[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.

The periplasmic expression and purification of AA15 lytic polysaccharide monooxygenases from insect species in Escherichia coli.

Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.

Foam fractionation of a recombinant biosurfactant apolipoprotein.

Locusta migratoria apolipophorin III (apoLp-III) possesses the ability to exist as a water soluble amphipathic α-helix bundle and a lipid surface seeking apolipoprotein. The intrinsic ability of apoLp-III to transform phospholipid vesicles into reconstituted discoidal high-density lipoproteins (rHDL) has led to myriad applications. To improve the yield of recombinant apoLp-III, studies were performed in a bioreactor. Induction of apoLp-III expression generated a protein product that is secreted from E. coli into the culture medium. Interaction of apoLp-III with gas and liquid components in media produced large quantities of thick foam. A continuous foam fractionation process yielded a foamate containing apoLp-III as the sole major protein component. The yield of recombinant apoLp-III was ~0.2 g / liter bacterial culture. Mass spectrometry analysis verified the identity of the target protein and indicated no modifications or changes to apoLp-III occurred as a result of foam fractionation. The functional ability of apoLp-III to induce rHDL formation was evaluated by incubating foam fractionated apoLp-III with phosphatidylcholine vesicles. FPLC size exclusion chromatography revealed a single major population of particles in the size range of rHDL. The results described offer a novel approach to bioreactor-based apoLp-III production that takes advantage of its intrinsic biosurfactant properties.

Genome-wide identification of lipases in silkworm (Bombyx mori) and their spatio-temporal expression in larval midgut.

Lipases play crucial roles in food digestion by degrading dietary lipids into free fatty acids and glycerols. The domesticated silkworm (Bombyx mori) has been widely used as an important Lepidopteran model for decades. However, little is known about the lipase gene family in the silkworm, especially their hydrolytic activities as digestive enzymes. In this study, a total of 38 lipase genes were identified in the silkworm genome. Phylogenetic analysis indicated that they were divided into three major groups. Twelve lipases were confirmed to be expressed in the midgut at both transcriptional and translational levels. They were grouped into the same gene cluster, suggesting that they could have similar physiological functions. Quantitative real-time PCR (qRT-PCR) analyses indicated that lipases were mainly expressed in anterior and middle midgut regions, and their expression levels varied greatly along the length of midgut. A majority of lipases were down-regulated in the midgut when larvae stopped feeding. However, a unique lipase gene (Bmlip10583) showed low expression level during feeding stage, but it was significantly up-regulated during the larvae-pupae transition. These results demonstrated that expression of silkworm lipases was spatially and temporally regulated in the midgut during larval development. Taken together, our results provide a fundamental research of the lipase gene family in the silkworm.

Characterization of E93 in neometabolous thrips Frankliniella occidentalis and Haplothrips brevitubus.

Insect metamorphosis into an adult occurs after the juvenile hormone (JH) titer decreases at the end of the juvenile stage. This generally coincides with decreased transcript levels of JH-response transcription factors Krüppel homolog 1 (Kr-h1) and broad (br), and increased transcript levels of the adult specifier E93. Thrips (Thysanoptera) develop through inactive and non-feeding stages referred to as "propupa" and "pupa", and this type of distinctive metamorphosis is called neometaboly. To understand the mechanisms of hormonal regulation in thrips metamorphosis, we previously analyzed the transcript levels of Kr-h1 and br in two thrips species, Frankliniella occidentalis (Thripidae) and Haplothrips brevitubus (Phlaeothripidae). In both species, the transcript levels of Kr-h1 and br decreased in the "propupal" and "pupal" stages, and their transcription was upregulated by exogenous JH mimic treatment. Here we analyzed the developmental profiles of E93 in these two thrips species. Quantitative RT-PCR revealed that E93 expression started to increase at the end of the larval stage in F. occidentalis and in the "propupal" stage of H. brevitubus, as Kr-h1 and br mRNA levels decreased. Treatment with an exogenous JH mimic at the onset of metamorphosis prevented pupal-adult transition and caused repression of E93. These results indicated that E93 is involved in adult differentiation after JH titer decreases at the end of the larval stage of thrips. By comparing the expression profiles of Kr-h1, br, and E93 among insect species, we propose that the "propupal" and "pupal" stages of thrips have some similarities with the holometabolous prepupal and pupal stages, respectively.

