Morphological and odorant-binding protein 1 gene intron 1 sequence variations in Anopheles stephensi from Jaffna city in northern Sri Lanka.

Three Anopheles stephensi biotypes have historically been differentiated through variations in the mode numbers of egg ridges and adult spiracular indices. Anopheles stephensi odorant-binding protein 1 gene (AsteObp1) sequences in Iran and Afghanistan have been recently interpreted to suggest that the three biotypes are sibling species. AsteObp1 intron 1 sequences, mode numbers of egg ridges and spiracular indices of An. stephensi in Jaffna city in Sri Lanka were therefore investigated in field-collected mosquitoes and short-term laboratory colonies established from them. AsteObp1 intron 1 sequences revealed the region to be polymorphic with four unique sequences, ASJF1-4, present in both short-term laboratory colonies and field-collected An. stephensi. The spiracular index did not relate to the mode number of egg ridges in Jaffna An. stephensi. The results suggested that numbers of egg ridges, spiracular indices and AsteObp1 intron 1 sequences were not useful for differentiating An. stephensi biotypes in Jaffna. It is proposed that the observed differences between An. stephensi mosquitoes in Jaffna now result from normal population variance in the context of rapidly changing bionomics in India and northern Sri Lanka.

Epidemiological and virological factors determining dengue transmission in Sri Lanka during the COVID-19 pandemic.

With the onset of the COVID-19 pandemic in early 2020 there was a drastic reduction in the number of dengue cases in Sri Lanka, with an increase towards the end of 2021. We sought to study the contribution of virological factors, human mobility, school closure and mosquito factors in affecting these changes in dengue transmission in Sri Lanka during this time. To understand the reasons for the differences in the dengue case numbers in 2020 to 2021 compared to previous years, we determined the association between the case numbers in Colombo (which has continuously reported the highest number of cases) with school closures, stringency index, changes in dengue virus (DENV) serotypes and vector densities. There was a 79.4% drop in dengue cases from 2019 to 2020 in Colombo. A significant negative correlation was seen with the number of cases and school closures (Spearman's r = -0.4732, p <0.0001) and a negative correlation, which was not significant, between the stringency index and case numbers (Spearman's r = -0.3755 p = 0.0587). There was no change in the circulating DENV serotypes with DENV2 remaining the most prevalent serotype by early 2022 (65%), similar to the frequencies observed by end of 2019. The Aedes aegypti premise and container indices showed positive but insignificant correlations with dengue case numbers (Spearman r = 0.8827, p = 0.93). Lockdown measures, especially school closures seemed to have had a significant impact on the number of dengue cases, while the vector indices had a limited effect.

Regional Variation in Dengue Virus Serotypes in Sri Lanka and Its Clinical and Epidemiological Relevance.

Dengue is a significant health concern in Sri Lanka, but diagnosis of the infecting dengue virus (DENV) serotype has hitherto been largely restricted to the Colombo district in the western province. Salinity tolerant Aedes vectors are present in the island's northern Jaffna peninsula, which is undergoing rapid groundwater salinization. Virus serotypes were determined by RT-qPCR in 107 and 112 patients diagnosed by NS1 antigen positivity from the Jaffna district in 2018 and 2019, respectively, and related to clinical characteristics. DENV1 and DENV2 were the most common serotypes in both years. Infections with multiple serotypes were not detected. DENV1 was significantly more prevalent in 2019 than 2018, while DENV3 was significantly more prevalent in 2018 than 2019 among the Jaffna patients. Limited genomic sequencing identified DENV1 genotype-I and DENV3 genotype-I in Jaffna patients in 2018. Dengue was more prevalent in working age persons and males among the serotyped Jaffna patients. DENV1 and DENV2 were the predominant serotypes in 2019 in the Colombo district. However, DENV1 and DENV3 were significantly more prevalent in Colombo compared with Jaffna in 2019. The differences in the prevalence of DENV1 and DENV3 between the Jaffna and Colombo districts in 2019 have implications for dengue epidemiology and vaccination. Salinity-tolerant Aedes vector strains, widespread in the Jaffna peninsula, may have contributed to differences in serotype prevalence compared with the Colombo district in 2019. Significant associations were not identified between virus serotypes and clinical characteristics among Jaffna patients.

Transcriptomic, proteomic and ultrastructural studies on salinity-tolerant Aedes aegypti in the context of rising sea levels and arboviral disease epidemiology.

BACKGROUND: Aedes aegypti mosquito, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development to become adult mosquitoes in fresh water (FW). It has recently been observed to develop in coastal brackish water (BW) habitats of up to 50% sea water, and such salinity tolerance shown to be an inheritable trait. Genomics of salinity tolerance in Ae. aegypti has not been previously studied, but it is of fundamental biological interest and important for controlling arboviral diseases in the context of rising sea levels increasing coastal ground water salinity. RESULTS: BW- and FW-Ae. aegypti were compared by RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4), proteomics of cuticles shed when L4 metamorphose into pupae, and transmission electron microscopy of cuticles in L4 and adults. Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 showed different mRNA levels in BW and FW L4 tissues. The salinity-tolerant Ae. aegypti were also characterized by altered L4 cuticle proteomics and changes in cuticle ultrastructure of L4 and adults. CONCLUSIONS: The findings provide new information on molecular and ultrastructural changes associated with salinity adaptation in FW mosquitoes. Changes in cuticles of larvae and adults of salinity-tolerant Ae. aegypti are expected to reduce the efficacy of insecticides used for controlling arboviral diseases. Expansion of coastal BW habitats and their neglect for control measures facilitates the spread of salinity-tolerant Ae. aegypti and genes for salinity tolerance. The transmission of arboviral diseases can therefore be amplified in multiple ways by salinity-tolerant Ae. aegypti and requires appropriate mitigating measures. The findings in Ae. aegypti have attendant implications for the development of salinity tolerance in other fresh water mosquito vectors and the diseases they transmit.

