In silico identification, characterization, and expression analysis of RNA recognition motif (RRM) containing RNA-binding proteins in Aedes aegypti.

RNA-binding proteins (RBPs) are the proteins that bind RNAs and regulate their functioning. RBPs in mosquitoes are gaining attention due to their ability to bind flaviviruses and regulate their replication and transmission. Despite their relevance, RBPs in mosquitoes are not explored much. In this study, we screened the whole genome of Aedes aegypti, the primary vector of several pathogenic viruses, and identified the proteins containing RNA recognition motif (RRM), the most abundant protein domain in eukaryotes. Using several in silico strategies, a total of 135 RRM-containing RBPs were identified in Ae. aegypti. The proteins were characterized based on their available annotations and the sequence similarity with Drosophila melanogaster. Ae. aegypti RRM-containing RBPs included serine/arginine-rich (SR) proteins, polyadenylate-binding proteins (PABP), heteronuclear ribonucleoproteins (hnRNP), small nuclear ribonucleoproteins (snRNP), splicing factors, eukaryotic initiation factors, transformers, and nucleolysins. Phylogenetic analysis revealed that the proteins and the domain organization are conserved among Ae. aegypti, Bombyx mori, and Drosophila melanogaster. However, the gene length and the intron-exon organization varied across the insect species. Expression analysis of the genes encoding RBPs using publicly available RNA sequencing data for different developmental time points of the mosquito life cycle starting from the ovary and eggs up to the adults revealed stage-specific expression with several genes preferentially expressed in early embryonic stages and blood-fed female ovaries. This is the first database for the Ae. aegypti RBPs that can serve as the reference base for future investigations. Stage-specific genes can be further explored to determine their role in mosquito growth and development with a focus on developing novel mosquito control strategies.

Seasonal abundance and infection of Japanese encephalitis vectors from Gorakhpur district, Uttar Pradesh, India.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a major public health problem in many states of India. Uttar Pradesh state contributes 75% of the total cases reported. A longitudinal study was undertaken to find out the seasonal abundance, infection in the JE vectors in Gorakhpur region of Uttar Pradesh, and intervention strategies like indoor residual spraying and long-lasting insecticidal nets were adopted. METHODS: The vector abundance was monitored from July 2013 to August 2016 at fortnightly intervals and identified using standard mosquito identification keys and screened for viral infection. Intervention measures like indoor residual spraying with lambda-cyhalothrin 10% WP at 25 mg/m2 in 58 gramasabha in Bhathat Block and long-lasting insecticidal nets were distributed @ 2 LLIN for each household with 5-6 members in 5 villages of Korabar block. RESULTS: A total of 5,36,609 mosquitoes constituting 34 species and 10 genera were collected during this study period. Among the JE vector, peak abundance of Culex tritaeniorhynchus was recorded just before the JE season from July to October in all the blocks. There was a marked reduction in the density of Cx. tritaeniorhynchus from July compared to baseline year 2014 in Bhathat Block where indoor residual spraying (IRS) was performed in June 2015. The vector density declined in 5 villages of Khorabar Block where long-lasting impregnated bed nets (LLINs) were distributed during July 2016. INTERPRETATION & CONCLUSION: The present study provided knowledge about the seasonal JE vector density and JE virus infection in mosquitoes during the monsoon season in Gorakhpur region of Uttar Pradesh. IRS and the personal protection measure like LLINs were distributed to interrupt the JE transmission in this area which gave encouraging results.

DNA barcoding and molecular evolution of mosquito vectors of medical and veterinary importance.

