Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

A method for rearing the yellow-spotted longicorn beetle, <Emphasis Type="Italic">Psacothea hilaris</Emphasis> (Coleoptera: Cerambycidae), to stabilize the last larval instar

The larvae of Psacothea hilaris (Pascoe) grown continuously under a long day in the laboratory pupate after the 4th, 5th, or 6th instar. This developmental polymorphism has complicated studies on the control of metamorphosis in P. hilaris. Since pupation in P. hilaris is known to be suppressed under a short day, a change in the photoperiod from a short to a long day with appropriate timing may assist in obtaining physiologically homogeneous larvae that pupate at the next molt. When the photoperiod was changed from 12-h light:12-h dark to 15-h light:9-h dark at the beginning of the 5th instar, the pupation rate at the next (5th) molt reached 90%, which was significantly higher than the proportion of larvae that pupated after the 5th instar when the larvae were grown continuously under a long day (52%). This rearing technique will expedite studies on the control of metamorphosis in P. hilaris.     

Degradation of haemoglobin in Chironomus during metamorphosis

The degradation of haemoglobin (Hb) during the metamorphosis of Chironomus pallidivitatus was studied by means of spectrophotometry, disc electrophoresis, and isotopic tracer methods. Hb concentration was maximal during the late fourth instar larva and exhibited a continual decline throughout the pharate adult periods; in the mature adult Hb is almost entirely absent. Coupled with this reduction in Hb is an increase in the quantity of bile pigment (tentatively identified as bililatrenes) most probably representing products of the Hb degradation. These pigments are found in considerable quantity within the meconium at the time of adult emergence.Larval, ⁵⁹Fe-labelled, Hb injected into late fourth instar larvae was found to be most concentrated in the midgut portion of the early pharate adult. The quantity of ⁵⁹Fe-haemoglobin in the midgut of early pharate adult was at least five times the concentration found in late pharate pupae. Spectrophotometric and electrophoretic analysis of the midgut contents of untreated larvae showed that Hb was one of the major soluble proteins in the midgut at the time of pupation. This rapid intestinal uptake of Hb suggests that the intestine is involved in the mechanism for the degradation and removal of the Hb, no longer required in the adult stage, a seemingly important step in the metamorphosis of this insect.     

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

Background

Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.

Results

Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.

Conclusion

Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.     

Purification,characterization and developmental changes in the titer of a new larval serum protein of the silkworm,Bombyx mori

A new protein was found as a major component of hemolymph proteins up to day 1 of the last larval instar of Bombyx mori, and was named Bombyx mori larval serum protein (BmLSP). The BmLSP was purified to homogeneity by ammonium sulfate precipitation, CM-cellulose column chromatography and gel permeation chromatography. The molecular weight of BmLSP was estimated to be 30,000 by SDS-PAGE and 25,000 by gel permeation chromatography. The amino acid composition of BmLSP was similar to that of 30 kDa proteins which are the major serum proteins in the older last (fifth) instar larvae. The 20 NH₂-terminal amino acids were sequenced and found to be quite different from those of the 30 kDa proteins. Developmental changes in BmLSP titer were followed throughout post-embryonic life by Western blotting using a specific antiserum against BmLSP. Within 1 day after larval hatching, BmLSP appeared in the hemolymph and remained at an almost constant level until day 1 of the last instar. On day 2 of the last instar, the BmLSP level suddenly fell and then gradually decreased toward larval-pupal metamorphosis. Thus, BmLSP is a true larval serum protein and is different from proteins stored for metamorphosis.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

Aldehyde oxidase activity in the tumorous-head strain of Drosophila melanogaster

Gross aldehyde oxidase activity from the egg-stage through 10-day-old adults and distribution of the enzyme in eye-antennal imaginal discs in third instar larvae were determined for the tumorous-head strain of Drosophila melanogaster. Aldehyde oxidase activity of several laboratory strains was measured for comparative purposes. Aldehyde oxidase activity was 100% higher during embryogenesis in tuh(ASU) eggs than in Oregon-R-C eggs. A second period of elevated aldehyde oxidase activity was observed during metamorphosis where tuh(ASU) pupae averaged 65% more enzyme activity than Oregon-R-C. Therefore, during determination and differentiation of the eye-antennal imaginal disc, the tuh(ASU) strain possesses a high aldehyde oxidase activity. Wild-type Drosophila melanogaster antennal imaginal discs are aldehyde oxidase positive, whereas attached eye imaginal discs are apparently aldehyde oxidase negative. A sample of eye-antennal imaginal discs from tuh(ASU) third instar larvae revealed that either one or both eye discs of 64% of the larvae were aldehyde oxidase positive. Aldehyde oxidase activity may be correlated with the homoeotic transformation in parts of the eye disc.     

