Genetic diversity and population structure of malaria vector mosquitoes Anopheles subpictus, Anopheles peditaeniatus, and Anopheles vagus in five districts of Sri Lanka.

BACKGROUND: Although Sri Lanka is considered as a malaria-free nation, the threat of re-emergence of outbreaks still remains due to the high prevalence and abundance of malaria vectors. Analysis of population genetic structure of malaria vectors is considered to be one of the vital components in implementing successful vector control programmes. The present study was conducted to determine the population genetic structure of three abundant malaria vectors; Anopheles subpictus sensu lato (s.l.), Anopheles peditaneatus and Anopheles vagus from five administrative districts in two climatic zones; intermediate zone (Badulla and Kurunegala districts) and dry zone (Ampara, Batticoloa and Jaffna districts) of Sri Lanka using the mitochondrial gene, cytochrome c oxidase subunit I (COI). METHODS: Adult mosquitoes of An. subpictus s.l., An. peditaeniatus, and An. vagus were collected from five study sites located in five districts using cattle baited traps and backpack aspirators. Representative samples of each species that were morphologically confirmed were selected from each locality in generating COI sequences (> 6 good quality sequences per species per locality). RESULTS: Anopheles subpictus s.l. specimens collected during the study belonged to two sibling species; An. subpictus 'A' (from all study sites except from Jaffna) and An. subpictus 'B' (only from Jaffna). The results of haplotype and nucleotide diversity indices showed that all the three species are having high genetic diversity. Although a high significant pairwise difference was observed between An. subpictus 'A' and 'B' (Fst> 0.950, p < 0.05), there were no significant genetic population structures within An. peditaeniatus, An. vagus and An. subpictus species A (p > 0.05), indicating possible gene flow between these populations. CONCLUSIONS: Gene flow among the populations of An. peditaeniatus, An. vagus and An. subpictus species A was evident. Application of vector control measures against all mosquito species must be done with close monitoring since gene flow can assist the spread of insecticide resistance genes over a vast geographical area.

Molecular characterization of Anopheline (Diptera: Culicidae) mosquitoes from eight geographical locations of Sri Lanka.

BACKGROUND: Genus Anopheles is a major mosquito group of interest in Sri Lanka as it includes vectors of malaria and its members exist as species complexes. Taxonomy of the group is mainly based on morphological features, which are not conclusive and can be easily erased while handling the specimens. A combined effort, using morphology and DNA barcoding (using the markers cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) region, was made during the present study to recognize anophelines collected from eight districts of Sri Lanka for the first time. METHODS: Cytochrome c oxidase subunit I and ITS2 regions of morphologically identified anopheline mosquitoes from Sri Lanka were sequenced. These sequences together with GenBank sequences were used in phylogenetic tree construction and molecular characterization of mosquitoes. RESULTS: According to morphological identification, the field-collected adult mosquitoes belonged to 15 species, i.e., Anopheles aconitus, Anopheles annularis, Anopheles barbirostris, Anopheles culicifacies, Anopheles jamesii, Anopheles karwari, Anopheles maculatus, Anopheles nigerrimus, Anopheles pallidus, Anopheles peditaeniatus, Anopheles pseudojamesi, Anopheles subpictus, Anopheles tessellatus, Anopheles vagus, and Anopheles varuna. However, analysis of 123 COI sequences (445 bp) (16 clades supported by strong bootstrap value in the neighbour joining tree and inter-specific distances of >3%) showed that there are 16 distinct species. Identity of the morphologically identified species, except An. subpictus, was comparable with the DNA barcoding results. COI sequence analysis showed that morphologically identified An. subpictus is composed of two genetic entities: An. subpictus species A and species B (inter-specific K2P distance 0.128). All the four haplotypes of An. culicifacies discovered during the present study belonged to a single species. ITS2 sequences (542 bp) were obtained for all the species except for An. barbirostris, An. subpictus species B, An. tessellatus, and An. varuna. Each of these sequences was represented by a single species-specific haplotype. CONCLUSIONS: The present study reflects the importance and feasibility of COI and ITS2 genetic markers in identifying anophelines and their sibling species, and the significance of integrated systematic approach in mosquito taxonomy. Wide distribution of malaria vectors in the country perhaps indicates the potential for re-emergence of malaria in the country.

Efficacy of the insect growth regulator novaluron in the control of dengue vector mosquitoes Aedes aegypti and Ae. albopictus.

