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The distribution and titre of the diapause-associated protein (DAP), which accumulates in the fat body of diapausing larvae of the southwestern corn borer, Diatraea grandiosella , were determined using an antibody raised to the purified protein. Immunoblotting and immunodiffusion showed that the highest concentration of DAP was present in the fat body. Small amounts of the protein were present in the haemolymph and even lower levels were detected in other tissues. Rocket immunoelectrophoresis showed that DAP accumulated in the fat body at the beginning of diapause, reached a plateau, and gradually declined towards the end of diapause. Similarly, the titre of DAP in last instar non-diapausing larvae treated with a JH analogue increased, reached a plateau and then declined. Double immunodiffusion and immunoblotting using the DAP-antibody and extracts of the fat body of D. grandiosella and the southern cornstalk borer, Diatraea crambidoides , and the sugarcane borer, Diatraea saccharalis , revealed the partial immunochemical identity of the DAP of D. grandiosella and a protein present in these two species. Denaturing electrophoresis with immunoblotting showed that the DAP-related proteins of D. crambidoides and D. saccharalis have apparent molecular weights of 33,000 and 36,000, respectively, as compared to a molecular weight of 35,000 for DAP of D. grandiosella . A dot blot analysis showed that no cross reaction occurred between the “diapause proteins” present in the haemolymph of adults of the Colorado potato beetle, Leptinotarsa decemlineata , and the DAP of D. grandiosella .

The larval fat body of the southwestern corn borer, Diatraea grandiosella , was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [ 3 H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [ 3 H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [ 3 H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.

The southwestern corn borer, Diatraea grandiosella , enters diapause as an immaculate mature larva which is a polymorphic variant of the spotted non-diapause larva. Because dormant immaculate larvae could be obtained by treating last stage non-diapause larvae with a juvenile hormone mimic (JHM), experiments were conducted to determine whether these hormonally induced immaculate larvae (HIL) were physiologically comparable to the normal environmentally induced immaculate diapause larvae (EIL). Comparative data obtained about pupation rates, response to JHM, metabolic reserves, oxygen consumption, and the state of spermatogenesis of HIL and EIL led to the conclusion that the HIL were physiologically similar to the EIL. The results demonstrate that the developmental programme of non-diapause larvae could be switched and ‘diapause’ induced solely by the topical application of JHM. We believe that the data further support our hypothesis that the larval diapause of D. grandiosella is initiated and maintained by the juvenile hormone.

1. 1. The proteins of the larval fat body of the southwestern corn borer, Diatraea grandiosella, were subjected to isoelectric focusing and disc electrophoresis in polyacrylamide gels to examine the diapause-associated protein (DAP). 2. 2. The fat bodies of last instar pre-diapausing larvae, as well as those of non-diapausing larvae which had been treated with juvenile hormone or a juvenile hormone mimic, were shown to store DAP. 3. 3. The protein fraction has an isoelectric point of 5.9, and appears to be a single polypeptide unit with a molecular weight of about 35,000. 4. 4. The protein was resistant to hydrolysis at 37°C by midgut enzymes and trypsin. 5. 5. De novo synthesis of DAP was demonstrated both in vitro and in vivo in the fat body of pre-diapausing larvae using radioactive leucine. 6. 6. Isolated fat bodies of 33-day old last instar pre-diapausing larvae incubated in macromolecule-free Grace's medium incorporated [3H]leucine selectively into DAP. 7. 7. Experiments employing [14C] and [3H]leucine and cycloheximide, an inhibitor of peptide bond formation, confirmed the synthesis of DAP in vivo. 8. 8. DAP was released unchanged from the fat body of mid-diapausing larvae in vitro. Since the protein does not accumulate in the haemolymph in vivo it may be structurally modified before or after release in vivo. 9. 9. The possible diapause-related functions of DAP are appraised.

A photothermogram constructed for a central Missouri population (ca. 39°N latitude) of the sunflower moth, Homoeosoma electellum (Hulst) predicted that this population has a seasonal life cycle of 2 complete and a partial third generation per year, and that daylengths of less than 13 h 30 min light/day and mean temperatures of less than 20°C induce the mature larval diapause. A test of the predicted life cycle revealed that larvae entered diapause when they were exposed from their first instar onwards to natural conditions in central Missouri beginning September 15, 1982. Sunflower moths obtained from northwest Texas (ca. 35°N latitude) and northeast South Dakota (ca. 45°N latitude) displayed shorter critical photoperiods for diapause induction at 20°C (12 h 30 min light/day and 12 h 15 min light/day, respectively) than did those from Missouri. The population of the sunflower moth obtained from South Dakota does not, therefore, appear to be adapted to local conditions, and moths might disperse from lower latitudes to establish transiet populations each year.

The effect of hormone treatments on larvae of the southwestern corn borer, Diatraea grandiosella , was examined to explore endocrine interactions which regulate its mature larval diapause. This species is especially suitable for investigating the endocrine control of larval diapause because it ecdyses from a spotted to an immaculate morph at the onset of diapause, and the immaculate morph may undergo up to three stationary ecdyses during diapause. The response of prediapause larvae to a β-ecdysone injection showed that the larvae have the potential to transform into the immaculate morph early in the final larval instar, but under normal conditions this ecdysis occurs after larvae reach maturity. Since a high rate of pupation occurred among early diapause larvae which received a head ligature, followed 17 days later by a β-ecdysone injection, diapause larvae retain active corpora allata. Since a head ligature prevented diapause larvae from responding to repeated topical applications of a juvenile hormone (JH) mimic or JH 1, the intermediate titer of JH associated with larval diapause may inhibit the synthesis or transport of ecdysiotropin, or its release from the corpora cardiaca. Current results suggest, therefore, that an interaction between the cerebral neurosecretory system and the corpora allata regulates the initiation, maintenance, and termination of this larval diapause.

