Smartt, C. | Search bioRxiv

bioRxiv2021

Activation of the autophagy pathway affects Dengue virus infection in Aedes aegypti

Mosquito-borne Dengue virus (DENV) has caused major disease worldwide, impacting 50 to 100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology and developing new control strategies becomes an important issue to eliminate DENV. We focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential anti-viral target in the mosquito. To understand the role played by autophagy in Ae. aegypti, we examined the expression of the pathway in vitro (Aag-2 cell) and in vivo (Ae. aegypti). The results indicated that DENV infection in Aag-2 cells caused the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) protein levels to increase which indicated the activation of the autophagy pathway. Rapamycin and 3-Methyladenine were used to activate or suppress the autophagy pathway, respectively. Rapamycin treatment decreased the virus titer in the Aag-2 cells, but the 3-Methyladenine treatment did not affect DENV titer. In Ae. aegypti, microinjected rapamycin increased the DENV titer after one-day infection and was significantly different compared to the control group titer. Two ATG genes, ATG4 and ATG12, were expressed differentially under the rapamycin treatments. Although the results differed between in vitro and in vivo studies, findings from both support the interaction between autophagy and DENV. Our studies revealed the activation of the autophagy pathway through rapamycin could be related to DENV infection in the mosquito. The possibility of autophagy being associated with different antiviral mechanisms at different extrinsic incubation times and tissues in Ae. aegypti is discussed. Author SummaryDengue virus (DENV) has been a great threat to public health and has not developed an efficient method to stop the transmission. To understand the complex interaction between virus and mosquito, we investigate the autophagy pathway and its role during the infection process. We noticed the induction of autophagy pathways from DENV infection in Aag-2 cells and blood meal from Ae. aegypti. Moreover, activation of the autophagy pathway from rapamycin could alter the DENV titer. Our results indicated the autophagy pathway is associated with DENV and could be crucial during the DENV infection. Furthermore, we proved the practicality of small molecules in altering the autophagy pathway in mosquitoes, and thus the usage of small molecules as possible mosquito pathogen vaccines should be evaluated.

molecular biology↗

bioRxiv2020

Combination of kdr mutation and detoxification gene expression associated with high tolerance for permethrin in a resistant Aedes aegypti population

Aedes aegypti, as one of the vectors transmitting several arboviruses, is a main target in mosquito control programs. Permethrin remains the major adulticide used to control these mosquitoes. The increasing percentage of permethrin resistant Aedes aegypti has become an important issue around the world. Knockdown resistance (kdr) is one of the major mechanisms related to permethrin resistance. On the other hand, detoxification genes including cytochrome P450 monooxygenases (P450) and glutathione S-transferases (GSTs) are also suggested as permethrin resistance apparatus. Here we selected a permethrin resistant (p-s) Aedes aegypti population from Florida and compared its mortality after exposure, median lethal dose (LD50), adult survivorship and larval development to several field populations. We used allele-specific PCR genotyping of the S989P, V1016I and F1534C sites in the sodium channel gene and gene expression analysis of several p450 and GSTs genes before and after permethrin exposure to determine their involvement in permethrin sensitivity between Ae. aegypti populations. Results indicated the p-s population had the highest resistance to permethrin based on LD50 and the mortality test. The larval development time did not significantly differ between the populations, however the p-s adults survived longer than the other populations. In the genotype study, p-s population had mostly homozygous mutations in all three mutant sites of the sodium channel gene. Detoxification gene expression studies showed that two p450 genes, AAEL009124 (CYP6N12) and, AAEL009127 (CYP6M11), were upregulated and, accession # AAEL006802, AAEL014891 (CYP6P12) and AAEL014619 (CYP9J22) were downregulated after 120 minutes of permethrin exposure in the p-s population. These results suggest that in highly permethrin resistant Aedes aegypti populations both kdr mutations and xenobiotic metabolism might be involved. Involvement of multiple mechanisms to achieve resistance to permethrin supports the need for implementing comprehensive mosquito control measures, such as an integrated pest management strategy, so that selection pressure for resistance is decreased without compromising control efforts while new methodologies are being developed. Author summaryPyrethroids have been applied as a major type of insecticide targeted at Aedes aegypti, a key vector in the transmission of several flaviviruses. Resistance to pyrethroids has emerged and has become a worldwide threat to mosquito control. Pyrethroid resistance usually occurs with knockdown resistance (kdr) where the voltage gated sodium channel is mutated. We selected a permethrin resistant (p-s) Aedes aegypti population from Florida and, along with two other field populations, examined three mutation sites, S989P, V1016I and F1534C. The data showed the p-s population had the most homozygous mutations which correlated to the permethrin resistance level. Besides kdr, detoxification genes also have been identified to have pyrethroid metabolizing abilities. We found two cytochrome P450 monooxygenases genes, CYP6N12 and CYP6M11, were overexpressed in the p-s population after permethrin exposure, suggesting a role in resistance to permethrin. Together, our results provide information about potential mechanisms used in major vectors with high insecticide resistance.

molecular biology↗