Insect Growth Regulator (IGR) novaluron is an alternative to synthetic neuro-inhibitory insecticides. Present study was designed to assess appropriate dosages of novaluron for dengue vector control. Larvae of Aedes aegypti and Ae. albopictus were exposed to a concentration series of novaluron (Rimon EC10) for two fixed exposure periods of 7-days and 14-days to determined LC50 and LC99 values. Inhibition of adult emergence (IE50 and IE99) was determined by a 14-day exposure. Semi-field experiments were conducted by exposing cohorts of Ae. aegypti larvae to IE99, 2 × IE99 and 10 × IE99 novaluron concentrations in water storage buckets (10 L) and plastic barrels (200 L). For the 7-day exposure, LC50 values were 0.047-0.049 ppm and LC99 were 0.144-0.151 ppm. For 14-day exposure, these values were 0.002-0.005 ppm and 0.006-0.01 ppm respectively. For both species, IE99 was 0.001 ppb under semi-field conditions, and was effective for nearly 2 months. Novaluron concentration 0.01 ppb was effective up to 3 months, with an IE of 89-95%. Authorities should critically review a reduction of the presently recommended field dosage of 200 ppm novaluron by × 100 or more. This would provide the same efficacy but mitigate environmental pollution, development of vector resistance, and financial losses.

Breeding Habitat Preference of the Dengue Vector Mosquitoes Aedes aegypti and Aedes albopictus from Urban, Semiurban, and Rural Areas in Kurunegala District, Sri Lanka.

Elimination of vector mosquito larvae and their breeding environments is an effective strategy in dengue disease control. Present study examined larval density and water quality in breeding habitats and container preference of dengue vectors Ae. aegypti and Ae. albopictus. Larval surveys were conducted monthly in urban, semiurban, and rural sites in Kurunegala, Sri Lanka, from January 2019 to December 2021. Larval densities were recorded under the following three categories: type of container (16 types), type of material (6 types), and location (indoor/outdoor). Breeding preference ratios (BPRs) were calculated using Index of Available Containers and the Index of Contribution to Breeding Sites. Out of 19,234 wet containers examined, larval stages were found in 1,043 habitats. Ae. albopictus larvae were in all three areas whereas Ae. aegypti larvae were restricted to urban areas. Highest number of wet containers and highest positivity were reported from urban followed by semiurban. In general, discarded nondegradable items were the most frequent and mostly positive breeding sites. For Ae. aegypti, the most preferred breeding sites were gutters and concrete slabs. Ae. albopictus mostly preferred concrete slabs in urban areas and tyres in semiurban and rural areas. Material types such as rubber and concrete were mostly preferred by Ae. aegypti whereas ceramic was preferred by Ae. albopictus. Although plastic was the most available material type in all study sites, preference to plastic was low except for urban Ae. albopictus. Both species preferred urban indoor breeding habitats although outdoor breeding was preferred by Ae. albopictus in rural areas. Larval densities of Ae. aegypti and semiurban Ae. albopictus significantly correlated with the BPR of the container type and material type. Dengue vector larvae were found in a 6.7-9.4 pH range. Total dissolved solids and alkalinity positively correlated with preference. Information generated can be successfully used in waste management and public education for effective vector control.

Weather-Based Prediction Models for the Prevalence of Dengue Vectors Aedes aegypti and Ae. albopictus.

Dengue is an important vector-borne disease transmitted by the mosquitoes Aedes aegypti and Ae. albopictus. In the absence of an effective vaccine, vector control has become the key intervention tool in controlling the disease. Vector densities are significantly affected by the changing weather patterns of a region. The present study was conducted in three selected localities, i.e., urban Bandaranayakapura, semiurban Galgamuwa, and rural Buluwala in the Kurunegala district of Sri Lanka to assess spatial and temporal distribution of dengue vector mosquitoes and to predict vector prevalence with respect to changing weather parameters. Monthly ovitrap surveys and larval surveys were conducted from January to December 2019 and continued further in the urban area up to December 2021. Aedes aegypti was found moderately in the urban area and to a lesser extent in semiurban but not in the rural area. Aedes albopictus had the preference for rural over urban areas. Aedes aegypti preferred indoor breeding, while Ae. albopictus preferred both indoor and outdoor. For Ae. albopictus, ovitrap index (OVI), premise index (PI), container index (CI), and Breteau index (BI) correlated with both the rainfall (RF) and relative humidity (RH) of the urban site. Correlations were stronger between OVI and RH and also between BI and RF. Linear regression analysis was fitted, and a prediction model was developed using BI and RF with no lag period (R 2 (sq) = 86.3%; F = 53.12; R 2 (pred) = 63.12%; model: Log10 (BI) = 0.153 + 0.286 ∗ Log10 (RF); RMSE = 1.49). Another prediction model was developed using OVI and RH with one month lag period (R 2 (sq) = 70.21%; F = 57.23; model: OVI predicted = 15.1 + 0.528 ∗ Lag 1 month RH; RMSE = 2.01). These two models can be used to monitor the population dynamics of Ae. albopictus in urban settings to predict possible dengue outbreaks.

Genetic diversity and population structure of Phlebotomus argentipes: Vector of Leishmania donovani in Sri Lanka.

