Fine-scale monitoring of insecticide resistance in Aedes aegypti (Diptera: Culicidae) from Sri Lanka and modeling the phenotypic resistance using rational approximation.

BACKGROUND: The unplanned and intensified use of insecticides to control mosquito-borne diseases has led to an upsurge of resistance to commonly used insecticides. Aedes aegypti, the main vector of dengue, chikungunya, and Zika virus, is primarily controlled through the application of adulticides (pyrethroid insecticides) and larvicides (temephos). Fine spatial-scale analysis of resistance may reveal important resistance-related patterns, and the application of mathematical models to determine the phenotypic resistance status lessens the cost and usage of resources, thus resulting in an enhanced and successful control program. METHODS: The phenotypic resistance for permethrin, deltamethrin, and malathion was monitored in the Ae. aegypti populations using the World Health Organization (WHO) adult bioassay method. Mosquitoes' resistance to permethrin and deltamethrin was evaluated for the commonly occurring base substitutions in the voltage-gated sodium channel (vgsc) gene. Rational functions were used to determine the relationship between the kdr alleles and the phenotypic resistant percentage of Ae. aegypti in Sri Lanka. RESULTS: The results of the bioassays revealed highly resistant Ae. aegypti populations for the two pyrethroid insecticides (permethrin and deltamethrin) tested. All populations were susceptible to 5% malathion insecticide. The study also revealed high frequencies of C1534 and G1016 in all the populations studied. The highest haplotype frequency was detected for the haplotype CC/VV, followed by FC/VV and CC/VG. Of the seven models obtained, this study suggests the prediction models using rational approximation considering the C allele frequencies and the total of C, G, and P allele frequencies and phenotypic resistance as the best fits for the area concerned. CONCLUSIONS: This is the first study to our knowledge to provide a model to predict phenotypic resistance using rational functions considering kdr alleles. The flexible nature of the rational functions has revealed the most suitable association among them. Thus, a general evaluation of kdr alleles prior to insecticide applications would unveil the phenotypic resistance percentage of the wild mosquito population. A site-specific strategy is recommended for monitoring resistance with a mathematical approach and management of insecticide applications for the vector population.

Genetic diversity and phylogeography of Phlebotomus argentipes (Diptera: Psychodidae, Phlebotominae), using COI and ND4 mitochondrial gene sequences.

BACKGROUND: Phlebotomus argentipes complex is the primary vector for cutaneous leishmaniasis, a burgeoning health concern in contemporary Sri Lanka, where effective vector control is important for proper disease management. Understanding the genetic diversity of the P. argentipes population in Sri Lanka is vital before implementing a successful vector control program. Various studies have indicated that genetic divergence, caused by genetic drift or selection, can significantly influence the vector capacity of arthropod species. To devise innovative control strategies for P. argentipes, exploring genetic diversity and phylogeography can offer valuable insights into vector competence, key genetic trait transfer, and impact on disease epidemiology. The primary objective is to analyze the genetic diversity and phylogeography of the P. argentipes complex in Sri Lanka, based on two mitochondrial genomic regions in modern representatives of P. argentipes populations. METHODOLOGY: A total of 159 P. argentipes specimens were collected from five endemic areas of cutaneous leishmaniasis and identified morphologically. Two mitochondrial regions (Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4) were amplified using the total DNA and subsequently sequenced. Partial sequences of those mitochondrial genes were utilized to analyze genetic diversity indices and to explore phylogenetic and phylogeographic relationships. PRINCIPAL FINDINGS: Among five sampling locations, the highest genetic diversity for COI and ND4 was observed in Hambantota (Hd-0.749, π-0.00417) and Medirigiriya (Hd-0.977, π-0.01055), respectively. Phylogeographic analyses conducted using COI sequences and GenBank retrieved sequences demonstrated a significant divergence of P. argentipes haplotypes found in Sri Lanka. Results revealed that they have evolved from the Indian ancestral haplotype due to historical- geographical connections of the Indian subcontinent with Sri Lanka. CONCLUSIONS: Utilizing high-mutation-rate mitochondrial genes, such as ND4, can enhance the accuracy of genetic variability analysis in P. argentipes populations in Sri Lanka. The phylogeographical analysis of COI gene markers in this study provides insights into the historical geographical relationship between India and P. argentipes in Sri Lanka. Both COI and ND4 genes exhibited consistent genetic homogeneity in P. argentipes in Sri Lanka, suggesting minimal impact on gene flow. This homogeneity also implies the potential for horizontal gene transfer across populations, facilitating the transmission of genes associated with traits like insecticide resistance. This dynamic undermines disease control efforts reliant on vector control strategies.