Further evidence is presented to demonstrate the involvement of juvenile hormone (JH) in regulating diapause in the final larval stage of the southwestern corn borer. Diatraea grandiosella. JH titres in the haemolymph were measured throughout the entire diapause period. Additional results showed that actively secreting corpora allata are necessary to maintain diapause because allatectomized larvae terminated diapause prematurely. A topical application of JH mimic 2 days after the allatectomy prevented this premature termination of diapause. Intact nervous connections between the brain and the corpora allata were necessary for the maintenance of JH secretion. Other surgical work showed that the brains of nondiapausing larvae exhibited a higher ecdysiotropic activity than those of pre-, early-or mid-diapausing larvae. A single application of a JH mimic was more effective in maintaining a diapause-like state in nondiapausing larvae than were repeated topical applications of C18-JH or an implantation of active corpora allata, suggesting that JH was more rapidly metabolized than was the JH mimic. The oxygen consumption of diapausing larvae which had received repeated topical applications of JH mimic was not significantly elevated over that of the controls indicating that treated larvae maintained a low metabolic rate even though they reverted to the spotted morph. A single application of 0.03 μg JH mimic/larva was sufficient to prolong diapause, thereby confirming that JH is necessary for diapause maintenance.

Pre-diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were found to store an electrophoretically discrete protein fraction which makes up about 20% of the soluble proteins of the fat body of newly diapaused individuals. This fraction appears to be restricted to the larval fat body and haemolymph. It accumulates in the fat body of pre-diapausing larvae and reaches a maximum titre in newly diapaused individuals. The protein is utilized gradually during diapause so that only traces of it remain after diapause has terminated. The protein fraction was found to accumulate in the fat body of non-diapausing larvae which had been treated with a juvenile hormone (JH) mimic. This treatment induces a diapause-like state. The involvement of JH in the accumulation and utilization of the protein was investigated by examining the proteins of the fat body after larvae had been allatectomized, or had received a JH mimic application, or had received both treatments. The results showed that non-diapausing larvae began to accumulate the protein fraction within 8 days of receiving a single application of JH mimic. Allatectomy of pre-diapausing larvae was shown to suppress the accumulation of the protein. This suppression was partially reversed by a topical application of JH mimic. JH mimic treatments and allatectomy of diapausing larvae were also shown to cause a decrease in the titre of most of the proteins of the fat body including the diapause-associated protein. The diapause-associated protein has a molecular weight of about 41,000, and contains 15–17 different amino acid residues, of which aspartate, leucine, lysine, and glutamate predominate, comprising about 43, 37, 35 and 32 residues per molecule, respectively. We suggest that diapauselin is a suitable name for this protein. Our findings provide the necessary framework for undertaking a more detailed analysis of the hormonal regulation of diapauselin synthesis and utilization.

The possible involvement of juvenile hormone (JH) in controlling the mature larval diapause of the European corn borer, Ostrinia nubilalis, was examined using biological and chemical assays for JH titres, topical applications of JH mimic, and injections of 20-hydroxy-ecdysone. Bioassays of extracts of larval haemolymph showed that (1) 4th instar pre-diapausing larvae had a higher JH titre (ca. 1450 Galleria Units (GU)/ml) than equivalent non-diapausing larvae (ca. 340 GU/ml), and that (2) 5th instar pre-diapausing larvae contained a JH titre of ca. 320 GU/ml, which declined to ca. 90 GU/ml in newly-diapaused larvae. Chemical assasys carried out on extracts of whole larvae showed that early diapausing larvae contained an extremely low titre of JH. In addition, the application of JH mimic or 20-hydroxy-ecdysone or both agents to diapausing larvae failed to reveal the presence of a functional JH titre during diapause. The application of JH mimic to early 5th instar non-diapausing larvae produced moribund larval-pupal intermediates rather than supernumerary larvae. Our results, therefore, suggest that although JH may control some phases of diapause induction, it is not involved in maintaining diapause.

The organization of the retrocerebral gland system in larvae of six species of Lepidoptera belonging to the family Pyralidae was compared using light and electron microscopy. We have demonstrated for the first time the presence of separate corpora cardiaca and corpora allata in the following economically important borers: the southwestern corn borer, Diatraea grandiosella, the sugar cane borer, Diatraea saccharalis, the European corn borer, Ostrinia nubilalis, and the rice stalk borer, Chilo plejadellus. In these species a long nervus corporis allati (ca. 300 μm) runs from the corpus cardiacum to the corpus allatum which is attached to the duct of the mandibular gland. The identity of the corpora allata of D. grandiosella was confirmed by transplantation. Corpora allata removed from pre-diapausing larvae and implanted into the haemocoele of early last stage non-diapausing larvae led to a high incidence of supernumerary larval rather than pupal ecdyses.

