Seasonal metabolic flexibility is correlated with microclimate variation in horned larks and house sparrows

Maximum and minimum metabolic rates in birds are flexible traits and such flexibility can be advantageous in variable climates. The climatic variability hypothesis (CVH) posits that more variable climates should result in greater metabolic flexibility for geographically distinct populations. Whether the CVH applies to sympatric species occupying microclimates differing in variability is unknown. Microclimates of open habitats are likely more variable than those of sheltered habitats. If the CVH extends to microclimates, we expect birds from open habitats to show greater flexibility than those from sheltered habitats. To test this extension of the CVH, we compared seasonal variation in microclimates and metabolic rates for sympatric horned larks Eremophila alpestris, which occupy open habitats, and house sparrows Passer domesticus, which occupy sheltered habitats. We measured operative temperature (T_e, an integrative measure of the thermal environment), summit metabolic rate (M_sum, maximal cold-induced metabolic rate), and basal metabolic rate (BMR, minimal maintenance metabolic rate) in summer and winter. For both winter and summer, daily minimum T_e was similar between open and sheltered habitats but maximum T_e was higher for open habitats. Winter microclimates, however, were colder for open than for sheltered habitats after accounting for convective differences. Both species increased M_sum in winter, but seasonal M_sum flexibility was greater for larks (43%) than for sparrows (31%). Winter increases in BMR were 92.5% and 11% for larks and sparrows, respectively, with only the former attaining statistical significance. Moreover, species * season interactions in general linear models for whole-organism metabolic rates were significant for BMR and showed a similar, although not significant, pattern for M_sum, with greater seasonal metabolic flexibility in horned larks than in house sparrows. These results suggest that extending the CVH to sympatric bird species occupying different microclimates may be valid.     

Subtle direct effects of rising atmospheric CO2 on insect eggs

Ocean acidification is an important consequence of rising levels of atmospheric CO₂. The chemistry of acidification is, however, general and may disturb pH in terrestrial systems. The present study examines the effects of rising CO₂ on insect eggs, which may be vulnerable to acidification because they are small, have (at least initially) poorly developed physiological systems, and support important developmental events. Newly‐laid eggs of the moth Manduca sexta are exposed to levels of CO₂ between 0 and 2200 p.p.m., in air, and effects on yolk pH, total developmental time, and survival are measured. Altered CO₂ has no effect, over several hours, on the pH of egg yolk, suggesting that yolk fluids are well buffered. By contrast, there is a large developmental change in yolk pH. Eggs exposed to eight different levels of CO₂ for the duration of development show a small but significant parabolic response in development time. Eggs develop fastest at intermediate levels of CO₂, between 400 and 1200 p.p.m., and slower at 0, 1600 and 2000 p.p.m. These results suggest that future rises in CO₂ may not have strong direct effects on insect development.     

Chemical and immunological properties of lipophorins from seven insect orders

The major insect hemolymph lipoprotein, lipophorin, was isolated from adults of eight insect species representing seven insect orders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to compare their respective apoprotein components. In all species examined lipophorin was composed of at least two apoproteins, apolipophorin I (M_r ～ 250,000) and apolipophorin II (M_r ～ 78,000), and two species had a third apoprotein, apolipophorin III (M_r ～ 17,000). The density of each isolated lipophorin was determined from the refractive index of KBr following density gradient centrifugation. Immunoblotting with anti-larval Manduca sexta apolipophorin I and II of the apoproteins separated by SDS-PACE indicated cross reactivity between anti-M sexta apoLp-ll and apoLp-ll in all species tested. Anti-M sexta apoLp-l exhibited no cross reactivity for any species tested. Fluorescent lectin staining of the apoproteins separated on SDS-PAGE gels revealed the presence of covalently bound carbohydrate residues.     

Effects of Midgut-Protein-Preparative and Ligand Binding Procedures on the Toxin Binding Characteristics of BT-R1, a Common High-Affinity Receptor in Manduca sexta for Cry1A Bacillus thuringiensis Toxins

The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M. sexta larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R₁ binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419–3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the M. sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R₁. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. sexta the cadherin-like BT-R₁ protein is a common high-affinity receptor protein for the Cry1A family of toxins.     

