| The relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids are widely used as biological control agents in sustainable agricultural programs. Females of the gregarious endoparasitoid Pteromalus puparum (Hymenoptera:Pteromalidae) inject venom along with eggs into their hosts. P. puparum does not inject polydnaviruses during oviposition. For this reason, P. puparum and its pupal host, the small white butterfly Pieris rapae (Lepidoptera:Pieridae), comprise an excellent model system for studying the influence of an endoparasitoid venom on the biology of the pupal host. P. puparum venom suppresses the immunity of its host, although the suppressive mechanisms are not fully understood. In this study, we investigated the molecular mechanism of the venom suppression to the innate immune responses of P. rapae pupa. The main results have been shown below.(1) Venom could influence gene transcripts levels in the two main immunity-conferring tissues, hemocytes and fat body of host pupa. At 1 h post-venom injection, we recorded significant decreases in transcript levels of 217 EST clones (revealing 113 genes identified in silico, including 62 unknown contigs) derived from forward subtractive libraries of host hemocytes and in transcript levels of 288 EST clones (221 genes identified in silico, including 123 unknown contigs) from libraries of host fat body. These genes are related to insect immune response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, stress response and transcriptional and translational regulation. We verified the reliability of the suppression subtractive hybridization (SSH) data with semi-quantitative RT-PCR analysis of a set of randomly selected genes. This analysis showed that most of the selected genes were down-regulated after venom injection.(2) Venom could down-regulate the transcripts levels of cecropin and lysozyme genes, and inhibit the antimicrobial activity of pupal hemolymph. The inhibition zone results showed that compared with negative control, Sephadex A-50 beads injection could induce the antimicrobial activity of pupal hemolymph, whereas venom could inhibit it. The full length cDNAs of cecropin and lysozyme were screened from the forward subtractive library, respectively. Multiple sequences alignment results represented that the sequences of cecropin and lysozyme of P. rapae were similar to these of other lepidopteran species, respectively. Relative quantitative real time PCR (rq-rtPCR) results indicated that all of Micrococcus luteus, Escherichia coli, and beads injections up-regulated the transcripts levels of both cecropin and lysozyme genes, whereas venom could down-regulate them. At 8 h post beads challenge, there was a peak of the transcripts level of cecropin gene, while the peak of lysozyme gene displaying at 24 h post challenge. After the peak, the transcripts levels of these 2 genes both came down. The transcripts levels of these 2 genes were higher in granulocytes and fat body than other tissues. Double-strand RNA (dsRNA) injection could down-regulate the transcripts levels of these 2 genes and the antimicrobial activity of pupal hemolymph. It is implicated that the cecropin and lysozyme possess the antimicrobial capability. Time course effect results showed that during 0-8 h post treatments, venom could effectively inhibit the transcripts levels of these 2 genes. After 8 h, the suppressive effect of the venom began to decrease. Dose effect results represented that the higher concentration of venom, the stronger inhibition. In addition, recombinant cecropin and lysozyme have no significant effect on the emergence rate of P. puparum offspring.(3) Venom could down-regulate the expression levels of prophenol oxidase activating proteinase (PAP) genes and inhibit the prophenol oxidase (proPO) activation in pupal hemolymph, but not suppress the expression level of proPO gene and not influence phenol oxidase (PO) activity directly. The PO activity results showed that compared with negative control, Sephadex A-50 beads injection could induce the PO activity of pupal hemolymph, but parasitization not. Venom could inhibit it by targeting to the proPO activating cascade not directly suppress the PO activity. The full length cDNAs of PAP1, PAP2 and proPO subunit 1 were isolated, respectively, by library screening and RACE reaction. Multiple sequences alignment results represented that the sequences of PAP1 and PAP2 as well as proPO subunit 1 of P. rapae were similar to the PAPs and proPO subunits sequences of other lepidopteran species, respectively. The rq-rtPCR and western blotting results indicated that all of M. luteus, E. coli, and beads injections up-regulated the transcripts and expression levels of these 3 genes, whereas venom could only down-regulate of PAP1 and PAP2 genes, but not of proPO subunit 1 gene. At 4 