Hemolymph Of Fifth Instar Larvae Research Articles

Involvement of Sep38β in the Insecticidal Activity of Bacillus thuringiensis against Beet Armyworm, Spodoptera exigua (Lepidoptera).

The p38 mitogen-activated protein kinases (MAPKs) are associated with insect immunity, tissue repair, and the insecticidal activity of Bacillus thuringiensis (Bt). Here, a p38 MAPK family gene (Sep38β) was identified from Spodoptera exigua. Among the developmental stages, the transcription level of Sep38β was the highest in egg, followed by that in prepupa and pupa. Sep38β expression peaked in Malpighian tubules and the hemolymph of fifth instar larvae. Knockdown of Sep38β or injection of SB203580 (a p38 MAPK inhibitor) significantly downregulated the SeDUOX expression and reactive oxygen species (ROS) level in the midgut, accounting for deterioration of the midgut to scavenge pathogens and enhancement of Bt insecticidal activity. In conclusion, all the results demonstrate that Sep38β regulates the immune-related ROS level in the insect midgut, which suppresses the insecticidal activity of Bt against S. exigua by 17-22%. Our study highlights that Sep38β is essential for insect immunity and the insecticidal activity of Bt to S. exigua and is a potential target for pest control.

Circadian clock regulates developmental time through ecdysone and juvenile hormones in Bombyx mori.

The circadian clock plays an integral role in hormone biosynthesis and secretion. However, how the circadian clock precisely coordinates hormonal homeostasis to maintain normal animal development remains unclear. Here, we show that knocking out the core clock gene Cryptochrome 1 (Cry1) significantly delays the developmental time in Bombyx mori. This study focuses on the ecdysone and juvenile hormone signalling pathways of fifth instar larvae with the longest developmental time delay. We found that the mutant reduced prothoracicotropic hormone synthesis in the brain, and could not produce sufficient ecdysone in the prothoracic gland, resulting in a delayed peak of 20-hydroxyecdysone titre in the hemolymph of fifth instar larvae, prolonging developmental time. Moreover, further investigation revealed that the mutant enhanced juvenile hormone biosynthesis and signalling pathway and that this higher juvenile hormone titre also resulted in prolonged developmental time in fifth instar larvae. Our results provide insights into the molecular mechanisms by which the circadian clock regulates animal development by maintaining hormonal homeostasis.

Trehalose transporter-like gene diversity and dynamics enhances stress response and recovery in Helicoverpa armigera

Trehalose is a primary sugar and its distribution across the insect body, regulated by trehalose transporters (TRETs), is essential for sugar metabolism and energy homeostasis. The large diversity of Tret-like sugar transporters (ST), belonging to SLC2A transporter family, in polyphagous insects probably contributes to their extremely adaptive nature. We aim to study spatio-temporal expression dynamics and functional relevance of ST transcript variants in the lepidopteran model organism, Helicoverpa armigera. Identification of 69 putative Tret-like HaST transcript variants from databases and their digital gene expression analysis indicated tissue and development-specific expression patterns. Phylogenetic and sequence similarity network analysis of HaSTs signify evolutionary divergence, while motif and structure analysis depicted conserved signatures. In vitro gene expression validation for selected genes depicts that HaST09 and 69 are fat body and haemolymph-specific. While, HaST06, 30, 36 and 57 are developmental stage or sex-specific. HaST69 has high expression in the haemolymph of fifth instar larvae. In the presence of trehalose metabolism inhibitors and abiotic stress, HaSTs expression show dysregulation, indicating their possible association with trehalose metabolism and stress recovery. In vivo gene silencing of HaST69 resulted in reduced trehalose accumulation in the insect body, suggesting its plausible role in sugar metabolism. The overall understanding of HaST diversity and expression dynamics highlights their putative roles in sugar transport during adaptation and stress recovery of insects.

The Key Role of Fatty Acid Synthase in Lipid Metabolism and Metamorphic Development in a Destructive Insect Pest, Spodoptera litura (Lepidoptera: Noctuidae).

Fatty acid synthase (FAS) is a key enzyme in the lipid synthesis pathway, however, its roles in insects remain largely unknown. Here, we firstly identified two FAS genes from the transcriptome dataset of the general cutworm Spodoptera litura, which is a destructive insect pest of many crops. Both SlFAS1 and SlFAS2 were highly expressed in third instar larvae and in their fat bodies. Then, we successfully silenced SlFAS1 in third instar larvae and the content of α-linolenic acid and triglyceride was significantly decreased. Besides that, the effect of FAS on the metamorphic development in S. litura was evaluated. The results indicate that after silencing SlFAS1, the survival rates of S. litura larvae decreased significantly compared to the control groups. Silencing SlFAS1 in fifth instar larvae resulted in more malformed pupae and adults, and the emergence rates were significantly reduced. Furthermore, the ecdysone content in the haemolymph of fifth instar larvae was significantly decreased after silencing SlFAS1. In addition, knocking down SlFAS1 significantly alters the expression of other key genes in the lipogenesis pathway, implying that FAS has an impact on the lipogenesis pathway. The present study deepens the understanding of FAS in insects and provides novel potential targets for managing insect pests.

