Amylase/proteinase ratios in larval midguts of ten stored-product insects

J. E. Baker Stored-Products Insects Research and Development Laboratory, US Department of Agriculture, Agric. Research Service, Savannah, GA 31403, USA

Keywords: stored-product insects, digestion, midgut enzymes, amylase, proteinase, trypsin, aminopeptidase, foods, biochemical adaptation

Abstract

Amylase/proteinase ratios in homogenates of isolated midguts of 4 granivorous beetles, Sitophilus oryzae (L.), Sitophilus granarius (L.), Tenebrio molitor L., and Tribolium castaneum (Herbst), which feed primarily on cereals or cereal products, were extremely high relative to those of Dermestes maculatus DeGeer, Attage- nus unicolor (Brahm) (= A. megatoma (E)), Anthrenus flavipes LeConte, and Tineola bisselliella (Hummel). The latter 4 species, which can feed and develop on diets of animal products or foods with relatively high protein content, had higher general proteinase (caseinolytic activity) and aminopeptidase activity and much higher proteinase/amylase ratios than the granivorous coleopterans. Larvae of the moths Anagasta kuehniel- la (Zeller) and Plodia interpunctella (Hubner) had much lower amylase levels and higher proteinase levels than S. oryzae, S. granarius, T. molitor, or T. castaneum. These moth larvae also feed on cereals and cereal products but have more varied feeding habits than the Coleoptera. The enzymology related to initial stages of digestion of large food polymers reflects the biochemical adaptation of these stored-product insects to their preferred foods.

Introduction

Successful adaptation of an insect to a particular food as a source of nutrients essential for growth, development, reproduction, and population main- tenance requires a unique combination of be- havioral, physiological, and biochemical processes (Slansky, 1982). Subsequent to behavioral events leading to food finding and consumption, digestive processes convert ingested food polymers into smaller, utilizable nutrients. Although the complex of enzymes involved in digestion for a given insect species generally corresponds to types of food con- sumed, there have been no specific, comparative studies of levels of major digestive enzymes among species that feed on a variety of different foods.

Species with a wide variety of feeding habits are found among stored-product insects. Included are granivorous species with relatively high carbohy-

Entomol. exp. appl. 40, 41-46 (1986). ~�9 Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands.

drate diets and species that feed on animal products with high protein content. Quantitative compari- sons of specific activities and ratios of midgut en- zymes among ten of these species were therefore conducted. Although enzymes associated with ter- minal stages of digestion of oligopeptides and oligosaccharides in phytophagous species are bound to specific regions of the midgut, enzymes associated with initial hydrolysis of large protein and carbohydrate polymers are soluble and are found in lumen contents (Terra et al., 1979; Ferreira & Terra, 1982; Baker et al., 1984). Since ratios and activities of these latter enzymes are most likely to reflect the degree of adaptation to food or diet components, midgut levels of amylase, general pro- teinase activity, trypsin, and soluble aminopepti- dase in larvae of these species were determined. Results were expressed in terms of specific activities or ratios of activities based on protein or on fresh larval weight.

42

Materials and methods

Insects

Larvae of the following species that attack ani- mal products were analyzed: hide beetle, Dermestes maculatus, reared on Gainesburger | dog food; black carpet beetle, Attagenus unicolor, reared on Purina Lab Chow | with 507o brewer's yeast; and the furniture carpet beetle, Anthrenusflavipes, and the webbing clothes moth, Tineola bisselliella, both reared on woolen cloth dusted with brewer's yeast. Larvae of the following granivorous species were analyzed: yellow mealworm, Tenebrio molitor, and the red flour beetle, Tribolium castaneum, both reared on a mixture of white flour, white cornmeal, and brewer's yeast, 10:10:1.5 (v/v); granary weevil, Sitophilus granarius, and rice weevil, S. oryzae, both reared on the starch-based artificial diet of Baker & Mabie (1973); and the Indianmeal moth, Plodia interpunctella, and Mediterranean flour moth, Anagasta kuehniella, both reared on the diet of Silhacek & Miller (1972). Bombyx mori L. was also included in the study. Cultures were main- tained at 28 ~ or 30~ and 50-60~ RH with a 12:12 LD photoperiod except Sitophilus larvae which were reared under continuous darkness.

