Fusion of spectral and topographic features for land use mapping using a machine learning framework for a regional scale application.

This study investigated the dynamics of land use and land cover (LULC) modelling, mapping, and assessment in the Kegalle District of Sri Lanka, where policy decision-making is crucial in agricultural development where LULC temporal datasets are not readily available. Employing remotely sensed datasets and machine learning algorithms, the work presented here aims to compare the accuracy of three classification approaches in mapping LULC categories across the time in the study area primarily using the Google Earth Engine (GEE). Three classifiers namely random forest (RF), support vector machines (SVM), and classification and regression trees (CART) were used in LULC modelling, mapping, and change analysis. Different combinations of input features were investigated to improve classification performance. Developed models were optimised using the grid search cross-validation (CV) hyperparameter optimisation approach. It was revealed that the RF classifier constantly outstrips SVM and CART in terms of accuracy measures, highlighting its reliability in classifying the LULC. Land cover changes were examined for two periods, from 2001 to 2013 and 2013 to 2022, implying major alterations such as the conversion of rubber and coconut areas to built-up areas and barren lands. For suitable classification with higher accuracy, the study suggests utilising high spatial resolution satellite data, advanced feature selection approaches, and a combination of several spatial and spatial-temporal data sources. The study demonstrated practical applications of derived temporal LULC datasets for land management practices in agricultural development activities in developing nations.

Taxonomy, Bio-Ecology and Insecticide Resistance of Anopheline Vectors of Malaria in Sri Lanka.

The objective of this review was to update the current knowledge on major malaria vectors in Sri Lanka and their bio-ecology and insecticide resistance status. Relevant data were collected through a comprehensive literature search performed using databases such as PubMed, NIH, Google Scholar and Web of Science. Sri Lanka had been endemic to malaria for centuries. However, due to a coordinated public health effort last indigenous malaria case was reported in 2012 and the island nation was declared free of malaria in 2016. Although 25 anopheline mosquitoes have been reported so far on the island, only Anopheles culicifacies and An. subpictus have been established as primary and secondary vectors of malaria respectively. Both vector species exist as a species complex, and the sibling species of each complex differ in their bio-ecology and susceptibility to malaria parasites and insecticides. The article provides a comprehensive and updated account of the bio-ecology and insecticide resistance of malaria vectors and highlights the challenges ahead of retaining a malaria-free status.

Microbiological quality of different tea grades produced in diverse agro-climatic regions in Sri Lanka.

In Sri Lanka, several tea grades are produced in factories located in different agro-climatic regions within three geographical elevations. The study aimed to determine the microbial quality of different tea grades and composite tea samples obtained from factories situated at diverse locations. The average APC, yeast & mould counts and coliforms in different tea grades ranged from 3.4 × 103 to 2.0 × 104 cfu/g, 4.8 × 102 to 2.5 × 103 cfu/g and 0.005 to 3.9 × 101 Most Probable Number (MPN)/g respectively. The tea samples collected from different factories had mean values of APC and yeast & mould as 5.3 × 103±1.3 × 103 cfu/g and 9.7 × 102±1.9 × 102 cfu/g. Escherichia coli (E.coli) and Salmonella were not detected either in tea grades or in composite samples. The identified microorganisms in tea samples belong to phyla Firmicutes, Proteobacteria, Ascomycota, Basidiomycota and Zygomycota. The samples collected from the mid country elevation had the highest counts of APC and yeast & mould counts were high in the low country elevation. More than 70 % of the tested samples comply with the SLTB guidelines given for the microbiological quality of black tea. The distribution of bacterial, yeast and mould and coliform densities of tea were significantly variable with respect to geographic areas.

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.

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.

Persistence and potential risks of tetracyclines and their transformation products in two typical different animal manure composting treatments.

