Amino acid and protein changes in the haemolymph of developing fourth instar Chironomus tentans

The concentration of free amino acids, total soluble protein, and haemoglobin in the haemolymph of fourth instar Chironomus tentans was investigated.The concentration of the free amino acid pool increases between the early (15.7 mM/l) and mid-(33.9 mM/l) fourth larval stages followed by a decline during the late (16.9 mM/l) fourth larval period. Alanine, serine, and the amides of aspartic acid and glutamic acid are the predominant free amino acids at all stages. Physiological fluid analysis of late fourth instar haemolymph detected 32 ninhydrin positive components including 18 common amino acids plus homoarginine, ornithine, citrulline, β-alanine, α-aminoadipic acid, α-aminoisobutyric acid, and sarcosine.The concentration of total soluble protein steadily increases during fourth instar larval development to a maximum of $\text{9.3}\text{g}\text{100}\text{ml}$ followed by a decline during the pharate pupal period. A similar pattern of variation occurs in haemoglobin content which comprises from 51 to 66% of Chironomus tentans haemolymph protein.The mM percentage of individual amino acids of total haemolymph protein varies little during the fourth instar. At all stages alanine and aspartic acid are the predominant amino acids.     

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.     

Developmental changes of storage proteins and biliverdin-binding proteins in the haemolymph and fat body of the common cutworm,Spodoptera litura

The concentrations of three storage proteins (SL-1,SL-2 and SL-3, hexamers of 70-80kDa subunits) and two biliverdin-binding proteins (BP-A and BP-B, dimers of 165kDa) in the haemolymph and fat body during larval and pupal development of Spodoptera litura were determined by immunodiffusion tests using polyclonal antisera. SL-1 and SL-2 (methionine-rich) first appeared in the haemolymph of one-day-old sixth (final) instar larvae, prominently increased in the haemolymph during the later feeding period and were almost totally sequestered by the fat body after gut purge. SL-3 (arylphorin) was first detected in the haemolymph during the molting period to the final larval ecdysis, increased in concentration throughout the entire feeding period of the final larval instar and was partly sequestered by the fat body several hours later than the other storage proteins. BP-A showed nearly the same pattern in the haemolymph as SL-3: BP-B increased during feeding period and decreased during molting period and attained a maximum level during the penultimate larval instar, however its concentration decreased considerably and remained low in the final larval instar. BP-A was partly and BP-B was almost totally sequestered by the fat body 8 h after sequestration of SL-1 and SL-2, rendering the fat body blue in colour. These facts suggest an additional function of biliverdin-binding proteins as amino acid storage proteins and the results show a differential uptake mechanism for these proteins by the fat body.     

斜纹夜蛾线粒体复合物ⅢFe-S蛋白基因克隆、序列分析及在不同发育阶段的表达特征

Fe-S蛋白(rieske iron-sulfur protein, RISP)是线粒体复合物Ⅲ的关键蛋白亚基之一, 在呼吸链电子传递中起着重要作用。本文利用RT-PCR技术和RACE方法获得了斜纹夜蛾Spodoptera litura (Fab.)线粒体复合物ⅢRISP基因, 该基因序列全长4 413 bp, 含有3个内含子; 开放阅读框长816 bp, 编码271个氨基酸。经氨基酸序列比对, 与鳞翅目家蚕Bombyx mori和小菜蛾Plutella xylostella的Fe-S蛋白一致性较高, 分别为83%和79%, 与其他目昆虫的RISP一致性较低。qRT-PCR分析结果表明, SlitRISP基因在斜纹夜蛾不同发育阶段的表达水平存在显著差异, 即幼虫期和成虫期的表达量显著高于卵期和蛹期, 其中幼虫期又以第4和5龄幼虫表达水平显著高于初孵幼虫以及第1, 2, 3和6龄幼虫。斜纹夜蛾SlitRISP基因的克隆成功为今后对其功能研究以及作为靶标设计新型高效杀虫剂提供了基础。     

Characterization of cuticular proteins in the red flour beetle, Tribolium castaneum