Genetic diversity, phylogenetic and phylogeographic analysis of Anopheles culicifacies species complex using ITS2 and COI sequences.

Anopheles culicifacies is the major vector of malaria in Sri Lanka and the Indian subcontinent which is characterized as a species complex with five sibling species provisionally designated as A, B, C, D and E. The current study was carried out to understand the phylogenetic and phylogeographic relationships between the sibling species of the species complex while observing their genetic diversity and genetic differentiation. Thirty-five ITS2 and seventy-seven COI sequences of An. culicifacies species complex reported from different geographical locations of Asia and China at the NCBI public database were used for the analysis. Bayesian likelihood trees were generated for the phylogenetic analysis. The divergence of the species complex was obtained from the Bayesian phylogeographic model in BEAST. There were two clades of the sibling species of An. culicifacies species complex as A, D and B, C and E in both phylogenetic and phylogeographic analysis using ITS2 sequences. Based on the highly divergent COI sequences and the high mutation rate of the mitochondrial genome, there were four and three clades in both phylogenetic and phylogeographic analysis using COI sequences. The diversification of An. culicifacies species complex was obtained as ranging from 20.25 to 24.12 Mya and 22.37 to 26.22 Mya based on ITS2 and COI phylogeographic analysis respectively. There was a recent diversification of the sibling species A and D than the sibling species B, C and E. Low haplotype diversity was observed in the sequences reported from Sri Lanka in both ITS2 and COI analysis that can be due to bottlenecks resulting from the intense malaria control efforts. A high genetic differentiation was achieved for some populations due to the large geographical distance. The high genetic diversity based on the five sibling species implies the possibility of maintaining a relatively high effective population size despite the vector control efforts.

Genetic Variation in the Domain II, 3' Untranslated Region of Human and Mosquito Derived Dengue Virus Strains in Sri Lanka.

Genetic variations in dengue virus (DENV) play a distinct role in epidemic emergence. The DENV 3' UTR has become a recent interest in research. The objective of the study was to examine the genetic variation in the domain II, 3' UTR region of human and mosquito-derived DENV. DENV-infected human sera were orally infected to laboratory reared Aedes aegypti mosquitoes. The domain II, 3' UTR of each human- and mosquito-derived sample was amplified. The nucleotide sequence variation, phylogenetic and secondary structure analysis was carried out incorporating respective regions of so far recorded Sri Lankan and the reference genotype strains of the DENV3 and DENV1 serotypes. The human- and mosquito-derived domain II, 3' UTR were identical in nucleotide sequences within the serotypes isolated, indicating the conserved nature of the region during host switch. The sequence analysis revealed distinct variations in study isolates compared to so far recorded Sri Lankan isolates. However, despite single nucleotide variations, the maintenance of structural integrity was evident in related strains within the serotypes in the secondary structure analysis. The phylogenetic analysis revealed distinct clade segregation of the study sequences from so far reported Sri Lankan isolates and illustrated the phylogenetic relations of the study sequences to the available global isolates of respective serotypes.

Compensatory Base Changes Reveal Sexual Incompatibility among Members of the Anopheles subpictus Sensu Lato (Diptera: Culicidae) Species Complex in Sri Lanka.

The mosquito Anopheles (Cellia) subpictus sensu lato (s.l.) is a major secondary vector of malaria in Sri Lanka. The sibling species composition in this species complex in Sri Lanka remains debatable. Compensatory base changes (CBCs) in the secondary structures of internal transcribed spacer 2 (ITS2) are reliable sources to predict sexual incompatibility among closely related species. The objective of the present study was to investigate the An. subpictus s.l. populations in Sri Lanka using the CBC analysis. Mosquito DNA was amplified and sequenced for the ITS2 region. The sequences were annotated using ITS2 Database. ITS2 secondary structures were constructed and analyzed for CBCs using various bioinformatics tools. The ITS2 regions consisted of two different lengths, 575 bp and 480 bp. The two CBCs and three hemi CBCs identified in the present study suggest that there may be at least two sexually incompatible sibling species. In conclusion, it is likely that there may be only two reproductively isolated sibling species in the An. subpictus species complex in Sri Lanka. However, due to high divergence of ITS2 in these species, it is reasonable to assume that they may be undergoing a speciation event to separate as a distinct species.