Mosquitoes (Diptera: Culicidae) are a key threat for millions of people worldwide, since they act as vectors for devastating pathogens and parasites. The standard method of utilisation of morphological characters becomes challenging due to various factors such as phenotypical variations. We explored the complementary approach of CO1 gene-based identification, analysing ten species of mosquito vectors belonging to three genera, Aedes, Culex and Anopheles from India. Analysed nucleotide sequences were found without pseudo genes and indels; they match with high similarity in nucleotide Basic Local Alignment Search Tool (BLASTn) search. The partial CO1 sequence of Anopheles niligricus was the first time record submitted to National Center for Biotechnology Information (NCBI). Mean intra- and interspecies divergence was found to be 1.30 and 3.83 %, respectively. The congeneric divergence was three times higher than the conspecifics. Deep intraspecific divergence was noted in three of the species, and the reason could be explained more accurately in the future by improving the sample size across different locations. The transitional and transversional substitutions were tested individually. Ts and Tv substitutions in all the 1st, 2nd and 3rd codons were estimated to be (0.44, 99.51), (40.35, 59.66) and (59.16, 40.84), respectively. Saturation of the sequences was resolved, since both the Ts and Tv exhibited a linear relationship suggesting that the sequences were not saturated. NJ and ML tree analysis showed that the individuals of the same species clustered together based on the CO1 sequence similarity, regardless of their collection site and geographic location. Overall, this study adds basic knowledge to molecular evolution of mosquito vectors of medical and veterinary importance and may be useful to improve biotechnological tools employed in Culicidae control programmes.

Genetic deviation in geographically close populations of the dengue vector Aedes aegypti (Diptera: Culicidae): influence of environmental barriers in South India.

Mosquitoes are vectors of devastating pathogens and parasites, causing millions of deaths every year. Dengue is a mosquito-borne viral infection found in tropical and subtropical regions around the world. Recently, dengue transmission has strongly increased in urban and semiurban areas, becoming a major international public health concern. Aedes aegypti (Diptera: Culicidae) is a primary vector of dengue. Shedding light on genetic deviation in A. aegypti populations is of crucial importance to fully understand their molecular ecology and evolution. In this research, haplotype and genetic analyses were conducted using individuals of A. aegypti from 31 localities in the north, southeast, northeast and central regions of Tamil Nadu (South India). The mitochondrial DNA region of cytochrome c oxidase 1 (CO1) gene was used as marker for the analyses. Thirty-one haplotypes sequences were submitted to GenBank and authenticated. The complete haplotype set included 64 haplotypes from various geographical regions clustered into three groups (lineages) separated by three fixed mutational steps, suggesting that the South Indian Ae. aegypti populations were pooled and are linked with West Africa, Columbian and Southeast Asian lineages. The genetic and haplotype diversity was low, indicating reduced gene flow among close populations of the vector, due to geographical barriers such as water bodies. Lastly, the negative values for neutrality tests indicated a bottle-neck effect and supported for low frequency of polymorphism among the haplotypes. Overall, our results add basic knowledge to molecular ecology of the dengue vector A. aegypti, providing the first evidence for multiple introductions of Ae. aegypti populations from Columbia and West Africa in South India.

Genetic differentiation among Aedes aegypti populations from different eco-geographical zones of India.

The present study explicitly evaluated the genetic structure of Aedes aegypti Linn, the vector of dengue, chikungunya, and Zika viruses, across different geo-climatic zones of India and also elucidated the impact of ecological and topographic factors. After data quality checks and removal of samples with excess null alleles, the final analysis was performed on 589 individual samples using 10 microsatellite markers. Overall findings of this study suggested that, Ae. aegypti populations are highly diverse with moderate genetic differentiation between them. Around half of the populations (13 out of 22) formed two genetic clusters roughly associated with geographical regions. The remaining nine populations shared genetic ancestries with either one or both of the clusters. A significant relationship between genetic and geographic distance was observed, indicating isolation by distance. However, spatial autocorrelation analysis predicted the signs of long-distance admixture. Post-hoc environmental association analysis showed that 52.7% of genetic variations were explained by a combination of climatic and topographic factors, with latitude and temperature being the best predictors. This study indicated that though overall genetic differentiation among Ae. aegypti populations across India is moderate (Fst = 0.099), the differences between the populations are developing due to the factors associated with geographic locations. This study improves the understanding of the Ae. aegypti population structure in India that may assist in predicting mosquito movements across the geo-climatic zones, enabling effective control strategies and assessing the risk of disease transmission.

Predation of Aedes aegypti Eggs By Foraging Ants Solenopsis invicta, Myrmicaria brunnea, Diacamma rugosum, and Monomorium minimum.