We are characterizing the cuticular proteins of Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) to determine their role in the function of the exoskeleton. Based on qualitative analyses of cuticles, we focused on the sodium dodecyl sulfate (SDS)-extractable proteins. A small-scale cuticle "mini-prep" procedure was devised that yields preparations virtually free of contaminating cellular material compared to hand-dissected preparations, as assessed by fluorescent microscopy using DAPI to stain nuclei. Proteins extracted in 1% SDS from various developmental stages (last larval instar, pupal, adult) were analyzed by one-dimensional denaturing polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. The cuticular protein profiles show both similarities and differences among the stages examined. The amino acid composition, glycosylation, and partial amino acid sequence of several abundant cuticular proteins indicate similarity to cuticular proteins of other insects.     

Purification and characterization of epidermis-origin hemolymph protein in Galleria mellonella

Epidermis-origin hemolymph protein (EOHP) was identified and purified from the last instar larval hemolymph of Galleria mellonella by anion exchange chromatography, chromatofocusing chromatography, and Sephadex G-100. The EOHP has a native molecular mass of 47 kDa and is composed of one subunit. The isoelectric point of the EOHP was determined to be 5.3. The amino acid composition of the EOHP was rich in aspartic acid, glutamic acid and lysine, but poor in tyrosine, methionine, and tryptophan. EOHP is present in hemolymph over the period from the 4th instar larvae to the adult stage examined. Concentration of EOHP is high during the larval stage but gradually decreased during the developmental stage from pupal to adult stage. EOHP is present in the cuticle, fat bodies and trachea but not in hemocytes, fore gut, mid gut and hind gut.     

Cloning,partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.     

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.     

Developmental expression of three genes for larval cuticular proteins of the tobacco hornworm, Manduca sexta

Three cDNA clones coding for the 12.8, 13.3, and 14.6 kDa larval cuticular proteins of the tobacco hornworm, Manduca sexta, were isolated and characterized. Hybridization to abdominal epidermal RNA from different stages showed that the genes for the 12.8 and 13.3 kDa proteins were expressed only during larval life. By contrast, the gene for the 14.6 kDa protein was expressed throughout the segment during the feeding, growing larval stages, then only in the flexible intersegmental regions during the deposition of endocuticle in the pharate pupa and adult. Quantitative RNA dot blot hybridizations showed that the RNA for each protein disappeared during the larval molt when the ecdysteroid titer was high, then reappeared during the preecdysial deposition of endocuticle. All disappeared when the epidermis became pupally committed at the onset of wandering. Exposure of the fourth instar epidermis to 20-hydroxyecdysone (20HE) in vitro under conditions that lead to the formation of a new larval cuticle by 48 hr caused the disappearance of these RNAs by 18 hr. Exposure of Day 2 fifth instar epidermis to 20HE in vitro caused a depression of these RNAs which in the case of the RNAs coding for the 12.8 and 13.3 kDa proteins was partially prevented by simultaneous exposure to methoprene, a juvenile hormone (JH) mimic. By contrast, the RNA for the 14.6 kDa protein was suppressed by exposure to methoprene alone. Thus, each of these larval cuticular genes is turned off by high ecdysteroid; the presence or absence of JH determines whether or not this suppression is permanent in some or all cells.     