Abundant residues of tetracyclines in animal manures and manure-derived organic fertilizers can pose a substantial risk to environments. However, our knowledge on the residual levels and potential risk of tetracyclines and their transformation products (TPs) in manure and manure-derived organic fertilizers produced by different composting treatments is still limited. Herein, the occurrence and distribution of four veterinary tetracyclines (tetracycline, oxytetracycline, chlortetracycline, and doxycycline) and ten of their TPs were investigated in paired samples of fresh manure and manure-derived organic fertilizers. Tetracyclines and TPs were frequently detected in manure and manure-derived organic fertilizer samples in ranging from 130 to 118,137 µg·kg-1 and 54.6 to 104,891 µg·kg-1, respectively. Notably, the TPs concentrations of tetracycline and chlortetracycline were comparable to those of the parent compounds, with 4-epimers being always dominant and retained antibacterial potency. Based on paired-sampling strategy, the removal efficiency of tetracyclines and TPs in thermophilic composting was higher than that in manure storage. Toxicological data in the soil environment and the data derived from equilibrium partitioning method, indicated that tetracyclines and some TPs like 4-epitetracycline, 4-epichlortetracycline and isochlortetracycline could pose median to high ecological risk to terrestrial organisms. Total concentrations of TPs in manure-derived organic fertilizers were significantly correlated with the absolute abundance of tet(X) family genes, which provide evidence to evaluate the effects of TPs on the levels of antibiotic resistance in the environment. Among them, the 4-epitetracycline could pose ecological risk and retain antibacterial potency. Our findings emphasize the importance of monitoring and controlling the prevalence of tetracyclines and their TPs in livestock-related environments.

Breeding site selection and co-existing patterns of tropical mosquitoes.

We investigated the physicochemical properties and the biotic interactions of breeding sites of tropical mosquito species. Field sampling was done in 12 study areas in Sri Lanka covering areas with secondary natural forests and human settlements. A total of 226 breeding sites were investigated to determine the biotic interactions and physiochemical properties of breeding water (pH, Conductivity, Dissolved Oxygen, Total Dissolved Solids and Temperature). A total of 80.5% of breeding sites from both habitats were positive for mosquito larvae of seven genera and 24 species. Orthopodomyia flavithorax (297) and Aedes albopictus (295) were dominated in tree holes of Alstonia macrophylla, Vateria copallifera and Artocarpus nobilis. Diversity indices showed that the diversity of mosquitoes is high in wet zone habitats of Sri Lanka compared to dry and intermediate zone habitats. Aedes albopictus coexisted with 11 different mosquito species while it avoided larvae of Culex fuscanus, Cx. uniformis and Tripteroides affinis. Strong positive associations were reported between Ae. albopictus and Ar. subalbatus while larvae of Or. flavithorax mosquitoes were not co-occurred with the larvae of Ae. vittatus, Ae. aegypti, Cx. sitiens, Ar. subalbatus, Anopheles spp and Tr. affinis. The findings identified the breeding adaptability and tolerance to a wide range of physiochemical properties of tropical mosquito communities.

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.

Use of transcriptional age grading technique to determine the chronological age of Sri Lankan Aedes aegypti and Aedes albopictus females.