h post beads challenge, there were peaks of the transcripts and expression levels of each gene, and after the peaks, the levels of these 3 genes came down. The transcripts and expression levels of PAP1 and PAP2 genes were higher in granulocytes and fat body than other tissues, while proPO subunit 1 gene only in hemocytes. The dsRNA injection could down-regulate the transcripts and expression levels of these 3 genes and the PO activity of pupal hemolymph. It is implicated that PAP1, PAP2, proPO subunit 1 play important roles in host hemolymph melanization. Time course effect results showed that during 0-8 h post treatments, venom could effectively inhibit the transcripts levels of PAP1 and PAP2 genes. After 8 h, the suppressive effect of the venom began to decrease. Dose effect results represented that the higher concentration of venom, the stronger inhibition to the transcripts levels of PAP 1 and PAP2.(4) Venom could down-regulate the expression level of scavenger receptor gene, and inhibit the phagocytosis of pupal hemocytes. The phagocytosis rate results showed that compared with negative control, both venom injection and parasitization could suppress the phagocytosis rate of pupal hemocytes. The full length cDNA of scavenger receptor was isolated by library screening and RACE reaction. Multiple sequences alignment result represented that the sequence of scavenger receptor of P. rapae was similar to other lepidopteran species. The rq-rtPCR and western blotting results indicated that all of Micrococcus luteus, Escherichia coli, and beads injections up-regulated the transcripts and expression levels of scavenger receptor gene, whereas venom could down-regulate them. At 4 h post beads challenge, there were peaks of the transcripts and expression levels of scavenger receptor gene. After the peaks, the levels of the gene came down. The transcripts and expression levels of the gene were highest in granulocytes. Immunolocation and western blotting results showed that scavenger receptor was located in cytoplasm and cellular membrane, and could not be secreted into the pupal plasma. Venom could suppress the gene expression in granulocytes. The dsRNA injection could down-regulate the transcripts and expression levels of the gene, the phagocytosis and encapsulation rates of pupal hemocytes. It is implicated that the scavenger receptor not only takes part in the phagocytosis, but also in encapsulation. Time course effect results showed that during 0-8 h post treatments, venom could effectively inhibit the transcripts levels of the gene. After 8 h, the suppressive effect of the venom began to decrease. Dose effect results represented that the higher concentration of venom, the stronger inhibition.(5) Venom could down-regulate the expression level of C-type lectin gene, and it resulted in the down-regulations of the antimicrobial, PO activities, the phagocytosis and encapsulation rates. The full length cDNA of C-type lectin was isolated by library screening and RACE reaction. Multiple sequences alignment result represented that the sequence of C-type lectin of P. rapae was similar to other lepidopteran species. The rq-rtPCR and western blotting results indicated that all of Micrococcus luteus, Escherichia coli, and beads injections up-regulated the transcripts and expression levels of C-type lectin gene, whereas venom could down-regulate them. At 4 h post beads challenge, there were peaks of the transcripts and expression levels of C-type lectin gene. After the peaks, the levels of the gene came down. The transcripts and expression levels of the gene were higher in granulocytes. Immunolocation and western blotting results showed that C-type lectin was expressed in granule of the granulocytes not in cytoplasm, and could be secreted into the pupal plasma. After 8 h, the C-type lectin could bind to the cellular membrane of the challenged plasmocytes. Venom could suppress the gene expression in granulocytes. The dsRNA injection could down-regulate the transcripts and expression levels of the gene, the antimicrobial and PO activities, as well as the phagocytosis and encapsulation rates. It is implicated that the C-type lectin may take part in the non-self recognition. Time course effect results showed that during 0-8 h post treatments, venom could effectively inhibit the transcripts levels of the gene. After 8 h, the suppressive effect of the venom began to decrease. Dose effect results represented that the higher concentration of venom, the stronger inhibition. Taken together, these results above have revealed the molecular mechanisms of the venom suppression on the innate immune responses of P. rapae pupa on a large extent. They provide some insight into the mechanism of the P. puparumlP. rapae parasitoid system. |
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