Development of secretory ability in the spermatheca of the migratory grasshopper, Melanoplus sanguinipes

During the first week of adult life, the protein content of the spermatheca increases by about 45% but the carbohydrate content does not change significantly. Incorporation of radiolabelled leucine into protein is high in newly emerged females but by day 1 has declined to about two-thirds the initial level, where it then remains. With SDS-PAGE about 30 protein bands separate, including six glycoprotein fractions. All bands are present throughout sexual maturation and except for one (at 25 Kd), show little change in quantity during this period. Allatectomy or hormone replacement therapy has little effect on the parameters measured and it is concluded that the development of secretory activity in the spermatheca is not controlled by juvenile hormone.     

The neuro-endocrine control of protein metabolism in the migratory grasshopper, Melanoplus sanguinipes

In normal females, distinct fluctuations in the protein content of the fat body and haemolymph are evident during each gonotrophic period. These fluctuations partly reflect changes in the protein requirements of the developing oocytes. Almost one half of the total protein deposited in the mature ovary is sequestered during the final stages of vitellogenesis when protein accumulated in the fat body and haemolymph is rapidly depleted. Although similar amounts of protein are deposited in the ovary during the first and subsequent gonotrophic periods, significantly less extraovarian protein is present throughout the latter periods.The accumulation of large amounts of protein in the fat body and haemolymph of ovariectomized females suggests that most yolk protein is of extraovarian origin. As the total protein content of these insects is comparable to that of vitellogenic females, ovariectomy apparently has no immediate effect on protein synthesis.Allatectomy or cautery of the median neurosecretory cells (mNSC) prevents vitellogenesis. Although protein gradually accumulates in the fat body and haemolymph of allatectomized females, the total protein content of these insects is significantly lower than that of controls. Treatment of allatectomized females with juvenile hormone analogue leads to a temporary but significant increase in the protein content of the fat body. However, the subsequent decline in fat body protein is paralleled by a pronounced increase in the protein content of the ovary. These findings suggest that the corpora allata (CA) stimulate both yolk protein synthesis in the fat body and its uptake into the ovary. The total protein content of mNSC-cauterized females is less than that of allatectomized females. This observation supports the proposal that the mNSC have not only an allatotropic effect but also a direct effect on protein synthesis.     

Accumulation of secretory proteins in the accessory reproductive glands of the male migratory grasshopper, Melanoplus sanguinipes: A developmental study

Production of secretion in the accessory reproductive glands of male Melanoplus sanguinipes has been examined by electrophoresis and radiolabelling. The secretion of each group of tubules (long hyaline glands, white glands, short hyaline glands, and seminal vesicles) can be resolved into more than 20 protein bands and includes several glycoproteins and, in the long hyaline and white glands only, lipoproteins. Each group of tubules has a characteristic pattern of synthesis and accumulation of proteins; that is, specific proteins appear in the secretion at particular times during sexual maturation. In allatectomized insects, the long hyaline glands accumulate very little secretion; the white glands and short hyaline glands accumulate about one-third the normal amount; and accumulation in the seminal vesicles is not affected by the operation. Allatectomy exerts its effect by inhibiting the synthesis of particular proteins. The observations are discussed in terms of juvenile hormone-specific protein synthesis in the accessory reproductive glands.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Protein release from the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study

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 effect of starvation on haemolymph metabolites,fat body and ovarian development in Oxya japonica (Acrididae: Orthoptera)

Haemolymph volume does not change significantly during 96 hr of starvation. Haemolymph and fat body metabolites are depleted significantly in insects denied food for 96 hr. The percentage of oöcyte resorption is increased considerably by starvation. Differences in the utilization of lipid and protein under conditions of starvation between adult Oxya and adult locusts may be attributed to the migratory habit of the latter.     

