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iGABAR, a member of the Cys-loop ligand-gated ion channel superfamily, is a significant target of the insecticide ivermectin (IVM). GRD is the potential subunit of the insect iGABAR. However, little information about GRD in Ae. aegypti has been reported. In this study, we involved cloning and characterizing the iGABAR subunit GRD of Ae. aegypti (Ae-GRD). Sequence analysis indicated that Ae-GRD, as part of the cysteine-loop ligand-gated ion channel family, is similar to other insect GRD. RNA interference (RNAi) was employed to explore IVM resistance in Ae. aegypti, resulting in a significant reduction in Ae-GRD expression (p < 0.05), and the mortality of Ae. aegypti adults with Ae-GRD knockdown was significantly decreased after exposure to ivermectin. Bioinformatics prediction identified miR-71-5p as a potential regulator of Ae-GRD. In vitro, dual-luciferase reporter assays confirmed that Ae-GRD expression was regulated by miR-71-5p. Microinjection of miR-71-5p mimics upregulated miR-71-5p expression and downregulated Ae-GRD gene expression, reducing mortality by 34.52% following IVM treatment. Conversely, microinjection of a miR-71-5p inhibitor decreased miR-71-5p expression but did not affect the susceptibility to IVM despite increased Ae-GRD expression (p < 0.05). In conclusion, Ae-GRD, as one of the iGABA receptor subunits, is a potential target of ivermectin. It may influence ivermectin resistance by modulating the GABA signaling pathway. The inhibition of Ae-GRD expression by miR-71-5p decreased ivermectin resistance and consequently lowered the mortality rate of Ae. aegypti mosquitoes. This finding provides empirical evidence of the relationship between Ae-GRD and its miRNA in modulating insecticide resistance, offering novel perspectives for mosquito control strategies.

Anaplasmosis is a serious infection which is transmitted by ticks and mosquitos. There are very few reports and studies that have been carried out to understand the prevalence, distribution, and epidemiological profile of Anaplasma spp. infection in dogs in Hainan province/island. In the present study, we have tried to understand the prevalence, distribution, and occurrence of Anaplasma spp. infections in dogs (n = 1051) in Hainan Island/Province to establish a surveillance-based study. The confirmed positive samples by Polymerase chain reaction (PCR) were subjected to capillary sequencing for further strain-specific confirmation, followed by the construction of phylogenetic trees to determine their genetic relations. Various statistical tools were used to analyze related risk factors. There were three species of Anaplasma detected from the Hainan region; namely, A. phagocytophilum, A. bovis, and A. platys. The overall prevalence of Anaplasma is 9.7% (102/1051). A. phagocytopihum was prevalent in 1.0% of dogs (11/1051), A. bovis was found in 2.7% of dogs (28/1051), and A. platys in 6.0% of dogs (63/1051). Our surveillance-based study conducted to understand the occurrence and distribution pattern of Anaplasma spp. in Hainan will help in designing effective control measures along with management strategies so as to treat and control the infection in the area.

Serotonin (5-HT) plays a vital role in many physiological processes in insects, regulating physiological activities such as growth and movement through multiple 5-HT receptors (5-HTRs), which were potential targets for some new insecticides. However, the specific function of individual 5-HTRs in Ae. aegypti is still unclear. In this study, we investigated the function of the 5-HT7A receptor during Ae. aegypti development. 5-HTR7A transcripts were detected at all stages of development by real-time PCR. The results indicated that the gene expression was highest in the limbs (p < 0.01). We also generated 5-HTR7A mutant mosquitoes using CRISPR-mediated gene editing. The mutants had an abnormal phenotype at the larval stage, including an aberrant head-to-chest ratio and decreased motor activity. The mutant pupae developed abnormally, and most died (56.67%) (p < 0.0001). Using external stimuli to larvae and pupae with abnormal phenotypes, we found the mutant G1 and G2 generations responded to external stimuli in a longer time than the wild-type (WT) mosquitoes, and most of the mutants were 2 to 3 s slower than the WTs to respond to external stimuli (p < 0.01). Due to higher mortality, mutant larvae and pupae had fewer numbers than the WTs. The egg hatching rate of mutant G1 and G2 generations was lower than that of the WTs (p < 0.01). The expression level of 5-HTR7A in the mutants decreased by about 65% compared with the control group using real-time PCR (p < 0.05). In all, the 5-HT7A receptor plays an important role in the metamorphosis, development and motor function of Aedes aegypti.

Polymerase chain reaction (PCR) is a unique technique in molecular biology and biotechnology for amplifying target DNA strands, and is also considered as a gold standard for the diagnosis of many canine diseases as well as many other infectious diseases. However, PCR still faces many challenges and issues related to its sensitivity, specificity, efficiency, and turnaround time. To address these issues, we described the use of unique ZnO nanoflowers in PCR reaction and an efficient ZnO nanoflower-based PCR (nanoPCR) for the molecular diagnosis of canine vector-borne diseases (CVBDs). A total of 1 mM of an aqueous solution of ZnO nanoflowers incorporated in PCR showed a significant enhancement of the PCR assay with respect to its sensitivity and specificity for the diagnosis of two important CVBDs, Babesia canis vogeli and Hepatozoon canis. Interestingly, it drastically reduced the turnaround time of the PCR assay without compromising the yield of the amplified DNA, which can be of benefit for veterinary practitioners for the improved management of diseases. This can be attributed to the favorable adsorption of ZnO nanoflowers to the DNA and thermal conductivity of ZnO nanoflowers. The unique ZnO nanoflower-assisted nanoPCR greatly improved the yield, purity, and quality of the amplified products, but the mechanism behind these properties and the effects and changes due to the different concentrations of ZnO nanoflowers in the PCR system needs to be further studied.