Comparative analyses of transcriptional responses of Dectes texanus LeConte (Coleoptera: Cerambycidae) larvae fed on three different host plants and artificial diet.

Dectes texanus is an important coleopteran pest of soybeans and cultivated sunflowers in the Midwestern United States that causes yield losses by girdling stems of their host plants. Although sunflower and giant ragweed are primary hosts of D. texanus, they began colonizing soybeans approximately 50 years ago and no reliable management method has been established to prevent or reduce losses by this pest. To identify genes putatively involved when feeding soybean, we compared gene expression of D. texanus third-instar larvae fed soybean to those fed sunflower, giant ragweed, or artificial diet. Dectes texanus larvae differentially expressed 514 unigenes when fed on soybean compared to those fed the other diet treatments. Enrichment analyses of gene ontology terms from up-regulated unigenes in soybean-fed larvae compared to those fed both primary hosts highlighted unigenes involved in oxidoreductase and polygalacturonase activities. Cytochrome P450s, carboxylesterases, major facilitator superfamily transporters, lipocalins, apolipoproteins, glycoside hydrolases 1 and 28, and lytic monooxygenases were among the most commonly up-regulated unigenes in soybean-fed larvae compared to those fed their primary hosts. These results suggest that D. texanus larvae differentially expressed unigenes involved in biotransformation of allelochemicals, digestion of plant cell walls and transport of small solutes and lipids when feeding in soybean.

Decoding the Reproductive System of the Olive Fruit Fly, Bactrocera oleae.

In most diploid organisms, mating is a prerequisite for reproduction and, thus, critical to the maintenance of their population and the perpetuation of the species. Besides the importance of understanding the fundamentals of reproduction, targeting the reproductive success of a pest insect is also a promising method for its control, as a possible manipulation of the reproductive system could affect its destructive activity. Here, we used an integrated approach for the elucidation of the reproductive system and mating procedures of the olive fruit fly, Bactrocera oleae. Initially, we performed a RNAseq analysis in reproductive tissues of virgin and mated insects. A comparison of the transcriptomes resulted in the identification of genes that are differentially expressed after mating. Functional annotation of the genes showed an alteration in the metabolic, catalytic, and cellular processes after mating. Moreover, a functional analysis through RNAi silencing of two differentially expressed genes, yellow-g and troponin C, resulted in a significantly reduced oviposition rate. This study provided a foundation for future investigations into the olive fruit fly's reproductive biology to the development of new exploitable tools for its control.

Sage controls silk gland development by regulating Dfd in Bombyx mori.

Silk gland is an organ that produces and secretes silk proteins. The development of the silk gland is essential for high silk production yield and silk quality. Although Sage reportedly plays a pivotal role in embryonic silk gland development, the mechanism underlying its action remains unclear. Our study aimed to determine the genes downstream of Sage through which it regulates the development of the silk gland. After chromatin immunoprecipitation and sequencing, Dfd was identified as a downstream target gene of Sage and it was confirmed that Sage could inhibit Dfd expression by competing with SGF1. When Dfd was knocked down through RNA interference (RNAi), the number of cells in the middle silk gland decreased, and the posterior silk gland was straightened. Simultaneously, the expression of Ser1 and silk fibroin genes was no longer strictly regional. These changes eventually led to an alteration in the composition of the Dfd RNAi cocoon. In conclusion, our research contributes to a deeper understanding of the development of silk glands.

Consequences of 'no-choice, fixed time' reciprocal host plant switches on nutrition and gut serine protease gene expression in Pieris brassicae L. (Lepidoptera: Pieridae).