Hormonal control of lipid synthesis in the fat body of the adult female tsetse fly, Glossina morsitans

Aqueous extracts of brain, thoracic ganglion or corpora cardiaca of female Glossina morsitans were shown to contain a substance which inhibited the synthesis of lipid from l[U-¹⁴C] leucine by fat cells incubated in vitro. The highest concentration of this substance was found in the corpora cardiaca; approximately 1 × 10^?6 gland pairs μl^?1 were required for maximum inhibition. At concentrations greater than 1 × 10^?4 gland pairs μl^?1 the lipid synthesis inhibiting factor (hereafter referred to as the LSIF) was inactivated by the presence of a substance which could be removed by gel filtration. The concentration of LSIF in the corpora cardiaca and midbrain varied throughout the reproductive cycle of the female. Net release of LSIF from the midbrain occurred between the 2nd and 7th day of the 9-day reproductive cycle. Net release from the corpora cardiaca began on day 5 and continued until the end of the interlarval period on day 9. Results are consistent with the hypothesis that LSIF is synthesised mainly in the medial neurosecretory cells of the midbrain whereas the corpora cardiaca are the site of storage and release into the haemolymph. LSIF was present in midbrain and corpora cardiaca extracts from male G. morsitans but at lower concentrations than in females. No variation in LSIF concentration could be correlated with the feeding cycle. LSIF activity was not detected in fresh haemolymph but was found at high concentration in boiled haemolymph, suggesting the presence of an inhibitor which was inactivated at high temperature. Preliminary investigations into the nature of LSIF have shown it to be inactivated by proteolytic enzymes and to be recoverable in a single peak from a Sephadex G15 column.Results support the view that LSIF is a peptide hormone which, in conjunction with an inhibitor, controls the lipid synthetic ability of the fat cells of the adult female tsetse fly throughout the reproductive cycle.     

Hormone stimulated lipolysis and proline synthesis in the fat body of the adult tsetse fly, Glossina morsitans

G. morsitans fat cells incubated in vitro with l-[U-¹⁴C]-leucine incorporated the radiolabel, mainly into triglycerides. Aqueous extracts of corpora cardiaca, midbrain, or thoracic ganglion stimulated the release of radiolabelled material from prelabelled fat cells in vitro. Corpora cardiaca extracts were the most active, approx. 1 × 10^?3 gland pairs/μl elicited the maximal response. At concentrations above 1 × 10^?3 gland pairs/μl the activity of corpora cardiaca extracts was inhibited by a substance which could be removed by gel filtration. The stimulatory factor in nervous-tissue extracts was destroyed by proteolytic enzymes and was recoverable in a single peak by Sephadex G15 gel filtration. Results suggest that it is a peptide hormone produced mainly by the median neurosecretory cells of the midbrain with the corpora cardiaca being the site of storage and release. No hormone was detectable in fresh haemolymph, but it was found at high concentration in boiled haemolymph, implying the presence of a heat labile inhibitor.Under the in vitro conditions used the hormone stimulated the synthesis of proline from alanine and the hydrolysis of triglycerides to free fatty acids. The probable functions of the hormone are to stimulate proline synthesis in response to demand for flight and/or to mobilise lipid for larval nutrition. The relative importance of these apparent functions in vivo could not be determined.     

Uric acid levels during last larval instar of Manduca sexta, an abrupt transition from excretion to storage in fat body

Levels of uric acid in the whole body of the tobacco hornworm, Manduca sexta increased steadily for the 9 days of the fifth instar. However, concentrations in the haemolymph were lowest during the transition from the feeding stage to the wandering stage (days 3, 4), the time when there was a switch from uric acid excretion by the Malpighian tubule-hindgut system to storage in the fat body. Haemolymph volumes, determined for larvae between 2 and 6 days into the fifth instar by isotope dilution with [¹⁴C]-inulin, were used to calculate rates of incorporation of uric acid into Malpighian tubules and fat body of larvae injected with [¹⁴C]-uric acid. These labelling studies indicated that the Malpighian tubules ceased to remove uric acid from the haemolymph some time between the last 6 hr of day 3 of the fifth instar and the first 18 hr of day 4. At the same period, fat body removed significant quantities of uric acid from the haemolymph. The times of initial decreases and increases in levels of uric acid in haemolymph and fat body, respectively, indicated that storage in the fat body started before cessation of elimination via the Malpighian tubule-hindgut system.     

Immunological analysis of apolipophorin-III in the haemolymph,ovaries, and testes of the fall webworm,Hyphantria cunea (Drury)

Apolipophorin-III (apoLp-III) was purified from the haemolymph of adult Hyphantria cunea (Drury) by KBr density gradient ultracentrifugation, gel filtration (Sephadex G-100) and ion exchange chromatography (CM-52), and its characteristics, molecular weight, tissue distribution, and sites of synthesis were examined. Molecular weight of apoLp-III was estimated to be 18 kDa. By electrophoretic analysis on 10% gels of male and female haemolymph from diverse developmental stages, apoLp-III was shown to be present in all stages. Western blotting was carried out to show that purified free apoLp-III is identical to apoLp-III associated with adult lipophorin. Immunological analysis also showed that apoLp-III is present in the ovary and the testis and in the case of testis, apoLp-III is heavily accumulated in the cyst. ApoLp-III is synthesized in larval and adult fat body but not in adult testis. Autoradiography following incubation of [¹⁴C]apoLp-III with testis showed that apoLp-III was taken up into testis. © 1996 Wiley-Liss, Inc.     

