused tiny \pan oc-ciir 1ati.rally on the a!idonien of Spodoptc abdomin,il seqiicnt$ o f Scoti<c anc! hfJrnc5tr.z

i Supcr1,1rv'le (iiiorpholufiii:~115 pcrfei.t larva<,

Reactioris of initn,ztwre stages of noctuid moths t o juvenoid, 91

1.ip. 2. Thc larval-pupd i n t e rmcd in to of A u t o g r a p h gmnrnLz L. (figs. n- c) .und .SpodoptcBra lirtoralis (Boisd.) (figs. f-j). The intermediates wcrc classified as follows: a, b, and f =.

scorc 4 ; c and g :=- score 3: d and h =-: score -7; c, i, and j = score 1

i d s maintained the general larval appearance but their mouth parts and nnwnnae were partly differentiated (cffect 4). This effect was common with high doses of juvciioids in A. gc?mnrz (figs. 23, b) and S. ipsilon, less frequent i t s .Ya littoralis (fig. 2f), and rare in M . brassicae.

The less affected insects were characterized by the presence of pupal ci:t!cle which appe;u-ed to spread accortlirig to species specific patterns. In 1'1. ,yriirzi~iu, pupal cuticle occurrcd on the pronotum of insccts with con- silierably metamorphosed appcndagcs and wing disks. In slightl}. less in- flt1::nced nninials it appearcd also on thc tiicsonotuni arid in othcrs both there aixi on the last nbdoniinal tergites (fig. I c ) ; finally in some i t also sprcad to*.\ a d s the middle of the body (fig. 2d). Pupd cuticle always occupied wel! ouilined regions clearly separated from the adjacent areas of larval cuticle. Ti:(: only arcas of Inrvnl cuticle in thc least affected, most pupal-like insects, oc,.urrcd i n intcrscgmcntal rcgions of the middle bod!, segments (fig. 2e).

92 F. Sehnal, M . M . Metwally , I . GelbiC

In the remaining species, pupal cuticle occurred in animals without everted wings. In S. ipsilon and M . brassicae i t formed tiny spots scattered within the larval cuticle either on the last three abdominal tergites (in S. ipsilon also on pronotum) or on all body tergites. In less affected animals the spots were fused into solid regions but an exact delineation between larval and pupal cuticular areas was often impossible to make. In S. littoralis, pupal cuticle first appeared on lateral parts of the 4th-8th abdominal segments (fig. 2f). The last areas of larval cuticle in the least affected individuals of all three species occurred mostly on the meso- and metathorax and first two or three abdominal segments. They were located laterally in S. ipsilon and M . brassicae but dorsally and ventrally in S. littoralis. Pupae of S. littoralis frequently retained reduced larval prolegs.

The distinction between larval and pupal cuticles was easy in A. gamma but often difficult or impossible to make in the other species. In S. ipsilon, the normal larval cuticle forms ‘pimples’ which are responsible for the rugose appearance of larval body surface. The intermediates often possessed this rough cuticle but the centres of pimples were tanned like pupal cuticle. In M . brassicae and S. littoralis, the cuticle of intermediates sometimes appeared similar to larval cuticle in texture and lack of tanning but it was nearly as hard as a typical pupal cuticle. We consider these cuticular types as transient forms between larval and pupal cuticles.

Table 5. Morphological effects of juvenoids XIV (S. littoralis) and XVI (remaining species) administered to larvae at indicated days of the last instar1

Application Autographa Scotra Mamertra Spodoptera Day Dose E P A E P A E P A E P A

1 2OOpg 4.5 100 0 1+4 84 16 - - - l f 4 100 0 3 100 0 1 30 16 0 0 10

2-3 2 0 0 p g 4 100 0 2.5 90 10 - - - 4 2 0 0 p g 3 100 0 3 80 0 - - - 5 2 0 0 p g 2 100 0 3 100 0 - - - 6 2 0 0 u g 1.8 100 0 2 100 0 - - - - - - 7 2 o o p g - - - - - - - - 2.2 30 33 -

- - 0 0 0 - - - - - - 8 200 Icg -

1 20,ug 4.5 100 0 1 16 33 0 0 90 1 5 ? 2 2 0 u g 4 100 0 1 20 74 4 10 90 1.3 30 ?