BACKGROUND: Aedes aegypti and Ae. albopictus are important vectors of human diseases such as dengue, chikungunya, and zika. In Sri Lanka, they have been responsible for transmitting dengue virus. One of the most important parameters influencing the likelihood of arbovirus transmission is the age structure of the mosquito population. However, mosquito age is difficult to measure with accuracy. This study aims to construct multivariate calibration models using the transcriptional abundance of three age-responsive genes: Ae15848 (calcium-binding protein), Ae8505 (structural component of cuticle), and Ae4274 (fizzy cell cycle/cell division cycle 20). METHODS: The transcriptional age-grading technique was applied to determine the chronological age of Ae. aegypti and Ae. albopictus female mosquito populations from Sri Lanka using the age-responsive genes Ae15848, Ae8505, and Ae4274. Furthermore, Ae. aegypti samples obtained from colonies reared at two temperatures (23 and 27 °C) were used to investigate the influence of temperature on this age-grading technique. Expression levels of these three genes were quantified using reverse transcription qualitative PCR (qRT-PCR), and results were normalized against the housekeeping gene ribosomal gene S17 (RpS17). RESULTS: The expression of Ae15848 and Ae8505 decreased with the age of mosquitoes and showed the most significant and consistent change while expression of Ae4274 increased with age. The multivariate calibration models showed > 80% correlation between expression of these age-responsive genes and the age of female mosquitoes at both temperatures. At 27 °C the accuracy of age predictions using the models was 2.19 (± 1.66) days and 2.58 (± 2.06) days for Ae. aegypti and Ae. albopictus females, respectively. The accuracy of the model for Ae. aegypti at 23 °C was 3.42 (± 2.74) days. CONCLUSIONS: An adult rearing temperature difference of 4 °C (23-27 °C) did not significantly affect the age predictions. The calibration models created during this study could be successfully used to estimate the age of wild Ae. aegypti and Ae. albopictus mosquitoes from Sri Lanka.

Observing the use of knee arthroplasty appropriateness tools in clinical practice: do appropriateness criteria tools predict surgeon decision-making?

OBJECTIVE: The primary aim of this study was to evaluate the agreement between surgeons and two validated total knee arthroplasty (TKA) appropriateness tools, and secondarily to compare Australian appropriateness rates to those reported internationally. METHODS: A consecutive sample of patients from one public hospital arthroplasty clinic and a convenience sample from private rooms of surgeons in New South Wales, Australia (n = 11), referred for surgical opinion regarding TKA were enrolled over 1 year. Surgeons applied appropriateness tools created by Escobar et al. and the American Academy of Orthopaedic Surgeons (AAOS). Correlation between the appropriateness tools and surgeon's decisions were evaluated. RESULTS: There were 368 patients enrolled, and contrasting rates of being "appropriate" for surgery were identified between the Escobar (n = 109, 29.6%) and AAOS (n = 292, 79.3%) tools. Surgeon agreement with the Escobar tool was substantial (ĸ = 0.61, 95%CI: 0.53-0.69) compared to slight with the AAOS tool (ĸ = 0.11, 95%CI: 0.06-0.16). Of those advised against TKA (n = 179, 48.6%), the AAOS tool suggested many patients (n = 111, 62.0%) were "appropriate" compared to the Escobar tool (n = 12, 6.7%). CONCLUSIONS: Surgeons rated patients seeking opinion for TKA as appropriate over half the time, however the AAOS tool had low correlation with surgeons as opposed to the Escobar tool. This was illustrated by both tools rating a majority of patients to be operated on as appropriate, but only the AAOS tool considering most patients not chosen for surgery to be appropriate. When comparing previously published appropriateness rates, appropriateness in Australia, USA, Spain and Qatar was found to be similar.

Blood parasites of bird communities in Sri Lanka and their mosquito vectors.

Avian blood parasites have been shown to have significant health effects on avifauna worldwide. Sri Lanka, a tropical island rich with resident and migratory birds, has not been properly evaluated for avian blood parasites or their vectors. We investigated the presence of avian haemoparasites in Sri Lankan birds and the potential mosquito vectors of those pathogens. Blood samples were collected from local/migratory birds captured by standard mist nets from Anawilundawa bird sanctuary, Hanthana mountain range, and the University of Peradeniya park. Mosquitoes were collected from Halgolla forest reserve and the forest patches in Kurunegala and Gampola areas in addition to the above mist-netting localities. Part of the mitochondrial cytochrome b (cytb) gene was amplified and sequenced to detect the presence of haemoparasites from avian blood samples (86) and mosquito samples (480). Blood parasites of the two genera, i.e., Haemoproteus (4 species; Haemoproteus sp. 1-4) and Plasmodium (5 species; Plasmodium sp. 1-5) were identified from seven bird species (four resident and three migratory). Among these, three bird species (Red-vented bulbul (3/16), Asian Brown flycatcher (1/1), and India pitta (1/1)) were positive for Plasmodium spp., while four (Yellow-browed bulbul (1/4), oriental white-eye (1/4), brown-headed Barbet (1/4), and Indian blue robin (1/1)) were positive for Haemoproteus spp. Two mosquito species were also positive for Plasmodium (3) and Haemoproteus (1) species. Phylogenetic analysis and haplotype networks created using positive sequences of haemoparasites showed that a Plasmodium clade was shared by Cx nigropunctatus mosquitoes and the migratory bird, Indian pitta. The majority (85%) of the Plasmodium and Haemoproteus sequences of this study were not linked to the well-characterized species suggesting the distinct nature of the lineages. Associations between mosquito species and blood parasites of birds suggest the possible vector status of these mosquitoes.