Isolation and Entomotoxic Properties of the Xenorhabdus nematophilus F1 Lecithinase

Xenorhabdus spp. and Photorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar. Substrate specificity studies showed that Photorhabdus spp. exhibited a broad lipase activity, while most of the Xenorhabdus spp. secreted a specific lecithinase. Xenorhabdus spp. occur spontaneously in two variants, phase I and phase II. Only the phase I variants of Xenorhabdus nematophilus and Xenorhabdus bovienii strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth). Five enzymatic isomers responsible for this activity were separated from the supernatant of a X. nematophilus F1 culture in two chromatographic steps, cation-exchange chromatography and C₁₈ reverse-phase chromatography. The substrate specificity of the X. nematophilus F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated. The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae. None of the isomers exhibited toxicity with the insects tested, Locusta migratoria, Galleria mellonella, Spodoptera littoralis, and Manduca sexta. The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.     

Isolation,identification, and synthesis of Mas-MG-MT I,a novel peptide from the larval midgut of Manduca sexta (lepidoptera: Sphingidae)

A five-residue myotropic peptide, Manduca sexta midgut myotropin I (Mas-MG-MT I), was isolated from an extract of 800 midguts of fifth instar larvae of the tobacco hornworm, Manduca sexta. It was purified by reverse phase and normal phase HPLC. Myotropic activity was screened by a heterologous Locusta migratoria oviduct bioassay. Sequence analysis, amino acid composition analysis, and comparison of candidate synthetic peptides in the amide and acid form revealed the following primary structure: Ala-Glu-Pro-Tyr-Thr-NH₂. This is the first fully identified peptide isolated directly from the midgut of an insect species. Few significant sequence homologies with known vertebrate and invertebrate peptides have been found. © 1995 Wiley-Liss, Inc.     

Toxicity of plumbagin and juglone to the eggs of the cotton stainer,Dysdercus koenigii

Toxicity of two plumbaginoids viz., plumbagin and juglone to the eggs of the cotton stainer,Dysdercus koenigii was studied by a residual film technique. Of these two, plumbagin showed the higher toxicity against different-age eggs with LC₅₀ ranging from 0.0044 to 0.0066%. Eggs showed low susceptibility in the middle of embryogenesis. The toxicity of plumbaginoids, especially plumbagin, is discussed in relation to mode of action and prospects of its use as an ovicide in control of the insect.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Studies on the carbohydrate moiety of vitellogenin from the tobacco hornworm,Manduca sexta

Vitellogenin can be isolated in large quantities from the hemolymph of the tobacco hornworm, Manduca sexta by a combination of KBr density gradient ultracentrifugation, gel permeation and cation exchange chromatography. Glycopeptides generated by exhaustive pronase digestion of vitellogenin were separated by gel permeation chromatography on a Bio-Gel P-6 column. The major glycopeptide was shown to bind to concanavalin A-Sepharose but not to lentil lectin-Sepharose and was sensitive to treatment with endo-β-N-acetylglucosaminidase H. Analysis of the glycopeptide by high field proton NMR spectroscopy revealed that the primary structure is of the high mannose class containing nine mannose and two N-acetylglucosamine units. These results suggested that N-glycosylation of insect proteins, as in mammals, yeasts and plants, involves the en bloc transfer of an oligosaccharide containing the unit (Glu)₃(Man)₉(GlcNAc)₂ from a lipid intermediate to an asparagine residue followed by removal of glucose residues. Processing to more complex structures does not seem to occur in M. sexta vitellogenin. In vitro uptake with isolated M. sexta follicles showed that deglycosylation had no significant effect on uptake of ¹²⁵I-labeled vitellogenin.     