2.3 60 40 1.5 100 0 4 2 O u g 3 100 0 3 78 16 1.5 40 40 0 0 100 5 2 0 p g 2 80 20 2.4 84 16 1.5 40 0 0 0 100

3 20 ,ug 3 100 0 - - -

6 2Opg - - - 1.5 66 0 0 0 0 - - - 7 2 0 p g - - - - - - - - 1.5 33 66 -

0 0 100 - - -. - - - - - 8 20 rcg -

’ In controls, the pupae ecdysed on day 7 in Spodoptera, on day 8 in Mamestra, on day 9 in A u t o g r a p h , and on day 12 in Scotza. Explanation of abbreviations: E, average morphological effect established according to table 4 ; P, per cent of treated larvae (at least 10 in each group) morphologically affected; A, per cent of treated larvae which produced normal adults.

Most intermediates of A. gamma ecdysed well but intermediates of other species often failed to escape from the old exuvia and died during ecdysis. In addition, many of those which did ecdyse had a hindgut prolapse or deformed abdomen (swollen tip of abdomen without sclerites covered with thin integument) and died very shortly after ecdysis. None of the intermediates of any of the noctuids examined was able to form a pupa.

Reactions of immature stages of noctuid moths to juvenoids 93

The morphological effects of juvenoids depended on the age of treated larvae (table 5). Caterpillars at the start of the last instar responded only to high doses and produced either imperfect (effect 4), nearly perfect (effect 4.5) and perfect superlarvae (effect 5) or defective pupae (effect 1). Applications of juvenoids within the second half of the feeding period were most effective and various larval-pupal intermediates were produced. Treatments performed towards the end of the instar, when the larvae transformed into pharate pupae, did not affect formation of the pupa but often inhibited the following pupal-adult transformation.

The action of juvenoids on the pupal-adult transformation was also examined in tests on pupae 12 hrs after ecdysis (table 6 ) . The most affected insects possessed an adult-like head but lacked scales on the thorax and

Table 6 . Activities of selected juvenoids on the pupal-adult transformation’

Compound Scot ia Spodoptera

I11 0.1 VIII 0.5 XVI 1 XIX 1 XXIII 1 XXIX < 10

5 10 0.1 2 5 5

Juvenoids were administered to freshly ecdysed pupae. Figures in the table indicate amounts in microgrammes which inhibited adult emergence in 50 O i o of treated insects. I

abdomen. The least influenced individuals appeared externally as adults with slightly defective mouth parts but did not emerge from the pupal exuvia.

3.3 Comparison of the activities of selected juvenoids on noctuids

The activities of compounds listed in fig. 1 were assayed on freshly laid eggs and on the last instar larvae in the second half of the feeding period. Table 7, which summarizes the results, records also the activities on the larvae of Galleria rnellorzella; these data will be considered in the Discussion at the end of this paper.

Freshly laid eggs treated with 0.1-1 O / o solutions of the effective juvenoids did not hatch whereas equivalent treatments of eggs with 1 O / o solution of olive oil in acetone had no effect. From this we concluded that 1 O / o and lower concentrations of juvenoids did not cause suffocation of the embryo but exerted some other ovicidal effect. It was not determined, however, if this effect was always due to a morphogenetic action of juvenoids on embryo- genesis rather than to a non-specific toxicity. The most potent ovicides among aliphatic juvenoids were the C-17 Cecropia juvenile hormone (111), the 2.4-dodecadienoates XIV-XVI, and 10-oxadodecenoate XX. From aromatic substances, the highest activity was found in epoxygeranyl ethers of p-ethylphenol (XXIII) and p-chlorphenol (XXVIII). Structures of these ovicidal juvenoids show no distinct common features which would permit conclusions about the relationship between the chemical structure and the biological activity.

94 F . Sehnal, M . M. Metwally , I . Gelbic'

In the tests on last instar larvae (table 7) the morphogenetic response guaranteed that we were dealing with juvenilizing effects. In the group of aliphatic juvenoids, the methyl farnesoate I and most of the farnesane-like dienoates 11-V and dodecenoates VI-XIII had little activity. The highest

Table 7. Doses of juvenoids inhibiting hatching in 50 Oio of treated eggs (EC-50) and affecting morphologically 50 " / o of treated larvae (ED-50)

EC-501 ED-502 Mamestra Spodoptera Galleria Alrtographa Scotin Mamestra Spodoptera