Rapid adaptive responses were evident from reciprocal host-plant switches on performance, digestive physiology and relative gene expression of gut serine proteases in larvae of crucifer pest P. brassicae transferred from cauliflower (CF, Brassica oleracea var. botrytis, family Brassicaceae) to an alternate host, garden nasturtium, (GN, Tropaeolum majus L., family Tropaeolaceae) and vice-versa under laboratory conditions. Estimation of nutritional indices indicated that larvae of all instars tested consumed the least food and gained less weight on CF-GN diet (significant at p≤0.05) as compared to larvae feeding on CF-CF, GN-GN and GN-CF diets suggesting that the switch to GN was nutritionally less favorable for larval growth. Nevertheless, these larvae, especially fourth instars, were adroit in utilizing and digesting GN as a new host plant type. In vitro protease assays conducted to understand associated physiological responses within twelve hours indicated that levels and properties of gut proteases were significantly influenced by type of natal host-plant consumed, change in diet as well as larval age. Activities of gut trypsins and chymotrypsins in larvae feeding on CF-GN and GN-CF diets were distinct, and represented shifts toward profiles observed in larvae feeding continuously on GN-GN and CF-CF diets respectively. Results with diagnostic protease inhibitors like TLCK, STI and SBBI in these assays and gelatinolytic zymograms indicated complex and contrasting trends in gut serine protease activities in different instars from CF-GN diet versus GN-CF diet, likely due to ingestion of plant protease inhibitors present in the new diet. Cloning and sequencing of serine protease gene fragments expressed in gut tissues of fourth instar P. brassicae revealed diverse transcripts encoding putative trypsins and chymotrypsins belonging to at least ten lineages. Sequences of members of each lineage closely resembled lepidopteran serine protease orthologs including uncharacterized transcripts from Pieris rapae. Differential regulation of serine protease genes (Pbr1-Pbr5) was observed in larval guts of P. brassicae from CF-CF and GN-GN diets while expression of transcripts encoding two putative trypsins (Pbr3 and Pbr5) were significantly different in larvae from CF-GN and GN-CF diets. These results suggested that some gut serine proteases that were differentially expressed in larvae feeding on different species of host plants were also involved in rapid adaptations to dietary switches. A gene encoding nitrile-specifier protein (nsp) likely involved in detoxification of toxic products from interactions of ingested host plant glucosinolates with myrosinases was expressed to similar levels in these larvae. Taken together, these snapshots reflected contrasts in physiological and developmental plasticity of P. brassicae larvae to nutritional challenges from wide dietary switches in the short term and the prominent role of gut serine proteases in rapid dietary adaptations. This study may be useful in designing novel management strategies targeting candidate gut serine proteases of P. brassicae using RNA interference, gene editing or crops with transgenes encoding protease inhibitors from taxonomically-distant host plants.

Coordinative mediation of the response to alarm pheromones by three odorant binding proteins in the green peach aphid Myzus persicae.

Odorant binding proteins (OBPs) play an essential role for insect chemosensation in insect peripheral nervous systems of antennae. Each antennal sensilla contains more than one OBP at high concentrations but the interactions and cooperation between co-localized OBPs are rarely reported. In present study, we cloned, expressed and purified eight OBPs of the green peach aphid Myzus persicae. The effects of knocking down the expression of these OBP genes by RNAi on the electrophysiological and behavioural responses of M. persicae to the aphid alarm pheromone, (E)-ß-farnesene (EßF) were investigated. The results showed that the aphids could still be repelled by EßF when the expression of each of three OBP genes was individually knocked down. However, the simultaneous knockdown of MperOBP3/7/9 expression significantly reduced the electrophysiological response and the repellent behaviours of M. persicae to EßF than the single OBP gene knockdown (P < 0.05). Rather than a normal saturation binding curve of individual OBP, the binding curve of MperOBP3/7/9 is bell-shaped with a higher affinity for the fluorescent probe N-phenyl-1-naphthylamine (1-NPN). The competitive binding assays confirmed that MperOBP3, MperOBP7, MperOBP9 and MperOBP3/7/9 mixture exhibited a stronger binding affinity for EßF, than for sex pheromones and plant volatiles with a dissociation constant of 2.5 µM, 1.1 µM, 3.9 µM and 1.0 µM, respectively. The competitive binding curve of MperOBP3/7/9 mixture to EßF is shallow without bottom plateau, suggesting a conformational change and a rapid dissociation after the displacement of all 1-NPN (in vivo after the saturation binding of all OBPs by EßF). The interaction between OBPs and formation of a heterogeneous unit may facilitate the delivery EßF to the OR at electrophysiological and behavioural levels during insect odorant signal transduction thus mediate M. persicae response to the alarm pheromone EßF.

Gut bacteria are essential for normal cuticle development in herbivorous turtle ants.

