Isolation and characterization of a larval hemolymph protein in Locusta migratoria

A high-molecular-weight protein, M_r 500,000, has been isolated and characterized from the hemolymph of the migratory locust, Locusta migratoria. It is composed of six seemingly identical subunits of apparent M_r 78,000. It contains low concentrations of carbohydrate and lipid, but high percentages of aspartate and glutamate as well as high proportions of hydrophobic amino acid residues. An antiserum, developed against this purified hemolymph protein, does not react in the double-diffusion test or after immunoblotting with purified lipophorin or cyanoprotein, two other major proteins in locust hemolymph. The concentration of this larval specific protein in the hemolymph of Locusta was examined during the last larval instar and in adult males by quantitative rocket immunoelectrophoresis. Its concentration increases in the second half of the fifth instar, concommitant with an increase in total protein. The protein is detectable by immunological techniques in adults, although its concentration is very low at this stage.     

Host plant urease in the hemolymph of the silkworm, Bombyx mori

Urease activity was detected in the hemolymph of the silkworm, Bombyx mori from the beginning of spinning to the pharate adult stage if the larvae were reared on mulberry leaves throughout the 5th-instar (the last larval instar). In contrast, no urease activity was detected in the hemolymph of insects fed artificial diets, resulting in accumulation of urea during the spinning stage. To identify the hemolymph urease, the enzyme was highly purified from the hemolymph of the spinning larvae that had been reared on mulberry leaves and the properties of the purified enzyme were compared with those of the mulberry leaf urease. Four out of six monoclonal antibodies raised against jack bean seed urease cross-reacted equally with the silkworm hemolymph urease and the mulberry leaf urease. Under reducing conditions, the hemolymph urease and the mulberry leaf urease migrated at 90.5 kDa on SDS-PAGE gels. The first 20 N-terminal sequence of the hemolymph urease revealed complete identity with that of the leaf urease. The optimum pH for activity and Km value for urea were almost the same for the two enzymes. In conclusion, these two ureases are very likely identical, strongly suggesting that the mulberry leaf urease passes through the larval gut wall into the hemolymph without being digested. In addition, oral administration of mulberry leaf urease just before spinning induced considerable urease activity in the hemolymph of the larvae, but the same treatment did not induce enzyme activity in the hemolymph of the larvae three days before the onset of spinning. These results suggest that the silkworm larvae acquire the host plant urease specifically at the end of the feeding stage in order to degrade urea accumulated in the hemolymph.     

Insect apolipophorin III. Purification and properties

The hemolymph of adult Manduca sexta (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin III. Apolipophorin III was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin III was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin III is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr = 34,000.     

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.     

Storage proteins are present in the hemolymph from larvae and adults of the Colorado potato beetle.

The protein composition of larval and adult hemolymph from the Colorado potato beetle, Leptinotarsa decemlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein 1, which occurs in the hemolymph of last instar larvae and short-day adults, appeared to be a storage protein. This protein dissociated into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a hexamer. Diapause protein 1 is composed of approximately 82,000 subunits. The amino acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed. Topical application of 1 microgram pyriproxyfen, a juvenile hormone analog, to last instar larvae and short-day adults suppressed the appearance of this protein in the hemolymph. Pyriproxyfen prematurely induced vitellogenin, when applied to last instar larvae. A larval specific protein was also identified in the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M(r) approximately 82,000) and its suppression by pyriproxyfen suggests that this protein is a storage protein as well.     

Viresin. A novel antibacterial protein from immune hemolymph of Heliothis virescens pupae.

Immune hemolymph was collected from fifth instar larvae and 1-day-old pupae of Heliothis virescens after injection of prepupae with live Enterobacter cloacae. Induction of antibacterial activity against Escherichia coli K12 D31 was 7.5 times greater in pupal than in larval immune hemolymph. Lysozyme activity of immune pupal hemolymph against Micrococcus lysodeikticus was 11 times greater when compared with lysozyme activity of immune larval hemolymph. Early pupal immune response with regard to antibacterial activity was much greater than larval immune response in H. virescens. Normal pupal hemolymph showed an increase in antibacterial activity and lysozyme that was induced during metamorphosis. Antibacterial protein was isolated together with lysozyme by gel filtration chromatography and then separated from lysozyme by sequential electrophoresis with a native acid gel and SDS gel. Molecular mass of antibacterial protein was estimated to be 12 kDa. The N-terminal amino acid sequence of 12-kDa protein was different from those of antibacterial molecules found in other insects and has not been identified before. A sample containing 12-kDa protein was negative for immunoblotting with anti-synthetic cecropin B antibody. We have named the novel 12-kDa antibacterial protein viresin. Viresin showed antibacterial activity against several Gram-negative bacteria including E. cloacae but not against Gram-positive bacteria.     

