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Although the third-stage larvae of Angiostrongylus cantonensis (AcL3) are thought to initiate infection by penetrating the epithelium of the small intestine, the mode of intestinal invasion remains obscure. Considering the inaccessibility of the gut tract and the need to sacrifice animals for this type of study, we devised an in vitro cell-parasite co-culture system to examine the initial cellular and molecular events between AcL3 and host epithelia. No apoptosis augmentation was detected in enterocytes after introduction of larvae. A significant increase in dead cells was detected in IEC-6, NCM460 and 293T after incubating for 4 h, with AcL3 wounding rat small intestinal epithelial cells IEC-6 more rapidly. Under a scanning electron microscope (SEM), cell gap opening was visualized in the IEC-6 monolayer treated with AcL3. Loosening of the extracellular matrix (ECM) of the monolayer was found to be involved in the parasite-cell interactions. Pretreating the AcL3 with a protease inhibitor attenuated its penetration ability of the artificial intestine barrier. In conclusion, AcL3 broke through the intestinal barrier of the host with the assistance of mechanical injury and the opening of a cell gap, but without causing apoptosis. The interaction platform presented here may provide direct insight into the cellular and molecular events during worm invasion of host enterocytes.

An olfactory co-receptor gene AalbOrco was isolated and described from Aedes albopictus using the RACE technique. AalbOrco contained typical structures of seven transmembrane domains and Nin-Cout. Sequence analysis demonstrated that AalbOrco is very similar to Orco orthologs from other dipterans, especially in the C-terminus. AalbOrco was expressed at significantly higher levels in embryos compared with larvae, however, AalbOrco transcripts were found to greatly increase in number from the larval to the pupal stage and were at the highest level at the adult stage. Since AalbOrco is abundantly expressed in adults and is distributed in chemosensory organs such as the antenna, maxillary palps and proboscis suggests that AalbOrco may play an important role in locating hosts. In addition, AalbOrco is also highly expressed in male abdomens as compared to female abdomens, however, no expression was detected in the ovipositor and the copulatory organ. SEM (scanning electron microscopy) and immunolocalization showed that AalbOrco is located in the cell bodies of olfactory sensory neurons (OSNs) under antennal cuticula and the dendrites of neurons in sensilla trichodea. Moreover, fluorescence signals were detected in some neuronal cell bodies and sensilla T2 from the labellum but were scarcely detected in male maxillary palps. The transcription level of AalbOrco in female adults decreased by 55.7% after the larvae continued to ingest exogenous AalbOrco dsRNA expressed bacterially until eclosion. Blood-sucking behavior was impaired by 44.7% compared to the control groups, although pupation and emergence rate were not significantly different between the control and treatment groups. We conclude that AalbOrco has the general characteristics of Orco orthologs and plays an important role in host-locating behavior. AalbOrco performs the olfactory function in antennae as well as in female maxillary palps.

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.