The juvenile hormone (JH) binding activity of the plasma of pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella , was examined. The source of the high affinity JH binding protein was investigated, and the protein was purified and partially characterized. The total JH binding activity for JH-I and JH-III increased in the plasma of pre-diapausing larvae, reached a plateau at the beginning of diapause, and remained relatively high throughout diapause. Neither the total JH binding activity nor the activity of the high affinity JH binding protein appeared to be related to the JH titre present in the plasma. The fat body of pre-diapausing, early diapausing, and mid-diapausing larvae was found to release a high affinity JH binding protein when it was incubated in vitro in Grace's medium. The maintenance of the relatively high levels of high affinity JH binding protein in the plasma of diapausing larvae may, therefore, depend upon the continued synthesis and release of the protein from the fat body. The high affinity JH binding protein present in the plasma of diapausing larvae was purified using gel filtration, ion exchange chromatography, isoelectric focusing and electrophoresis. The purified protein was subjected to denaturing electrophoresis and found to consist of a single polypeptide chain with an apparent molecular weight of about 30,000, and to exhibit a dissociation constant of 4.6 × 10 −7 M at 4°C for JH-I.

The effect of carbohydrates on feeding behaviour and growth and development of the southwestern corn borer, Diatraea grandiosella, was examined. A feeding behavioural biossay based on establishment of first stage larvae on test or control stations within 24 hr was employed. A nutritional bioassay evaluated larval growth rates and metamorphic success of larvae reared on test or control diets. The principal results showed that glucose, fructose, sucrose, and dextrins were feeding stimulants and permitted optimum growth and development. A dietary supplement of amylopectin allowed reasonably good growth while amylose was not a utilizable carbohydrate. The pentoses, arabinose, ribose, and xylose, as well as galactose, mannose, and sorbose, were found to inhibit larval growth. Since of all these sugars only xylose acted as a feeding deterrent it was proposed that at high concentrations they or metabolic derivatives were toxic. The results are discussed in relation to those obtained with several other insect species.

The effect of daylength and temperature on the regulation of the larval diapause of a central Missouri population of the sunflower moth, Homoeosoma electellum, was examined. Fully grown fourth-instar larvae exhibit a facultative diapause. Measurements of the effect of photoperiod on diapause induction revealed critical photoperiods of about 13 h 30 min light/day at 20°C, and between 11 h 45 min and 12 h light/day at 23°C. Third and fourth-instar larvae were shown to be the main sensitive stages for diapause determination. Daylength was also shown to be an important regulator of the rate of diapause development. A short day of LD 10:14 h permitted only a low rate of diapause development, whereas long days of LD 14:10 h and LD 16:8 h accelerated diapause development at 25 and 30°C. When long days were alternated with short days at 30°C the accelerating effect of long days on diapause development was not found. Systematic transfers of chilled diapausing larvae revealed an accelerated diapause development in groups transferred from 10 to 30°C LD 10:14 h, but diapause development was not accelerated in groups transferred from 10 to 30°C LD 16:8 h.

The effect of various combinations of photoperiod and temperature on the induction and termination of the mature larval diapause of a Missouri strain of the southwestern corn borer. Diatraea grandiosella, was examined. Larval exposure to regimes in which the low phase of a 30°:23°C thermoperiod coincided with a scotophase of 10 to 14 hr duration led to high incidence of diapause. Larval exposure to 30°:24°C, 33°:21°C, and 36°:18°C thermoperiods with half cycles of 12 hr in continuous darkness yielded a diapause incidence of 16%, 22%, and 59%, respectively, whereas exposure to a 30°:24°C thermoperiod in continuous illumination yielded a completely nondiapause generation. Larval exposure to one of a series of 36°:18°C thermoperiods in which the duration of the high phase was increased in 2 hr increments from 0 to 24 hr in continuous darkness showed that “short-day” thermoperiods yielded a high incidence of diapause. However, no clearly defined critical thermoperiod was observed. An examination of photoperiodic and thermoperiodic effects on diapause development showed that, in general, those combinations of temperature and light cycles which were diapause inductive also retarded diapause development. The relationship between seasonal photoperiods and thermoperiods in southeastern Missouri was examined.

The source of the lipophorin present in the larval haemolymph of the southwestern corn borer, Diatraea grandiosella , was examined in vitro . Although lipophorin was shown to be one of several proteins released from cultured fat body and midgut, only fat body was shown to synthesize lipophorin. Fat body, incubated in a medium containing [ 3 H]leucine, was shown to release radiolabelled lipophorin using immunoprecipitation. Similar studies using midguts incubated in a medium containing [ 3 H]leucine did not reveal any synthesis of lipophorin. Lipophorin was isolated by density-gradient ultracentrifugation from media in which the fat bodies of about 600 diapausing larvae had been incubated for 4 hr. The isolated lipophorin had a peak density of 1.11 g/ml, and contained various lipids including diacylglycerol, triacylglycerol, sterol, hydrocarbon, free fatty acid, phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin.

The release of protein from the perivisceral fat body of non-diapausing, pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella , was examined in vitro . Time course studies showed a selective release of proteins into macromolecule-free Grace's medium. The rate of release of individual proteins differed. The release of some proteins was partially inhibited by the incorporation of potassium cyanide (10 −2 M) and ouabain (5 × 10 −3 M) into the medium. During a 5 min incubation a single major high molecular weight protein fraction was released at a high rate from the fat body of both non-diapausing and diapausing larvae. A low molecular weight protein (the diapause-associated protein) was also released readily from the fat body of diapausing larvae. Although most proteins released from the fat body in vitro appeared to be present in the haemolymph in vivo , one notable exception was the absence of the diapause-associated protein from the haemolymph. The method holds promise for facilitating further studies of protein release from insect fat body.