Preparation of midguts

Larvae were removed from cultures, weighed in- dividually, and midguts dissected under cold 1~ NaCI. Midguts devoid of food particles were dis- carded. Midguts that were full of food particles were arranged into replicates of 5 or 10 midguts, depending on species. Dissected midguts (including lumen contents) were homogenized in 0.5 ml to 2.0 ml distilled H20 depending on species, cen- trifuged for 30 min at 5300 g, and the supernatant decanted and used for the enzyme assays. Three replicates were prepared for each species. In most cases, larvae in the final instar were selected.

Assays

Amylase was assayed with soluble starch as sub- strate by using the dinitrosalicylic acid (DNS) procedure of Bernfeld (1955) as modified by Baker (1983). One unit of amylase activity was defined as the amount of enzyme producing 1/~mole maltose

hydrate/min at 30 ~ Maltose hydrate was used as the standard.

Trypsin-like and aminopeptidase activity were measured by using o~-N-benzoyl-DL-arginine-p-ni- troanilide and L-leucine-p-nitroanilide, respectively (Baker, ~1981). One unit of activity was the amount of enzyme that hydrolyzed 1 t~mole substrate/min at 30~ A molar extinction value of 8800 at 410 nm was used.

General proteinase activity was measured with 1 ml 1~ casein in buffer as substrate. The reaction was stopped with addition of 3 ml 10~ trichloroa- cetic acid (TCA). Absorbance at 280 nm of TCA soluble material after filtration was determined. One unit of caseinolytic activity was defined as the amount of enzyme producing 1.0 AU280 nm/min at 30 ~ in a 4.02 ml volume. Caseinolytic activity was determined on separate groups of larval mid- guts compared to samples used to analyze amylase, trypsin, and aminopeptidase.

Protein was estimated with the procedure of Lowry et al. (1951). Bovine serum albumin was used as the standard.

Determination of p H optima

Optimum pH for enzyme activity of each species with each substrate was taken from literature values (if available) or was estimated from pH curves es- tablished at 1.0 pH unit intervals. Citrate (pH 4.0, 5.0), phosphate (pH 6.0 and 7.0), tris (pH 8.0), diol (pH 9.0, and 10.0), and carbonate (pH 11.0) buffers of 50 mM concentration were used.

Results and discussion

Activities of amylase, trypsin-like proteinase, and aminopeptidase in midguts of tested species are given in Tables 1 and 2. Larvae of the four granivorous beetles, S. oryzae, S. granarius, T. mo- litor, and T. castaneum had much higher amylase levels individually and as a group than the other species. Highest amylase activities based on fresh weight and midgut protein were found in larvae of S. oryzae. Adults of S. oryzae also have an extreme- ly active amylase with levels 3-fold and 8-fold higher than that found in adults of S. granarius or S. zeamais Motschulsky, respectively (Baker & Woo, 1984). Amylase was barely detectable in A.

43

Table 1. Activities of midgut homogenates prepared from ten species of stored product insects against amylase (starch), trypsin (BApNA), and aminopeptidase (LpNA) substrates. Means _+ S.E.

Species Larval weight (mg)

Units/g fresh body weight

Starch a BApNA b LpNA b

D. maculatus 36.8_+0.8 7.2_+ 2.2 1.4_+0.1 0.2_+0.02 A. unicolor 6.7_+0.2 158.9_+ 10.8 7.6_+0.3 1.4_+0.1 A. fldvipes 2.9 _+ 0.2 0.01 0.4 _+ 0.1 1.0 _+ 0.1 T. bisselhella 1.4+0.1 9.4_+ 0.6 1.3_+0.1 7.1_+0.1 T. molitor 35.0_+ 1.3 795.6_+ 61.7 0.2_+0.02 0.1 _+0.03 S. granarius 3.8 _+ 0.1 1300.0 _+ 147.0 2.4 _+ 0.2 0.6 _+ 0.04 S. oryzae 2.6 _+ 0.1 2049.0 _+ 237.0 1.3 _+ 0.05 0.5 _+ 0.02 T. castaneum 2.4 _+ 0.1 837.6 _+ 111.1 1.5 _+ 0.1 0.6 _+ 0.03 P. interpunctella 10.0_+ 0.6 34.5 _+ 4.9 9.2_+ 0.9 2.9_+ 1.5 A. kuehniella 12.0 _+ 0.5 206.5 _+ 15.1 25.9 _+ 3.1 1.4 _+ 0.03 B. mori 2.7_+0.1 c 0.1_+ 0.01 0.7-+0.1 1.5-+0.1

a 1 unit = 1.0 #mole maltose hydrate/min at 30 ~ b 1 unit = 1.O#mole/minat 30~

Larval weight (g).

