A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.     

cDNA cloning and mRNA expression of L-3,4-dihydroxyphenylalanine decarboxylase gene homologue from the silkworm,Bombyx mori

l-3,4-Dihydroxyphenylalanine decarboxylase (DDC) cDNA, from Bombyx mori that contains an open reading frame of 1437 bp encoding 478 amino acids, was cloned and characterized. Expression analyses of B. mori DDC mRNA by Northern and in situ hybridization indicated that expression of silkworm DDC expression is possibly controlled by neuropeptide hormones in tissue- and stage-specific manners.     

Regulation of genes for ubiquitination and SUMO-specific protease involved in larval development of the silkworm,Bombyx mori

Protein modifications with highly conserved small proteins, such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO), regulate various cellular processes; however, the contribution of these protein modifications to larval development in insects has not yet been elucidated. We investigated the regulation of genes for these protein modifications in the posterior silk gland (PSG) during larval development of the silkworm Bombyx mori. We found that several genes encoding enzymes (E1, E2, and E3) for ubiquitination and SUMO-specific protease were upregulated by 20-hydroxyecdysone (20E), and, consistently, increases in ubiquitinated proteins were observed during the fourth molting stage. An injection of 20E into larvae at the fourth feeding stage induced higher expression levels of these E1, E2, and E3 genes and ecdysis approximately one day earlier than in mock-treated larvae. The expression of the fibroin heavy-chain gene (fibH) was simultaneously suppressed approximately one day earlier in 20E-injected larvae. The treatment of cultured PSG with 20E also induced these genes, which could be categorized into at least two types: those induced by a high dose of 20E, or by a pulse of 20E. In contrast to the 20E treatment, the administration of PR-619, an inhibitor of Ub- and SUMO-specific proteases in larvae, delayed ecdysis and prolonged the expression of fibH. These results suggest that the regulation of genes for ubiquitination and SUMO-specific protease is involved in the larval development of B. mori.     

Purification,characterization and developmental changes in the titer of a new larval serum protein of the silkworm,Bombyx mori

A new protein was found as a major component of hemolymph proteins up to day 1 of the last larval instar of Bombyx mori, and was named Bombyx mori larval serum protein (BmLSP). The BmLSP was purified to homogeneity by ammonium sulfate precipitation, CM-cellulose column chromatography and gel permeation chromatography. The molecular weight of BmLSP was estimated to be 30,000 by SDS-PAGE and 25,000 by gel permeation chromatography. The amino acid composition of BmLSP was similar to that of 30 kDa proteins which are the major serum proteins in the older last (fifth) instar larvae. The 20 NH₂-terminal amino acids were sequenced and found to be quite different from those of the 30 kDa proteins. Developmental changes in BmLSP titer were followed throughout post-embryonic life by Western blotting using a specific antiserum against BmLSP. Within 1 day after larval hatching, BmLSP appeared in the hemolymph and remained at an almost constant level until day 1 of the last instar. On day 2 of the last instar, the BmLSP level suddenly fell and then gradually decreased toward larval-pupal metamorphosis. Thus, BmLSP is a true larval serum protein and is different from proteins stored for metamorphosis.     

The role of eclosion hormone in the larval ecdyses of Manduca sexta

Each larval moult in Manduca sexta consists of an identical series of developmental and behavioural events leading up to ecdysis. Injections of eclosion hormone into staged larvae in any instar resulted in the premature elicitation of the larval pre-ecdysis behaviour, comprising a rhythmic sequence of muscle contractions, followed by the larval ecdysis behaviour.A marked depletion of eclosion hormone stores form the ventral chain of ganglia coincided with each larval ecdysis and in the moult to the fifth instar, eclosion hormone activity appeared in the blood at the onset of the pre-ecdysis behaviour.Responsiveness to eclosion hormone for pre-ecdysis and ecdysis behaviour developed about 12 and 6 hr before normal ecdysis, respectively. Elicitation of ecdysis behaviour by exogenous hormone inhibited both subsequent behavioural responses to eclosion hormone and endogenous hormonal release.In conclusion, the behavioural programme involved in each larval ecdysis appears to be controlled by the eclosion hormone.     

Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria

Femur cuticle from fifth instar larvae of the desert locust, Schistocerca gregaria, has been characterized with respect to composition, rate of deposition, and rate of sclerotization. The results are compared with those from adult cuticle of the same species. The protein compositions of the two types of cuticle are very similar, but the rates of deposition of both protein and chitin are different. The main difference is, however, that sclerotization is restricted to the first day after ecdysis in larval cuticle, whereas in adult cuticle sclerotization continues for at least a couple of weeks. The result is that the endocuticle remains untanned in the larvae, whereas in the adults the whole cuticle becomes tanned.     

Induction of supernumerary larval moulting in the tobacco hornworm Manduca sexta: interaction of bisacylhydrazine ecdysteroid agonists with endogenous Juvenile Hormone

Abstract When given in a critical dietary dose range, the insecticidal bisacylhydrazine ecdysteroid agonists RH‐5849 or tebufenozide (RH‐5992) cause fifth stage Manduca sexta (L.) larvae to moult to a supernumerary sixth‐stage giant larva. The effect is dependent on exposure to the chemicals immediately after the previous ecdysis. Previous removal of the corpora allata does not interfere with the induction of premature moulting by RH‐5849 but completely prevents the formation of supernumerary larvae. The juvenilizing effect is therefore due to the interaction of the moult‐promoting effect of the ecdysteroid agonists with the high titre of endogenous Juvenile Hormone that is present just after ecdysis to the fifth stage in this insect. The ecdysteroid agonists themselves appear to have no intrinsic Juvenile Hormone‐agonist properties. Sixth‐stage larvae resulting from exposure to critical dietary concentrations of RH‐5849 are morphologically completely larval in character. When transferred to diet without the ecdysteroid agonist, they feed normally and gain weight, growing much larger than control fifth stage insects. At the end of the supernumerary stage, they cease to feed, wander in the usual way, and form a normal pupal cuticle but then die as pharate pupae without shedding the sixth‐stage larval cuticle.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, <Emphasis Type="Italic">Papilio xuthus</Emphasis>

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.     

Structure and expression of mRNA for a pupal cuticle protein of the silkworm,Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.     

A Cuticle Protein Gene from the Japanese Oak Silkmoth, <Emphasis Type="Italic">Antheraea yamamai</Emphasis>: Gene Structure and mRNA Expression

A cuticle protein gene, AyCP12, from the Japanese oak silkmoth, Antheraea yamamai, was isolated and characterized. The gene spans 1107 bp and consists of one intron and two exons coding for a 112 amino acid polypeptide with a predicted molecular mass of 12,163 Da and a pI of 4.4. The AyCP12 protein contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the AyCP12 cDNA is most homologous to another silkmoth, A. pernyi, cuticle protein ApCP13 (82% protein sequence identity). Northern blot analysis revealed that AyCP12 showed the epidermis-specific expression.     

Temporal pattern of changes in mitotic frequency in the epidermis and other larval tissues of Bombyx mori

Temporal changes in mitotic frequency were examined in various tissues through late larval life of Bombyx mori. From the second larval ecdysis to the third and from the third larval ecdysis to the fourth, there was a definite temporal change of mitotic pattern in each tissue. In the epidermis as well as in the tracheal epithelium, mitoses began to appear about 1 day after an ecdysis, and showed a maximum 1 to 2 days after an ecdysis. In the fat body, mitoses were observed continuously through the instars, and the mitotic frequency showed a maximum state just before an ecdysis. In the abdominal muscle the frequency was highest at about the middle of the period between two successive ecdyses. Furthermore, epidermal mitoses coincided with the time when the density of epidermal nuclei per unit area decreased to a half. This suggests that epidermal mitoses may be initiated by some process related to the increase in cell size.     