I in. I1 in. I11 0.01 IV in. V in. VI in. VII in. VIII 0.5 IX in. X in. XI in. XI1 in. XI11 1 XIV 0.8 xv 1 XVI 0.5 XVII in. XVIII ? XIX in. xx 1 XXI in. XXII 0.7 XXIII 0.5 XXIV ? xxv in. XXVI in. XXVII in. XXVIII 0.5 XXIX 1 xxx 1 XXXI 1 XXXII 0.5

0.5 in. 5 5 0.5 0.05

in. 80 0.5 8 ? 1 0.5 80 0.5 5 0.1 100 0.1 in.

in. in. 1 in.

in. 10 0.01 0.08 0.1 0.1 1 5 0.5 5 ? in. 0.7 0.5 0.05 8 1 500

in. in. 0.01 0.6 ? in. 5 in.

in. in. 1 >loo 0.1 100

in. 0.08 0.5 3 0.5 in. 0.5 in.

in. in. ? ?

30 200 ? ? 5 200

10 100 10 in. 10 30 10 300

>zoo in. in. in. in. in. 50 in. 5 100 1 50 0.05 50

50 >200 100 ?

1 80 20 ? in. in. in. in. 80 200

200 in. in. in.

? ? in. in.

100 in. 8 200

>zoo >zoo >zoo ?

200 in.

200 ?

200 ?

>200 >zoo

100 in.

100 in. in. in. in. 80 80 5

in. in.

>200 >200

in. in.

>200 in. in. in.

>200 in.

100 200

? in.

in. >loo

500 >200

80 80

100 10 80

>zoo in. in. 60

6 2 0.8

>loo 200

8 >200

200 in. 50

200 in. in. in. in. 10 70 in.

>loo

1 EC-50 values indicate concentrations of juvenoids (in O/o) in acetone which inhibited hatching of 50 O / o of eggs treated within 12 hrs aRer deposition. Juvenoids with EC-50 higher than 1 "/a are considered as ineffective (in.). * ED-50 values indicate doses of juvenoids in niicrogramnies per specimen which affected 50 O/o of treated insects. Juvenoids, which did not cause any effect at 200 ,ug/ specimen were considered as inactive (in.).

activity among these compounds - except for M . bvassicae - was found in ethyl 11-chloro-2-dodecenoate VIII; the corresponding isopropyl ester IX was little active and the N,N-diethylamide X was inactive. Compounds XI and XI1 with additional substituents on C-7 were also inactive. On the other hand, the 2,+dodecadienoates XIV-XVI with farnesane skeleton possessed the highest activities of all compounds tested. Second in activity to these dienoatcs was the 5-oxa-2-dodecenoate XIX. The remaining 5-oxa and 10- oxa esters XVII, XVIII, and X X showed moderate activities.


The aromatic juvenoids were mostly inactive. Significant activity was found only in epoxygeranyl and epoxycitronellyl ethers of p-ethylphenol (XXIII and XXX) and in the methylenedioxybenzene derivative XXIX. The last mentioned compound nearly matched the activity of the most potent aliphatic juvenoids.

The four noctuids we examined were in most instances sensitive to identical compounds but the effective doses greatly varied. For example, A . gamma responded to 0.05 pg of XVI whereas S. littoralis required about ten times, M . brassicae about fie times, and S. ipsilon about 500 times higher doses. These relationships between species sensitivity were different with different compounds.

4 Discussion

4.1 Phase forms in the development of caterpillars

The phenotype of locusts is known to be dependent on population density. The extreme forms of this phenotype variation are called gregarious and solitary phases. The phases differ from one another in coloration, propor- tions of different body parts, rate of development and behaviour. Develop- ment of the phase characteristics is controlled hormonally (STAAL 1961; GIRARDIE and JOLY 1967).

The existence of similar phases in Lepidoptera was first suggested by FAURE (1943a, b) who noticed that crowding induces dark coloration in armyworms Laphygma (Spodoptera) exigue, L. exempta, and Spodoptera abyssinia. The effect of population density on the pigmentation of caterpillars has been confirmed in various species (cf. review by IWAO 1968). LONG (1 953) demonstrated that the population density influenced also the rate of development, number of larval instars, body size, mortality, and possibly also behaviour. Several authors showed that crowding of caterpillars induced pupal diapause and affected also the morphology, size lon- gevity, and fertility of adults (IWAO 1968).

