Differential synthesis of bacteria-induced proteins of Manduca sexta larvae and pupae

The range of inducible antibacterial and other associated haemolymph proteins in Manduca sexta larvae and pupae was examined by high resolution two-dimensional (2D) isoelectric focusing-polyacrylamide gel electrophoresis. Twenty-two major inducible proteins were consistently resolved on gels of haemolymph from bacteria-injected larvae. Haemolymph from bacteria-injected pupae showed a different pattern of induced proteins. The proteins of the two stages include those which (i) are induced in both stages, (ii) those which are exclusively induced in either larvae or pupae, (iii) those which are inducible in larvae, but consititutively present in pupae, and, (iv) those which are induced in larvae, and which are present at intermediate levels but may be induced to higher levels in pupae.The antibacterial activity of the haemolymph from larvae and pupae was compared on acidpolyacrylamide gels, and the apparent M_r and pI of the inducible proteins determined. Certain of the inducible proteins appear to resemble the cecropin and attacin proteins of Hylophora cecropia.     

Effects of methoprene,a juvenile hormone analogue,on the larval and pupal stages of the yellow fever mosquito, Aedes aegypti

Exposure of early fourth-instar larvae of Aedes aegypti to the juvenile hormone analogue Altosid ZR15^® (methoprene) significantly increased the concentration of carbohydrates in the haemolymph of late fourth-instar larvae and reduced the haemolymph carbohydrate concentration of 24-h-old pupae relative to controls. Such treatment also effected a decline in haemolymph amino nitrogen levels of the pupal stage and a depletion of haemolymph proteins in late fourth-instar larvae as well as pupae. Two of nine protein fractions in the haemolymph of larvae were significantly depleted following methoprene treatment. Fourteen soluble protein fractions were present in the haemolymph of control pupae; two of these were missing from the pupae which were treated as larvae with methoprene. A further protein fraction, common to the haemolymph of both treated and control pupae, was significantly reduced in concentration as a consequence of exposure to methoprene. The juvenile hormone analogue impaired the capacity of the fat bodies of late fourth-instar larvae and pupae to synthesise proteins, resulting in a lowered concentration of fat body proteins. Glycogen levels in the fat bodies of treated larvae were significantly lower than in controls and glycogenolysis was suppressed due to an overall depletion of glycogen phosphorylase and, in pupae, a lowered ratio of active: inactive enzyme. The data are consistent with the proposition that the juvenile hormone analogue elicits neuroendocrinological changes in the target insect.     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

Induced immunity in Antheraea assama Ww larvae against flacherie causing Pseudomonas aeruginosa AC-3

This study reports for the first time the induction of immunity in Antheraea assama Ww larvae against bacterial flacherie. In silkworms group of disease caused by bacteria are collectively called "flacherie." This refers to the flaccid condition of the larvae due to the infections of bacterial strains pathogenic to muga silkworm. Antibacterial activity against pathogenic Pseudomonas aeruginosa AC-3 causing flacherie, was induced by injection of heat-killed cells of the same strain. Experiments on larval survivability and viable cell count revealed peak immune response on third day. Comparison of the amount of food ingested, excreta produced and larval weight of the saline-injected control, live bacteria-challenged larvae and heat-killed bacteria-injected larvae "(vaccinated)" confirmed the development of immunity against bacterial infection in the "vaccinated" set. The haemolymph of A. assama larvae was analyzed for proteins associated with bacterial infection. Out of the total 32 detected proteins, eleven (A1-2, A15-20, A22-23, and A29) were constitutively synthesized in both the control and live bacteria-injected larvae. Four inducible proteins A4, A9-10, and A21 were detected in the haemolymph of the live bacteria-injected larvae. Synthesis of rest of the proteins varied between the control and their live bacteria-injected counterparts. General protein profile of "vaccinated" larvae injected with live bacteria were found to be similar to that of the saline-injected control.     