Immunological analysis of lipophorin in the haemolymph,ovaries, and testes of the fall webworm,Hyphantria cunea (Drury)

Lipophorin (LP) was purified from haemolymph in last instar larvae of Hyphantria cunea (Drury) by KBr density gradient ultracentrifugation and gel filtration. LP is composed of Apo-LP I and Apo-LP II with molecular weights of 230 kDa and 80 kDa, respectively. The level of haemolymph LP in early pupae was somewhat greater than in last instar larvae. In males, this LP concentration is maintained throughout pupal development, whereas the level of haemolymph LP decreases in female pupae beginning at day 7, coincident with the onset of vitellogenesis in the fall webworm. In both male and female adults, haemolymph LP concentrations were dramatically increased in comparison to their pre-adult levels. Actually, LP was found in the ovary by immunodiffusion, tandem-crossed immunoelectrophoresis, and Western blotting. Location of LP in the ovary was also traced by immunogold labelling. Also, LP appeared in small amounts in protein yolk bodies of the ovary at an early stage of vitellogenesis, when nurse cells are bigger than the oocyte, but in greater amounts at those stages when the oocyte is larger than nurse cells—that is, when vitellogenesis is actively taking place. This fact clearly reveals that LP is synthesized by fat body and released into the haemolymph, and then taken up by the growing ovary during vitellogenesis. Also, LP was detected in testes by immunological analysis. Western blotting showed that LP was present in testicular fluid but not in the peritoneal sheath and cysts. To test whether LP is also synthesized in testes, testes and fat body tissues were cultured in vitro, indicating that fat body synthsizes LP but testes do not. The result showed that the haemolymph LP itself is taken up into the testes. © 1994 Wiley-Liss, Inc.     

Culture of fat body of Periplaneta americana: Tissue development and establishment of cell lines

Long-term culture of larval fat body from Periplaneta americana was carried out. The cultures in a chemically defined medium show a strict dependance of the fat body on oxygen. The cultures with serum supplementation give rise to numerous cell migrations. We have studied the development of the different cell types and especially that of the mycetocytes and adipocytes. The mycetocytes can be cultured provided that they remain associated with the adipocytes. In these mycetocytes, the progressive loss of the symbiotes is related to the lipid load. The adipocytes continue to store lipids and glycogen for over 6 months. Then, the selection of different phenotypes gives rise to a homogeneous and adipocyte-like cell population. After 7 months, these cells can be subcultured regularly and a new cell line from P. americana has been established which is the only one isolated from explants of the insect fat body.     

Vitellogenin titre in haemolymph of Locusta migratoria in normal adults,after ovariectomy,and in response to methoprene

Vitellogenin in the haemolymph of Locusta migratoria was assayed by rocket immunoelectrophoresis to elucidate aspects of its regulation. In many normal adult females, vitellogenin first appeared on days 5–9, rose quickly to peak levels, and declined before a second vitellogenic cycle; in others, it appeared later and built up more slowly. The timing of first appearance of vitellogenin, and proportions of early and late-developing individuals, differed markedly in groups from the same colony assayed in different years, suggesting effects of both genetic and environmental variation. Average peak levels of vitellogenin were 25–30 mg/ml. After ovariectomy, vitellogenin appeared near the normal time and increased for several weeks to about 300 mg/ml; haemolymph volume also increased greatly, so that the total haemolymph-vitellogenin pool reached about 300 mg/individual, or 100 times the normal amount. After ovariectomy, no cyclicity of vitellogenin accumulation was apparent. These results show that the ovary is not required for stimulation of vitellogenin synthesis, and suggest that normal cycling may depend on inhibition by the mature ovary. Females treated with ethoxyprecocene on day 1 of adult life to inactivate the corpora allata did not produce vitellogenin, but were induced to do so with the juvenile hormone analogue, methoprene. After injection of 150 μg of methoprene in mineral oil, there was one day lag, then vitellogenin increased in the haemolymph to the normal peak level and declined slowly to zero during 5 weeks; after a second injection of methoprene, vitellogenin re-appeared more rapidly, with less lag, reflecting accelerated secondary hormonal stimulation of vitellogenin synthesis in the fat body. Adult males showed no detectable haemolymph vitellogenin even after injection of large doses of methoprene.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