All evidence gathered in ecological experiments showed a clear parallel between the phases of locusts and the effect of crowding in caterpillars and lead LONG (1953) to suggest that the biological principles of the effect of crowding are identical in locusts and caterpillars. Our results confirm his assumption by demonstrating that the larval coloration of M . brassicae and S. littoralis, the species which were used in ecological studies (cf. IWAO 1968), is affected by juvenoids. Similar observation on S. littoralis was also reported by ABDALLAH et al. (1974) and GAWAAD et al. (1974). Thus, the larval coloration in normal development is partly controlled by the juvenile hormone in a similar way as in locusts (JOLY 1952; NBMEC et al. 1970). Fur- thermore, OGURA and SAITO (1972) established that the occurrence of black gregarious pigmentation in ligated caterpillars of Leucania separata is stim- ulated by hormonal factor(s) from the brain-corpora cardiaca complex and from the suboesophageal ganglion. This is again in accordance with the observation that neurohormone(s) control(s) the black pigmentation in locusts (cf. review by ROWELL 1971). Complexity of the control of coloration is seen from the variation of the effects of juvenoids on the pigmentation of A. gamma in our experiments.

96 F. Sehnal, M. M . Metwally, I . GelbiE

The foregoing argument permits us to conclude that phase variations in Orthoptera and Lepidoptera are controlled in identical ways. In both orders, they occur only in some families and have apparantly different economic significance and various amplitude in different species. Information available on the phase coloration in caterpillars is summarized in table 8. Lepidopterans showing the phase variation belong to families Sphingidae, Bombycidae, Saturniidae, Noctuidae, Notodontidae, and Geometridae, respectively, and many of them are known to migrate either as larvae (march- ing columns of armyworms) or adults (e. g. A. gamma).

Table 8. Selected list of lepidopterous species showing the phase dimorphism'

Factors affecting Author Family t h e coloration

(subfamily) CrE!d- juvenoids Species

I ~ o m b y x mori L. Bombycidae Temper- humidity KIGUCHI, 1972

Celerio euphorbiae L. Manduca sexta Johannson Saturnia pavonia L. Autographa (Plwsia) gamma L.

Heliothis virescens (F.) Spodoptera littoralis (Boisd.)

Leucania separata Walk. Diataraxia oleracea L. Orthosia crwda Schiff. Mamestra brassicae L.

ature Sphingidae Yes Yes Sphingidae ? Yes Saturniidae Yes ? Noctuidae Yes Uncertain (Plusiinae) (Noctuinae) ? Yes (Amphipyrinae) Yes Yes

(Acronyctinae) Yes ? (Melanchrinae) Yes ? (Cuculliinae) Yes ? (Hadeninae) Yes Yes

SEHNAL, 19722 TRUMAN et al., 1973 LONG, 1953 LONG, 1953; present study STAAL, 1972p ZAHER and MOUSSA, 1961; present study IWAO, 1962 LONG, 1953 LONG, 1953 HIRATA, 1966; present study

Additional species in which crowding affects the coloration of caterpillars are listed in

Discussion at the First International Zoecon Conference on Insect Hormones, the review by IWAO (1968).

Monterey Beach, California, April 1972.

Juvenoids were reported to induce some colour alterations which cannot be unambiguously classified as phase changes. WELLINGTON (1969) noticed a reduction of melanization in both caterpillars and adults of Malacosoma californicum pluwiale (Lasiocampidae) when the larvae were treated with juvenoids. In Pieris rapae (Pieridae) (HIDAKA and OHTAKI 1963) and Cele- rio euphorbiae (Sphingidae) (SEHNAL, unpubl.) administration of juvenoids towards the end of the last larval instar reduced the dark pigmentation in pupae.

4.2 Role of the juvenile hormone in the last instar caterpillars

Our results demonstrate that administration of juvenoids to caterpillars frequently induces prolongation of the last larval instar. This effect occurs in all noctuids which have been studied in this paper and also in Heliothis virescens (BENSKIN and VINSON 1973). Administration of juvenoids or implantation of corpora allata appears to prolong, under certain conditions, the last larval instar in all Lepidoptera but the body weight increase is parti-


cularly conspicuous in noctuids. The length of the last larval instar may nearly double in Chilo suppresalis (FUKAYA 1962), Galleria mellonella (SEHNAL and GRANGER 1975), Bombyx mori (AKAI and KOBAYASHI 1971), and Cerura vinula (HINTZE-PODUFAL 1971). In Plodia interpunctella (STAAL 1972), Cydia (Carpocapsa) pornonella (GELBIE and SEHNAL 1973), and Hyalophora cecropia (RIDDIFORD 1972) the instar may be prolonged for up to several months with insects eventually dying without undergoing pupation.