Preliminary Studies on the Humoral Immune Response of Mulberry Silkworm, Bombyx mori Larvae Fed on Different Mulberry Varieties

Diseases infecting silkworms cause 30-40% cocoon crop loss. The silkworm, Bombyx mori L. defense against pathogens is provided by open circulatory system through humoral and cellular response. Three mulberry varieties, Morus alba var. Rosa, M. alba var. Morittiana and M. alba var. Laevigata, were used to study the effect of host plant and bacterial contagious infection on the humoral immune of B. mori larvae. The specific activity of related enzymes in haemolymph of fifth instar larvae was estimated. Results indicated that mulberry varieties and bacterial contagious infection had significant effects on enzymes activity values in larval haemolymph. Moreover, M. alba var. laevigata was superior in the humoral immune response.

Differential Count of Haemocyte from the Haemolymph of Muga Silkworm (Antheraea assamensis H.) reared on the host plant

Five types of circulating haemocytes have been identified from the haemolymph of 5th instar larvae of the Muga Silkworm (Antheraea assamensis) reared on Som leaves (Machilus bombycina) at Govt. Basic Muga Seed Farm, Khanapara, Assam, India. Haemocytes were identified by means of light microscopy as prohaemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Differential count of haemocyte from the haemolymph of fifth-instar larvae of Muga Silkworm were also studied and calculated. Among them, prohaemocytes were found to be the most abundant cell types in the haemolymph, whereas spherulocytes the rarest.

Silkworm storage protein Bm30K-19G1 has a certain antifungal effects on Beauveria bassiana.

Storage proteins in the 30 K family are ubiquitous in the hemolymph of insects and play important roles in adult metamorphosis, development, egg formation, carrier transport and even host immunity. Some studies have shown that the 30 K proteins can inhibit apoptosis and have certain antifungal effects. The silkworm protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in hemolymph of fifth instar larvae. Our previous transcriptome sequencing, real-time PCR analysis and proteomic studies showed that the expression level of the 30 K protein gene was significantly up-regulated in the silkworm infected with Beauveria bassiana. In this study, the ORF sequence of Bm30K-19G1 was amplified by PCR. The sequence is 1311 bp in length and encodes a 436 amino acid peptide. Bm30K-19G1 was expressed in all tested tissues of fifth instar larvae, with highest expression in fat body and the lowest expression in the midgut. Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+) as vector and E. coli Arctic Express (DE3) as the expression bacterium strain. The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B. bassiana. Bm30K-19G1 also inhibited the growth and reproduction of B. bassiana in vivo; the median lethal time of infected silkworms was postponed by 6.4 h and the time for death of all infected larvae was postponed by 10 h. The results indicated that the silkworm storage protein 30K-19G1 is an antifungal protein against B. bassiana and help to elucidate the molecular mechanism of silkworm resistance against B. bassiana.

Expression and Functional Analysis of Storage Protein 2 in the Silkworm,Bombyx mori

Storage protein 2 (SP2) not only is an important source of energy for the growth and development of silkworm but also has inhibitory effects on cell apoptosis. Endothelial cell (EC) apoptosis is an important contributing factor in the development of atherosclerosis; therefore, study of the antiapoptotic activity of SP2 on ECs provides information related to the treatment of atherosclerosis and other cardiovascular diseases. In this study, the sp2 gene was cloned and expressed in Escherichia coli to produce a 6xHis-tagged fusion protein, which was then used to generate a polyclonal antibody. Western blot results revealed that SP2 levels were higher in the pupal stage and hemolymph of fifth-instar larvae but low in the egg and adult stages. Subcellular localization results showed that SP2 is located mainly on the cell membrane. In addition, a Bac-to-Bac system was used to construct a recombinant baculovirus for SP2 expression. The purified SP2 was then added to a culture medium for human umbilical vein ECs (HUVECs), which were exposed to staurosporine. A cell viability assay demonstrated that SP2 could significantly enhance the viability of HUVEC. Furthermore, both ELISA and flow cytometry results indicated that SP2 has anti-apoptotic effects on staurosporine-induced HUVEC apoptosis.