Immune responses of the argasid tick Ornithodoros moubata moubata induced by infection with the filarial worm Acanthocheilonema viteae

Investigations were undertaken to determine whether the tick Ornithodoros moubata moubata mounted a detectable immune response to primary and secondary infections with Acanthocheilonema viteae. Uninfected control tick survival rate was 70%, but only 45% in the primary infection group. Post-secondary infection survival rate (82%) was comparable to controls, indicating that these selected ticks had some protective advantage. Mean A. viteae infective larvae recovery from ticks with secondary infections was 31.4% lower than expected, suggesting the development of immunity. SDS-PAGE of haemolymph for proteins induced post-primary infection yielded a stronger signal at 45 kDa than controls, which was further elevated post-secondary infection. Proteins at 48, 22 and 16 to 18 kDa were detected in haemolymph from infected ticks but not seen from controls. The direct effect of haemolymph on microfilarial viability was examined using a novel in vitro assay; in these preliminary trials no differences were observed in parasite viability when exposed to haemolymph from infected or uninfected groups of ticks.     

Major haemolymph proteins inCeratitis capitata: Biosynthesis and secretion during development

Summary The accumulation of major haemolymph proteins (a group of proteins immunologically related to Calliphorin) their biosynthesis in vivo and in organ culture as well as their secretion, has been studied during the late larval stages and white pupae of the Mediterranean fruit flyCeratitis capitata. The accumulation of major haemolymph proteins in the haemolymph, shows a twenty fold increase from the 4-day old larvae to the white pupae stage, while in the fat body there is only a seven fold increase. It is evident from the in vivo and organ culture studies, that the major haemolymph proteins are synthesized during the late larval stage and their synthesis declines abruptly during the stage of white pupae. It seems also that each polypeptide has its own characteristic developmental kinetics of synthesis. The major haemolymph proteins are synthesized in the fat body and are very quickly secreted into the haemolymph.     

Exoproteinases of the type A in pathogenesis of insect bacterial diseases

Immune inhibitors produced in infected larvae of Galleria mellonella by such entomopathogens as Pseudomonas aeruginosa, Serratia marcescens and Heterorhabditis bacteriophora effectively blocked in vitro bactericidal activity of insect haemolymph against Escherichia coli D31, both in Galleria mellonella and Pieris brassicae pupae previously vaccinated with Enterobacter cloacae. Even at a trace concentration, the extracellular proteinases, by proteolytic degradation, totally destroyed the activity of cecropin peptides from Galleria and cecropin-like and attacin-family proteins from Pieris, but no ability to destroy antibacterial activity was shown by extracts obtained from Galleria larvae killed by massive doses of bacterial saprophytes. It is suggested that by blocking antibacterial immune response of the host, the proteinases help the bacteria to multiply in the haemolymph, thus they could be considered an important factor in the pathogenesis of bacterial diseases of insects.     

Diapause and development in the tobacco budworm,Heliothis virescens: A comparison of haemolymph ecdysteroid titres

The signal to induce diapause in H. virescens comes early in development (prior to the third instar in most insects), but the signal to break diapause can come shortly after entrance into diapause at pupation. Haemolymph ecdysteroid titres in both diapause-bound and non-diapause-bound Heliothis virescens larvae were similar in the first two thirds of the last-larval instar, when similar changes in morphology and behaviour occurred. However, the number of stepwise increases in titre and the timing of the steps was different in the two groups of larvae. Haemolymph ecdysteroid titres in the last third of the instar were approx, five times higher in non-diapause than in diapause-bound larvae. In diapausing pupae, haemolymph ecdysteroid titres dropped to levels found in larvae which had completed two thirds of the last instar. When diapausing pupae were warmed to break diapause, haemolymph ecdysteroid titres rose again. However, 2 of the 4 high ecdysteroid levels detected in pupae developing after diapause break were considerably lower than those detected for non-diapause pupae.     