Altered tyrosine metabolism and melanization complex formation underlie the developmental regulation of melanization in Manduca sexta

The study of hemolymph melanization in Lepidoptera has contributed greatly to our understanding of its role in insect immunity. Manduca sexta in particular has been an excellent model for identifying the myriad components of the phenoloxidase (PO) cascade and their activation through exposure to pathogen-associated molecular patterns (PAMPs). However, in a process that is not well characterized or understood, some insect species rapidly melanize upon wounding in the absence of added PAMPs. We sought to better understand this process by measuring wound-induced melanization in four insect species. Of these, only plasma from late 5th instar M. sexta was unable to melanize, even though each contained millimolar levels of the putative melanization substrate tyrosine (Tyr). Analysis of Tyr metabolism using substrate-free plasmas (SFPs) from late 5th instar larvae of each species showed that only M. sexta SFP failed to melanize with added Tyr. In contrast, early instar M. sexta larvae exhibited wound-induced melanization and Tyr metabolism, and SFPs prepared from these larvae melanized in the presence of Tyr. Early instar melanization in M. sexta was associated with the formation of a high mass protein complex that could be observed enzymatically in native gels or by PO-specific immunoblotting. Topical treatment of M. sexta larvae with the juvenile hormone (JH) analog methoprene delayed pupation and increased melanizing ability late in the instar, thus linking development with immunity. Our results demonstrate that melanization rates are highly variable in Lepidoptera, and that developmental stage can be an important factor for melanization within a species. More specifically, we show that the physiological substrate for melanization in M. sexta is Tyr, and that melanization is associated with the formation of a PO-containing protein complex.     

Insect eggs exert rapid control over an oxygen-water tradeoff

In terrestrial environments, the exchange of respiratory gases exacts a water cost: obtaining oxygen or carbon dioxide requires losing water. Insect eggs should be especially sensitive to this tradeoff-because they are unable to forage for water, have high surface area-to-volume ratios, and experience large temperature-driven changes in oxygen demand. Previous work from our laboratory, on eggs of a common hawk-moth, Manduca sexta, has shown that, during development, metabolic rate and water loss rates rise in parallel. These correlative data suggest that eggshell conductance increases to accommodate increasing metabolic demand. Here, we test this idea experimentally by subjecting eggs of M. sexta to 15, 21 (normoxia) and 35% oxygen for 24h, while measuring rates of metabolism (as carbon dioxide emission) and water loss. Hypoxia depressed egg metabolic rates, but led to pronounced, rapid increases in water loss. By contrast, hyperoxia had no significant effect on metabolism or water loss. These data demonstrate that insect eggs actively participate in balancing oxygen gain and water loss, and that they use tissue oxygen status, or some correlate of it, as a cue for increasing eggshell conductance. Rapid control over conductance may allow eggs to conserve water during an initial period of low metabolic demand, thereby deferring water costs of respiratory gas exchange until late in development.     

中国南方害虫生物防治50周年回顾

综述了中国南方50年来害虫生物防治的研究与应用概况,特别是大卵（蓖麻蚕卵）繁殖赤眼蜂防治甘蔗螟虫的成功先例,为后来国内应用柞蚕卵繁殖赤眼蜂提供了宝贵经验。应用平腹小蜂防治荔枝蝽,解决了荔枝生产的主要害虫问题;引进澳洲瓢虫、孟氏隐唇瓢虫、松突圆蚧花角蚜小蜂均是我国生物防治成功的例证;以生物防治为主的水稻害虫综合防治研究坚持了20多年,从生产实践和理论方面均取得显著的成绩;在昆虫病原微生物的研究与应用方面,利用苏云金杆菌以色列变种防治蚊虫、松毛虫质多角体病毒的发现和应用、斜纹夜蛾核多角体病毒的分离以及中试和工厂化生产、昆虫病原线虫的大量繁殖与应用,取得了一批有应用价值的成果;在分子生物学研究方面,利用基因重组扩大了病原微生物的毒力与杀虫谱。总结和继承我国乃至世界上最早的以虫治虫（黄蚁防治柑桔害虫）的经验,对促进我国生物防治事业的发展具有重要意义。     