The diseases transmitted by Aedes mosquitoes, such as dengue, chikungunya, yellow fever, and Zika, are ever-increasing. Rapid and unplanned urbanization adversely impacts various endemic species such as ants and facilitates the breeding of Aedes mosquitoes. We have observed the predatory potential of ants over Aedes eggs in urban breeding habitats, and their impact on Aedes mosquito breeding was determined by a field experiment that mimicked the natural breeding habitats. It was found that 99.4% of eggs were removed from the experimental containers by foraging ants in 4 days. The present study demonstrates the role of ants as a natural regulator, limiting Aedes mosquito breeding.

Chikungunya virus isolated in Lakshadweep islands in the Indian Ocean: evidence of the Central/East African genotype.

Virological investigation was carried out to determine the etiology of suspected Chikungunya fever among humans reported in the Lakshadweep islands in the Indian Ocean. Three out of 23 acute sera samples showed cytopathological changes in Vero cell lines. Further, indirect immunofluorescence antibody test and reverse transcriptase-polymerase chain reaction studies demonstrated the Chikungunya virus etiology during the episode. E1 gene sequence analysis has confirmed the involvement of the Central/East African genotype of the Chikungunya virus.

Natural vertical transmission of dengue virus in Aedes albopictus (Diptera: Culicidae) in Kerala, a southern Indian state.

The natural occurrence of vertical transmission of dengue viruses in Aedes albopictus (Skuse) mosquitoes was examined in the state of Kerala in southern India. Adults and larvae of Ae. albopictus collected from Kerala were screened for dengue viruses by using enzyme-linked immunosorbent assay with dengue-specific monoclonal antibodies. The possibility of the vertical transmission of dengue virus in Ae. albopictus was further evidenced by the detection of the virus in field-collected adult males as well as females emerged from field-collected larvae. Two pools, one pool from the adult males and one pool from the emerged females derived from field-collected larvae, were collected in the relatively hot months of June and March, respectively, and found to be positive for dengue virus antigen. Dengue serotype 2 virus was isolated from field-collected male adults in Kerala. These findings suggest that dengue virus is maintained in Ae. albopictus mosquitoes during the dry season by vertical transmission in nature.

The treatment of visceral leishmaniasis (kala-azar) in India: no obvious signs of long-term success.

In India, the eastern state of Bihar is particularly badly affected by visceral leishmaniasis (VL). It was in Bihar in the 1980s that the first clear signs of resistance to pentavalent antimonials, which had then been the standard antileishmanial treatment for several decades, were observed. New drugs and new formulations of old drugs have since been developed for the treatment of VL. However, despite some initial signs of benefit after each major revision in the method of treatment of VL in India, the VL-related case fatality rates recorded in India since the 1970s show no clear evidence of long-term success. In fact, the most recent data indicate that such rates have stabilised or even increased, probably because of the continued usage of sodium stibogluconate in northern Bihar.

Prediction of 3-dimensional structure of salivary odorant-binding protein-2 of the mosquito Culex quinquefasciatus, the vector of human lymphatic filariasis.

Olfaction of insects is currently recognized as the major area of research for developing novel control strategies to prevent mosquito-borne infections. A 3-dimensional model (3D) was developed for the salivary gland odorant-binding protein-2 of the mosquito Culex quinquefasciatus, a major vector of human lymphatic filariasis. A homology modeling method was used for the prediction of the structure. For the modeling, two template proteins were obtained by mGenTHERADER, namely the high-resolution X-ray crystallography structure of a pheromone-binding protein (ASP1) of Apis mellifera L., [1R5R:A] and the aristolochene synthase from Penicillium roqueforti [1DI1:B]. By comparing the template protein a rough model was constructed for the target protein using MODELLER, a program for comparative modelling. The structure of OBP of the mosquito Culex quinquefasciatus resembles the structure of pheromone-binding protein ASP1 of Apis mellifera L., [1R5R:A]. From Ramachandran plot analysis it was found that the portion of residues falling into the most favoured regions was 86.0%. The predicted 3-D model may be further used in characterizing the protein in wet laboratory.