Neuropeptide F and the corn earworm, Helicoverpa zea: A midgut peptide revisited

The neuropeptide Y family of peptides is implicated in the regulation of feeding across a broad range of animals, including insects. Among vertebrates, neuropeptide Y exerts its actions mainly centrally, whereas peptide YY and pancreatic polypeptide arise from digestive tissues. Among invertebrates, neuropeptide F (NPF) is the sole counterpart of the NPY family. Shared features of NPF sequences derived for Lepidoptera indicate that the midgut peptide ( Hez-MP-I) of the corn earworm, Helicoverpa zea, characterized more than a decade ago, is a carboxyl fragment of a full-length NPF. An antibody to Hez-MP-I was used to characterize the peptide's distribution in tissues of larvae, pupae, and adults. Immunostaining demonstrated NPF-related material both in nervous tissues and in abundant endocrine cells of the midgut. Radioimmunoassay of Hez-MP-I in the head, midgut and hemolymph of fifth instar larvae revealed concentration changes corresponding to development and feeding state. As with the vertebrate homologs, NPF may arise both centrally and peripherally to modulate the physiology of feeding and digestion of Lepidoptera.

Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraeamylitta

An inhibitor of Aspergillus oryzae fungal protease was purified to homogeneity from the hemolymph of fifth instar larvae of Antheraea mylitta by ammonium sulfate precipitation, anion exchange and gel filtration (FPLC) chromatography, and termed as AmFPI-1. The extent of purification was checked by two-dimensional gel electrophoresis, and the molecular weight of purified inhibitor was determined by SDS-PAGE as 10.4 kDa. Fifteen N-terminal amino acid sequences of this protein were determined, and degenerate oligonucleotides were synthesized on the basis of these sequences. A cDNA library of A. mylitta integument was constructed, and protease inhibitor cDNA was partially amplified by PCR using degenerate oligonucleotides and CDS primers. A full-length inhibitor cDNA clone obtained by screening the library with PCR amplified DNA as probe was sequenced. The cDNA consists of 543 nucleotides with an ORF of 315 bp and encodes a protein of 105 amino acids. The sequence exhibits similarity to several Bombyx mori ESTs, and in particular to N-terminal amino acid sequence of an inducible serine protease inhibitor (ISPI-1) from Galleria mellonella indicating its relatedness to ISPI-1 of G. mellonella. The presence of this protease inhibitor in the hemolymph may play an important role as a natural defense system against invading microorganisms.

Selective secretion of free glycine, a neutralizer against a plant defense chemical, in the digestive juice of the privet moth larvae

The larva of the privet moth, Brahmaea wallichii (Brahmaeidae) is a specialist feeder of the privet tree, Ligustrum obtusifolium (Oleaceae). A very high concentration (50 mM or 0.4%) of free glycine, found in the digestive juice of the larvae, works as a neutralizer against the very strong protein-denaturing activity of privet leaves that is caused by oleuropein, an iridoid that functions in chemical defense. Concentration of free glycine was high in the anterior region of the midgut lumen and low in the posterior region. To examine if some glycine-specific secretion mechanism exists, injection experiments were performed using 15N-labeled amino acids. When 13 μmol (1 mg) of 15N-glycine was injected into hemolymph of fifth instar larvae of B. wallichii, a high concentration of 15N (5 mM or 75 μg/g midgut content) was detected in the anterior parts of the midgut lumen 1 h after injection. 15N-NMR data indicated at least 60% of the 15N found in midgut lumen existed as 15N-glycine. Approximately, 25% of the injected 15N-glycine was estimated to have moved from the hemolymph to the midgut lumen. In contrast, no 15N was detected in the midgut lumen when 13 μmol of 15N-labeled alanine, lysine and glutamate were injected into hemolymph. Glycine was the only amino acid whose concentration was higher in the midgut lumen (50 mM) than in the hemolymph (22 mM). These data suggest the existence of some active and glycine-specific secretory mechanism in the midgut of B. wallichii.