A water-soluble protein specific to the adult cuticle in Tenebrio: Its use as a marker of a new programme expressed by epidermal cells

The water-soluble proteins of abdominal cuticle of larvae, pupae and adults subjected to electrophoresis, revealed a specific pattern for each stage. An antibody against an adult specific 18 kdaltons protein was produced. Electrophoresis analysis of SDS slab gel and Western-blots revealed that the 18 kdaltons adult protein is not present in the water-soluble fraction from haemolymph or fat body of pupae which secreted pre-ecdysial adult cuticle. Immuno-histochemical methods showed that the cross-reaction of the immune serum was limited to the apical part of the adult epidermis and to the cuticle lamellae which are not stabilized. Therefore, the 18 kdaltons protein is specific for the adult cuticle and originates in the epidermis.     

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda (J. E. Smith), associated with parasitism by the braconid parasitoid Cotesia marginiventris (Cresson)

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda, associated with parasitism by the parasitoid Cotesia (= Apanteles) marginiventris were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis. As early as hour 4 after parasitization treatment, several electrophoretically slow-migrating, high-molecular-weight proteins were detected in the host's haemolymph. These proteins were detected earlier in haemolymph from parasitized larvae than in haemolymph from control larvae, and their concentrations were higher in heavily parasitized host larvae (≥ 3 eggs/host) than in lightly parasitized larvae (1 egg/host). Additionally, unique proteins that migrated electrophoretically with bovine serum albumin appeared in the haemolymph of parasitized larvae at hour 8 after parasitization treatment and were evident in haemolymph collected through to hour 64.     

The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis

Fat bodies from diapausing fifth-instar larvae of Ostrinia nubilalis were incubated in vitro at 5 or 23°C in Grace's medium and the glycerol contents of the organ and incubation medium determined. Fat bodies from diapausing larvae chilled 3 weeks at 5°C secreted glycerol into the medium at 5°C at a net rate of approx. 0.75 nmol/mg fat body dry wt/h for at least 96 h while the tissue levels remained essentially constant. Depending upon the experiment, from 6 to 15 times more glycerol was produced in 24 h at 5°C by these fat bodies than by those taken from diapausing unchilled larvae and incubated at either 5 or 23°C. A minimal chilling period of 10–12 days was recognized as necessary for chilled larval fat bodies to demonstrate rates of glycerol synthesis greater than those of unchilled larvae and the lag showed a temporal correlation with changes in haemolymph glycerol concentrations. These results suggest that this response to chilling by O. nubilalis is relatively slow. While incubation, at 23°C, of fat bodies from previously chilled larvae did not result in cessation of glycerol secretion, the rate of its appearance in the culture medium decreased during the 24-h incubation period. Although the ability of chilled fifth-instar larvae to accumulate glycerol is not dependent upon the diapause state results show that clearance of glycerol from the haemolymph by rewarmed O. nubilalis is related to diapause intensity.     

不同家蚕品系的耐热性特征

研究了家蚕Bombyx mori 3个品系(Pure Mysore,NB4D2和CSR2）5龄幼虫和蛹在不同温度（35,38和40℃）下的耐热性,采用Probit分析测定了它们在各温度下的LT50值和置信限。结果表明：多化性品系Pure Mysore在高温下的存活率高于两个二化性品系NB4D2和CSR2,而两个二化性品系中NB4D2表现出更好的耐热性。家蚕幼虫接触38℃高温6 h和40℃高温3 h后,其血淋巴中出现90,70和29 kDa的热激蛋白条带。在恢复过程中,NB4D2和CSR2的血淋巴中未见29 kDa蛋白条带,而Pure Mysore幼虫的血淋巴中29 kDa蛋白仍然表达。当幼虫置于高温下时,血淋巴中90和70 kDa蛋白表达,但是检测不到29 kDa蛋白。研究认为热激蛋白表达与热带家蚕不同品系的耐热性以及与同一品系不同发育阶段的耐热性具有相关性。     

Diet-Induced Over-Expression of Flightless-I Protein and Its Relation to Flightlessness in Mediterranean Fruit Fly,Ceratitis capitata