The properties of the high-affinity low molecular weight juvenile hormone (JH) binding protein present in the hemolymph of larvae of five species of pyralid moths, a noctuid moth, and a sphingid moth were compared. The pyralid moths exhibit a facultative diapause as last-instar larvae. The species employed were the southwestern corn borer, Diatraea grandiosella, the southern cornstalk borer, Diatraea crambidoides, the sugarcane borer, Diatraea saccharalis, the European corn borer, Ostrinia nubilalis, the sunflower moth, Homoeosoma electellum, the cabbage looper, Trichoplusia ni, and the tobacco hornworm, Manduca sexta. The binding characteristics of the proteins were determined using saturation binding assays and competitive binding assays. The dissociation constants of JH I, JH II, and JH III for the binding protein of all the species varied from 0.8 x 10−7 M to 2.8 x 10−7 M. Calibrated gel filtration showed that the binding protein of all the species had apparent molecular weights ranging from 29,000 to 31,000. Electrophoresis in 7% acrylamide gels revealed that the relative mobilities of the binding proteins ranged from 0.33 to 0.43. Isoelectric focusing showed that the binding proteins had isoelectric points between 4.4 and 5.0.

The binding of tritiated juvenile hormone I, and the juvenile hormone mimics, methoprene and hydroprene (alkyl-3,7,11-trimethyl-2,4-dodecadienoates), to proteins of the haemolymph of larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. The techniques of analytical isoelectric focusing, disc electrophoresis and gel filtration were employed. Juvenile hormone was shown to bind to a high affinity low molecular weight protein in the haemolymph of diapausing larvae. This protein has an apparent dissociation constant of about 3.1 × 10 −7 M at 4°C, an isoelectric point of about 4.85, and a molecular weight of about 3 × 10 4 . Juvenile hormone was also bound to at least one low affinity high molecular weight protein fraction. High juvenile hormone binding activity was found in the haemolymph of newly diapaused larvae. Methoprene and hydroprene bound selectively to a different protein fraction present in the haemolymph of pre-diapausing larvae. This protein fraction had a low relative mobility when subjected to electrophoresis at pH 8.9. A preliminary study of the binding of tritiated juvenile hormone I to proteins of the haemolymph of pre-diapausing larvae of the southern cornstalk borer, Diatraea crambidoides, was also conducted in vitro. A low molecular weight binding protein with identical electrophoretic properties to that of D. grandiosella was present. The high molecular weight binding protein fraction of the two species, however, had different electrophoretic properties.

A comparative study was undertaken of southwestern corn borers (Diatraea grandiosella) collected from south central Mexico (19°N latitude) and southeast Missouri (37°N latitude). All the life stages of the Mexican insects were found to be larger or heavier, or both, than were those of the Missouri insects. Mexican larvae grew at a higher rate and attained a significantly heavier body weight than did Missouri larvae. Although both Mexican and Missouri larvae underwent ecdyses during diapause, Mexican larvae ecdysed more frequently than did the Missouri larvae at 23°C light:dark 12 h:12 h. Larvae that ecdysed most frequently did not necessarily spend longer in diapause. Electrophoresis of the fat body and haemolymph proteins of Mexican and Missouri larvae revealed similar patterns. The fat body of diapausing Mexican larvae contained substantial amounts of a diapause-associated protein which has been characterized previously from the fat body of Missouri Larvae. Double immunodiffusion confirmed that the diapause-associated protein of the Mexican larvae was identical to that present in the Missouri larvae. Smaller amounts of this protein appear to be present in the fat body of diapausing Mexican larvae than are present in that of diapausing Missouri larvae.

Disc electrophoresis was used to examine and characterize the esterases present in the fat body, haemolymph, and midgut of last stage larvae of the southwestern corn borer, Diatraea grandiosella . Significant temporal changes were observed in the pattern of the 4 major esterases of the fat body and 3 major esterases of the haemolymph. These changing profiles presumably relate, in part, to a requirement for the degradation of juvenile hormone (JH) in preparation for metamorphosis. The binding capacity of esterases present in the larval midgut towards JH I and three JH mimics (alkyl-3,7,11-trimethyl-2,4-dodecadienoates) was also examined. The midgut of last stage nondiapausing larvae was shown to contain a carboxylesterase which bound all three JH mimics. Another esterase which bound JH I, but not the mimics, was also present. An esterase with a similar electrophoretic mobility was detected in the haemolymph and integument. Since the JH I binding esterase did not bind the JH mimics, the mimics do not appear to synergize JH by inhibiting its ester hydrolysis.

A comparative study of the fat body of diapausing and non-diapausing larvae of the corn borer, Diatraea grandiosella , was undertaken using the electron microscope and the oxygen electrode. The electron microscopic results showed a shift from a synthetic to a storage function taking place in a 1 to 2 day period during the final instar of non-diapausing larvae, and in a 4 to 8 day period in that of pre-diapausing larvae. This transition was characterized by a decrease in the number of mitochondria and amount of rough endoplasmic reticulum, and by an increase in the number of proteinaceous granules and lysosomes. In vitro measurements using the oxygen electrode showed that the fat body is a normal aerobic respiratory tissue. The tissue reacted in a predictable manner to inhibitors of oxidative metabolism, including malonate, rotenone, oligomycin, and antimycin, and to the uncoupler, dinitrophenol. During the last instar the observed decrease in the respiratory rate of the fat body coincided with the observed ultrastructural changes in its cells. The fat body of 75 day old environmentally induced and juvenile hormone induced diapausing larvae consumed 90% and 78% less oxygen, respectively than that of 14 day old non-diapausing larvae.