EL-IBRASHY (1971, 1973) postulated that allatectomy of the last instar larvae of noctuids leads to a decreased activity of prothoracic glands which in turn results in reduced feeding and smaller pupal size. The information available now, however, supports a different conclusion. It seems that an increased titre of the juvenile hormone or juvenoids inhibits, possibly in- directly via the brain hormones, either the function of prothoracic glands or the action of ecdysone. Consequently, the moulting process is delayed and the insects continue to grow. Pupation apparently ensues only when the titre of the juvenilizing substance falls under an effective threshold. This happens later than normally after administration of juvenoids or aRer implantation of extra corpora allata but precociously in allatectomized larvae as it was shown by EL-IBRASHY (1973) in the case of S. ipsilon. The slight delay of pupation which was observed by EL-IBRASHY (1971) and EL-IBRASHY and SHEHATA (1971) in allatectomized larvae of S. littoralis, was probably caused by the inflicted injury and by the removal of the corpora cardiaca.

There is increasing evidence that corpora allata continue to function, though at a reduced rate, in the last larval instar of many Lepidoptera. The role of the juvenile hormone may be to postpone pupation until the insect either reaches sufficient body size or meets favourable environmental conditions. These goals are achieved through an interplay of several hormones. In noctuids, production of the juvenile hormone seems to be linked with a temporary block of ecdysone secretion or activity but mechanisms controlling food intake and digestion continue to function. Therefore, the larvae treated with juvenoids grow without undergoing ecdyses. In Chilo suppresalis, juvenile hormone appears to impede production of ecdysone and circumvent food consumption; these insects enter a state of diapause during which they neither moult nor grow (FUKAYA 1962; FUKAYA and KOBAYASHI 1966). Diapausing last instar larvae of Diatraea grandiosella, however, maintain a high titre of both juvenile hormone and ecdysone. These larvae undergo stationary moults without any increase in body size (YIN and CHIPPENDALE 1973).

4.3 Pattern of the larval-pupal transformation in epidermis

The juvenile hormone and juvenoids inhibit imaginal differentiation only when administered prior to a critical period of determination (cf. SLAMA et al. 1974). Morphological effects elicited by applying juvenoids to the last instar larvae reveal when the critical period for the larval-pupal transformation occurs in different regions of epidermis. Regions which have passed the critical period do not respond to juvenoids and produce pupal cuticle whereas regions which have not yet passed the critical period do

98 F. Sehnal, M . M . Metwally, I . GelbiE

respond and produce larval cuticle. Examination of various larval-pupal intermediates showes that the larval-pupal transformation starts on the pronotum in A. gamma, on the pronotum and last abdominal tergites in S. ipsilon, on lateral body regions in S. littoralis, and more or lesse simul- taneously on many tergites in M . brassicae. Further progress is directed towards the middle of the body and the last areas maintaining larval determination are intersegmental membranes in A. gamma, central portions of several abdominal tergites and sternites in S. littoralis, and lateral regions of mesothorax, metathorax, and first three abdominal segments in M . brassicae and S. ipsilon. In A. gamma, groups of epidermal cells appear to undergo the transformation coherently and in one determinative step - the intermediates always possess well outlined regions of pupal cuticle next to areas of dis- tinctly larval cuticle. By contrast, epidermal cells of other species seem to differentiate asynchronously and in several steps each of them being sensitive to juvenoids. The intermediates of these species show mozaics composed of tiny spots of larval and pupal cuticles as well as areas of cuticle with features transient between larval and pupal types.

4.4 Chemical structure and the activity of juvenoids on caterpillars

Although noctuid larvae tolerate huge doses of juvenoids they are respon- sive to the same compounds as other Lepidoptera. The following discussion defines several features in the structure of juvenoids which seem to be responsible for the degree of their activity on noctuids and other moths.