Insecticide susceptibility of the sand fly leishmaniasis vector Phlebotomus argentipes in Sri Lanka.

BACKGROUND: Leishmania donovani-induced and sand fly-transmitted leishmaniasis is a growing health problem in Sri Lanka. Limited knowledge on biological and behavioral characteristics of probable vector Phlebotomus argentipes hinders disease control. Here, insecticide susceptibility patterns of P. argentipes were investigated with exploration of probable underlying resistance mechanisms. METHODS: Adult sand flies were collected using standard cattle baited net traps and CDC light traps from selected sites in four districts. Adult F1 progeny of P. argentipes were exposed to different concentrations of DDT, malathion, deltamethrin and propoxur using WHO susceptibility bioassay kits. Post-1-h knockdown and post-24-h mortality were recorded and analyzed. Metabolic enzyme activity and the sensitivity of the acetylcholinesterase target-site were determined by biochemical assays using wild-caught flies. Extracted fly DNA samples were tested for the presence of knockdown-resistance (kdr) type mutations. RESULTS: The LC100 values for DDT, malathion, propoxur and deltamethrin were 0.8-1.5%, 0.9-2.0%, 0.017-0.03% and 0.007% respectively. Insecticide-susceptibility levels were higher than the discriminating dosages established for Aedes mosquitoes, except for malathion. The lowest susceptibility levels (except for deltamethrin) were detected in the Mamadala population, whereas the highest levels were detected in the Mirigama population. The percentage of knocked-down sand flies was < 75% at any tested concentration, including those, which exhibited 100% mortality after 24 h. Elevated activity levels of glutathione S-transferase (3%, 7%, 12.5% and 14%) and esterase (2%, 5%, 5.5% and 6.5%) were detected in flies that originated from Mirigama, Pannala, Thalawa and Mamadala respectively, while monooxygenase quantities remained below the cut-off level. Ten to 34.5% of flies were heterozygous for acetylcholinesterases target-site insensitivity, associated with organophosphate and carbamate resistance. Pyrethroid-resistance-associated L1014F kdr-type mutation in the voltage gated sodium channel gene was detected in 30/53 flies. CONCLUSIONS: Populations of P. argentipes in Sri Lanka are largely susceptible to common insecticides, except for malathion (used extensively in the past for malaria control). Their insecticide susceptibility appears negatively associated with past malaria endemicity of the study sites, with signs of early insecticide tolerance. Presence of insecticide target site insensitivity in a notable proportion of flies and enhanced insecticide metabolizing enzyme activities imply potential future challenges for leishmaniasis control, with a call for urgent proactive measures for its containment.

Development of the major arboviral vector Aedes aegypti in urban drain-water and associated pyrethroid insecticide resistance is a potential global health challenge.