麦红吸浆虫3-羟基-3-甲基戊二酰辅酶A还原酶基因SmHMGR的克隆及在滞育与发育变态过程中的表达动态

【目的】3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是保幼激素(JH)合成途径的限速酶。麦红吸浆虫Sitodiplosis mosellana是一种典型的专性幼虫滞育昆虫。本研究旨在探讨HMGR基因在麦红吸浆虫滞育和发育变态过程中的作用。【方法】通过RT-PCR和RACE技术克隆麦红吸浆虫滞育前幼虫HMGR基因全长cDNA序列;利用生物信息学软件分析HMGR基因核苷酸和其编码的蛋白氨基酸序列特性;采用qPCR技术测定其在麦红吸浆虫滞育不同时期3龄幼虫及不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹和后蛹以及雌雄成虫)中的mRNA表达水平。【结果】克隆获得一条麦红吸浆虫HMGR基因全长cDNA序列,命名为SmHMGR(GenBank登录号: MG876766)。该基因全长2 548 bp,其中开放阅读框长2 328 bp,编码775个氨基酸,预测的蛋白分子量为84.16 kD,理论等电点为8.29。序列分析发现该基因编码的蛋白具有HMGR蛋白家族典型的HMG-CoA-reductase-classⅠ催化功能域及其他保守功能基序;序列比对和系统发育分析表明,SmHMGR与达氏按蚊Anopheles darling等长角亚目(Nematocera)昆虫HMGR的相似性最高、亲缘关系最近。SmHMGR在麦红吸浆虫滞育前的3龄早期幼虫中表达量显著升高,进入滞育后一直维持较高水平,并在滞育后静息阶段的当年12月至翌年1月达到最高。SmHMGR在蛹期表达量低于幼虫期,预蛹期表达量最低;在雌成虫中表达量显著高于在蛹和雄成虫中的表达量。【结论】SmHMGR的表达与麦红吸浆虫发育密切相关,可能在滞育诱导、维持及滞育后静息状态的维持及生殖中发挥作用,其表达量的降低可能参与了幼虫到蛹的变态。     

Trypsinogen-like cDNAs and quantitative analysis of mRNA levels from the Indianmeal moth, Plodia interpunctella

Two cDNA fragments encoding full-length trypsinogen-like proteins were cloned from larvae of two strains (RC688s and HD198r) of the Indianmeal moth, Plodia interpunctella (Hübner), which differed in their sensitivity to Bacillus thuringiensis protoxins. One cDNA fragment contained 874 nucleotides, including a 780-nucleotide open reading frame that encoded a trypsinogen-like protein (PiT2b). Another cDNA fragment amplified from both P. interpunctella strains contained 864 nucleotides including a 780 bp open reading frame encoding a second trypsinogen-like protein (PiT2c). The cDNA sequence of PiT2b shared 89% sequence identity with PiT2a, a trypsinogen-like protein cloned previously from this species. The cDNA sequences of PiT2a and PiT2c shared 83% identity. The cDNA sequence identity between PiT2b and PiT2c was 80%. The cDNA for PiT2b from strain RC688s was different at six nucleotide positions from that of PiT2b from strain HD198r. Five nucleotide replacements occurred in the open reading frame leading to amino acid changes at all five positions. There were five nucleotide differences in the cDNAs for PiT2c trypsinogen-like proteins from the two strains. Two nucleotide substitutions in the open reading frame resulted in replacements of two amino acid residues in the deduced protein sequences. Amino acid sequences for PiT2a and PiT2b shared 84% identity, but only 50% identity was observed between PiT2c and the other two trypsinogen-like proteins. The deduced amino acid sequences for PiT2b and PiT2c included both signal and zymogen activation peptides and amino acid sequence motifs which are conserved in seven homologous trypsinogen-like proteins from other insects. Typical features of the putative trypsinogen-like proteins from P. interpunctella included the serine proteinase active site triad (His(81), Asp(133), and Ser(233)), three pairs of cysteine residues for disulfide bridges, and three residues, Asp(227), Gly(250), and Gly(260), that help to confer trypsin-like specificity to the enzymes. Quantitative RT-PCR analyses showed that, in fourth instar larvae, RC688s had 1.6-fold higher PiT2a trypsinogen-like mRNA than did HD198r. Expression of PiT2b mRNA was 3.4-fold higher in HD198r than in RC688s. Expression of PiT2c mRNA was 2.8-fold higher in RC688s than in HD198r. Mean accumulation levels of mRNAs for all three trypsinogen-like proteins were slightly higher in RC688s than in HD198r based on total RNA, and 1.3-fold higher in RC688s than in HD198r based on wet weight of larval body tissues.     