The low activity of farnesoate esters (I) for various Lepidoptera (GELBIE and SEHNAL 1973; present study) is probably due to the presence of three isolated double bonds. Interestingly, farnesoate derivatives with longer carbon chain possess high activities for Bombyx mori (OHTAKI et al. 1972), Chilo suppresalis and Galleria mellonella (MITSUI et al. 1973). Compounds with farnesane carbon skeleton and two isolated double bonds (11-IV) are more active than the farnesoate (cf. table 7) and those with two conjugated double bonds are highly active - alkyl 3.7.1 I-trimethyl-2.4-dodecadienoates (XIV-XVI) and their derivatives appear to be the most potent acyclic juvenoids for Lepidoptera (HENRICK et al. 1973; GELBIE and SEHNAL 1973; TRUMAN et al. 1973; AZARYAN et al. 1974; present results).

Additional substituents on C-7 appear to decrease the activity of farnesane-like juvenoids. For example, the 7.1 I-dichloro-3.7.1 I-trimethyl-2- dodecenoates (XI) have low activity or are inactive in Adoxophyes orana (ABDALLAH 1972), Cydia pomonella (GELBIE and SEHNAL 1973), Ostrinia nubialis (LEWIS et al. 1973) and also in our species. On the other hand, a substitution just on C-11 mostly increases the activity. The increase is par- ticularly significant in ethyl 1 ~-chloro-3.7.11-trimethyl-2-dodecenoate (VIII) which is fairly active on all caterpillars (GELBIE and SEHNAL 1973; NOVAK K. and SEHNAL 1973; NOVAK V. and SEHNAL 1973; VARJAS and SEHNAL 1973; present report).

The functional group of juvenoids is often decissive for their species specificity. Galleria mellonella differs from noctuids by a higher sensitivity to ethyl ll-methoxy-3.7.1~-trimethyl-2.4-dodecadienoate (XV) than to corresponding isopropyl ester (XVI). The isopropyl ester is also highly active


on Choristoneura furniferana (RETNAKARAN 1973). Both G. mellonella and noctuids resist the action of N.N-dialkylamides or aliphatic juvenoids.

Synthetic juvenile hormones (11,111) belong to the most active juvenoids for Cydia pomonella (GEL& and SEHNAL 1973), Manduca sexta (TRUMAN et al. 1973), G. mellonella (present report), and also the noctuid Heliothis wirescens (BENSKIN and VINSON 1973; GUERRA et al. 1973). Noctuids examined in the present study, however, were barely sensitive to these compounds.

It is interesting to note that juvenoids with etheric oxygen replacing the methylene group in C-5 or C-10 positions of the farnesane skeleton (XVII-XIX) possess equal or higher activities than the corresponding farnesane-like compounds.

The activity of aromatic juvenoids is influenced both by the length and branching of the aliphatic side chain and by the para substituent on aromatic ring. High activities are found in aromatic ethers of epoxygeraniol, epoxy- citronellol and their homologues su& as 6.7-epoxy-3-ethyl-7-methyl-2- nonenol (LEWIS et al. 1973; JACOBSON et al. 1972). Monotopic benzene derivatives such as I-(9.1O-epoxy-5.IO-dimethyl-l.4-decadienyl)-benzene and related compounds are also fairly active (CHANG et al. 1972; MORO- HOSHI et al. 1972). Effective p-substituents on aromatic ring are alkyls and the methylenedioxy group, in certain cases also chlorine (LEWIS et al. 1973; present results) and alkoxy and acetoxy moieties (JACOBSON et al. 1972). The l.2-(methylenedioxy)-4-/(6.7-epoxy-3.7-dimethyl-2-octenyl)oxy/-benzene (XXIX) and 1-ethyl-~-/(6.7-epoxy-~.~-dimethyl-2-octenyl)oxy/-benzene (XXIII) are possibly the most active aromatic juvenoids on various Lepidoptera (ABDALLAH 1972; JACOBSON et al. 1972; MOROHOSHI et al. 1972; RICHMOND 1972; BENSKIN and VINSON 1973; GUERRA et al. 1973; GELBIC and SEHNAL 1973; TRUMAN et al. 1973).

Acknowledgements

We express our gratitude to all who provided chemicals, insects, and rearing procedures for our study: to the Institute of Organic Chemistry and Biochemistry in Prague and Zoecon Corporation in Palo Alto, and to Drs. M. HAFEZ, 2. HOSTOUNSKP, I. NOVAK, L. VARJAS and L. WEISMANN. Critical reading of the manuscri t by Drs. V. N. BUROV, I. NOVKK, M. R O M A ~ K , G. H. STAAL, and J. H. WILLIS is gratefuty acknowledged.