BACKGROUND: Aedes aegypti were found developing in the water in open public drains (drain-water, DW) in Jaffna city in northern Sri Lanka, a location where the arboviral diseases dengue and chikungunya are endemic. METHODS: Susceptibilities to the common insecticides dichlorodiphenyltrichloroethane (DDT), malathion, propoxur, permethrin and deltamethrin and activities of the insecticide-detoxifying enzymes carboxylesterase (EST), glutathione S-transferase (GST) and monooxygenase (MO) were compared in adult Ae. aegypti developing in DW and fresh water (FW). RESULTS: DW Ae. aegypti were resistant to the pyrethroids deltamethrin and permethrin, while FW Ae. aegypti were susceptible to deltamethrin but possibly resistant to permethrin. Both DW and FW Ae. aegypti were resistant to DDT, malathion and propoxur. Greater pyrethroid resistance in DW Ae. aegypti was consistent with higher GST and MO activities. CONCLUSIONS: The results demonstrate the potential for insecticide resistance developing in Ae. aegypti adapted to DW. Urbanization in arboviral disease-endemic countries is characterized by a proliferation of open water drains and therefore the findings identify a potential new challenge to global health.

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.

Susceptibility to common insecticides and detoxifying enzyme activities in Anopheles sundaicus (sensu lato) after cessation of indoor residual spraying of insecticides in the Jaffna Peninsula and its surroundings in northern Sri Lanka.

BACKGROUND: Sri Lanka has been malaria-free since 2013 but re-introduction of malaria transmission by infected overseas travelers is possible due to a prevalence of potent malaria vectors. Knowledge of the insecticide resistance status among Anopheles vectors is important if vector control has to be reintroduced in the island. The present study investigated the insecticide susceptibility levels and resistance mechanisms of Anopheles sundaicus (sensu lato) (previously classified as Anopheles subpictus species B) an important malaria vector in the Jaffna Peninsula and it surroundings in northern Sri Lanka after indoor residual spraying of insecticides was terminated in 2013. RESULTS: Species-specific PCR assays identified An. sundaicus (s.l.) in four locations in the Jaffna and adjacent Kilinochchi districts. Bioassays confirmed that An. sundaicus (s.l.) collected in Kilinochchi were completely susceptible to 0.05% deltamethrin and 5% malathion and resistant to 4% dichlorodiphenyltrichloroethane (DDT), whereas those from Jaffna were relatively susceptible to all three insecticides. Kilinochchi populations of An. sundaicus (s.l.) showed significantly higher glutathione S-transferase activity than population from Jaffna. However, Jaffna An. sundaicus (s.l.) had significantly higher Propoxur-resistant acetylcholinesterase activity. Activities of non-specific esterases and monooxygenases were not significantly elevated in An. sundaicus (s.l.) collected in both districts. CONCLUSIONS: The susceptibility to malathion and deltamethrin in An. sundaicus (s.l.) suggests that they can be still used for controlling this potential malaria vector in the Jaffna Peninsula and adjacent areas. Continuing country-wide studies on other malaria vectors and their insecticide susceptibilities are important in this regard.

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.

Diversity, distribution, abundance, and feeding pattern of tropical ornithophilic mosquitoes.

Bird-biting mosquitoes act as bridge vectors of diverse pathogens of emerging infectious diseases. In this study, we report for the first time the abundance, diversity, distribution, and feeding pattern of bird-biting mosquitoes on an island where avifaunal diversity is rich. Monthly mosquito collections were done at six different habitats in three different climatic zones using bird-baited traps over a year. Collected mosquitoes were identified using morphological and molecular tools. A total of 2,655 bird-biting mosquitoes of eight genera and 25 species were identified. Of these, 52% were Culex species, which represents 35% of the Culex species in the country. The most abundant species were Culex sitiens, Cx. pseudovishnui, Cx. nigropunctatus and Cx. quinquefasciatus, whereas the latter two were common to all habitats. The highest abundance was reported in lowland forests (49.6%), while it was lowest in highland forests (22.3%). Highest species similarity was reported from highland forests. Seasonal variations of the most abundant species were significantly different in selected habitats (p< 0.05). Two distinct biting peaks were identified, from 06:00 to 21:00 and 22:00 to 02:00. The biting nature of identified ornithophilic mosquitoes suggests the potential vector status of these mosquitoes.

DNA barcoding of morphologically characterized mosquitoes belonging to the subfamily Culicinae from Sri Lanka.