The hormonal mechanism which controls the larval diapause of the southwestern corn borer was examined. The onset of this facultative mature larval diapause is marked by a transition from a spotted to an immaculate larval form, and during diapause individuals may undergo one or more stationary larval ecdyses. Experiments were designed to uncover the nature of the humoral mechanism regulating this diapause state. The finding that injecting diapause larvae with 20-hydroxyecdysone only brought about a stationary larval ecdysis suggests that diapause was not maintained by the lack of ecdysone. Neck ligations performed on larvae which had just entered diapause resulted in a premature termination of diapause, and larval-pupal ecdysis occurred in the thoraco-abdominal section, suggesting that a cephalic factor was necessary for the maintenance of diapause. This finding was further supported by the discovery that injecting 20-hydroxyecdysone into the thoraco-abdominal section of previously ligated diapause larvae also resulted in a premature termination of diapause and larval-pupal ecdysis, indicating that ecdysone only initiated the pupal moulting cycle when the cephalic factor was absent. Further experiments led to the conclusion that the juvenile hormone is the cephalic factor. Topical treatment with a juvenile hormone mimic caused non-diapause mature larvae to become immaculate and enter diapause. Periodical topical application of this mimic to diapause larvae prolonged diapause and increased the number of stationary larval ecdyses. These findings suggest that the initiation and maintenance of diapause are regulated by juvenile hormone titre. Results indicate that larvae retain a high titre of juvenile hormone until the last stages of diapause. Injection of 20-hydroxyecdysone into early or middiapause larvae only caused stationary larval ecdyses, while the same injection into larvae in the late stages of diapause caused some of them to pupate. Histological studies of the neurosecretory cells, corpus cardiacum-allatum complex, and prothoracic glands showed that the endocrine system was not inactive during diapause. A new hypothesis is therefore proposed which recognizes the existence of hormonal activity during larval diapause and emphasizes the principal regulatory role of juvenile hormone.

The digestion, absorption, and transport of [4-14C]cholesterol and glycerol tri [1-14C]oleate were examined in the midguts of fifth instar larvae of the southwestern corn borer, Diatraea grandiosella. The results indicate that the anterior region of the midgut is the most active zone for the absorption and transport of lipids into the haemolymph. The middle and posterior regions of the midgut appear to serve as temporary storage sites for absorbed lipid. Cholesterol was absorbed into the midgut cells without prior esterification and released into the haemolymph as free sterol. Although absorbed sterol was not esterified in the anterior region of the midgut, a substantial portion was esterified in the middle and posterior regions. Analysis of midgut contents and faeces showed that triolein was hydrolyzed in the lumen. Free fatty acids and unhydrolyzed triolein were the main labelled lipids in the faeces. In the midgut tissue radioactive [14C] from triolein was incorporated into phospholipids and triglycerides. The anterior region had the highest titre of labelled phospholipids, diglycerides and free fatty acids, and the middle and posterior regions had the highest titre of labelled triglycerides. The results suggest that free fatty acids and possibly diglycerides are absorbed. Diglycerides were the major labelled lipid in the haemolymph.

The major protein fraction of the larval hemolymph of the southwestern corn borer, Diatraea grandiosella, was shown to be composed of approximately six native proteins, and accounted for up to 55% of the plasma proteins. The apparent molecular weights of these proteins ranged from 350,000 to 500,000, as determined by nondenaturing electrophoresis at a neutral pH. Lower apparent molecular weights were obtained when the major protein fraction was subjected to electrophoresis at a high pH under nondenaturing conditions, indicating that these proteins dissociated under alkaline conditions. The isoelectric points of the major hemolymph proteins fell between 5.6 and 5.9. Denaturing electrophoresis and two-dimensional electrophoresis showed that the native proteins were composed of subunits having apparent molecular weights of 78,000, 84,000, and 87,000. These subunits were the major labeled polypeptides found in the hemolymph of feeding last instar larvae 24 h after they had been injected with [3H]leucine. They were also the major polypeptides synthesized when the fat body of last instar prediapausing larvae was incubated in vitro for 4 h in Grace's medium containing [3H]leucine. Immunoelectrophoresis confirmed that the major hemolymph proteins were present in the larval fat body. In addition, these proteins were the major proteins present in the hemolymph of diapasuing larvae.

Disc electrophoresis was used to examine and characterize the esterases and lipase-like enzymes present in the midgut cells and digestive fluid of larvae of the southwestern corn borer, Diatraea grandiosella. Seven esterases and two lipase-like enzymes were detected in the midgut cells, whereas only four esterases and one lipase-like enzyme were detected in the digestive fluid. Although actively feeding non-diapausing and prediapausing larvae had similar patterns of esterases, the titres of the slow-migrating esterases decreased during starvation and diapause. Genetic polymorphism was found among the rapidly-migrating carboxylesterases. The results also show that the major lipase-like enzyme of the midgut cells is not secreted in the lumen. Lipolysis in the intestinal lumen appears to be carried out by a slow-migrating enzyme secreted only from the posterior section of the midgut.

1. 1. The functional properties of 3 lipoprotein fractions, designated lipoproteins 2–4, 5, and 8–9, were examined in the haemolymph of feeding and diapausing larvae of the southwestern corn borer, Diatraea grandiosella. 2. 2. The lipid composition of the 3 fractions varied in last instar diapause-programmed larvae, reflecting a change from the feeding and storage of lipids in pre-diapausing larvae to the utilization of lipids during diapause. 3. 3. Lipoproteins 2–4 and 5 carried diglycerides as their major neutral lipid class, whereas lipoprotein 8–9 carried free fatty acids, triglycerides, hydrocarbons, and polar lipids as its major lipid classes. 4. 4. Lipoprotein 5 of early diapausing larvae fed corn tissue contained plant-derived pigments, including a derivative of α-carotene. 5. 5. Lipoprotein 8–9 was released in vitro from the fat body, and constituted a major protein fraction of the haemolymph of larvae, pupae, and adults. 6. 6. The protein components of lipoprotein 8–9 had the following characteristics: apparent molecular weights of about 5.2 × 105; isoelectric points of 5.5 and 5.65; and aspartate, glutamate, tyrosine and phenylalanine as their principal amino acids.