Table 2. Specific activities of midgut homogenates prepared from larvae of ten species of stored product insects against amylase (starch), proteinase (casein), trypsin (BApNA), and aminopeptidase (LpNA) substrates.

Species Substrate a

Starch Casein BApNA LpNA

D. maculatus 0.8 _+ 0.3 122.7_+ 3.0 153.1_+ 6.5 25.6+_ 1.9 A. unieolor 8.4 ___ 0.8 171.7-+24.8 399.4-+ 31.8 72.1_+ 4.6 A. flavipes 0.03-+ 0.01 86.0_+ 15.2 62.2_+ 8.9 173.7+20.8 T. bisselliella 0.5 _+ 0.04 138.3-+24.9 73.5_+ 6.4 398.9_+ 9.9 T. molitor 47.0 _+ 8.3 10.8_+ 2.1 14.6_+ 1.1 4.3_+ 1.3 S. granarius 104.4 _+10.0 25.8_+ 3.1 19.0_+ 0.8 50.4_+ 4.3 S. oryzae 137.3 _+ 8.1 9.4_+ 4.4 3.2_+ 0.1 32.9_+ 0.9 T. eastaneum 40.7 -+ 4.8 24.9_+ 8.2 16.0_+ 1.4 30.7_+ 1.1 P. lnterpunetella 1.9 -+ 0.1 33.3_+ 3.2 522.8_+ 24.8 164.9_+ 1.5 A. kuehniella 12.2 _+ 0.8 39.3_+ 5.8 1533.9_+172.2 82.6_+ 0.5 B. mori 0.01_+ 0.001 - 58.0_+ 4.0 129.0_+11.1

a Mean _+ S.E. of duplicate or triplicate analyses of midguts dissected from 3 replicate groups of larvae per species. Depending on species, 5 to 10 midguts per replicate were dissected and homogenized in a final volume of 0.5 ml to 2.0 ml. Casein, BApNA, LpNA

- milliunits/mg protein; starch - units/mg protein.

f lav ipes b u t it was r e l a t ive ly ac t ive in A. unicolor, w h i c h feeds o n w o o l as well as o t h e r a n i m a l a n d

p l a n t p r o d u c t s . A m y l a s e s in t h e 2 p h y c i t i n e m o t h s ,

P. interpunctella a n d A. kuehniella, as well as in T

bisselliella, were m o s t ac t ive in a l k a l i n e ( p H 9.0)

b u f f e r s . A m y l a s e a c t i v i t y in A. kuehniella was

m u c h h i g h e r t h a n t h a t in P. interpunctella b u t it

was m u c h lower in t h e s e g r a n i v o r o u s l e p i d o p t e r a n s

t h a n in t h e g r a n i v o r o u s c o l e o p t e r a n s . T h e a l k a l i n e

a m y l a s e o f B. mori ( K a n e k a t s u , 1978) was re la t ive ly inac t ive .

C o m p a r e d to ac t i v i t y in o t h e r t e s t ed species ,

t r y p s i n - l i k e a c t i v i t y a g a i n s t B A p N A in A. kuehniel-

la was e x t r e m e l y h igh . A c t i v i t y levels were 18.5 a n d

3 .4 - fo ld h i g h e r t h a n in D. maculatus a n d A.

unicolor, respect ively . L a r v a e o f P. interpunctella a l so ac t ive ly h y d r o l y z e d B A p N A . Dermestes

maculatus, A. unicolor, A. flavipes, a n d T. bissel- liella h a d s i g n i f i c a n t l y h i g h e r ac t iv i t i e s a g a i n s t

B A p N A t h a n d i d t h e f o u r g r a n i v o r o u s c o l e o p t e -

rans . Lowes t ac t iv i t i e s a g a i n s t B A p N A a n d c a s e i n

were f o u n d in S. oryzae.

44

Although larvae of the moths A. kuehniella and P. interpunctella had highest levels of activity against BApNA, D. maculatus, A. unicolor, A. flavipes, and T. bisselliella had much higher activi- ty against casein, a general proteinase substrate. Proteinases in these latter species have been exten- sively studied (Baker, 1981, 1982; Ward, 1972, 1975). The complex of endopeptidases and exopep- tidases in these wool and hide-infesting species results in relatively rapid hydrolysis of casein. How- ever, since enzymes that actively hydrolyzed BAp- NA in midgut homogenates of A. kuehniella and P. interpunctella did not hydrolyze casein to a similar extent, the exact role of the hydrolases with trypsin- like (BApNA) specificity in protein digestion in lar- vae of these moth species has yet to be clarified.