The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

Diapause phenomena in non-diapausing last instar larvae,pupae, and pharate adults of the Colorado beetle

From the first day of the last (fourth) larval instar no trace of juvenile hormone (JH) can be detected in the haemolymph by Galleria bioassay. Three specific diapause proteins, which are also found in diapausing adults, appear in the haemolymph. These proteins disappear towards the end of the pupal stage. Study of the ultrastructure of the fat body revealed the formation from lysosomes of proteinaceous bodies which are also characteristic for adult diapause. The behaviour of last instar larvae and pupae resembles that of prediapausing and diapausing adults respectively. Injection of synthetic JH delays the appearance of the diapause proteins in the haemolymph and of proteinaceous bodies in the fat body for 2 to 3 days. The absence of JH seems to trigger off these diapause phenomena.     

Effects of aminoethylation of cysteine residues on the structural and functional activities of the Escherichia coli 30 S ribosomal proteins

The purified 30 S ribosomal proteins from Escherichia coli strain Q13 were chemically modified by reaction with ethyleneimine, specifically converting cysteine residues to S-2-aminoethylcysteine residues. Proteins S1, S2, S4, S8, S11, S12, S13, S14, S17, S18 and S21 were found to contain aminoethylcysteine residues after modification, whereas proteins S3, S5, S6, S7, S9, S10, S15, S16, S19 and S20 did not. Aminoethylated proteins S4, S13, S17 and S18 were active in the reconstitution of 30 S ribosomes and did not have altered functional activities in poly(U)-dependent polyphenylalanine synthesis, R17-dependent protein synthesis, fMet-tRNA binding and Phe-tRNA binding. Aminoethylated proteins S2, S11, S12, S14 and S21 were not active in the reconstitution of complete 30 S ribosomes, either because the aminoethylated protein did not bind stably to the ribosome (S2, S11, S12 and S21) or because the aminoethylated protein did not stabilize the binding of other ribosomal proteins (S14). The functional activities of 30 S ribosomes reconstituted from a mixture of proteins containing one sensitive aminoethylated protein (S2, S11, S12, S14 or S21) were similar to ribosomes reconstituted from mixtures lacking that protein. These results imply that the sulfhydryl groups of the proteins S4, S13, S17 and S18 are not necessary for the structural or functional activities of these proteins, and that aminoethylation of the sulfhydryl groups of S2, S11, S12, S14 and S21 forms either a kinetic or thermodynamic barrier to the assembly of active 30 S ribosomes in vitro.     

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

Fatty acid profile of the cabbage looper, Trichoplusia ni, and the effect of diet and rearing conditions

The fatty acids, palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) were measured by GLC in Trichoplusia ni (Hübn.) from larvae reared on three different diets. The effect of rearing temperature and humidity on the levels of these fatty acids in pupae and pharate adults was determined. T. ni tended to accumulate 18:3 somewhat in proportion to its level in the diet. The respective levels (relative percentage) of this fatty acid in the diet and in the pupae were: bean leaves, 73·6 and 56·7; the diet based on lima beans, 10·2 and 3·5; and a meridic diet containing wheatgerm oil, 5·6 and 1·8. Pupae from larvae reared on a diet marginal in 18:3 content produced adults with deformed wings when reared at 30°C and normal wings when reared at 23 or 24°C, but there was no difference in their tissue level of 18:3. The phospholipids of last instar larvae, pharate pupae, pupae, pharate adults, and emerged adults are made up mostly of 18:0, 18:2, and 18:3 while the triglycerides of these stages contain relative large quantities of 16:0, 16:1, and 18:1. Pupal rearing temperature did not appear to influence the level of 18:3 in these two fractions enough to account for the degree of deficiency expressed as adult wing deformity. Both high (95%) and low (20%) relative humidity have an adverse effect on wing development but this effect can be overcome by 18:3 supplementation of the larval diet.