Effect of ace inhibitors and TMOF on growth, development, and trypsin activity of larval Spodoptera littoralis

Angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of cleaving dipeptide or dipeptideamide moieties at the C-terminal end of peptides. ACE is present in the hemolymph and reproductive tissues of insects. The presence of ACE in the hemolymph and its broad substrate specificity suggests an important role in processing of bioactive peptides. This study reports the effects of ACE inhibitors on larval growth in the cotton leafworm Spodoptera littoralis. Feeding ACE inhibitors ad lib decreased the growth rate, inhibited ACE activity in the larval hemolymph, and down-regulated trypsin activity in the larval gut. These results indicate that S. littoralis ACE may influence trypsin biosynthesis in the larval gut by interacting with a trypsin-modulating oostatic factor (TMOF). Injecting third instar larvae with a combination of Aea-TMOF and the ACE inhibitor captopril, down-regulated trypsin biosynthesis in the larval gut indicating that an Aea-TMOF gut receptor analogue could be present. Injecting captopril and enalapril into newly molted fifth instar larvae stopped larval feeding and decreased weight gain. Together, these results indicate that ACE inhibitors are efficacious in stunting larval growth and ACE plays an important role in larval growth and development.     

Purification,properties, and titer of a hemolymph trophic factor in larvae and pupae of Manduca sexta

Purification of a hemolymph protein (hemolymph trophic factor, or HTF) from last instar larvae of Manduca sexta was achieved using Sephadex G15-120 gel filtration and DEAE anion exchange chromatography. Homogeneity was visualized using SDS gel electrophoresis and ampholytic chromatofocusing. HTF was estimated to be a tetrameric protein with a molecular weight of 286 K and a Stokes' radius of 55.3 × 10⁻⁸ cm by agarose bead gel filtration; chromatofocusing suggests an isoionic point > 10. Polyclonal antibodies to HTF were prepared in rabbits and an ELISA was developed. The ELISA was used to titer HTF during the last larval instar and day 1 and 14 of the pupal stage and estimates a maximum of 1.5 mg/ml larval hemolymph on day 6 with a smaller larval peak of 0.75 mg/ml at day 3 and titers of 0.70 and 0.35 mg/ml on the 2 pupal days, respectively. ELISA of aqueous extracts of larval fat body, epidermis, and cuticle demonstrate that HTF comprises nearly a third of the soluble fat body protein and is a lesser component of epidermis and cuticle. The physiological role of HTF has not yet been determined.     

The effects of boric acid-induced oxidative stress on antioxidant enzymes and survivorship in Galleria mellonella

Larvae of the wax moth, Galleria mellonella (L.), were reared from first instar on a diet supplemented with 156, 620, 1,250, or 2,500 ppm boric acid (BA). The content of malondialdehyde (MDA, an oxidative stress indicator), and activities of the antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] were determined in the fat body and hemolymph in the 7th instar larvae and newly emerged pupae. Relative to control larvae, MDA was significantly increased in larval hemolymph, larval and pupal fat body, but decreased in the pupal hemolymph. Insects reared on diets with 156- and 620-ppm BA doses yielded increased SOD activity but 1,250- and 2,500-ppm doses resulted in decreased SOD activity in larval hemolymph. SOD activity was significantly increased but CAT was decreased in the larval fat body. High dietary BA treatments led to significantly decreased GST activity. However, they increased GPx activity in larval hemolymph. Dietary BA also affected larval survival. The 1,250- and 2,500-ppm concentrations led to significantly increased larval and pupal mortality and prolonged development. In contrast, the lowest BA concentration increased longevity and shortened development. We infer that BA toxicity is related, at least in part, to oxidative stress management.     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.     

The ontogeny of multiple hemoglobins in Chironomus thummi (Diptera): The effects of a compound with juvenile hormone activity

The multiple hemoglobins (Hbs) of Chironomus thummi show distinct and significant ontogenetic changes during development from the third instar through the fourth instar and metamorphosis into the pupa. A total of nine Hbs are resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hbs 2 and 3, which are stage specific for the fourth instar, are first detected on the fourth day of this stage by electrophoresis and immunoprecipitation. Hb 4 is the predominant Hb species in the early and middle fourth instar, but during the late fourth instar and prepupa, Hb 1 predominates. The concentrations of Hbs 5–9 remain relatively constant in middle instars and decrease during later development. The Hb content of larval hemolymph exhibits changes that coincide with developmental stages; molting is characterized by low Hb content, whereas, the hemolymph of intermolt animals contains relatively high levels of Hbs. Treatment of fourth instars with a juvenile hormone analog, Altosid, prolongs this stage and inhibits the progress of normal development resulting in the formation of larval-pupal intermediates. Altosid also appears specifically to inhibit the accumulation of soluble hemolymph proteins related to pupation and metamorphosis, without affecting the concentration of Hb. Most significantly, it induces the precocious appearance of Hbs 2 and 3, which remain elevated above control levels in the late larval and prepupal stages. The present results strongly suggest that Altosid stimulates the appearance and accumulation of larval-specific proteins in vivo, while it inhibits the appearance of pupation-related proteins.     