Molecular identification of glucose-regulated protein 78 (grp78) gene from the Indian meal moth,Plodia interpunctella,and its regulation by nutrient uptake

Glucose-regulated protein 78 (GRP78) is a member of the HSP70 family of proteins and is localized in the endoplasmic reticulum (ER) within cells. GRP78 and its gene has been identified in only a few species of insects, and its role is not clear. Here, we identified full-length grp78 cDNA from the Indian meal moth, Plodia interpunctella, and demonstrated the role of grp78 in developmental and physiological processes of the insect. The deduced amino acid sequence of GRP78 contained highly conserved functional motifs of the HSP70 family and the C-terminal motif of KDEL, which is characteristic of ER-localized HSP70. It also showed high identity (93–94%) with GRP78 and related HSP70 proteins of lepidopteran species. Gene expression analysis showed that grp78 mRNA levels were high in the egg, feeding larval, and adult stages, but low in the molting, wandering larval, and pupal stages of development. In a tissue comparison of fifth instar P. interpunctella, grp78 level was higher in the gut than in the integument or fat bodies. Grp78 level decreased greatly when fifth-instar larvae were starved for 48 h, but recovered within 3–6 h after re-feeding. Our data suggest that grp78 is highly associated with dietary energy conditions during development and may play an important role in the nutritional physiology of insects.     

Development of the larval ovary in the moth, Plodia interpunctella

The morphogenesis of ovaries and the organization of germ cells within them were visualized during the larval stages of the moth, Plodia interpunctella. The germ cells were observed by utilizing confocal microscopy coupled with immuno-fluorescent staining for the alpha-crystallin protein 25 (alphaCP25). The alphaCP25 was previously shown to be specific to germ cells of pupae and adults, and this study shows that alphaCP25 is present in larval germ cells as well. A cluster of 28 germ cells that stain for alphaCP25 was found in the gonads of newly hatched first instar larvae. The founding germ cells became segregated into four clusters, most likely by somatic cell intrusion, around the beginning of the second instar. Division of the primary germ cells began by the end of the second instar and the formation of all cystoblasts appeared to be completed within the four ovarioles by the end of the third instar. Within the ovarioles of third instar larvae, the germ cells were organized with a distal cap of seven germ cells which was segregated from the majority of the germ cells. The main body of germ cells was arranged around a central germ cell-free core as a spiral. Divisions of the cystoblasts to form cystocyte clusters were nearly completed during the fourth (last) larval instar. These features suggest that the strategy to produce follicles in moths is fundamentally different from the fruitfly, Drosophila. It appears that during the initial stages of ovary development in P. interpunctella, the primary germ cells undergo stage-complete divisions that are completed prior to the onset of the next set of divisions, which results in a complete complement of follicles available by the time of adult eclosion, while in Drosophila the primary germ cell divisions are initiated in the adult stage, and follicles are produced individually as resources are available.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

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.     

Storage proteins in vespid wasps: characterization,developmental pattern,and occurrence in adults

Wasps of family Vespidae contain three types of major proteins that have the size, amino acid composition, subunit composition, immunological reactivity, and pattern of occurrence characteristic of storage proteins. The three types of storage protein, which have been identified in other Hymenoptera, are very high density lipoprotein, high glutamine/glutamic acid protein, and hexamerin. The predominant pattern of occurrence for these proteins is as known from most or all Holometabola: synthesis during the last larval instar and utilization as an amino acid source during metamorphosis. Hexamerin also occurred in a large young adult female Monobia quadridens but not a small one, which suggests that carry-over into adult females is a reaction norm response to quantity of larval provisions, because these wasps could not have fed as adults. In two paper wasp species of the genus Polistes, hexamerin was present in large adult females which emerged during the colony cycle phase when reproductive females are typically produced, but not in adult female offspring that emerged earlier in the colony cycle or in adult females that were workers. It cannot be confirmed by these data that the hexamerin in the adult paper wasps represented carry-over from metamorphosis rather than post-emergence feeding, but the pattern of occurrence suggests that presence of storage protein may play a role in caste differentiation in paper wasps. No storage protein was found in any adult Vespula maculifrons, a yellowjacket wasp, suggesting that caste differentiation in vespine wasps does not incorporate storage protein as a component.