Phlebotomus argentipes is the vector of Leishmania donovani which causes the disease leishmaniasis, a neglected tropical disease and a growing health problem in Sri Lanka. A proper understanding of the population genetic structure of sand fly vectors is considered important prior to planning and implementation of a successful vector control program. Thus, the present study was conducted to determine the population genetic structure of sand fly vectors in Sri Lanka. Two mitochondrial genes namely Cytochrome c oxidase subunit 1 (Cox 1) and Cytochrome b (Cytb), and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. Analyses included maximum likelihood method, network analysis and DNA polymorphisms. The outcome revealed unique sequences of all genomic regions studied except the cox 1 gene had a relationship with sand flies isolated previously from Sri Lanka, India and Israel and cytb gene of 4 sand flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar. Furthermore, cox 1 gene and ITS 2 region analyses based on FST values indicated a possible gene flow between the study sites whereas cytb gene analysis favoured the existence of genetically distinct populations of P. argentipes in each of the study sites. Poor population differentiation of P. argentipes, a possible consequence of a gene flow, is indeed of concern due to the risk imposed by promoting the spread of functionally important phenotypes such as insecticide resistance across the country, making future vector control efforts challenging.

Insecticide resistance mechanisms with novel 'kdr' type gene mutations in the tropical bed bug Cimex hemipterus.

BACKGROUND: The tropical bed bug, Cimex hemipterus, is a serious indoor public health pest in tropical regions causing intense physical discomfort and mental distress to humans. At present, the application of insecticides is the major control strategy. The present study was designed to evaluate the development of resistance and resistance mechanisms in Cimex hemipterus from Kandy district, Sri Lanka. METHODS: The resistance status of the collected bed bugs was determined against the discriminative dosages of DDT, malathion, propoxur, deltamethrin and permethrin by conducting bioassays according to World Health Organization guidelines. Activities of insecticide metabolizing enzymes, i.e. esterases, glutathione S-transferases (GST) and monooxygenases, and the insensitivity of organophosphate/carbamate target site acetylcholinesterase (AChE), were evaluated by biochemical assays. Regions of the gene of the pyrethroid/DDT target site, the voltage-gated sodium channel regulatory protein (VGSC), were sequenced for possible kdr mutations. RESULTS: Survival percentages of bed bug population were 71, 68 and 51% for DDT, malathion and propoxur respectively. KT50 and KT90 values, calculated using log-probit mortality curves for deltamethrin were 62.55 and 123.96 h, respectively. These values were much higher for permethrin where KT50 was 201.10 h and the KT90 was beyond the detectable range. Results were compared with previous values reported for the same population in 2002. Resistance to propoxur has increased significantly from 11 to 51% with about a 20-fold increase in the number of individuals with elevated esterase mechanism. No significant change has occurred in malathion and DDT resistance, in GST and monooxygenase activities, and in AChE sensitivity for the past 14 years. Six kdr associated mutations (Y/L995H, V1010L, I1011F, L1014F, V1016E, L1017F/S) and a non-kdr associated mutation (A1007S mutation) were found from the α-region of the VGSC gene. Out of the kdr type mutations, only L1014F has been reported previously form C. hemipterus while the others have been reported from other insects. CONCLUSIONS: The bed bug population has developed high resistance to propoxur with increased esterase activities. KT50 for deltamethrin and permethrin has increased 125- and 20-fold, respectively, over the period 2002 to 2016. To the authors' knowledge, this is the first time that the possible involvement of a kdr type mutation in developing pyrethroid resistance in C. hemipterus has been shown in Sri Lanka.

Seasonal exposure of fish to neurotoxic pesticides in an intensive agricultural catchment, Uma-oya, Sri Lanka: linking contamination and acetylcholinesterase inhibition.

The annual cultivation pattern in the Uma-oya catchment in Sri Lanka is characterized by Yala and Maha rainfall periods and associated cropping. Two cultivation seasons were compared for pesticide residues: base flow, field drainage, and the runoff and supplementary sediment data for three sites in the catchment. Organophosphate and N-methyl carbamate pesticide analysis confirmed a higher concentration in the Yala season with low-flow conditions. Acetylcholinesterase (AChE) activity was measured by standard spectrometry in the brain, muscle, and eye tissues of three freshwater cyprinid fishes, Garra ceylonensis, Devario malabaricus, and Rasbora daniconius from three study sites during months overlapping two seasons in 2010 (December) and 2011 (July). Baseline AChE data were measured from fish samples from a forested reserve in the Knuckles. A 73% inhibition in muscle AChE activity in G. ceylonensis was associated with intense pesticide exposure months in the Yala season. The AChE inhibition more than 70% in G. ceylonensis eyes in both Yala (76%) and Maha (72.5%) seasons indicates particular sensitivity of eye tissue to inhibitors. The less dramatic AChE inhibition in the eye tissues in D. malabaricus and R. daniconius in both seasons indicates exemplary protective capacity of muscle AChE in fish. The highest inhibition of AChE (up to 60% in brain and up to 56% in muscle AChE activity in R. daniconius and up to 47.8% in brain and up to 64.6% in muscle AChE activity in D. malabaricus) occurred during the Yala season. Tissue AChE activity and physiological activity in fish were correlated. The results collectively indicate that AChE is a consistent biomarker for diffused contaminant exposure in agricultural catchments.