Regulation of synthesis of larval serum proteins after transplantation of larval fat body into adult Drosophila melanogaster

The larval serum proteins, LSP1 and LSP2, of Drosophila melanogaster are synthesized by the fat body during the third instar. We examined the potential for LSP synthesis by fat body implants in adult flies. Fat body from third instar donors will continue to synthesize LSPs in both males and females. Implants from late second instar larvae will start synthesizing LSP1 and LSP2 in females but only LSP1 in males, suggesting that regulation of these proteins is not the same and that the physiological milieu in the two sexes differs. The newly synthesized LSPs are secreted into the hemolymph for approximately 48 hr when secretion stops but synthesis continues. This sequence follows the pattern for LSP secretion in situ. Fat body from mid second instar larvae is variable in its ability to synthesize LSPs. LSPs are not detected in implants from first instar larvae despite there being a high level of protein synthesis in the implant and considerable growth of the fat body cells. We conclude that there is a critical stage of differentiation during the latter half of the second instar when the fat body becomes independent of the larval milieu and can synthesize LSPs in the adult.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

A larval specific lipoprotein: purification and characterization of a blue chromoprotein from Heliothis zea

The green color of the hemolymph of last instar larvae of the corn earworm, Heliothis zea (Boddie), is caused by the appearance of a blue chromoprotein. This protein (Mr approximately 560,000) is composed of 4 subunits (Mr approximately 150,000). It contains 8.4% lipid and has an equilibrium density of 1.26 g/ml. This protein is absent during all early larval stages, in pupae and in adult animals, but is a major hemolymph protein in 5th instar larvae. Although its physiological function remains unclear, this blue chromoprotein represents the first member of a new class of larval-specific insect lipoproteins.     

Divergence of larval resource acquisition for water conservation and starvation resistance in Drosophila melanogaster

Laboratory selection experiments have evidenced storage of energy metabolites in adult flies of desiccation and starvation resistant strains of D. melanogaster but resource acquisition during larval stages has received lesser attention. For wild populations of D. melanogaster, it is not clear whether larvae acquire similar or different energy metabolites for desiccation and starvation resistance. We tested the hypothesis whether larval acquisition of energy metabolites is consistent with divergence of desiccation and starvation resistance in darker and lighter isofemale lines of D. melanogaster. Our results are interesting in several respects. First, we found contrasting patterns of larval resource acquisition, i.e., accumulation of higher carbohydrates during 3rd instar larval stage of darker flies versus higher levels of triglycerides in 1st and 2nd larval instars of lighter flies. Second, 3rd instar larvae of darker flies showed ~40?h longer duration of development at 21°C; and greater accumulation of carbohydrates (trehalose and glycogen) in fed larvae as compared with larvae non-fed after 150?h of egg laying. Third, darker isofemale lines have shown significant increase in total water content (18%); hemolymph (86%) and dehydration tolerance (11%) as compared to lighter isofemale lines. Loss of hemolymph water under desiccation stress until death was significantly higher in darker as compared to lighter isofemale lines but tissue water loss was similar. Fourth, for larvae of darker flies, about 65% energy content is contributed by carbohydrates for conferring greater desiccation resistance while the larvae of lighter flies acquire 2/3 energy from lipids for sustaining starvation resistance; and such energy differences persist in the newly eclosed flies. Thus, larval stages of wild-caught darker and lighter flies have evolved independent physiological processes for the accumulation of energy metabolites to cope with desiccation or starvation stress.     

家蚕幼虫血细胞密度变化成因及血细胞密度与耐高温性的关系

[目的]血细胞是昆虫血淋巴免疫的主导者.调查家蚕Bombyx mori幼虫血细胞密度变化和成因、血细胞密度与家蚕抗性的关系,是研究家蚕血细胞相关的免疫调控和抗性育种的重要组成.[方法]用细胞计数板统计家蚕品种大造不同龄期(4龄第1-4天、5龄第1-8天和上蔟期)幼虫10 μL血淋巴中的血细胞数目并计算血细胞密度,利用I...