Seasonal adaptations of populations of the southwestern corn borer, Diatraea grandiosella , obtained from south-central Mexico (19°N latitude) and southeast Missouri (37°N latitude) were compared. Day length and temperature were found to serve as environmental cues to programme the larval diapause of both populations, but different critical values were observed. The critical day length for diapause induction was about 13 hr light/day for Mexican larvae and about 15 hr light/day for Missouri larvae, and was relatively stable at 20 to 30°C. Mexican larvae displayed a less-intense diapause than did Missouri larvae. Some diapausing Mexican larvae maintained at 25 or 30°C pupated in about 15 days, regardless of the day length to which they were exposed. The rate of diapause development of Mexican larvae was high at day lengths between 14 hr and 16 hr, whereas that of Missouri larvae was accelerated at day lengths of 16 hr at 25 and 30°C. Diapause development of Mexican larvae was virtually unaffected by chilling at 10°C, whereas that of Missouri larvae continued at a low rate at 10°C. Selection of Mexican larvae for diapause showed that only four generations were needed to significantly increase the incidence of diapause.

1. Lipophorin was isolated from the hemolymph of diapausing larvae of the southwestern corn borer,Diatraea grandiosella, and shown to have a density of 1:11 g/ml and a molecular weight of 520,000. 2. The holoprotein was comprised of apolipoprotein-I (Mr=231,000) and apolipoprotein-II (Mr=74,000), and contained about 38% lipid (28% neutral lipids, 10% polar lipids), and 3% carbohydrate. 3. The major neutral lipids present were diacylglycerol and sterol, while the major polar lipids present were phosphatidyl choline, phosphatidyl ethanolamine, and sphingomyelin. Carotenoids were also present. 4. Apolipophorin-I and apolipophorin-II were immunochemically distinct. The holoprotein was immunochemically identical to a protein present in the larval hemolymph of two other species ofDiatraea. 5. A mean concentration of 5 mg lipophorin/ml hemolymph was determined using rocket immunoelectrophoresis.

The proteins of the fat body of non-diapausing, pre-diapausing, and newly-diapaused larvae of the southwestern corn borer, Diatraea grandiosella , were examined. Since a low titre of juvenile hormone (JH) is present in the haemolymph throughout the final instar of non-diapausing larvae, the hormone does not appear to stimulate the pre-metamorphic synthesis of proteins. In contrast, the high titre of JH in the haemolymph during the final instar of pre-diapausing larvae appears to stimulate the synthesis of selected proteins. For example, pre-diapausing larvae store in their fat body a low molecular weight protein which has been named the ‘diapause-associated protein’. When non-diapausing larvae were treated topically with C 17 -JH or a JH mimic, from 50 to 70% entered a diapause-like state as fully grown larvae. These hormone-treated larvae accumulated the diapause-associated protein and a high molecular weight protein in their fat bodies. Both of these proteins were shown to be released from the fat body of newly-diapaused larvae in vitro , and may function in the haemolymph during diapause. The high molecular weight protein, isolated from the haemolymph, was shown to contain neutral and polar lipids, including biochromes. Its storage in the fat body and release into the haemolymph may be essential for the transport of lipids during diapause. The fat body proteins of newly-diapaused larvae of the southern cornstalk borer, Diatraea crambidiodes , were also examined electrophoretically. They were found to contain a similar protein pattern to that of D. grandiosella , including the presence of a diapause-associated protein.

The effect of dietary carbohydrates on feeding behaviour, food consumption, and survival of adult rice weevils, Sitophilus oryzae , was investigated. Weevils survived well on diets containing 72 per cent (w/w) cereal starches, dextrins, amylopectin, and glycogen but died prematurely on diets containing amylose, cellulose, inulin, mono-, and disaccharides. It was shown that dietary polysaccharides which permitted good survival were feeding stimulants while those which caused early mortality were either feeding deterrents or did not provide suitable physical feeding substrates. Starvation rather than digestive enzymatic lesions was therefore the principal cause of early adult mortality on the test diets. It was concluded that the branched-chain amylopectin portion of the cereal starches in the weevil's natural diet serves as both a feeding stimulant and an essential nutrient.

The effect of temperature and photoperiod on the onset and termination of the mature larval diapause of the southwestern corn borer, Diatraea grandiosella , was examined. The results showed that diapause induction was an extremely temperature-dependent process. Larvae reared at 23°C under short days all entered diapause whereas 90 per cent of those reared at 27°C developed continuously. A photoperiodic response was only demonstrable at 25°C, when diapause was instituted following larval exposure to daily photophases ranging from 8 to 14 hr. An examination of the sensitivity of immature larvae to low temperatures revealed that all instars showed some susceptibility to low temperature induction of diapause. No intermediate instar was found in which the developmental programme could be switched entirely from a diapause to a non-diapause one, or vice versa. Diapause development was also found to be primarily temperature regulated. The rate of termination of diapause at 30°C was significantly higher than that at 25 or 23°C. Continuous exposure to light rather than a daily photoperiod produced the highest rate of diapause development. It was also shown that a period of chilling (5°C) did not accelerate diapause development. The significance of the results in relation to the evolution and geographical distribution of the species is discussed.

