Gap junctions formed by two hemichannels from two neighboring cells are cell-to-cell communication channels; hemichannels are communication channels between intracellular and extracellular environments. Hemichannels are hexameric proteins formed by connexins, pannexins, innexins and vinnexins. Innexin-hemichannels (innexons) exist in the lepidopteran cell surface, but their component innexins and functions have not been reported. Recent studies by others have demonstrated that hemichannels, connexons and pannexons from vertebrates serve as regulators of apoptosis via inactivating the PI3K/Akt signaling pathway. Here, the apoptogenic properties of innexons are demonstrated using two innexin cDNAs, Spli-inx2 and Spli-inx3, which were isolated from hemocytes of lepidopteran Spodoptera litura. Alignment analysis revealed that these two genes belong to a conserved innexin family, as they contain the insect signature YYQWV motif at the beginning of the second transmembrane domain. Immunofluorescence showed that two fusion proteins, Inx2-V5 and Inx3-V5, were localized predominantly in the cell membrane, cytoplasm and also nuclei. Ectopic expression in Sf9 cells and over-expression of Inx2 and Inx3 in Spli221 cells promoted apoptosis. In the Spli221 cells, apoptotic cells presented remarkable membrane blebbing. This study also showed that Sf9 and Spli221 cells undergo low level apoptosis under normal culture conditions, but not Hi5 cells. In Hi5 stable cell lines, biotinylation was used to isolate surface proteins and confirm Inx2 and Inx3 localization in the cell membrane and also further data showed that Hi5 cells may activate the PI3K signaling pathway via phosphorylating molecular Akt downstream. This result suggests that innexon-promoted apoptosis may be involving the PI3K/Akt signaling pathway. These findings will facilitate further examinations of the apoptotic regulation by the PI3K/Akt signaling pathway and comparative studies of innexons, connexons, pannexons, and vinnexons.

Recent studies have demonstrated that hemichannels, which form gap junctions when paired from apposing cells, may serve additional roles when unpaired including cell adhesion and paracrine communication. Hemichannels in mammals are formed by connexins or pannexins, while in insects they are formed by pannexin homologues termed innexins. The formation of functional gap junctions by insect innexins has been established, although their ability to form functional nonjunctional hemichannels has not been reported. Here the characteristics of nonjunctional hemichannels were examined in three lepidopteran cell types, two cell lines (High Five and Sf9) and explanted hemocytes from Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Selective fluorescent dye uptake by hemichannels was observed in a significant minority of cells, using fluorescence microscopy and flow cytometry. Carbenoxelone, an inhibitor of mammalian junctions, disrupted dye uptake, while flufenamic acid and mefloquine did not. The presence of Ca(2+) and Mg(2+) in the media increased hemichannel activity. Additionally, lipopolysaccharide, a stimulator of immune activity in lepidopterans, decreased dye uptake. These results demonstrate for the first time the activity of nonjunctional hemichannels in insect cells, as well as pharmacological tools to manipulate them. These results will facilitate the further examination of the role of innexins and nonjunctional hemichannels in insect cell biology, including paracrine signaling, and comparative studies of mammalian pannexins and insect innexins.

Microplitis bicoloratus parasitism induction of apoptotic DNA fragmentation of host Spodoptera litura hemocytes has been reported. However, how M. bicoloratus parasitism regulates the host signaling pathways to induce DNA fragmentation during apoptosis remains unclear. To address this question, we performed a new RNAseq-based comparative analysis of the hemocytes transcriptomes of non-parasitized and parasitized S. litura. We were able to assemble a total of more than 11.63 Gbp sequence, to yield 20,571 unigenes. At least six main protein families encoded by M. bicoloratus bracovirus are expressed in the parasitized host hemocytes: Ankyrin-repeat, Ben domain, C-type lectin, Egf-like and Mucin-like, protein tyrosine phosphatase. The analysis indicated that during DNA fragmentation and cell death, 299 genes were up-regulated and 2,441 genes were down-regulated. Data on five signaling pathways related with cell death, the gap junctions, Ca²⁺, PI3K/Akt, NF-κB, ATM/p53 revealed that CypD, which is involved in forming a Permeability Transition Pore Complex (PTPC) to alter mitochondrial membrane permeabilization (MMP), was dramatically up-regulated. The qRT-PCR also provided that the key genes for cell survival were down-regulated under M. bicoloratus parasitism, including those encoding Inx1, Inx2 and Inx3 of the gap junction signaling pathway, p110 subunit of the PI3K/Akt signaling pathway, and the p50 and p65 subunit of the NF-κB signaling pathway. These findings suggest that M. bicoloratus parasitism may regulate host mitochondria to trigger internucleosomal DNA fragmentation. This study will facilitate the identification of immunosuppression-related genes and also improves our understanding of molecular mechanisms underlying polydnavirus-parasitoid-host interaction.