Mitochondrial metabolic genes provide phylogeographic relationships of global collections of Aedes aegypti (Diptera: Culicidae).

Phylogeographic relationships among global collections of the mosquito Aedes aegypti were evaluated using the mitochondrial Cytochrome C Oxidase 1 (CO1) and NADH dehydrogenase subunit 4 (ND4) genes including new sequences from Sri Lanka. Phylogeographic analysis estimated that Ae. aegypti arose as a species ~614 thousand years ago (kya) in the late Pleistocene. At 545 kya an "early" East African clade arose that continued to differentiate in East Africa, and eventually gave rise to three lineages one of which is distributed throughout all tropical and subtropical regions, a second that contains Southeast Asian/Sri Lankan mosquitoes and a third that contains mostly New World mosquitoes. West African collections were not represented in this early clade. The late clade continued to differentiate throughout Africa and gave rise to a lineage that spread globally. The most recent branches of the late clade are represented by South-East Asia and India/Pakistan collections. Analysis of migration rates suggests abundant gene flow between India/Pakistan and the rest of the world with the exception of Africa.

Gene Flow Patterns Among Aedes aegypti (Diptera: Culicidae) Populations in Sri Lanka.

In Sri Lanka, dengue is the most serious arboviral disease. Recent increases in dengue cases suggest a higher infection rate and spread of the disease to new areas. The present study explores gene flow patterns of Ae. aegypti, the main vector of dengue disease, among 10 collection sites including major ports and inland cities using variations at 11 microsatellite loci. Discriminant analysis of principal components (DAPC) and k-means clustering estimated eight genetic clusters. Analysis of Molecular Variance (AMOVA) estimated equal variances among cities and among collections in Colombo, Sri Lanka. Significant evidence, although weak, was detected for isolation by distance. Analysis of gene flow rates and directions using MIGRATE-n indicated that populations throughout the island served as a source of immigrants for Colombo with abundant gene flow among major commercial cities in Sri Lanka, which appear to receive migrant mosquitoes from throughout Sri Lanka. The observed patterns probably arise through human movement of Ae. aegypti during commerce from throughout Sri Lanka into Colombo increasing the risk of spread. The patterns uncovered in this study are significant for global health as Sri Lanka is situated along a key international shipping route.

Mitochondrial-COII sequence polymorphism reflects spatial genetic clustering of Anopheles culicifacies sibling species E in Sri Lanka.

BACKGROUND & OBJECTIVES: Malaria infects around 216 million people annually with estimated 445,000 deaths globally. Anopheles culicifacies is the vector of malaria in Sri Lanka, a complex of five morphologically identical sibling species of which precise identification using DNA-based methods is still under experimentation. This study was carried out in Sri Lanka to observe the utility of BCE-PCR assay based on mitochondrial Cytochrome Oxidase II (COII) developed in India, in sibling species B and E identification in Sri Lanka, to characterize nucleotide and corresponding amino acid sequences of COII region in major vector sibling species E in Sri Lanka and to analyze the spatial distribution pattern of sibling species E in Sri Lanka using microsatellite markers. METHODS: BCE-PCR was carried out for the samples to identify their sibling status. Sequencing of COII region was then carried out to investigate the genetic diversity of Sri Lankan sibling species E, sequences were aligned and compared; microsatellite genotyping was carried out and the spatial clustering pattern was analyzed. RESULTS: Identification of sibling species B and E using BCE-PCR was confusing due to the heterogeneity in the COII region of sibling species in Sri Lanka. Non-synonymous substitutions were detected in COII gene amongst sibling species E. Spatial distributed two clusters were detected in the studied population. INTERPRETATION & CONCLUSION: Existence of genetic variants among sibling species is suggested in Sri Lanka. Further, the pattern of sibling species identification in BCE-PCR was reflected in the spatial clustering of sibling E in Sri Lanka.

Microcystin-LR-induced cytotoxicity and apoptosis in human embryonic kidney and human kidney adenocarcinoma cell lines.

