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.     

Cloning and expression of apolipophorin-III from the common cutworm,Spodoptera litura

We have cloned apolipophorin-III (apoLp-III) cDNA from adult fat body of Spodoptera litura. The sequence encodes a 188 amino acid polypeptide including a 22 amino acid leader peptide. The circular dichroism spectrum from the purified apoLp-III indicated a considerable content of α-helix. Sequence alignment showed that S. Litura apoLp-III has a relatively high degree of sequence identity with the apoLps-III of lepidopteran, Manduca sexta (72%), Galleria mellonella (67%), Bombyx mori (60%). These alignments with four lepidopteran apoLps-III showed highly identical residues and conservative replacements at a degree of 86%. Levels of mRNA from last instar larval fat body and adult fat body were compared through Northern blot analysis using ³²P-labeled 704 bp apoLp-III cDNA probe. A 850 bp mRNA was detected in both stages and mRNA level of day 1 adult fat body was much higher than that of last instar larval fat body. The tissue-distribution of apoLp-III mRNA in adult ovary and testis was also examined and we confirmed the presence of apoLp-III mRNA in ovary and testis although apoLp-III was expressed in these tissues at very low levels compared with the adult fat body. Arch. Insect Biochem. Physiol. 39:166–173, 1998. © 1998 Wiley-Liss, Inc.     

Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838 bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24 kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49-46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.     

Photoreception in decapitated larvae of silkworm Bombyx mori

To investigate the photoreception that controls daily oscillations at the periphery in insects, we decapitated larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) by ligature, and observed rhythms in their peripheral tissues under several light conditions. We measured the mRNA expression of period (per) and timeless (tim), which are homologues of Drosophila clock genes that function in the core oscillator of the circadian clock system. The expression of both per and tim significantly changed in the midgut, Malpighian tubules and silk glands of decapitated larvae exposed to photophase and scotophase that were reversed from the original daily light–dark cycle under which the larvae were housed. Under constant darkness, the daily expression of tim mRNA persisted for at least one cycle in the midgut and silk gland. In addition, an appropriate light stimulus under constant darkness induced a significant phase shift in the endogenous timing system (probably a circadian clock) that determined peak levels of tim mRNA expression in the midgut and silk glands of decapitated larvae. Since light regulated the gene expression rhythm in peripheral tissues of decapitated silkworm larvae, neither the brain nor eyes were essential for photoreception to control daily oscillations in these tissues. Thus, peripheral tissues in insects might directly use light even at the larval stage.     

甘蓝夜蛾和八字地老虎肌动蛋白基因的克隆与序列分析

肌动蛋白是细胞骨架微丝的主要组成成分,在肌肉收缩、细胞骨架形成、细胞移动等方面起重要作用。以鳞翅目夜蛾科昆虫甘蓝夜蛾Mamestra brassicae L.和八字地老虎Agrotis c-nigrum 3龄幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到2种昆虫的肌动蛋白的cDNA序列,甘蓝夜蛾肌动蛋白的cDNA序列含有1441个碱基,而八字地老虎肌动蛋白的cDNA序列含有1411个碱基。2种昆虫的该基因的cDNA序列均包括1个1131个碱基的开放阅读框,编码1个含376个氨基酸的蛋白。甘蓝夜蛾肌动蛋白分子量约为41.8kDa;八字地老虎肌动蛋白分子量约为41.9kDa。Prosite软件分析结果表明,甘蓝夜蛾和八字地老虎肌动蛋白氨基酸序列中存在3个肌动蛋白特征片段。GenBank数据库搜索及序列比对结果表明,甘蓝夜蛾肌动蛋白属于肌肉特异型肌动蛋白,八字地老虎肌动蛋白属于细胞质特异型肌动蛋白。2个基因的cDNA序列已经登录GenBank并获得登录号,甘蓝夜蛾肌动蛋白cDNA序列登录号为EU035314,八字地老虎肌动蛋白cDNA序列登录号为EU035315。利用RT-PCR技术在八字地老虎4龄、5龄、6龄幼虫、蛹期4个不同发育阶段和6龄期的肠道、体壁、脂肪体3种不同组织中都检测到了肌动蛋白基因在mRNA水平的表达。     

Cloning of Immune Protein Hemolin cDNA from the Indianmeal Moth, Plodia interpunctella, and its High Induction During Development and by Bacterial Challenge

Partial cDNA of hemolin, an insect immune protein, was cloned from indianmeal moth, Plodia interpunctella, and the rates of hemolin mRNA expression were demonstrated in each stage of development and also by bacterial injections. A deduced amino acid sequence from the cloned hemolin cDNA was approximately 44‐54% similar to hemolins of 5 other moths. During development the level of hemolin mRNA was the highest at the time of the larval‐pupal matamorphosis. Hemolin was also rapidly induced by the injection of bacteria into the 4^th or the 5^th instar larvae. Hemolin induction rate by bacterial challenge was higher in the 5^th instar larvae than in 4^th instars. Our results suggest that hemolin could have multiple roles that act both on cellular processes during development and on the immune reactions for the resistance to pathogen invasion.     