About 16 electrophoretically-resolved proteins with aminopeptidase activity have been demon- strated in T. bisselliella (Ward, 1975) and this spe- cies had the highest activity against LpNA in this study. Of the 4 species that can develop on high protein diets, soluble aminopeptidase activity was lowest in D. maculatus. However, much of the pep- tidase activity in this species is insoluble and is bound to midgut cell fragments (Baker, unpub- lished observations). In general, soluble aminopep- tidase activity in the 3 fabric feeding insects, A. unicolor, A. flavipes, and T. bisselliella was higher than that found in the 4 granivorous beetles. Lowest aminopeptidase activity was found in T. molitor.

Ratios of carbohydrase to proteinase activity in larval midguts reflect the generalized feeding ranges of these stored product insect species (Ta- ble 3). The granivorous beetles, particularly S. ory- zae and S. granarius, whose larvae feed internally within cereal grains, have very high amylase/pro- teinase and amylase/aminopeptidase ratios. Cereals generally have a relatively high starch content. For instance, although wheat endosperm contains about 70~ of the total wheat protein (Kasarda et al., 1971), wheat kernels contain about 55~ starch and starch is by far the major nutrient is wheat en- dosperm (D'Appolonia et al., 1971). Assimilation efficiencies for S. oryzae and S. granarius feeding on intact wheat (Singh et al., 1976; Campbell et al., 1976) and corn (Gomez et al., 1982; 1983) range from about 75 to 80~ These high digestibility in- dices not only reflect the quality of cereals as food for these species but also are a measure of the phys- iological and enzymological adaptation of the granivorous beetles to these foods.

Larvae of A. kuehniella and P. interpunctella have much lower amylase and somewhat higher general proteinase activity than the 4 granivorous beetles. Larvae of A. kuehniella prefer to feed on flour or meal but like P interpunctella will feed on a variety of stored food products. Both lepidopte- ran species are omnivorous relative to Sitophilus and the specific activities and ratios of digestive en- zymes reflect the wider range of foodstuffs utilized.

Similar to the granivorous species, larvae of D.

Table 3. Ratios of amylase (starch), caseinolytic (casein), trypsin-like (BApNA), and aminopeptidase (LpNA) activities in isolated lar- val midguts of ten species of stored product insects.

Species Activity ratios a

Casein/s tarch b S ta rch /BApNA ~ B A p N A / L p N A ~

D. maculatus 153 5.1 7.0 A. unicolor 20.4 20.9 5.4 A. f lavipes 2866 0.03 0.4 T. bisselliella 276 7.2 0.2 T. molitor 0.23 3978 2.0 S. granarius 0.25 6500 0.3 S. oryzae 0.07 51225 0.1 T. castaneum 0.6 2792 0.5 P. interpunctella 17.0 3.7 3.2 A. kuehniella 3.2 7.9 18.5 B. mori 0.1 0.5

a Ratios based on units of amylase activity and milliunits of proteinase activity. b Units or mil l iuni ts /mg protein. c Units or mil l iuni ts /mg fresh weight.

maculatus, A. unicolor, A. flavipes, and T. bissel- liella with high proteinase/amylase ratios are well adapted to their particular feeding habits. Only a few moths in the Tineidae and several coleopteran species in the Dermestidae will successfully feed upon and are able to digest various keratinaceous materials. Assimilation efficiencies for several of these species feeding on woolen fabric or on fur range from 40 to 60% (Titschack, 1925; Baker, 1974; Baker & Schwalbe, 1975; Ghauvin & Geu- guen, 1978; Chauvin et al., 1981). These values are comparable to those of many herbivorous insects (Waldbauer, 1968) and again are a measure of the enzymological adaptation of this group of species to their food substrates.

Specific digestive hydrolases such as lipase in Solenopsis (Ricks & Vinson, 1972), proteinases in haemotophagous insects (Gooding, 1975), and the cellulase complex in xylophagous species (Martin & Martin, 1979; Bayon & Mathelin, 1980) are exam- ples of biochemical adaptations to the nutrient composition of the preferred foods of these species. In addition, this study has shown that ratios of en- zymes involved in initial phases of digestion of in- gested foods also reflect the general nutrient com- position of preferred foods of stored-product insects. The dramatic differences in ratios of mid- gut enzymes in species with such a wide range of feeding habits, as used in this study, are an exagger- ation of combinations of very high and very low specific activities of species feeding on relatively restricted diets. However, as feeding habits become less restricted, the relative proportions of enzymes involved in protein and carbohydrate digestion do not show as great a divergence and activity ratios approach unity.