Across the evolutionary history of insects, the shift from nitrogen-rich carnivore/omnivore diets to nitrogen-poor herbivorous diets was made possible through symbiosis with microbes. The herbivorous turtle ants Cephalotes possess a conserved gut microbiome which enriches the nutrient composition by recycling nitrogen-rich metabolic waste to increase the production of amino acids. This enrichment is assumed to benefit the host, but we do not know to what extent. To gain insights into nitrogen assimilation in the ant cuticle we use gut bacterial manipulation, 15N isotopic enrichment, isotope-ratio mass spectrometry, and 15N nuclear magnetic resonance spectroscopy to demonstrate that gut bacteria contribute to the formation of proteins, catecholamine cross-linkers, and chitin in the cuticle. This study identifies the cuticular components which are nitrogen-enriched by gut bacteria, highlighting the role of symbionts in insect evolution, and provides a framework for understanding the nitrogen flow from nutrients through bacteria into the insect cuticle.

Molecular characterization and expression of two enzymes from Dendroctonus armandi, with phloem feeding and juvenile hormone.

The Chinese white pine beetle, Dendroctonus armandi Tsai and Li, is a serious native pest in the Qinling Mountains of China. exo-Bevicomin, as the main component of bark beetle pheromone, is released by the female D. armandi. In this paper, we identified two genes encoding, (Z)-6-nonen-2-ol dehydrogenase and CYP6CR, that are known to be involved in xo-brevicomin synthesis to improve the understanding of exo-brevicomin biosynthesis in the Chinese white pine beetle. The two protiens had high homology with their orthologs in the exo-brevicomin biosynthesis pathway from D. ponderosae. The expression profiles of CYP6CR12 and DaZnoDH in D. armandi females are closely correlated with exo-brevicomin biosynthesis. The expression levels of CYP6CR12 and DaZnoDH are also regulated by feeding behavior and juvenile hormone levels. Since they are also expressed in males, CYP6CR12 and DaZnoDH are not only important for exo-brevicomin biosynthesis that this might be potential role for the semichemical biosysthesis pathways.

Functional analysis of a novel orthologous small heat shock protein (shsp) hsp21.8a and seven species-specific shsps in Tribolium castaneum.

Small heat shock proteins (sHSPs) are important modulators of insect survival. Previous research revealed that there is only one orthologous cluster of shsps in insects. Here, we identified another novel orthologous cluster of shsps in insects by comparative analysis. Multiple stress experiments and function investigation of Tchsp21.8a belonging to this orthologous cluster and seven species-specific shsps were performed in the stored-grain pest Tribolium castaneum. The results indicated that expression of Tchsp21.8a showed weak responses to different stresses. However, expressions of most species-specific shsps exhibited hyper-responses to heat stress, and expressions of all species-specific shsps displayed diverse responses during other stresses to protect beetles in a cooperative manner. Additionally, Tchsp21.8a and species-specific Tcshsp19.7 played important roles in the development of T. castaneum, and all Tcshsps had a certain impact on the fecundity. Our work created a comprehensive reliable scaffold of insect shsps that can further provide instructive insights to pest bio-control.

Molecular characterization of Antheraea mylitta arylphorin gene and its encoded protein.

Antheraea mylitta arylphorin protein was extracted from the silk gland of fifth instar larvae and purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The N-terminal sequencing of ten amino acids (NH2-SVVHPPHHEV-COOH) showed similarity with Antheraea pernyi arylphorin. Based on N-terminal and C-terminal A. pernyi arylphorin sequences, primers were designed, and A. mylitta arylphorin cDNA was cloned by RT-PCR from silk gland mRNA. Sequencing of complete cDNA including 25 nucleotides at 5' UTR (obtained by 5' RACE) showed that it consisted of an ORF of 2115 nucleotides which could encode a protein of 704 amino acids (predominantly aromatic residues) having molecular weight 83 kDa. Homology modelling was done using A. pernyi arylphorin as a template. Cloned arylphorin cDNA was expressed in E. coli and recombinant His-tagged protein was purified by Ni-NTA affinity chromatography. Analysis of tissue-specific expression of arylphorin by real-time PCR showed maximum expression in the fat body followed by silk gland and integument. 5' flanking region (759 bp) of arylphorin gene was amplified by inverse PCR and the full length gene (5359 nucleotides) containing five exons and four introns was cloned from the A. mylitta genomic DNA and sequenced. Polyclonal antibody was raised against purified arylphorin and more native arylphorin protein (500 kDa) was purified from the fat body by antibody affinity chromatography. Study of mitogenic effect of native and chymotrypsin hydrolysate of arylphorin on different insect cell lines showed that arylphorin could be used as serum substitute for in vitro cultivation of insect cells.

