Parasitism and Suitability of Trichogramma chilonis on Large Eggs of Two Factitious Hosts: Samia cynthia ricini and Antheraea pernyi.

Trichogramma, an effective biological control agent, demonstrates promise in environmentally sustainable pest management through its parasitic action toward insect eggs. This study evaluates the parasitism fitness and ability of T. chilonis with regard to two factitious host eggs, aiming to develop a cost-effective biological control program. While T. chilonis demonstrated the ability to parasitize both host eggs, the results indicate a preference for ES eggs over COS eggs. The parasitism and emergence rates of T. chilonis regarding ES eggs (parasitism: 89.3%; emergence: 82.6%) surpassed those for COS eggs (parasitism: 74.7%; emergence: 68.8%), with a notable increase in the number of emergence holes observed in the ES eggs compared to the COS eggs. Moreover, the developmental time of T. chilonis for ES eggs (10.8 days) was shorter than that for COS eggs (12.5 days), resulting in a lower number of dead wasps produced. Notably, no significant difference was observed in the female ratios between the two species. A comprehensive analysis was conducted, comparing the size and shell thickness of the two factitious hosts. The ES eggs exhibited smaller dimensions (length: 1721.5 µm; width: 1178.9 µm) in comparison to the COS eggs (length: 2908.8 µm; width: 2574.4 µm), with the ES eggshells being thinner (33.8 µm) compared to the COS eggshells (47.3 µm). The different host species had an effect on the body length of the reared parasitoids, with T. chilonis reared on COS hosts exhibiting a larger body length (female: 626.9 µm; male: 556.7 µm) than those reared on ES hosts (female: 578.8 µm; male: 438.4 µm). Conclusively, the results indicate that ES eggs present a viable alternative to COS eggs for the mass production of Trichogramma species in biological control programs.

Effects of Pyriproxyfen Exposure on Reproduction and Gene Expressions in Silkworm, Bombyx mori.

The silkworm, Bombyx mori Linnaeus, is an important economic insect and a representative model organism of Lepidoptera, which has been widely used in the study of reproduction and development. The development of the silkworm's reproductive gland is easily affected by many external factors, such as chemical insecticides. After the silkworm larvae were treated with different concentrations of pyriproxyfen, the results showed that the number of eggs and hatching rate of eggs in the silkworm can be reduced by pyriproxyfen, and the concentration effects were displayed. Pyriproxyfen exposure could affect the normal development of the ovary tissue by reducing the number of oocytes and oogonia in the ovaries of silkworm fed with pyriproxyfen. We employed qRT-PCR, to detect the expressions of genes related to ovary development (Vg, Ovo, Otu, Sxl-S and Sxl-L) and hormone regulation (EcR and JHBP2) in silkworm. Our study showed that the transcription levels of Vg, Ovo, Otu, Sxl-S and Sxl-L in the treatment group were lower than those in the control group (6.08%, 61.99%, 83.51%, 99.31% and 71.95%, respectively). The transcription level of ECR was 70.22% for the control group, while that of JHBP2 was upregulated by 3.92-fold. Changes of transcription levels of these genes caused by pyriproxyfen exposure ultimately affect the absorption of nutrients, energy metabolism, ovary development and egg formation of the silkworm, thus leading to reproductive disorders of the silkworm. In general, our study revealed the response of silkworm reproduction to pyriproxyfen exposure and provided a certain reference value for the metabolism of the silkworm to pyriproxyfen.

Comparative Proteomic Analysis Reveals Immune Competence in Hemolymph of Bombyx mori Pupa Parasitized by Silkworm Maggot Exorista sorbillans.