Acknowledgement

I thank S. M. Woo, Biological Technician, for technical assistance during this study. Mention of a proprietary product does not constitute a recom- mendation by the U.S. Department of Agriculture.

45

R~sum~

Rapports entre amylase et proteinase dans les in tes- tins de dix insectes de denrdes stock~es

Le rapport amylase sur prot6inase d'homog6nats de tube digestif moyen de 4 col6opt~res granivores: Sitophilus oryzae (L.), S. granarius (L.), Tenebrio molitor L. et Tribolium castaneum Herbst, qui s'ali- mentent surtout sur c6r6ales ou d6riv6s de c6r6ales, est extr~mement 61ev6 par rapport fl ceux de Dermes- tes maculatus DeGeer, Attagenus unicolor Brahm, Anthrenus flavipes LeConte et Tineola bisselliella Hummel.

Les 4 derni~res esp~ces, qui peuvent consommer et se d6velopper sur des produits animaux ou des aliments avec une teneur en prot6ines relativement 61ev6e, ont en g6n6ral des activit6s prot6inase (acti- vit6 cas6inolytique) et aminopeptidase plus 61ev6es et un rapport prot6inase sur amylase bien sup6rieur

celles des col6opt~res granivores. Les chenilles d'Anagasta kuehniella Zeller et Plo-

dia interpunctella Hfibner ont des teneurs en amy- lase beaucoup plus faibles et, en prot6inases plus 61ev6es que S. oryzae, S. granarius, T. molitor ou T. castaneum.

Ces chenilles consomment aussi les c6r6ales et leurs deriv6s, mais ont un r6gime alimentaire plus vari6 que les col6opt~res. Uenzymologie en relation avec les stades initiaux de digestion des gros poly- m~res des aliments reflbte l'adaptation biochimique de ces insectes des denr~es fl leurs aliments pr6f6r~s.

References

Baker, J. E., 1974. Influence of nutrients on the utilization of woolen fabric as a food for larvae of Attagenus megatoma (F.) (Coleoptera: Dermestidae). J. stored Prod. Res. 10: 155-160.

Baker, J. E., 1981. Resolution and partial characterization of the digestive proteinases from larvae of the black carpet beetle. In: G. Bhaskaran, S. Friedman & J. G. Rodriguez (eds.), Cur- rent Topics in Insect Endocrinology and Nutrition. Plenum, New York: 283-315.

Baker, J. E., 1982. Application of capillary thin layer isoelectric focusing in polyacrylamide gel to the study of alkaline pro- teinases in stored-product insects. Comp. Biochem. Physiol. 71B: 501-506.

Baker, J. E., 1983. Properties of amylases from midguts of lar- vae of Sitophilus zeamais and Sitophilus granartus. Insect Bi- ochem. 13: 421-428.

46

Baker, J. E. & J. A. Mabie, 1973. Growth responses of larvae of the rice weevil, maize weevil, and granary weevil on a meridic diet. J. Econ. Entomol. 66: 681-683.

Baker, J. E. & C. P. Schwalbe, 1975. Food utilization by larvae of the furniture carpet beetle, Anthrenusflavipes. Entomol. exp. appl. 18: 213-219.

Baker, J. E. & S. M. Woo, 1985. Purification, partial characteri- zation, and postembryonic levels of amylases from Sitophilus oryzae and Sitophilus granarius. Arch. Insect Biochem. Phys- iol. 2: 415-428.

Baker, J. E., S. M. Woo & M. A. Mullen, 1984. Distribution of proteinases and carbohydrases in the midgut of larvae of the sweetpotato weevil Cylas formicarius elegantulus and re- sponse of proteinases to inhibitors from sweet potato. En- tomol, exp. appl. 36: 97-105.

Bernfeld, E, 1955. Amylases, c~ and ~. In: S. E Colowick & N. O. Kaplan (eds.), Methods in Enzymology. Academic Press, New York: 149-158.

Bayon, C. & J. Mathelin, 1980. Carbohydrate fermentation and by-product adsorption studied with labelled cellulose in Oryctes nasicornis larvae (Coleoptera: Scarabaeidae). J. In- sect Physiol. 26: 833-840.

