Variation in Parasitoid Virulence of Tetrastichus brontispae during the Targeting of Two Host Beetles.

In host-parasitoid interactions, antagonistic relationship drives parasitoids to vary in virulence in facing different hosts, which makes these systems excellent models for stress-induced evolutionary studies. Venom compositions varied between two strains of Tetrastichus brontispae, Tb-Bl and Tb-On. Tb-Bl targets Brontispa longissima pupae as hosts, and Tb-On is a sub-population of Tb-Bl, which has been experimentally adapted to a new host, Octodonta nipae. Aiming to examine variation in parasitoid virulence of the two strains toward two hosts, we used reciprocal injection experiments to compare effect of venom/ovarian fluids from the two strains on cytotoxicity, inhibition of immunity and fat body lysis of the two hosts. We found that Tb-Onvenom was more virulent towards plasmatocyte spreading, granulocyte function and phenoloxidase activity than Tb-Blvenom. Tb-Blovary was able to suppress encapsulation and phagocytosis in both hosts; however, Tb-Onovary inhibition targeted only B. longissima. Our data suggest that the venom undergoes rapid evolution when facing different hosts, and that the wasp has good evolutionary plasticity.

Identification of Novel ARSB Genes Necessary for p-Benzoquinone Biosynthesis in the Larval Oral Secretion Participating in External Immune Defense in the Red Palm Weevil.

External secretions, composed of a variety of chemical components, are among the most important traits that endow insects with the ability to defend themselves against predators, parasites, or other adversities, especially pathogens. Thus, these exudates play a crucial role in external immunity. Red palm weevil larvae are prolific in this regard, producing large quantities of p-benzoquinone, which is present in their oral secretion. Benzoquinone with antimicrobial activity has been proven to be an active ingredient and key factor for external immunity in a previous study. To obtain a better understanding of the genetic and molecular basis of external immune secretions, we identify genes necessary for p-benzoquinone synthesis. Three novel ARSB genes, namely, RfARSB-0311, RfARSB-11581, and RfARSB-14322, are screened, isolated, and molecularly characterized on the basis of transcriptome data. To determine whether these genes are highly and specifically expressed in the secretory gland, we perform tissue/organ-specific expression profile analysis. The functions of these genes are further determined by examining the antimicrobial activity of the secretions and quantification of p-benzoquinone after RNAi. All the results reveal that the ARSB gene family can regulate the secretory volume of p-benzoquinone by participating in the biosynthesis of quinones, thus altering the host's external immune inhibitory efficiency.

Functional conservation and division of two single-carbohydrate-recognition domain C-type lectins from the nipa palm hispid beetle Octodonta nipae (Maulik).

As an invasive pest, the complete and effective innate immune system is crucial for the nipa palm hispid beetle Octodonta nipae (Maulik) to adjust to new environments. C-type lectins (CTLs) are large families of carbohydrate-binding proteins that possess one or more characteristic carbohydrate-recognition domains (CRD) and function as pattern-recognition receptors, which play important roles in mediating humoral and cellular immunity. In the present study, for the first time, we report two CTL-Ss (single-CRD CTLs) from O. nipae (Maulik) (designated OnCTL1 and OnCTL2). The two CTL-Ss share high identity at conserved amino acids associated with conserved carbohydrate binding sites Gln-Pro-Asp (QPD) motifs and clearly show a 1:1 orthologous relationship in insects, which endow them with functional conservation and diversification. mRNA abundance analysis showed that OnCTL1 was upregulated upon Staphylococcus aureus and Escherichia coli challenge at 6 and 12 h, while OnCTL2 underwent no changes upon E. coli challenge and was even downregulated after S. aureus infection. Knockdown of OnCTL1 significantly decreased the transcripts of two key serine proteases (prophenoloxidase activating factors), OnPPAF1 and OnPPAF3, followed by the reduction of haemolymph phenoloxidase activity; it also increased the expression of Defensin2B. In contrast, silencing of OnCTL2 significantly decreased the expression of Defensin2B and Attacin3C, the encapsulation index, and the phagocytosis rate compared to the dsEGFP group. The spreading results showed that more irregularly shaped plasmatocytes and lower levels of aggregation were found in OnCTL2-silenced pupae than in the dsOnCTL1 and dsEGFP groups. We can infer from the results of this study that the two OnCTLs play important roles in the immune system and generate a functional division: OnCTL1 seems to function more in humoral immunity including mediating bacterial recognition and activating the phenoloxidase cascade, and OnCTL2 plays a greater role in enhancing cellular immunity. These observations could replenish information on the functional diversification of insect CTLs, and also provide valuable information to unravel the immunity in O. nipae.