BACKGROUND: Vectors of mosquito-borne diseases in Sri Lanka, except for malaria, belong to the subfamily Culicinae, which includes nearly 84% of the mosquito fauna of the country. Hence, accurate and precise species identification of culicine mosquitoes is a crucial factor in implementing effective vector control strategies. During the present study, a combined effort using morphology and DNA barcoding was made to characterize mosquitoes of the subfamily Culicinae for the first time from nine districts of Sri Lanka. Cytochrome c oxidase subunit 1 (cox1) gene from the mitochondrial genome and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. RESULTS: According to morphological identification, the field collected adult mosquitoes belonged to 5 genera and 14 species, i.e. Aedes aegypti, Ae. albopictus, Ae. pallidostriatus, Aedes sp. 1, Armigeres sp. 1, Culex bitaeniorhynchus, Cx. fuscocephala, Cx. gelidus, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Cx. whitmorei, Mansonia uniformis and Mimomyia chamberlaini. Molecular analyses of 62 cox1 and 36 ITS2 sequences were exclusively comparable with the morphological identifications of all the species except for Ae. pallidostriatus and Aedes sp. 1. Although the species identification of Armigeres sp. 1 specimens using morphological features was not possible during this study, DNA barcodes of the specimens matched 100% with the publicly available Ar. subalbatus sequences, giving their species status. Analysis of all the cox1 sequences (14 clades supported by strong bootstrap value in the Neighbor-Joining tree and interspecific distances of > 3%) showed the presence of 14 different species. This is the first available DNA sequence in the GenBank records for morphologically identified Ae. pallidostriatus. Aedes sp. 1 could not be identified morphologically or by publicly available sequences. Aedes aegypti, Ae. albopictus and all Culex species reported during the current study are vectors of human diseases. All these vector species showed comparatively high diversity. CONCLUSIONS: The current study reflects the significance of integrated systematic approach and use of cox1 and ITS genetic markers in mosquito taxonomy. Results of DNA barcoding were comparable with morphological identifications and, more importantly, DNA barcoding could accurately identify the species in the instances where the traditional morphological identification failed due to indistinguishable characters of damaged specimens and the presence of subspecies.

Genotype and biotype of invasive Anopheles stephensi in Mannar Island of Sri Lanka.

BACKGROUND: Anopheles stephensi, the major vector of urban malaria in India, was recently detected for the first time in Sri Lanka in Mannar Island on the northwestern coast. Since there are different biotypes of An. stephensi with different vector capacities in India, a study was undertaken to further characterise the genotype and biotype of An. stephensi in Mannar Island. METHODS: Mosquito larvae were collected in Pesalai village in Mannar and maintained in the insectary until adulthood. Adult An. stephensi were identified morphologically using published keys. Identified adult An. stephensi were molecularly characterized using two mitochondrial (cox1 and cytb) and one nuclear (ITS2) markers. Their PCR-amplified target fragments were sequenced and checked against available sequences in GenBank for phylogenetic analysis. The average spiracular and thoracic lengths and the spiracular index were determined to identify biotypes based on corresponding indices for Indian An. stephensi. RESULTS: All DNA sequences for the Mannar samples matched reported sequences for An. stephensi from the Middle East and India. However, a single nucleotide variation in the cox1 sequence suggested an amino acid change from valine to methionine in the cox1 protein in Sri Lankan An. stephensi. Morphological data was consistent with the presence of the Indian urban vector An. stephensi type-form in Sri Lanka. CONCLUSIONS: The present study provides a more detailed molecular characterization of An. stephensi and suggests the presence of the type-form of the vector for the first time in Sri Lanka. The single mutation in the cox1 gene may be indicative of a founder effect causing the initial diversification of An. stephensi in Sri Lanka from the Indian form. The distribution of the potent urban vector An. stephensi type-form needs to be established by studies throughout the island as its spread adds to the challenge of maintaining the country's malaria-free status.