ON THE RATE OF OXYGEN CONSUMPTION BY FERTILIZED AND UNFERTILIZED EGGS : IV. CHAETOPTERHS AND ARBACIA PUNCTULATA

1. Unfertilized eggs of Chaetopterus consume about 2.4 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 2. In the 1st hour after fertilization, the fertilized eggs consume oxygen at about 53 or 54 per cent of this rate, which is about 1.3 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 3. For the first 6 hours after fertilization, at 21°C., the curve of the rate of oxygen consumption is slightly asymmetrically sigmoid. The prefertilization rate is regained between 4½ and 5 hours after fertilization. Soon after 6 hours, ciliary activity begins, and the rate of oxygen consumption rises rapidly. 4. The unfertilized eggs of Arbacia punctulata consume about 0.36–0.5 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. The absolute determination is difficult as these eggs are highly sensitive to shaking in the manometer vessels, and these difficulties are discussed. 5. The fertilized eggs of Arbacia punctulata consume oxygen at the rate of about 2.0 mm.³ O₂ per hour per 10 mm.³ 21°C. At 1 hour after fertilization the rate is already rising. 6. A comparison of the absolute rates of oxygen consumption, and the changes in rate at fertilization of these and a number of other eggs, together with a theoretical discussion, and a discussion of discrepancies in measurements on the eggs of Arbacia punctulata, is contained in the fifth paper of this series (21).     

Respiratory metabolism and thiabendazole susceptibility in developing eggs of Haemonchus contortus

Weston K. M., O'Brien R. W. and Prichard R. K. 1984. Respiratory metabolism and thiabendazole susceptibility in developing eggs of Haemonchus contortus. International Journal for Parasitology14: 159–164. The respiratory metabolism of and uptake of thiabendazole (TBZ) by unembryonated (8–16 cells) and embryonated eggs of Haemonchus contortus have been compared. Lipid, which forms the greatest energy reserve in the eggs, decreases during embryonation and seems to be the sole source of respiratory energy. Trehalose increases to the same extent as glycogen decreases during this development. Isocitrate dehydrogenase (NADP⁺) and lactate dehydrogenase were not detected in the unembryonated eggs, but were present after embryonation. In addition, the activities of citrate synthase and malate dehydrogenase significantly increased during embryonation. Respiratory enzymes involving cytochrome c oxidation and reduction were detected in eggs at both stages of development. However, in line with other results indicating an increased capacity for and utilization of aerobic metabolism, the rate of oxygen uptake more than tripled during development of the eggs. Although both unembryonated and embryonated eggs took up palmitate, its metabolism to CO₂ only occurred in the embryonated eggs.The unembryonated eggs, exposed to TBZ for 4 h, concentrated it 5.9 times and the embryonated eggs 2.1 times, which are in proportion to their respective lipid contents. Uptake of TBZ was dependent on the concentration in the incubation medium and appears to be a passive process.The studies indicate that the embryonated eggs have a greater capacity for, and do utilize aerobic metabolism to a greater extent than do unembryonated eggs. The reduced susceptibility of embryonated eggs to TBZ could be associated with this metabolic difference and/or with their reduced uptake of TBZ.     

Metabolic responses of Glossina pallidipes (Diptera: Glossinidae) puparia exposed to oxygen and temperature variation: Implications for population dynamics and subterranean life