Quantitative determination of the juvenile hormones in the haemolymph of Locusta migratoria during normal development and after implantation of corpora allata

Simultaneous quantitative determination of the three naturally occurring juvenile hormones in insects (JH-I, JH-II and JH-III) was performed on haemolymph samples of both normally developing locusts and locusts implanted with active corpora allata, using capillary gas chromatography with electron capture detection.In fourth instar female larvae, 24–48 hr after the third ecdysis, as well as in adult females, 18 days after the imaginal ecdysis, only JH-III was detected. In fifth instar female larvae JH-III was present in very low concentrations, if at all.After implantation of four pairs of corpora allata taken from young fourth instar female larvae or one pair or corpora allata taken from adult females into fifth instar female larvae 0–24 hr after ecdysis, an elevation of the JH-III titre was observed. Neither JH-I nor JH-II could be detected. The amount of JH-III, already elevated 2 hr after implantation, remained high for several days in comparison to that of control insects. On the third day after the subsequent moult the JH-III level was comparable to that of normally developing fifth instar larvae. Factors involved in the achievement of the haemolymph JH-titre are discussed.     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

Determination of ions, amino acids/amines in the haemolymph of fairy shrimps by capillary electrophoresis with indirect UV and laser-induced fluorescence detection

We identified and quantified the major inorganic ions present in a haemolymph of four species of fairy shrimp, to determine its ionic strength, using capillary electrophoresis, non-destructive micro-technique for the analysis of small amounts of body fluids. This is the first speciation and quantitative analysis of inorganic ions in the haemolymph of fairy shrimps.     

Characteristics of larvae of the southwestern corn borer, Diatraea grandiosella, obtained from populations present in tropical and temperate regions

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.     

Ovarian and fat body protein concentrations in Ips sexdentatus (Coleoptera: Scolytidae) parasitized by nematodes

The protein concentrations of fat body and ovaries in Ips sexdentatus either uninfected or infected by Parasitaphelenchus sp., P. sexdentati, or Contortylenchus diplogaster were measured at various stages of insect development, from preswarming maturation to the first oviposition (24 hr after mating). Weight variations of the fat body and ovaries in insects infected by C. diplogaster show the same evolution as those observed in uninfected insects, but at a much lower level. Fat body proteins in uninfected insects reach their minimum level during swarming, but they remain fairly constant throughout the maturation of the first egg. After dropping shortly after swarming, the ovarian protein level in such insects increases in two stages during ovarian maturation. The first stage, which corresponds to a slow protein incorporation, takes place during the first 12 hr after mating. During the second stage, i.e., beyond 12 hr, a significant level of proteins is rapidly incorporated into the ovaries. In insects infected by Parasitaphelenchus fat body proteins are reduced and protein incorporation into the ovaries is reduced; Parasitaphelenchus would thus affect at least some proteins required for ovarian maturation in their host. Fat body protein levels are even more affected by C. diplogaster than by Parasitaphelenchus, while incorporation into ovaries seems to be less affected in spite of slower ovarian growth. C. diplogaster might thus essentially act both upon proteins which are not required for the ovarian maturation of their host and upon nonproteinaceous substances that are required for such maturation. Results are discussed in relation to the possible mode of action of parasitic nematodes.     

An electrophoretic investigation of six species of darters in the subgenus Catonotus

The electrophoretic patterns of lactate dehydrogenase, phosphoglucomutase and total muscle proteins distinguished the species Etheostoma flabellare, E. kennicotti and E. squamiceps from each other and from three species of the E. virgatum group (E. virgatum, E. obeyense and E. barbouri), which all possessed identical patterns. Etheostoma flabellare, the species with the widest geographic range, possessed the greatest amount of genetic variation in all three protein systems studied. Patristic distances were calculated via an unrooted Wagner network. Using this method it was shown that E. flabellare was the most divergent species, E. kennicotti and E. squamiceps were less divergent, and the members of the E. virgatum group clustered closely.