Zusammenfassung

Reaktionen der Eier, Raupen und Puppen von Eden (Noctuidae) auf Juvenoide Versuche mit G , G l - bis loloigen Acetonlosungen von 32 ausgewahlten Juvenoidverbindungen bei frisch abgelegten Eiern von Marnestra brassicae und Spodoptera littoralis zeigten, dai3 28 dieser Substanzen das Eischliipfen verhinderten, Ein allgemeiner Zusammenhang zwischen der chemischen Struktur und der oviziden Wirkung konnte nicht festgestellt werden. Die mit 1-1 00 p g einiger Juvenoide behandelten Raupen des vorletzten Larvenstadiums farbten sich im letzten Stadium hell (solitare Farbung). Diese Wirkung auf die Larven- Plgmentierung war nicht eindeutig bei Autograph gamma. Die Verpuppung der behandelten Raupen aller untersuchten Arten (einschliefilich Scotia ipsilon) wurde verzogert und die entstandenen Puppen wogen wesentlich mehr als die Kontrollen. Diese Resultate weisen auf die wichtige Rolle des Juvenilhormons bei der Steuerung des Phasenwechsels, der Ent- wlklungslange und des Wachstumsumfangs im letzten Larvenstadium hin. Die mit hoheren Mengen der Juvenoide behandelten Raupen wandelten sich nicht zu Puppen, sondern ent-

100 F . Sehnal, M. M . Metwally , I . Gelbir

weder zu Riesenraupen (im Falle von S. ipsilon und S. littoralis) oder zu larval-pupalen Mischformen (bei allen Arten). Die Verteilung der larvalen und pupalen Cuticula in diesen Obergangsformen erwies, da8 das raumzeitliche Muster der pupalen Diff erenzierung der Epidermis artspezifisch ist. Bei S. ipsilon, M . brassicae und S. littoralis kamen auch inter- mediare Typen zwischen der larvalen und pupalen Cuticula vor. Die morphogenetische Wirksamkeit der Juvenoide wurde an den Raupen des zweiten Drittels des letzten Stadiums getestet. Die hochstaktive Substanz, ll-methoxy-3.7.11-trimethyl-dodeca-2.4-diencarbon- saure-isopropylester, beeinflugte 50 O/o der Tiere bei der Anwendung von 0,05 pgiTier im Falle von A. gamma, 0,s p g bei S. littoralis, 6 p g bei M . brassicae und 50 p g bei S. ipsilon. Die Empfindlichkeit dieser Arten gegeniiber verschiedenen Substanzen war unterschiedlich. Die pupal-adulte Umwandlung wurde nach der Applikation von kleinen Juvenoidmengen auf Praepuppen und frisch gehautete Puppen gehemmt.

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Inst i tut f u r Angewand te Zoologie, Universitat Munchen

Spermatogenesis and testicular development in the gypsy moth Porthetria dispar L.

With 4 figures

By H. S. SALAMA~

Abstract

The morphology of the testes and their development was investigated in the different developmental stages of the gypsy moth Porthetria dispar L. Succession of spermatogenesis was also studied. Spermatogonial cells were found in the testicular follicles of the first three larval instars. Primary spermatocytes first appeared in the fourth larval instar. In the fifth larval instar, prepupal and pupal stages, spermatids and spermatozoa were then formed. Secondry spermatocytes arise from primary spermatocytes by first meiotic division. In the early fifth larval instar, a second meiotic division of secondry spermatocytes took place producing spermatids with rounded heads and in a latter stage fully formed bundles of eupyrene and apyrene spermatozoa were formed. The percentage of developmental sper- matogenic stages was estimated in various insect stages.

1 Introduction

Studies on spermatogenesis of insects are of basic importance for evaluating the effects of chemosterilants, juvenile hormone analogues, irradiation or

Permanent address: Laboratory of Plant Protection, National Research Centre, Dokki, Cairo, Egypt.

2. a n g . En t . 81 (1976), 102-110 @ 1976 Verlag P a u l Pare), I l a m b u r g u n d Berlin ISSN 0044-2240 / ASTM-Coden: ZANEAE

reactions of immature stages of noctuid moths to juvenoids

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