An ecdysone-inducible putative "DEAD box" RNA helicase in the spruce budworm (Choristoneura fumiferana)

RNA helicases are a family of enzymes that unwind nucleic acid duplexes, such as RNA/RNA and RNA/DNA, in a 3' to 5' direction into single-stranded polynucleotides. A putative RNA helicase cDNA (CfrHlc64) was isolated from the spruce budworm, Choristoneura fumiferana. CfrHlc64 was 1998 nucleotides in length, and the deduced protein had 565 amino acids with a predicted molecular mass of 64 kDa. It contained eight functional motifs conserved in the "DEAD box" family of RNA helicases. The deduced amino acid sequence showed 10-50% identities to homologues of other species from bacteria to human. In vitro expression of the cDNA resulted in recombinant proteins of 64 kDa as expected from the deduced amino acid sequence. Northern blotting and RT-PCR analyses revealed the presence of CfrHlc64 mRNA in all developmental stages from embryo to adult. Higher levels of CfrHlc64 mRNA were detected in the fat body and midgut than in the epidermis of sixth instar larvae. The CfrHlc64 protein was distributed mainly in the fat body. Female adults expressed CfrHlc64 mRNA at higher levels than male adults. The nonsteroidal ecdysone agonist, tebufenozide, enhanced the expression of CfrHlc64 in a dose-dependent manner.     

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59 789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC posses an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE. Mol. Reprod. Dev. 51:265–273, 1998. © 1998 Wiley-Liss, Inc.     

Modification of the nutritional parameters and of midgut biochemical and absorptive functions induced by the IGR fenoxycarb in Bombyx mori larvae

Fifth instar larvae of B. Mori were topically or orally treated with increasing amounts of the Insect Growth Regulator (IGR) fenoxycarb in a single application, in order to determine its effects on the nutritional parameters, the midgut functional activities and the growth of the silk glands. The IGR affected in a dose-dependent manner the progress of the life cycle of the insect, causing a delay or inhibition of spinning, alteration of the feeding behaviour, decrease of the nutritional parameters, impairment of the growth of the silk glands, and an increased mortality during larval-pupal transformation. Measurement of leucine uptake into midgut brush border membrane vesicles and midgut histochemistry revealed a reduced absorption of leucine by the midgut and a large alteration of a number of midgut enzyme activities as a result of treatments with a high dose of fenoxycarb (2.5 μg). Treatments with a dose of 2.5 femto g/larva caused an increase in leucine uptake by the midgut, an increased weight of the cocoon shell, and a modification of some midgut enzyme activities. The lepidopteran midgut appears to be a larval organ that responds promptly to the exposure to fenoxycarb. The epithelial columnar cells modify their absorptive functions, at least with regard to amino acid uptake, as well as their metabolic activity, with a modification of the oxidative status of the cells that is detectable with a single dose of the chemical as low as few fg/larva. Arch. Insect Biochem. Physiol. 39:18–35, 1998. © 1998 Wiley-Liss, Inc.     

Feeding regulation by neuropeptide Y on Asian corn borer Ostrinia furnacalis

The Asian Corn Borer Ostrinia furnacalis is a major agricultural pest. In this study, a full‐length neuropeptide Y (npy) gene in O. furnacalis was sequenced and cloned from cDNA library, which contains an ORF of 273 bp by encoding 90 amino acid residues. The mature OfurNPY is composed of 29 amino acids with amidation in C‐terminal. The spatiotemporal expression analysis showed that npy highest expression level was in the midgut of the fifth instar larvae (the gluttony period). When the expression of npy was knocked down by feeding or injecting dsNPY, larval food consumption, body size, and body weight were significantly inhibited compared to controls. These results indicate that NPY is an important regulator in the control of feeding of O. furnacalis.     

Spatiotemporal expression pattern of an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus during early development,and its potential role in larval vertical migration

We have cloned and studied Hp‐ECPN, an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus. Hp‐ecpn cDNA was produced and found to contain a 1461‐bp open reading frame that encodes 486 amino acids. Accumulation of Hp‐ecpn mRNA and protein expression occurred at the 14 h postfertilization (hpf) swimming blastula stage and thereafter. The Hp‐ECPN protein was N‐glycosylated, and the amino acid sequence was similar to that of vertebrate encephalopsins. Whole‐mount immunohistochemistry revealed the presence of Hp‐ECPN in cells (ECPN cells) that appeared initially around the tip of the archenteron in 20 hpf early gastrulae. By the 54 hpf pluteus stage, ECPN cells had spread through the aboral ectoderm, and, by the eight‐arm pluteus stage, were restricted to the tips of the larval arms and the posterior end of the body. The number of ECPN cells increased under conditions of continuous light, but decreased under continuous dark. Knockdown of Hp‐ecpn mRNA using morpholino antisense oligonucleotides decreased the number of ECPN cells considerably, and inhibited the vertical swimming of the larvae. This suggested that Hp‐ECPN plays a role in photosensitive larval swimming vertical migration. In adult tissues, the ECPN cells were detected exclusively in tube feet.     

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M _r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression. Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998