The silkworm maggot, Exorista sorbillans, is a well-known larval endoparasitoid of the silkworm Bombyx mori that causes considerable damage to the silkworm cocoon crop. To gain insights into the response mechanism of the silkworm at the protein level, we applied a comparative proteomic approach to investigate proteomic differences in the hemolymph of the female silkworm pupae parasitized by E. sorbillans. In total, 50 differentially expressed proteins (DEPs) were successfully identified, of which 36 proteins were upregulated and 14 proteins were downregulated in response to parasitoid infection. These proteins are mainly involved in disease, energy metabolism, signaling pathways, and amino acid metabolism. Eight innate immune proteins were distinctly upregulated to resist maggot parasitism. Apoptosis-related proteins of cathepsin B and 14-3-3 zeta were significantly downregulated in E. sorbillans-parasitized silkworm pupae; their downregulation induces apoptosis. Quantitative PCR was used to further verify gene transcription of five DEPs, and the results are consistent at the transcriptional and proteomic levels. This was the first report on identification of possible proteins from the E. bombycis-parasitized silkworms at the late stage of parasitism, which contributes to furthering our understanding of the response mechanism of silkworms to parasitism and dipteran parasitoid biology.

Cloning and expression analysis of a peptidoglycan recognition protein in silkworm related to virus infection.

In this study, the full-length cDNA of a peptidoglycan recognition protein named BmPGRP-S3 was identified from the silkworm, Bombyx mori by rapid amplification of cDNA ends. It is 807 bp and comprises the following: a 5'-untranslated region (UTR) with a length of 112 bp, a 3'-UTR with a length of 92 bp including a poly-adenylation signal sequence (AATAAA) and a poly(A) tail. The longest open reading frame (ORF) of BmPGRP-S3 is 603 bp and encodes a polypeptide of 200 amino acids with a predicted molecular weight of 22.3 kDa including a PGRP domain. Sequence similarity and phylogenic analysis results indicated that BmPGRP-S3 belongs to the group of insect PGRPs and is closer to BmPGRP-S4 with the highest identity of 68%. Fluorescent quantitative real-time PCR results revealed that the mRNA transcripts of BmPGRP-S3 were presented in all of the tissues, but were highest in the midgut. In the silkworm larvae infected with B. mori cytoplasmic polyhedrosis virus (BmCPV), the relative expression level of BmPGRP-S3 was upregulated. The DNA segment of a mature BmPGRP-S3 peptide was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. Western blot results revealed that mature BmPGRP-S3 could be detected in the hemolymph and midgut which were the most important immune tissues in silkworm. All the results suggested that BmPGRP-S3 may play an important role in the immune response of silkworm to BmCPV infection and provided helpful information for further studying the function of BmPGRP-S3 in silkworm.

Cytoplasmic polyhedrosis virus-induced differential gene expression in two silkworm strains of different susceptibility.

Digital gene expression (DGE) was performed to investigate the gene expression profiles of 4008 and p50 silkworm strains at 48 h after oral infection with BmCPV. 3,668,437 clean tags were identified in the BmCPV-infected p50 silkworms and 3,540,790 clean tags in the control p50. By contrast, 4,498,263 clean tags were identified in the BmCPV-infected 4008 silkworms and 4,164,250 clean tags in the control 4008. A total of 691 differentially expressed genes were detected in the infected 4008 DGE library and 185 were detected in the infected p50 DGE library, respectively. The expression profiles identified some important differentially expressed genes involved in signal transduction, enzyme activity and apoptotic changes, some of which were verified using quantitative real-time PCR (qRT-PCR). These results provide important clues on the molecular mechanism of BmCPV invasion and resistance mechanism of silkworms against BmCPV infection.

Digital gene expression analysis in the midgut of 4008 silkworm strain infected with cytoplasmic polyhedrosis virus.

Digital Gene Expression was performed to investigate the midgut transcriptome profile of 4008 silkworm strain orally infected with BmCPV. A total of 4,498,263 and 4,258,240 clean tags were obtained from the control and BmCPV-infected larvae. A total of 752 differentially expressed genes were detected, of which 649 were upregulated and 103 were downregulated. Analysis results of the Kyoto Encyclopedia of Genes and Genomes pathway showed that 334 genes were involved in the ribosome and RNA transport pathways. Moreover, 408 of the 752 differentially expressed genes have a GO category and can be categorized into 41 functional groups according to molecular function, cellular component and biological process. Differentially expressed genes involved in signaling, gene expression, metabolic process, cell death, binding, and catalytic activity changes were detected in the expression profiles. Quantitative real-time PCR was performed to verify the expression of these genes. The upregulated expression levels of Calreticulin, FK506-binding protein, and protein kinase c inhibitor gene probably led to a calcium-dependent apoptosis in the BmCPV-infected cells. The results of this study may serve as a basis for future research not only on the molecular mechanism of BmCPV invasion but also on the anti-BmCPV mechanism of silkworm.