A Monoclonal Antibody to the Shield Beetle Cassida rubiginosa (Coleoptera, Chrysomelidae): A Tool for Predator Gut Analysis

Abstract We describe the development of a monoclonal antibody (MAb) to the hemolymph of fifth-instar larvae of the shield beetle, Cassida rubiginosa Muell. (Coleoptera, Chrysomelidae), to be used as a tool for predator gut analysis. The MAb reacts with all life stages of C. rubiginosa, shows no cross-reactions outside the genus Cassida (31 herbivorous insect species and 12 potential predators of C. rubiginosa were tested), and is less reactive with other Cassida species than with C. rubiginosa. Specificity tests were conducted using an indirect enzyme-linked immunosorbent assay. Western blot analysis following SDS–PAGE showed that the MAb reacts with a single protein of molecular weight 32 kDa. The immunoblot of a native PAGE showed a reaction with three bands, the monomer of 32 kDa and two bands with approximate molecular weights of 60 and 90 kDa. Using this MAb, we were able to unambiguously identify individuals of the model predator Nabis mirmicoides Costa (Hemiptera, Nabidae) after feeding on one C. rubiginosa last-instar larva. The MAb may be useful for predator gut analysis to identify the predator complex of C. rubiginosa.

Purification and characterization of two insecticyanin-type proteins from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini

Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as determined by gel-filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Native BBP-II had a molecular weight of 48 kDa estimated by gel-filtration. SDS–PAGE revealed a single band with a molecular weight of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight/mass spectrometry), respectively. On this basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs collected from the hemolymph is attributed to the presence of biliverdin IX γ, which is non-covalently and stoichiometrically bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl (95:5, v/v). One molecule of BBP-I contains one molecule of biliverdin IX γ, whereas BBP-II contains two molecules of biliverdin IX γ. The amino acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity. These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.

Insecticyanin of Agrius convolvuli: purification and characterization of the biliverdin-binding protein from the larval hemolymph.

Blue biliprotein, insecticyanin (INS), has been purified from the hemolymph of the sweet potato hornworm, Agrius convolvuli. The protein was efficiently isolated from the hemolymph of fifth instar larvae using three successive column chromatographic techniques: hydrophobic interaction chromatography, ion exchange chromatography and gel-filtration. The purified INS showed a native molecular weight of approximately 59,000 by gel-filtration. SDS-PAGE revealed a single band with Mr of approximately 26,000. Moreover, the molecular mass of INS was 21,213 by MALDI-TOF/MS. Thus, the native A. convolvuli INS molecule was assumed to be a trimer in solution. The blue coloration of A. convolvuli INS from the hemolymph was attributed to the presence of biliverdin IX gamma, due to the absorbance maxima at 360 and 695 nm, which was non-covalently bound with the apoprotein. Amino acid composition and N-terminal sequence of A. convolvuli INS is similar to M. sexta INS. A. convolvuli INS represents one of the biliverdin-binding proteins in lepidopteran insects.

Isolation and characterization of four serine proteinase inhibitors (serpins) from hemolymph of Manduca sexta

Four serine proteinase inhibitors have been isolated from hemolymph of fifth instar larvae of Manduca sexta. One of these, an inhibitor specific for elastase, has been previously shown to be a member of the serpin family of serine proteinase inhibitors. Of the three remaining inhibitors, two are specific for chymotrypsin and one for trypsin. The four inhibitors have molecular weights of approx. 47,000 and isoelectric points between 4.4 and 4.8. The four proteins have very similar amino acid compositions, and NH 2-terminal sequence analysis suggests that they represent members of a gene family.

The characterization of ferritin in an insect

In mammals, the iron storage protein ferritin is predominantly synthesized on free polysomes and accumulates in the cytosol but some is secreted and circulates in the blood as serum ferritin. In insect tissues, on the other hand, iron-containing holoferritin accumulates in the vacuolar system and can be secreted through the Golgi complex. The midgut can secrete it to the gut lumen and other tissues to the hemolymph. Ferritin was isolated from the midgut and hemolymph of fifth instar larvae of Calpodes ethlius, Lepidoptera, Hesperiidae. This holoferritin is stable to heat (75°C) or in the presence of SDS, proteinase K, or urea, has an Mr above 600,000, contains iron and resembles mammalian ferritins in appearance by electron microscopy. Calpodes ferritin is a glycoprotein having N-linked high-mannose oligosaccharides. It is not antigenically related to horse ferritin but is related to that from Manduca sexta, Lepidoptera, Sphingidae. In its native form, Calpodes ferritin has only 3 isoforms with a pI 6.5–7 suggesting a more uniform subunit composition than that in vertebrates. It has two principle subunits, with relative Mrs of 24,000 (L) and 31,000 (G) and two minor subunits with Mrs of 26,000 and 28,000 all of which cross-react with antibody to Manduca ferritin. The 24 kDa subunit is the only one that is not glycosylated. Iron injections induce an increase in the proportion of the 24 kDa subunit. We conclude that Calpodes has ferritin and that it is glycosylated like mammalian serum ferritin.

Stimulated synthesis of the female‐specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

AbstractApplication of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female‐specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.