The metabolic reserves of Magicicada cassini , a periodical cicada with a 17 year life cycle, were examined. Analysis of males and females which were collected shortly after emergence and just before death provided information about the nature and fate of the reserves which had been accumulated during the attenuated larval period. Results showed that young females had a mean weight of 565 mg compared with only 351 mg for equivalent males. Lipids and soluble proteins were the principal reserve materials. Glycogen was a relatively minor reserve of both sexes. While young females contained about twice as much soluble protein (110 mg) as the males (51 mg), they contained three times as much total lipid material (58–19 mg). Lipids were fractionated into neutral and phospholipids and the absolute amounts of each class were determined; triglycerides were the main component. Further analyses of old females provided information about the utilization of reserves for oogenesis. The data showed that even though the adults feed, lipids and soluble proteins stored during the larval stages are essential for egg maturation. The results are discussed in relation to the special requirements of an insect which has a protracted immature stage, yet a fleeting adult existence.

Feeding behaviour of newly hatched larvae of the Angoumois grain moth was investigated. A bioassay was developed based on larval response to various test materials which were added to corn starch and glycerol pellets placed in a circular arena. The distribution of larvae between two test and two control pellets in each arena was usually determined after 4 hr incubation to determine what factors influenced the initial phase of the insect's feeding behaviour. When components of a refined diet (casein, yeast, wheat germ, bran, and cholesterol) were assayed for their effect on feeding behaviour only wheat germ was found to evoke a strongly positive response. Further study revealed that the lipid portion of wheat germ contained potent arrestants and probably also contained biting incitants and feeding stimulants. An investigation into the nature of some of the active components in this lipid fraction revealed that triglycerides and, to a lesser extent, fatty acids served as arrestants. The classification of the measured behavioural response is discussed.

Larvae of the Angoumois grain moth readily established in refined diets moulded into pellets. A bioassay based on larval establishment, weight, and numbers of adults was used to determine the insect's major nutritional requirements. The results showed that test diets containing corn starch (49–64%), glycerol (15–19%), casein (9%), yeast (7·5%), and wheat germ (2·6%) permitted good establishment and normal development. No further improvements were obtained when diets were supplemented with a mineral or vitamin mixture, cholesterol, or ascorbic acid. It was concluded that yeast and wheat germ supplied the insect with adequate amounts of lipids, vitamins, and minerals. However, even the best diets lacked some undetermined growth promoting factor because larvae grew better in a natural product control diet.

The southwestern corn borer, Diatraea grandiosella , was reared on a meridic diet and its lipid requirements were determined. A dietary supplement of wheat germ oil was found to contain factors which were essential for larval growth and normal wing development. An investigation into the nature of these factors showed that linoleate or linolenate promoted normal wing development whereas β-sitosterol enhanced the larval growth rate. A study of the species fatty acid requirements revealed that dietary supplements of methyl linoleate or linolenate, but not methyl oleate or stearic acid, permitted normal wing development. The dosage required was low and as little as 0·03% methyl linoleate resulted in a high incidence of normal wing development. Dietary fatty acids, however, only marginally improved the larval growth rate. A study of sterol requirements revealed that (1) a dietary sterol was essential for larval growth, (2) β-sitosterol (0·18%) permitted a significantly higher larval growth rate than either cholesterol, 7-dehydrocholesterol, or stigmasterol. A dietary supplement of ergosterol did not support larval growth. The present results are discussed in relation to known lipid requirements and metabolism in other plant-feeding insects.

A simple method for rearing the Angoumois grain moth, Sitotroga cerealella , in an artificial diet is described. Although the larvae normally feed within grain kernels, they readily became established in pellets containing a mixture of five natural products. An evaluation of the growth rate in this medium revealed a marked sex difference. The males attained a maximum mean larval weight of 7·2 mg at 22 days compared with 12·3 mg for mature females at 24 days. Both sexes were found to pass through four larval instars. A study of the pupation rate showed that males reached 50 per cent pupation a full 5 days before females. While both sexes spent about 6·7 days in the pupal-pharate adult stage, the mean weight of newly-ecdysed female adults was 77 per cent greater than that of the males.

The total lipid content of the fat-body reached a maximum of about 4.5 mg. per insect in mature larvae and only changed slightly through the pharate pupal stage. The lipid content of the haemolymph (about 0.5 mg. per insect) also remained fairly constant at the onset of metamorphosis. During this period triglycerides formed about 90 per cent of the neutral fatbody lipids with 1,2-diglycerides and sterols comprising the other 10 per cent. In contrast, 1,2-diglycerides and sterols made up about 65 per cent of the haemolymph neutral lipids. Triglycerides, free fatty acids, sterol esters, and hydrocarbons were only minor components. The significance of these analytical results and the function of some of the lipid components are discussed.

The midgut lipids of the southwestern corn borer, Diatraea grandiosella Dyar, were examined during its metamorphosis from larva to pupa. Results showed that the highest midgut lipid content (approximately 0.38 mg. per insect) was found in actively feeding, 14-day larvae. Following pupation the midgut lipid content dropped to only 0.04 mg. per insect. Thin-layer chromatography revealed that five neutral lipid classes could generally be detected in midgut extracts. For example, 14-day larval midguts contained the highest concentration of neutral lipids in the following proportions: triglycerides, 42 per cent; sterol esters, 29 per cent; sterols, 16 per cent; 1,2-diglycerides, 6 per cent; and traces of fatty acids and hydrocarbons. The titres of triglycerides, diglycerides, and sterol esters varied more than those of the other constituents at different nutritional and developmental stages. These lipid classes, along with free sterols, are apparently involved in neutral lipid storage or transport in midgut cells. Results obtained from midguts of insects which had been starved or fed lipid-free diets supported this interpretation. Possible mechanisms for lipid absorption, storage, and transport were evaluated.