Microcystin-LR (MC-LR) is a potent hepatotoxin, and increasing evidence suggests that it might also induce kidney injury. The aim of the present work was to evaluate the cytotoxicity and possible apoptotic effects of MC-LR on a human embryonic kidney cell line (HEK-293) and human kidney adenocarcinoma cell line (ACHN). Cells were exposed for 24 h to pure MC-LR (1.0-200 µM) and the cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulphorhodamine B (SRB) cell viability assays. Cell viability in both cell lines was significantly decreased after treatment with MC-LR at 50 µM for 24 h (P<0.001). Moreover, MC-LR-treated ACHN and HEK-293 cells exhibited a marked dose-dependent loss of confluence as judged by phase-contrast microscopy. Similarly, fluorescence microscopic observations following acridine orange-ethidium bromide (AO/EB) staining confirmed that both cell types were undergoing apoptosis after treatment with MC-LR for 24 h. Expression of three apoptosis-related genes, Bax, Survivin and p53, was analysed by quantitative reverse transcriptase PCR analysis. Both Bax and p53 functioned as promoters of MC-LR-mediated apoptosis in ACHN and HEK-293 cells. The Survivin gene acted as a suppressor of apoptosis at lower MC-LR concentration (1 µM) and the gene was upregulated at higher MC-LR concentration (10 µM) (P<0.001). Significant increases of caspase 3 (P<0.0001) and caspase 9 (P<0.0001) activity were detected in both cell lines after exposure to MC-LR for 24 h, indicating the MC-LR induces cytotoxicity and a marked apoptosis in both ACHN and HEK-293 kidney cell lines.

Studies on the efficacy of Toxorhynchites larvae and three larvivorous fish species for the control of Aedes larval populations in water-storage tanks in the Matale district of Sri Lanka.

A study was conducted to compare the feeding efficacy of Toxorhynchites larvae (L3 & L4) and three larvivorous fish species on Aedes larvae. Ground-level cement water-storage tanks (20%–80%) and water-storing barrels (8.33%–54.55%) formed the majority of Aedes-positive outdoor containers. Ae. albopictus, Ae. macdougali and Ae. vittatus were recorded in water-storage tanks, with Ae. macdougali being dominant. In the laboratory, the consumption rate (time to devour 10 Ae. albopictus L3 larvae in a vessel of 78.57 cm2 of surface area) for Toxorhynchites was significantly lower (mean time of 330 minutes) than for any of the tested fish species, Poecilia reticulata (Guppy), Puntius bimaculatus (Ipilli Kadaya) and Rasbora caveri (Dandiya), which needed 16.67, 27.33 and 24 minutes respectively. There were no significant differences (P=0.062) between the consumption rates of the three fish species. A field study was carried out to determine the feeding efficacy of Toxorhynchites larvae, P. reticulata, P. bimaculatus and R. caveri on Aedes larval populations in outdoor cement tanks by noting the percentage reduction of Aedes larvae per 100 cm2 surface area after one week. Toxorhynchites larvae caused a 20%–83.33% reduction with 1–8 larvae per tank. A complete reduction (100%) was achieved with P. bimaculatus and R. caveri with 1–3 fish per tank. P. reticulata showed similar results, but with 90% reduction being achieved once with two fish per tank. There was a higher possibility of losing Tx. larvae than the fish species during the removal of water by the householders. The efficiency of the three fish species for consuming Aedes larvae was greater than that with Tx. larvae. It appears feasible to use Puntius bimaculatus, Rasbora caveri and Poecilia reticulata for controlling Aedes breeding in outdoor cement water-storage tanks in Sri Lanka.

Anopheles culicifacies sibling species B and E in Sri Lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides.

Members of the Anopheles culicifacies Giles complex (Diptera: Culicidae) are well established as the predominant vectors of malaria in Sri Lanka. Until recently, only sibling species B was reported to be present in Sri Lanka, which was surprising as species B is a poor vector of malaria in India. This was clarified by the identification through Y-chromosome morphology that what was reported as B on the island is really a mixture of B and E. The fecundity, longevity and insecticide resistance of B and E are of relevance to malaria transmission and its control and are reported in this study. The mean egg production of these two sibling species did not differ significantly. The mean age of wild mosquitoes was assessed by the Polovodova technique of observing ovarian dilatations. More of species E than B had three or more dilatations, i.e. had reached an age at which sporozoites could have developed to maturity, although the difference between the species was of borderline significance. Following feeding on Plasmodium vivax or Plasmodium falciparum infected blood, some females of species E developed oocysts but none of species B did so. Both sibling species were found fully susceptible in laboratory tests to lambdacyhalothrin and deltamethrin, but resistant to DDT and partially resistant to malathion.