Understanding the factors affecting insect gas exchange in subterranean environments is critical to understanding energy budgets and predicting mortality under field conditions. Here, we examine the metabolic rate (MR) responses of tsetse puparia, which remain underground for ca. 1 month in this life-stage, to varying oxygen and temperature. First, the effects of temperature and oxygen on puparial MR were investigated by ramping temperature from 15 to 35 °C under 10, 21 or 40% O₂. Overall, temperature was the dominant effect on puparial MR although O₂ had small but significant impacts. Second, critical O₂ concentration (P_CRIT) for MR of puparia was examined across a range of oxygen concentrations (0-40%). P_CRIT was 6% O₂ which is similar to P_CRIT in other basal arthropods but relatively high for inactive or subterranean insects. Third, we asked if puparia exposed to anoxia might experience oxygen debt, potentially indicative of anaerobic metabolism or cellular repair. Metabolic responses to anoxia were limited or insignificant, but MR was marginally elevated (∼15%) in anoxia-exposed (4 h) puparia by 12 h post-anoxia. Finally, we examined the ability of puparia to withstand water submersion, thus simulating flooding conditions frequently experienced in tropical soil habitats. Puparia were unable to survive submersion for >24 h suggesting limited flooding tolerance. These novel results suggest that soil conditions experienced by puparia should not be limiting for MR, except possibly under high temperature-low O₂ conditions. Due to a large safety margin between P_CRIT and soil oxygen levels and limited effects of oxygen on metabolism during temperature ramping experiments, we suggest that Glossina pallidipes puparia are not particularly susceptible to oxygen availability in their natural environment. However, soil flooding associated with tropical rainfall likely imposes strong selection on tsetse populations and may have had important effects for tsetse energy budgets and evolution.     

Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC–MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.     

Disruption of insect isoprenoid biosynthesis with pyridinium bisphosphonates

Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C₅), with the allylic diphosphate primer dimethylallyl diphosphate (DMAPP; C₅) to generate the C₁₅ prenyl chain (FPP) used for protein prenylation as well as sterol and terpene biosynthesis. Here, we designed and prepared a series of pyridinium bisphosphonate (PyrBP) compounds, with the aim of selectively inhibiting FPPS of the lepidopteran insect order. FPPSs of Drosophila melanogaster and the spruce budworm, Choristoneura fumiferana, were inhibited by several PyrBPs, and as hypothesized, larger bisphosphonates were more selective for the lepidopteran protein and completely inactive towards dipteran and vertebrate FPPSs. Cell growth of a D. melanogaster cell line was adversely affected by exposure to PyrPBs that were strongly inhibitory to insect FPPS, although their effect was less pronounced than that observed upon exposure to the electron transport disrupter, chlorfenapyr. To assess the impact of PyrBPs on lepidopteran insect growth and development, we performed feeding and topical studies, using the tobacco hornworm, Manduca sexta, as our insect model. The free acid form of a PyrBP and a known bisphosphonate inhibitor of vertebrate FPPS, alendronate, had little to no effect on larval M. sexta; however, the topical application of more lipophilic ester PyrBPs caused decreased growth, incomplete larval molting, cuticle darkening at the site of application, and for those insects that survived, the formation of larval–pupal hybrids. To gain a better understanding of the structural differences that produce selective lepidopteran FPPS inhibition, homology models of C. fumiferana and D. melanogaster FPPS (CfFPPS2, and DmFPPS) were prepared. Docking of substrates and PyrBPs demonstrates that differences at the −3 and −4 positions relative to the first aspartate rich motif (FARM) are important factors in the ability of the lepidopteran enzyme to produce homologous isoprenoid structure and to be selectively inhibited by larger PyrBPs.     

Identification of Bacillus thuringiensis Delta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in Insect Specificity

Cry1C domain III amino acid residues involved in specificity for beet armyworm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against S. exigua but resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved in S. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.     

Purification of insulin-like peptides from insect haemolymph and royal jelly

Insulin-like peptides from the haemolymph of the tobacco hornworm, Manduca sexta L. and from royal jelly of the honey bee, Apis mellifera L., were purified by acid extraction, ion-exchange chromatography, gel filtration and affinity chromatography. Quantities detected were 500 and 25 pg porcine insulin equivalents per gram of lyophilized material for haemolymph and royal jelly, respectively. M. sexta insulin was similar to vertebrate insulin in solubility, chromatographic, immunological and biological properties. Amino acid compositions were comparable except for serine, leucine and phenylalanine. The A. mellifera peptide had similar properties, but was not isolated in large enough quantity to determine its amino acid composition. These results demonstrated that insect insulin and vertebrate insulin are structurally similar.     

Silencing of Maternal Heme-binding Protein Causes Embryonic Mitochondrial Dysfunction and Impairs Embryogenesis in the Blood Sucking Insect Rhodnius prolixus

The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.