[Development of real-time fluorescent quantitative PCR method for detecting Bombyx mori cytoplasmic polyhedrosis virus].

A pair of specific primers were designed according to the published 118 bp conserved sequence of polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) in GenBank and a serial dilutions of a recombinant plasmid were prepared and used to generate standard curves, to establish a real-time fluorescent quantitative PCR method for detection of BmCPV. The results showed that the linear relationship between virus concentration (X) and Ct (Y) was Y = -3. 582 lgX + 38.748 with the correlation coefficient R2 = 0999. The method was very sensitive, specific and reproducible. It can be applied in the rapid detection of BmCPV and the prevalence investigation of this disease.

cDNA cloning and characterization of LASP1 from silkworm, Bombyx mori, involved in cytoplasmic polyhedrosis virus infection.

Full-length cDNA of a LIM and SH3 contained protein 1 (named BmLASP1) was identified from the silkworm, Bombyx mori, for the first time by rapid amplification of cDNA ends. The full-length cDNA of BmLASP1 is 2094 bp, consisting of a 5'-terminal untranslated region (UTR) of 117 bp, and a 3'-UTR of 610 bp with two poly-adenylation signal sequence AATAAA and a poly (A) tail. The BmLASP1 cDNA encodes a polypeptide comprising 455 amino acids, including a LIM domain, two nebulin domains and an SH3 domain. The theoretical isoelectric point is 7.07 and the predicted molecular weight is 51.8 kDa. BmLASP1 has no signal peptide but three potential N-glycosylation sites. Sequence similarity and phylogenic analyses indicated that BmLASP1 belonged to the group of insect LASP1 with a longer linker region which is different from vertebrate LASP1. The LASP1 in silkworm contained eight exons in its coding regions, and the last exon-intron boundary was conserved the same as in mammalian and Ciona intestinalis LASP1 genes. By fluorescent quantitative real-time polymerase chain reaction, the mRNA transcripts of BmLASP1 were mainly detected in the gonad, head, and spiracle, and slightly in the silk gland, vasa mucosa, midgut, fat body, and hemocytes. After silkworm larvae were infected by B. mori cytoplasmic polyhedrosis virus (BmCPV), the relative expression level of BmLASP1 was down-regulated in the midgut. This result suggested that BmLASP1 may play an important role in the response of silkworm to BmCPV infection.

Novel protein of IBP from silkworm, Bombyx mori, involved in cytoplasmic polyhedrosis virus infection.

In the present study, the full-length cDNA of a novel insulin-related peptide-binding protein (named BmIBP2) was identified from silkworm, Bombyx mori, using rapid amplification of cDNA ends. The full-length cDNA of BmIBP2 is 1293 bp, consisting of a 5'-terminal untranslated region (UTR) of 61 bp, and a 3'-UTR of 335 bp with a poly-adenylation signal sequence AATAAA and a poly (A) tail. The BmIBP2 cDNA encodes a polypeptide of 298 amino acids, including an IG domain and an IGc2 domain, with a theoretical isoelectric point of 5.73 and a predicted molecular weight of 33.1 kDa. The BmIBP2 also has a signal peptide of 23 amino acids and a potential N-glycosylation site. The sequence similarity and phylogenic analysis indicated that BmIBP2 belongs to the group of invertebrates IBP and is closer to IGFBP7 than to the other IGFBPs in vertebrates. These findings suggest that BmIBP2 is a putative homolog of vertebrate endocrine factor IGFBP7 and has a functional similarity. By fluorescent quantitative real-time polymerase chain reaction, mRNA transcripts of BmIBP2 were mainly detected in the midgut but were hardly detectable in the hemocytes, vasa mucosa, fat body, silk gland, head, testicle, ovary, and spiracle. After the silkworm larvae were infected by B. mori cytoplasmic polyhedrosis virus (BmCPV), a significant up-regulation in the relative expression level of BmIBP2 was found. All the results suggested that BmIBP2 is a novel protein that plays an important role in the insulin-signal pathway and in the immune response of silkworm to BmCPV infection.