Removal of gut symbiotic bacteria negatively affects life history traits of the shield bug, Graphosoma lineatum.

The shield bug, Graphosoma lineatum (Heteroptera, Pentatomidae), harbors extracellular Pantoea-like symbiont in the enclosed crypts of the midgut. The symbiotic bacteria are essential for normal longevity and fecundity of this insect. In this study, life table analysis was used to assess the biological importance of the gut symbiont in G. lineatum. Considering vertical transmission of the bacterial symbiont through the egg surface contamination, we used surface sterilization of the eggs to remove the symbiont. The symbiont population was decreased in the newborn nymphs hatched from the surface-sterilized eggs (the aposymbiotic insects), and this reduction imposed strongly negative effects on the insect host. We found significant differences in most life table parameters between the symbiotic insects and the aposymbiotics. The intrinsic rate of increase in the control insects (0.080 ± 0.003 day-1) was higher than the aposymbiotic insects (0.045 ± 0.007 day-1). Also, the net reproductive and gross reproductive rates were decreased in the aposymbiotic insects (i.e., 20.770 ± 8.992 and 65.649 ± 27.654 offspring/individual, respectively), compared with the symbiotic insects (i.e., 115.878 ± 21.624 and 165.692 ± 29.058 offspring/individual, respectively). These results clearly show biological importance of the symbiont in G. lineatum.

The microRNA pathway is involved in Spodoptera frugiperda (Sf9) cells antiviral immune defense against Autographa californica multiple nucleopolyhedrovirus infection.

The microRNA (miRNA) pathway is an epigenetic mechanism that plays important roles in various biological processes including host-virus interactions by regulating gene expression of the host and/or the virus. Previously, we showed that the cellular microRNAome in Spodoptera frugiperda (Sf9) cells is modulated following Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection suggesting that miRNAs may contribute in the cellular antiviral immunity. Here, we investigated the role of core components of the miRNA pathway in Sf9-AcMNPV interaction. Gene expression analyses showed that the expression levels of Dicer-1 (Dcr1), Argonaute-1 (Ago1) and Exportin-5 (Exp5) increased following AcMNPV infection particularly at 16 h post infection (hpi). Ran expression levels, however, decreased in response to virus infection. The expression levels of cellular miRNAs, miR-184 and let-7, also diminished at the post infection times further confirming differential expression of the cellular miRNAs following AcMNPV infection. To determine the role of the miRNA pathway in the interaction, we silenced key genes in the pathway using specific dsRNAs. RNAi of Dcr1, Ago1 and Ran enhanced viral DNA replication and reduced the abundance of miR-184 and let-7 underscoring the importance of the miRNA pathway in antiviral immunity in Sf9 cells. Suppression of the miRNA pathway in mock and infected cells had no effect on Ran expression levels suggesting miRNA-independent downregulation of this gene after virus infection. In conclusion, our results suggest the antiviral role of the miRNA pathway in Sf9 cells against AcMNPV. To modulate this immune response, AcMNPV represses host miRNAs likely through downregulation of Ran to enhance its replication in the host cells.

Small interfering RNA pathway contributes to antiviral immunity in Spodoptera frugiperda (Sf9) cells following Autographa californica multiple nucleopolyhedrovirus infection.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a well-known virus in the Baculoviridae family. Presence of the p35 gene in the AcMNPV genome as a suppressor of the short interfering RNA (siRNA) pathway is a strong reason for the importance of the siRNA pathway in the host cellular defense. Given that, here we explored the roles of Dicer-2 (Dcr2) and Argonaute 2 (Ago2) genes, key factors in the siRNA pathway in response to AcMNPV infection in Spodoptera frugiperda Sf9 cells. The results showed that the transcript levels of Dcr2 and Ago2 increased in response to AcMNPV infection particularly over 16 h post infection suggesting induction of the siRNA pathway. Reductions in the expression levels of Dcr2 and Ago2 by using specific dsRNAs in Sf9 cells modestly enhanced production of viral genomic DNA which indicated their role in the host antiviral defense. Using deep sequencing, our previous study showed a large number of small reads (siRNAs of ∼20 nucleotides) from AcMNPV-infected Sf9 cells that were mapped to some of the viral genes (hot spots). Down-regulation of Dcr2 in Sf9 cells resulted in enhanced expression levels of the selected virus hotspot genes (i.e. ORF-9 and ORF-148), while the transcript levels of virus cold spots (i.e. ORF-18 and ORF-25) with no or few siRNAs mapped to them did not change. Overexpression of AcMNPV p35 as a suppressor of RNAi and anti-apoptosis gene in Sf9 cells increased virus replication. Also, replication of mutant AcMNPV lacking the p35 gene was significantly increased in Sf9 cells with reduced transcript levels of Dcr2 and Ago2, highlighting the antiviral role of the siRNA pathway in Sf9 cells. Together, our results demonstrate that Dcr2 and Ago2 genes contribute in efficient antiviral response of Sf9 cells towards AcMNPV, and in turn, the AcMNPV p35 suppresses the siRNA pathway, besides being an antiapoptotic protein.

Gammaproteobacteria as essential primary symbionts in the striped shield bug, Graphosoma Lineatum (Hemiptera: Pentatomidae).

Many members of suborder Heteroptra harbor heritable symbiotic bacteria. Here we characterize the gut symbiotic bacterium in Graphosoma lineatum (Hemiptera: Pentatomidae) by using molecular phylogeny, real-time PCR analysis as well as light and electron microscopy observations. The microscopy observations revealed the presence of a large number of rod-shaped bacterial cells in the crypts. A very high prevalence (98 to 100%) of the symbiont infection was found in the insect populations that strongly supports an intimate association between these two organisms. Real-time PCR analysis also showed that the Gammaproteobacteria dominated the crypts. The sequences of 16sr RNA and groEL genes of symbiont showed high levels of similarity (93 to 95%) to Pantoea agglomeranse and Erwinia herbicola Gammaproteobacteria. Phylogenetic analyses placed G. lineatum symbiont in a well-defined branch, divergent from other stink bug bacterial symbionts. Co-evolutionary analysis showed lack of host-symbiont phylogenetic congruence. Surface sterilization of eggs resulted in increased pre-adult stage in the offspring (aposymbionts) in comparison to the normal. Also, fecundity, longevity, and adult stage were significantly decreased in the aposymbionts. Therefore, it seems that the symbiont might play a vital function in the host biology, in which host optimal development depends on the symbiont.