Haemolymph and midgut proteins of the southwestern corn borer, Diatraea grandiosella, were studied for developmental changes and possible relationships at the onset of metamorphosis. The protein content of both tissues was found to increase markedly during the last two larval instars before falling sharply during the pharate pupal stage. However, a comparative study of their rates of protein biosynthesis revealed that, unlike midgut cells, larval haemocytes were unable rapidly to synthesize protein. Further electrophoretic results indicated that midgut cells, like those of the larval fat body, have the potential to synthesize at least the two major haemolymph proteins. The observed decrease in protein content of the haemolymph during the pharate pupal stage could be partially attributed to the uptake of at least one of its proteins by the fat body. The origin and fate of haemolymph proteins in metamorphosing insects are discussed.

The Angoumois grain moth, Sitotroga cerealella , was reared in a refined medium moulded into pellets, and its larval and pupal metabolic reserves were examined. Results showed that lipids and proteins (65–86 per cent larval and pupal dry weights) formed the major reserves of both sexes and that glycogen was only a minor component. Mature female larvae weighed about 44 per cent more than males and therefore contained more reserves, especially in the form of lipids. Although the accumulation of storage materials was found to be essentially complete by the mature larval stage, their in vivo distribution changed during the pharate pupal stage. In mature larvae the haemolymph contained a high protein content and the fat body a high lipid content. Following the larval-pupal ecdysis, however, the fat body became the principal storage centre for protein, lipid, and glycogen. A thin-layer chromatographic study of the neutral lipid classes of whole larvae and pupae of both sexes revealed that both had similar compositions and that triglycerides (70–80 per cent) were the major component. Further investigation of the distribution of the neutral lipids showed that while triglycerides (85 per cent) were the principal neutral lipid class in the fat body, the haemolymph lipids were more evenly divided between triglycerides (38 per cent), diglycerides (15 per cent), free fatty acids (9 per cent), sterols (11 per cent), sterol esters (7 per cent), and hydrocarbons (20 per cent). The results are discussed in relation to the known contents and in vivo distribution of lipid, protein, and glycogen in other insects during metamorphosis.

Developmental changes in the fat body proteins of the southwestern corn borer, Diatraea grandiosella, were studied from the beginning of the fifth instar to the newly ecdysed pupal stage. The dry weight of the perivisceral fat body was found to increase markedly from a mean of 1·15 mg/insect at 10 days of age to 7·9 mg in the newly ecdysed pupa (16 days). This rise was partially attributed to the increase in fat body protein content which took place during the same period. The fat body protein content rose from about 0·25 mg/insect at 10 days to a maximum of about 1·7 mg/insect in the newly ecdysed pupa. The rate of protein biosynthesis during this period was estimated using leucine-14C as a tracer. Results showed that 14-day-old larval fat body, unlike that of newly ecdysed pupae, had a high rate of protein biosynthesis. The chronology of synthesis and accumulation of the major proteins was determined by subjecting the soluble fat body proteins to disk electrophoresis. A changing electrophoretic pattern was detected between 10 and 15 days, which coincided with the period of active protein biosynthesis. Analyses of 15-day-old mature larval fat body revealed the presence of three major soluble proteins whose titre greatly increased after pupal ecdysis. The relationship between fat body and haemolymph proteins and the role of the major fat body proteins in adult differentiation are discussed.

The relationship between the proteins of the haemolymph and fat body was examined during metamorphosis of the large white butterfly, Pieris brassicae . The protein analysis results showed that about 4·9 mg protein was lost from the haemolymph of an individual insect between the prepupal and newly ecdysed pupal stages, while during the same period the fat body gained about 5·5 mg protein. From the change in distribution of 14 C protein between these two tissues it was concluded that the fat body was storing the lost haemolymph protein at pupation. A more detailed examination using disk electrophoresis of labelled and unlabelled proteins from these two tissues showed that the fat body was in fact selectively storing two major haemolymph proteins. It was found that these proteins accumulated in the haemolymph during the fourth and fifth instars before being transferred to the fat body at the pharate pupal stage. No changes were detected in either haemolymph or fat body proteins during pupal diapause. However, the adult moulting cycle was marked by partial histolysis and release of degraded fat body proteins and the loss of another major protein from the haemolymph, not into the fat body, but presumably into the differentiating adult tissues. The changing roles that the fat body and haemolymph play in the synthesis, storage, translocation, or degradation of proteins during development are discussed.

1. 1. Following the ingestion of 14 C-leucine, the relative in vivo protein biosynthetic activities of the haemolymph and its adjacent tissues, fat body and midgut were examined in mid-fifth instar Pieris brassicae larvae. 2. 2. The results show that at this stage the fat body and midgut, but not the haemolymph, are active sites of protein biosynthesis. 3. 3. The possibility that the low level of radioactivity detected in the haemolymph proteins may have resulted from the slow release of labelled protein from either the fat body, or midgut, or both, into the haemolymph, is discussed.

Electrophoretic analyses showed that four major proteins were lost from the haemolymph of Sitotroga during the pharate pupal stage. These proteins were selectively taken up by the fat-body and probably serve as an amino-acid reserve for adult differentiation.