Cuticular sclerotization in larval and adult locusts, Schistocerca gregaria

The sclerotization of both larval and adult cuticle from the desert locust, Schistocerca gregaria, has been studied by measuring the incorporation of radioactive dopamine and N-acetyldopamine into the cuticle. The results are compared with the degree of sclerotization of the cuticle and the amount of sclerotizing enzyme present. The various parts of the cuticle differ considerably with respect to the degree of sclerotization: in adult locusts the mandibles and the dorsal mesothoracic cuticle contain about twenty times as much cross-linking material per mg cuticle than is present in the abdominal tergites and sclerites.The degree of sclerotization in the various types of cuticle is apparently not determined by the amounts of sclerotizing enzyme present, and the rate at which radioactive dopamine or N-acetyldopamine is incorporated into the cuticle appears also to be unrelated to the amount of enzyme.The degree of sclerotization of the various parts of the cuticle from fifth instar larvae corresponds with the amounts of labelled dopamine which are incorporated during the first day after ecdysis, whereas there is no correlation between sclerotization and the amounts of labelled dopamine which are incorporated in older larvae. The degree of sclerotization of adult cuticle after 1 day corresponds to the incorporation of dopamine during the first day. When older animals are compared only little correlation is observed. The relative rates of sclerotization in the various parts of the cuticle must therefore change as the adult insect grows older.The changes in the incorporation pattern during the development of the locust are discussed in relation to the physiological control of the sclerotization process.     

Cloning and expression of glucose regulated protein 78 (GRP78) in<Emphasis Type="Italic"> Fenneropenaeus chinensis</Emphasis>

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35°C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25°C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.     

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.     

Ultrastructural differences between larval and pupal cuticles ofPieris brassicae (lepidoptera)

Summary The study of larval and pupal cuticles ofPieris brassicae has revealed some differences in ultrastructural architecture. The lamellated cuticle of larvae is traversed by processes of epidermal cells connected with fibrils possibly acting as supports for the arched cuticle but never developing into a space system. In contrast pupal cuticles are traversed by a space system containing bundles of fibrils which are involved in the transport of lipid containing material.Ultrastructural changes occuring in the pupal cuticle few days after ecdysis are described.These results are discussed in relation to the present knowledge of cuticle structure.     

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster

Summary The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein from the Chinese oak Silkmoth, Antheraea pernyi.

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.     

Developmental attenuation of the pre-ecdysis motor pattern in the tobacco hornworm,Manduca sexta

Summary At the culmination of each molt, the larval tobacco hornworm exhibits a pre-ecdysis behavior prior to shedding its old cuticle at ecdysis. Both pre-ecdysis and ecdysis behaviors are triggered by the peptide, eclosion hormone (EH). Pre-ecdysis behavior consists of rhythmic abdominal compressions that loosen the old larval cuticle. This behavior is robust at larval molts, but at the larval-pupal molt the only comparable behavior consists of rhythmic dorso-ventral flexions of the anterior body. These flexions appear to be an attenuated version of the larval pre-ecdysis behavior because (1) they show the same EH dependence, and (2) the motor patterns recorded from EH treated, deafferented larval and pupal preparations are similar except that the pupal pattern is much weaker. Both patterns are characterized by rhythmic, synaptically-driven bursts of action potentials in motoneurons MN-2 and MN-3, which occur synchronously in all segments. However, the synaptic drive to the motoneurons and their resultant levels of activity are reduced during the pupal pre-ecdysis motor pattern, especially in posterior abdominal segments. Although the dendritic arbors of both motoneurons regress somewhat during the larval-pupal transformation, this does not appear to be the primary source of diminished synaptic drive because regression is greatest in the segments in which synaptic inputs remain the strongest. The developmental weakening of the pre-ecdysis motor pattern thus may be due to changes at the interneuronal level.Abbreviations A2, A3... abdominal segments 2, 3, etc. - ALE anterior lateral external muscle - day L3 third day of the 5th larval instar - day P0 the day of pupal ecdysis - DN _a anterior branch of the dorsal nerve - EH eclosion hormone - HPLC high performance liquid chromatography - TP tergopleural muscle     

Cloning, characterization and tissue specific expression of a sucrose synthase gene from tropical epiphytic CAM orchid Mokara Yellow

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic CAM orchid Mokara Yellow. The cDNA is 2748bp in length containing an open reading frame of 2447bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of M. Yellow sucrose synthase (Msus1) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli. Northern blot analysis showed that the expression pattern of Msus1 mRNA is tissue specific with highest levels in strong sinks such as expanding leaves and root tips, but not detectable in mature leaves and flowers. Incubation with sugars resulted in a significant increase in the steady-state Msus1 mRNA levels in shoots of seedlings.