Dynamic changes of gut bacterial communities present in larvae of Anoplophora glabripennies collected at different developmental stages

The Asian long-horned beetle, Anoplophora glabripennies (Motschulsky), is a destructive wood-boring pest that is capable of killing healthy trees. Gut bacteria in the larvae of the wood-boring pest is essential for the fitness of hosts. However, little is known about the structure of the intestinal microbiome of A. glabripennies during larval development. Here, we used Illumina MiSeq high-throughput sequencing technology to analyze the larval intestinal bacterial communities of A. glabripennies at the stages of newly hatched larvae, 1st instar larvae and 4th instar larvae. Significant differences were found in larval gut microbial community structure at different larvae developmental stages. Different dominant genus was detected during larval development. Acinetobacter were dominant in the newly hatched larvae, Enterobacter and Raoultella in the 1st instar larvae, and Enterococcus and Gibbsiella in the 4th instar larvae. The microbial richness in the newly hatched larvae was higher than those in the 1st and 4th instar larvae. Many important functions of the intestinal microbiome were predicted, for example, fermentation and chemoheterotrophy functions that may play an important role in insect growth and development was detected in the bacteria at all tested stages. However, some specific functions are found to be associated with different development stages. Our study provides a theoretical basis for investigating the function of the intestinal symbiosis bacteria of A. glabripennies.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of molecular markers for metamorphic competency and their response to starvation or feeding in the mosquito, Aedes aegypti (Diptera: Culicidae)

The nutritional condition of fourth instar larvae of the yellow fever mosquito, Aedes aegypti, governs female longevity and egg production, both are key determinants of pathogen transmission. As well, nutrition provisions larval growth and development and attains its greatest pace in the last larval instar in preparation for metamorphosis to an adult. These developmental processes are regulated by a complex endocrine interplay of juvenile hormone, neuropeptides, and ecdysteroids that is nutrition sensitive. We previously determined that feeding for only 24 h post-ecdysis was sufficient for fourth instar Ae. aegypti larvae to reach critical weight and accumulate sufficient nutritional stores to commit to metamorphosis. To understand the genetic basis of metamorphic commitment in Ae. aegypti, we profiled the expression of 16 genes known to be involved in the endocrine and nutritional regulation of insect metamorphosis in two ways. The first set is a developmental profile from the beginning of the fourth instar to early pupae, and the second set is for fourth instars starved or fed for up to 36 h. By comparing the two sets, we found that seven of the genes (AaegCYP302, AaegJHE43357, AaegBrCZ4, AaegCPF1-2, AaegCPR-7, AaegPpl, and AaegSlif) were expressed during metamorphic commitment in fourth instars and in fed but not starved larvae. Based on these results, the seven genes alone or in combination may serve as molecular indicators of nutritional and metamorphic status of fourth instar Ae. aegypti larvae and possibly other mosquito species in field and laboratory studies to gauge sub-lethal effects of novel and traditional cultural or chemical controls.     

Proteins of the fat body of non-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella: Effect of juvenile hormone

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 C₁₇-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.     

Morphological and biochemical characterizations of the great scallop Pecten maximus metamorphosis

In order to characterize Pecten maximus metamorphosis within a hatchery environment, the relationships existing among the various larval rearing parameters, the biochemical composition of the larvae and metamorphosis have been determined. Metamorphosis levels are correlated with the percentages of double ring larvae, as well as with the larval lipid content. A multiple regression incorporating the percentage of double ring larvae and larval lipid content shows that these two combined parameters explain 50 % of the total metamorphosis variance, with an equal relative importance for each of them. In an attempt to identify other possible endogenous markers, the kinetics of biogenic amines were also examined throughout larval and post-larval development. A steady increase in serotonin and dopamine levels was recorded during larval development while a sudden decrease in both molecules was noted during metamorphosis. It is suggested that these two amines may be used as indicators of larval competence for P. maximus metamorphosis.     

Effects of BmCPV Infection on Silkworm Bombyx mori Intestinal Bacteria

The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm.     

Effect of garlic oil on incorporation of amino acids into proteins of Culex pipiens quinquefasciatus Say larvae

Incorporation of [¹⁴C]leucine into proteins of 3rd instar Culex pipiens quinquefasciatus Say larvae increases linearly with time between 1 and 4 h. Garlic oil as well as the active larvacidal principle from it, viz. diallyl disulphide, inhibits significantly synthesis of the larval proteins. The maximum reduction in incorporation is observed during the first hour of treatment. The incorporation of [¹⁴C]phenylalanine is also inhibited by garlic oil and the effect is irreversible. Garlic oil does not seem to have any effect on proteins already labelled and it does not suppress substantially oxygen uptake by the larvae.     