Campbell, A., N. B. Singh & R. N. Sinha, 1976. Bioenergetics of the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae). Can. J. Zool. 54: 768-798.

Chauvin, G. & A. Gueguen, 1978. D6veloppement larvaire et bi- lan d'utilisation d'6nergie en fonction de l'hygrometrie chez Tinea pellionella L. (Lepidoptera: Tineidae). Can. J. Zool. 56: 2176-2185.

Chauvin, G., A. Gueguen & G. Vannier, 1981. Croissance, bilan d'6nergie et production d'eau metabolique chez la larvae de Tineola bisselliella. Can. J. Zool. 59: 297-304.

D'Appolonia, B. L., K. A. Gilles, E. M. Osman & Y. Pomeranz, 1971. Carbohydrates. In: Y. Pomeranz (ed.), Wheat Chemistry and Technology. St. Paul, Amer. Assoc. Cereal Chem.: 301- 392.

Ferreira, C. & W. R. Terra, 1982. Function of midgut caeca and ventriculus: Microvilli bound enzymes from cells of different midgut regions of starving and feeding Rhynchosciara americana larvae. Insect Biochem. 12: 257-262.

Gomez, L. A., J. G. Rodriguez, C. G. Poneleit & D. E Blake, 1982. Preference and utilization of maize endosperm variants by the rice weevil. J. Econ. Entomol. 75: 363-367.

Gomez, L. A., J. G. Rodriguez, C. G. Poneleit, D. F. Blake & C. R. Smith, Jr., 1983. Influence of nutritional characteristics of selected corn genotypes on food utilization by the rice weevil

(Coleoptera: Curculionidae). J. Econ. Entomol. 76: 728-732. Gooding, R. H., 1975. Digestive enzymes and their control in

haematophagous arthropods. Acta Tropica 32: 96-111. Kanekatsu, R., 1978. Studies on further properties for an alka-

line amylase in the digestive juice of the silkworm, Bombyx morl. J. Fac. Textile Sci. Technol. (Series E., No. 9) 76: 1-21.

Kasarda, D. D., C. C. Nimmo & G. O. Kohler, 1971. Proteins and the amino acid composition of wheat fractions. In: Y. Pomer- anz (ed.), Wheat Chemistry and Technology. St. Paul, Amer. Assoc. Cereal Chem.: 227-299.

Lowry, O. H., N. J. Rosebrough, A. L. Farr & R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

Martin, M. M. & J. S. Martin, 1979. The distribution and ori- gins of the cellulolytic enzymes of the higher termite, Macro- termes natalensis. Physiol. Zool. 52: 11-21.

Ricks, B. L. & S. B. Vinson, 1972. Digestive enzymes of the im- ported fire ant, Solenops~s richteri (Hymenoptera: Formici- dae). Entomol. exp. appl. 15: 329-334.

Silhacek, D. L. & G. L. Miller, 1972. Growth and development of the Indian meal moth, Plodia interpunctella (Lepidoptera: Phycitidae), under laboratory mass-rearing conditions. Ann. Entomol. Soc. Am. 65: 1084-1087.

Singh, N. B., A. Campbell & R. N. Sinha, 1976. An energy budget of Sitophilus oryzae (Coleoptera: Curculionidae). Ann. Entomol. Soc. Am. 69: 503-512.

Slansky, F., 1982. Insect nutrition: An adaptationist's perspec- tive. Florida Entomol. 65: 45-71.

Terra, W. R., C. Ferreira & A. G. DeBianchi, 1979. Distribution of digestive enzymes among the endo- and ectoperitrophic spaces and midgut cells of Rhynchosciara and its physiologi- cal significance. J. Insect Physiol. 25: 487-494.

Titschack, E., 1925. Untersuchungen fiber den Temperaturein- fluss auf die Kleidermotte. (Tineola bisselliella Hum.). Z. wiss. Zool. 124: 213-251.

Waldbauer, G. P., 1968. The consumption and utilization of food by insects. Adv. Insect Physiol. 5: 229-288.

Ward, C. W., 1972. Diversity of proteases in the keratinolytic lar- vae of the webbing clothes moth, Tineola bisselliella. Comp. Biochem. Physiol. 42B: 131-135.

Ward, C. W., 1975. Resolution of proteases in the keratinolytic larvae of the webbing clothes moth. Aust. J. Biol. Sci. 28: 1-23.

Accepted: June 30, 1985