Cloning and Immunosuppressive Properties of an Acyl-Activating Enzyme from the Venom Apparatus of Tetrastichus brontispae (Hymenoptera: Eulophidae).

Venom injected into the host plays vital roles in facilitating successful parasitization and development for parasitoid wasps, especially those devoid of polydnavirus, and the abundant venom proteins appear to be most likely involved in parasitization success. Previously, we found the four most abundant venom proteins, including 4-coumarate:CoA ligase-like 4 (4CL4-like), in the Tetrastichus brontispae (Hymenoptera: Eulophidae) venom apparatus. In this study, we cloned, expressed T. brontispae 4CL4-like (Tb4CL4-like) in Escherichia coli, and investigated its immunosuppressive properties. The deduced amino acid sequence for Tb4CL4-like shares high identity at conserved amino acids associated with the acyl-activating enzyme (AAE) consensus motif but shows only <40% identity with the members in the AAE superfamily. mRNA abundance analysis indicated that Tb4CL4-like was transcribed mainly in the venom apparatus. Recombinant Tb4CL4-like inhibited Octodonta nipae (Coleoptera: Chrysomelidae) pupal cellular encapsulation and spreading by targeting the hemocyte cytoskeleton and reduced the hemocyte-mediated phagocytosis of E. coli in vivo. Moreover, Tb4CL4-like exhibited greater affinity to palmitic acid and linolenic acid based on the molecular docking assay and is hypothesized to be involved in fatty acid metabolism. In conclusion, our results suggest that Tb4CL4-like may be an immunity-related AAE protein that is involved in the regulation of host immunity through fatty acid metabolism-derived signaling pathways.

Combination of label-free quantitative proteomics and transcriptomics reveals intraspecific venom variation between the two strains of Tetrastichus brontispae, a parasitoid of two invasive beetles.

The venom apparatus is a conserved organ in parasitoids that shows adaptations correlated with life-style diversification. Combining transcriptomics and label-free quantitative proteomics, here we explored the venom apparatus components of the endoparasitoid Tetrastichus brontispae (Eulophidae), and provide a comparison of the venom apparatus proteomes between its two closely related strains, T. brontispae-Octodonta nipae (Tb-On) and T. brontispae-Brontispa longissima (Tb-Bl). Tb-Bl targets the B. longissima pupa as its habitual host. However, Tb-On is an experimental derivative of Tb-Bl, which has been exposed to the O. nipae pupa as host consecutively for over 40 generation. Results showed that approximately 1505 venom proteins were identified in the T. brontispae venom apparatus. The extracts contained novel venom proteins, such as 4-coumarate-CoA ligase 4. A comparative venom proteome analysis revealed that significant quantitative and qualitative differences in venom composition exist between the two strains; although the most abundant venom proteins were shared between them. The differentially produced proteins were mainly enriched in fatty acid biosynthesis and melanotic encapsulation response. Six of these enriched proteins presented increased levels in Tb-On, and this result was validated by parallel reaction monitoring (PRM) analysis. Overall, our data reveal that venom composition can evolve quickly and respond to host selection.

[A study of forensic individual identification used STR locus with silver staining and multiplex PCR methods].

Amplification of short tandem repeat(STR) loci has become a useful tool for human identification applications. To improve throughput and efficiency for the forensic materials and gain foure and six STR locis multiplex methods with silver staining, CSF1PO,TPOX,THO1 and vWA(referred to as multiplex A), D18S51, D7S820, D13S317, D5S818, D3S1358 and Amelogenin(referred to as multiplex B) have been evaluated for use in a rape case. The products of multiplex amplication were separated in a denaturing polyacrylamide gel and analyzed with silver staining. Two multiplex amplications used in this case could provide a power of discrimination of approximately 2.43 x 10(-19). Silver staining was shown to be a validation methods for analysing the products of four and six multiplex amplications.