Activity of the prothoracic gland and its sensitivity to prothoracicotropic hormone in the penultimate and last-larval instar of Bombyx mori

The time course of secretion of ecdysone in vitro by the prothoracic glands of Bombyx mori was studied through the penultimate and last-larval instars. Ecdysone was produced by the glands in high amounts by the penultimate instar at 72 and 84 h while the glands in the last instar exhibited a high activity over 4 days around the time of gut purge and thereafter. The glands in the penultimate instar produced ecdysone at a low level throughout the instar before the sharp peak of activity, when they became inactive and remained so for the first 3 days of the last instar after when they regained secretory activity. Sensitivity of the glands to prothoracicotropic hormone varied in accord with the changes in their secretory activity. Inactive glands were not stimulated by 22K-prothoracicotropic hormone. In addition, glands with maximal activity in the penultimate instar were insensitive to 22K-prothoracicotropic hormone. These results suggest that the prothoracic glands in the penultimate and last-instar larvae are physiologically different.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

The metabolism of cycloartenol,lanosterol, 24-methylenecholesterol and fucosterol in Chlorella ellipsoidea

Lanosterol and cycloartenol labelled with tritium at C-2, and 24-methylenecholesterol and fucosterol labelled with tritium at C-2 and C-4 were fed to actively growing cultures of Chlorella ellipsoidea. Lanosterol and cycloartenol were converted to each of the five desmethyl sterols of C. ellipsoidea. Lanosterol was more efficiently incorporated than cycloartenol.Although there was some evidence for the reduction of the 24-methylene group, it was apparent that 24-methylene-cholesterol was converted primarily to the C₂₉ sterols, clionasterol and poriferasterol. Labelled fucosterol was reduced at the 24(28) double bond, producing clionasterol.     

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm,Bombyx mori

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.     

Disruptive developmental effects of benzyl-1,3-benzodioxole derivatives on unparasitized and parasitized Manduca sexta larvae

Treatment of tobacco hornworm larvae with the benzyl-1,3-benzodioxole derivative J-2710 immediately after ecdysis to the fourth instar disrupted development either during the moult to the fifth instar or shortly thereafter. Larvae given topical applications of 100 μg J-2710 in 1 μl acetone suffered 100% mortality, often after secreting moulting fluid in large pockets between the epidermis and the cuticle later in the fourth instar. Larvae that successfully ecdysed had abnormalities of the mouthparts and cervix that interfered with normal feeding, inhibiting growth in the fifth instar. Larvae of the gregarious endoparasitic wasp Cotesia congregata (=Apanteles congregatus) frequently failed to emerge from host Manduca sexta larvae treated with high doses of J-2710, particularly when the host failed to feed normally. Less potent disruptive effects on Manduca and Cotesia were seen after treatment of larvae with the derivatives J-3370 and J-2581.No anti-juvenile hormone action of J-2710 was observed. J-2710-treated M. sexta larvae showed no precocious metamorphosis and the developmental effects of J-2710 were not prevented by co-application of the juvenile hormone analogue methoprene in doses ranging from 1 to 100 μg/larva. Moreover, J-2710 had no effect on the action of methoprene in the black larval assay for juvenile hormone-like activity, unlike results reported to occur using the Galleria wax wound assay.     

Cloning and expression of a JHA-inducible glutathione S-transferase gene in the common cutworm Spodoptera litura (Lepidoptera: Noctuidae)

The juvenile hormone titer during the last instar is a crucial determining switch for metamorphosis in lepidopterans. We previously observed that the induction of glutathione S-transferase (GST) activity by a juvenile hormone analog (JHA) is reversely proportional to the occurrence of JHA-induced supernumerary instars following topical application of JHA to Spodoptera litura during the last instar period. In this paper, at least five JHA-induced GSTs were purified by glutathione-affinity chromatography, and one was further isolated for determination of the N-terminal sequence. The corresponding cDNA was cloned and named SlGST1 (GenBank accession no. AY506545). SlGST1 was classified into the epsilon class of GSTs by phylogenetic analysis. Northern blot analyses further showed that the SlGST1 in fat bodies can be induced by JHA, particularly in 0-day-old sixth-instar larvae, in which induction was much higher than that in 1- and 2-day-old sixth-instar larvae. To our knowledge, this is the first JHA-inducible GST to have been identified, and we believe that it will provide new insight into the role of GST in insect development, particularly during metamorphosis.