Identification of catalytically distinct arylalkylamine N-acetyltransferase splicoforms from Tribolium castaneum.

The assumption that structural or sequential homology between enzymes implies functional homology is a common misconception. Through in-depth structural and kinetic analysis, we are now beginning to understand the minute differences in primary structure that can alter the function of an enzyme completely. Alternative splicing is one method for which the activity of an enzyme can be controlled, simply by altering its length. Arylalkylamine N-acetyltransferase A (AANATA) in D. melanogaster, which catalyzes the N-acetylation of biogenic amines, has multiple splicoforms - alternatively spliced enzyme isoforms - with differing tissue distribution. As demonstrated here, AANAT1 from Tribolium castaneum is another such enzyme with multiple splicoforms. A screening assay was developed and utilized to determine that, despite only a 35 amino acid truncation, the shortened form of TcAANAT1 is a more active form of the enzyme. This implies regulation of enzyme metabolic activity via alternative splicing.

Immunological Function of the Antibacterial Peptide Attacin-Like in the Chinese Oak Silkworm, Antheraea pernyi.

BACKGROUND: Antibacterial peptides play important roles in the innate immune system of insects and are divided into four categories according to their structures. Although many antibacterial peptides have been reported in lepidopteran insects, the roles of an attacin-like gene in immune response of Antheraea pernyi remain unclear. OBJECTIVE: In this study, the cloning and immunological functions of an attacin-like gene from Antheraea pernyi were investigated. METHODS: The open reading frame of Ap-attacin-like gene was cloned by PCR using the specific primers and then was ligated to the pET-32a vector to construct the recombinant plasmids Ap-attacin- like-pET-32a. The recombinant Ap-attacin-like protein was expressed in E. coli (BL21 DE3) cells and purified by Ni-NTA affinity chromatography. The expression patterns of Ap-attacin-like in different tissues or under microorganism challenges were investigated by real-time PCR and western blotting. Finally, agar well diffusion assay was performed to determine the antimicrobial activity of the recombinant Ap-attacin-like proteins based on the inhibition rate. RESULTS: The expression level of Ap-attacin-like was highest in the fat body compared with the other examined tissues. The expression of Ap-attacin-like in the fat body was significantly elevated after E. coli, Beauveria bassiana, Micrococcus luteus or Nuclear Polyhedrosis Virus challenges. In addition, the recombinant Ap-attacin-like proteins had obvious antibacterial activity against E. coli. CONCLUSION: Ap-attacin-like was highly expressed in immune-related tissues and its expression level was significantly induced by different microorganism challenges, suggesting that Ap-attacin-like participated in the innate immunity of A. pernyi.

Effects of a Reserve Protein on Spodoptera frugiperda Development: A Biochemical and Molecular Approach to the Entomotoxic Mechanism.

Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda, considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda. The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes.

Functional expression of recombinant hybrid enzymes composed of bacterial and insect's chitinase domains in E. coli.

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

Molecular and Potential Regulatory Mechanisms of Melanin Synthesis in Harmonia axyridis.

Melanization is a common phenomenon in insects, and melanin synthesis is a conserved physiological process that occurs in epidermal cells. Moreover, a comprehensive understanding of the mechanisms of melanin synthesis influencing insect pigmentation are well-suited for investigating phenotype variation. The Asian multi-colored (Harlequin) ladybird beetle, Harmonia axyridis, exhibits intraspecific polymorphism based on relative levels of melanization. However, the specific characteristics of melanin synthesis in H. axyridis remains elusive. In this study, we performed gene-silencing analysis of the pivotal inverting enzyme, tyrosine hydroxylase (TH), and DOPA decarboxylase (DDC) in the tyrosine metabolism pathway to investigate the molecular and regulatory mechanism of melanin synthesis in H. axyridis. Using RNAi of TH and DDC genes in fourth instar larvae, we demonstrated that dopamine melanin was the primary contributor to the overall body melanization of H. axyridis. Furthermore, our study provides the first conclusive evidence that dopamine serves as a melanin precursor for synthesis in the early pupal stage. According to transcription factor Pannier, which is essential for the formation of melanic color on the elytra in H. axyridis, we further demonstrated that suppression of HaPnr can significantly decrease expression levels of HaTH and HaDDC. These results in their entirety lead to the conclusion that transcription factor Pannier can regulate dopamine melanin synthesis in the dorsal elytral epidermis of H. axyridis.