Identification and Functional Characterization of CYP4D2 Putatively Associated with ß-Cypermethrin Detoxification in Phortica okadai.

Phortica okadai, a polyphagous pest, serves as a vector for Thelazia callipaeda in China. Currently, there are no effective control strategies for this vector. Agricultural pest control may cause P. okadai to become a threat due to the development of pesticide resistance. Cytochrome P450s (CYP450) plays a significant role in detoxifying xenobiotics in insects. In this study, we performed RNA sequencing of P. okadai exposed to ß-cypermethrin for 0 and 1 h and then gene cloning of the five up-regulated CYP450 genes. Three CYP450 genes were successfully cloned, and their expression patterns in different developmental stages and in different tissues were analyzed by RT-qPCR. Pocyp4d2 was observed to have the highest expression in the midgut (fold change 2.82 for Pocyp4d2, 2.62 for Pocyp49a1, and 1.77 for Pocyp28d2). Functional analysis was carried out according to overexpression in S2 cells from the pfastbac1 vector and RNAi with siRNA. The results of the CCK8 assay indicated that the overexpression of the recombinant protein PoCYP4D2 suppressed the decrease in S2 cell viability due to ß-cypermethrin. The expression levels of PoCYP4D2 decreased significantly, and the mortality rates increased from 6.25% to 15.0% at 3 h and from 15.0% to 27.5% at 6 h after Pocyp4d2-siRNA injection. These results suggest that Pocyp4d2 may be an essential key gene in the metabolism of ß-cypermethrin in P. okadai. This study constitutes a foundation to explore further the functions of P. okadai CYP450 genes in insecticide metabolism.

Laboratory Culture and Life Cycle of Thelazia callipaeda in Intermediate and Definitive Hosts.

Human thelaziasis caused by Thelazia callipaeda is being increasingly reported worldwide. Notably, an epidemic trend is observed in Southwest China. Whether Phortica okadai found in Southwest China can act as a vector of T. callipaeda and human-derived T. callipaeda animal infections has not been widely reported. Here, P. okadai was maintained in a laboratory and experimentally infected with first-stage larvae collected from adult T. callipaeda that were isolated from infected human subjects. Dead P. okadai were subjected to PCR assay and dissected every two days to detect T. callipaeda. Subsequently, live flies were used to infect a rabbit. The infection procedures were performed once a day (20 min) for two weeks. The results show that L1 collected from the adult T. callipaeda could successfully parasitize P. okadai captured in Zunyi, a city in Southwest China, and developed into L3, and a rabbit was successfully infected with T. callipaeda using P. okadai as the intermediate host. The present study demonstrates a human-derived T. callipaeda infection in rabbits, through P. okadai, under laboratory conditions for the first time. These results provide insights into the transmission cycle of T. callipaeda and constitute a foundation to develop an effective treatment protocol for T. callipaeda infection.

Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction.

Macrophages are innate immune cells with essential roles in the immune response during helminth infection. Particularly, the direction of macrophage polarization could contribute to pathogen trapping and killing as well as tissue repair and the resolution of type 2 inflammation. This study establishes that the recombinant protein of Thelazia callipaeda macrophage migration inhibitory factor (T.cp-MIF) induces THP-1-derived macrophages to undergo M1 to M2 type dynamic polarization, using the methods of flow cytometry, real-time quantitative PCR, differential transcriptomic analysis and western blot. Interestingly, there was an increase in protein and mRNA expression of M1-type proteins and cytokines after the use of PI3K inhibitors, suggesting that the polarization state tends to favor the M1 type after M2 type inhibition. In conclusion, the dynamic polarization mechanism of T.cp-MIF-induced human THP-1-derived macrophages from M1 to M2 type is related to the binding of TLR4. It can first affect the M1 type polarization of macrophages by activating its downstream NF-κB pathway. Activation of the PI3K/Akt pathway and inhibition of NF-κB phosphorylation affects the M2 type polarization of macrophages.

Macrophage migration inhibitory factor of Thelazia callipaeda induces M2-like macrophage polarization through TLR4-mediated activation of the PI3K-Akt pathway.

Macrophage migration inhibitory factor (MIF), an immunoregulatory cytokine plays an important role in inflammation and the immune response, and has been described as having a potential role in immune evasion by parasites. Thelazia callipaeda, a vector-borne zoonotic eye worm with a broad host range, has been documented as an agent of ocular infection of thelaziosis. The ability of T. callipaeda to persist in an immunologically competent host has led to the suggestion that it has evolved specific measures to counter immune defenses. To date, whether the immune evasion of T. callipaeda is related to MIF and the possible related signaling pathway and molecular mechanism have remained unclear. In the present study, we examined the effect of T. callipaeda MIF (T. cp-MIF) on macrophages. We analyzed the antigenic epitopes of the candidate T. cp-MIF and found that it exhibited an ideal antigenic index. Morphology, Flow cytometry, and cytokine analysis showed that T. cp-MIF induced the dynamic polarization of THP-1 macrophages from the M1-like phenotype to the M2-like phenotype. The chemotaxis assay revealed an inhibitory effect of T. cp-MIF on THP-1 macrophages. Western blotting suggested that, compared to the control, THP-1 macrophages exposed to T. cp-MIF had higher TLR4 protein expression and the phosphatidylinositol 3'-kinase (PI3K) -Akt pathway activation. In conclusion, T. cp-MIF induces M2-like macrophage polarization through TLR4-mediated activation of the PI3K-Akt pathway, which might provide a basis for future research on how it affects the immune system of the host.