Aedes larval bionomics and implications for dengue control in the paradigmatic Jaffna peninsula, northern Sri Lanka.

BACKGROUND: The larval bionomics of Aedes across the Jaffna peninsula in northern Sri Lanka was investigated to obtain information needed for developing more effective larval source reduction measures to control endemic arboviral diseases. METHODS: The habitats of preimaginal stages of Aedes mosquitoes were surveyed, and ovitrap collections were carried out in densely populated areas of the Jaffna peninsula. Aedes larval productivities were analysed against habitat characteristics, rainfall and dengue incidence. Adults emerging from collected larvae were tested for dengue virus (DENV). RESULTS: Only Aedes aegypti, Ae. albopictus and Ae. vittatus were identified in the field habitat collections and ovitraps. Aedes aegypti was the predominant species in both the field habitat and ovitrap collections, followed by Ae. albopictus and small numbers of Ae. vittatus. Tires and open drains were the preferred field habitats for Ae. aegypti, although larval productivity was higher in discarded plastic containers. The three Aedes species differed in field habitat preferences. Concomitant presence of the three Aedes species was observed in the field habitats and ovitraps. Larval productivities were inversely correlated with the salinity of the field habitat. Rainfall in the preceding month significantly correlated with larval productivity in the field habitats. DENV serotype 2 was detected in Ae. aegypti collected from ovitraps in the city of Jaffna. High Breteau, House and Container indices of 5.1, 5.1 and 7.9%, respectively, were observed in the field habitat surveys and ovitrap indices of up to 92% were found in Jaffna city. CONCLUSIONS: Aedes larval indices in populated areas of the peninsula showed a high potential for dengue epidemics. Unacceptable littering practices, failure to implement existing dengue control guidelines, vertical transmission of DENV in vector mosquitoes and preimaginal development in brackish water and open surface drains, as well as in domestic wells that provide potable water, are serious constraints to the current Aedes larval source reduction methods used to control dengue in the Jaffna peninsula. Similar shortcomings in arboviral disease control are likely present in other resource-constrained tropical coastal zones worldwide.

Anopheline bionomics, insecticide resistance and transnational dispersion in the context of controlling a possible recurrence of malaria transmission in Jaffna city in northern Sri Lanka.

BACKGROUND: Malaria was eliminated from Sri Lanka in 2013. However, the influx of infected travelers and the presence of potent anopheline vectors can re-initiate transmission in Jaffna city, which is separated by a narrow strait from the malaria-endemic Indian state of Tamil Nadu. METHODS: Anopheline larvae were collected from different habitats in Jaffna city and the susceptibility of emergent adults to DDT, malathion and deltamethrin investigated. RESULTS: Anopheline larvae were found in wells, surface-exposed drains, ponds, water puddles and water storage tanks, with many containing polluted, alkaline and brackish water. Anopheles culicifacies, An. subpictus, An. stephensi and An. varuna were identified in the collections. Adults of the four anopheline species were resistant to DDT. Anopheles subpictus and An. stephensi were resistant while An. culicifacies and An. varuna were possibly resistant to deltamethrin. Anopheles stephensi was resistant, An. subpictus possibly resistant while An. varuna and An. culicifacies were susceptible to malathion. DNA sequencing showed a L1014F (TTA to TTC) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel protein in deltamethrin-resistant An. subpictus-a mutation previously observed in India but not Sri Lanka. CONCLUSION: Anopheles subpictus in Jaffna, like An. stephensi, may have recently originated in coastal Tamil Nadu. Besides infected overseas travelers, wind- and boat-borne carriage of Plasmodium-infected anophelines across the Palk Strait can potentially reintroduce malaria transmission to Jaffna city. Adaptation to diverse larval habitats and resistance to common insecticides in anophelines are identified as potential problems for vector control should this happen.

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.

Anthropogenic Factors Driving Recent Range Expansion of the Malaria Vector Anopheles stephensi.

The malaria vector Anopheles stephensi is found in wide tracts of Asia and the Middle East. The discovery of its presence for the first time in the island of Sri Lanka in 2017, poses a threat of malaria resurgence in a country which had eliminated the disease in 2013. Morphological and genetic characterization showed that the efficient Indian urban vector form An. stephensi sensu stricto or type form, has recently expanded its range to Jaffna and Mannar in northern Sri Lanka that are in proximity to Tamil Nadu state in South India. Comparison of the DNA sequences of the cytochrome oxidase subunit 1 gene in An. stephensi in Jaffna and Mannar in Sri Lanka and Tamil Nadu and Puducherry states in South India showed that a haplotype that is due to a sequence change from valine to methionine in the cytochrome oxidase subunit 1 present in the Jaffna and Mannar populations has not been documented so far in Tamil Nadu/Puducherry populations. The Jaffna An. stephensi were closer to Tamil Nadu/Puducherry populations and differed significantly from the Mannar populations. The genetic findings cannot differentiate between separate arrivals of the Jaffna and Mannar An. stephensi from Tamil Nadu or a single arrival and dispersion to the two locations accompanied by micro-evolutionary changes. Anopheles stephensi was observed to undergo preimaginal development in fresh and brackish water domestic wells and over ground cement water storage tanks in the coastal urban environment of Jaffna and Mannar. Anopheles stephensi in Jaffna was resistant to the common insecticides deltamethrin, dichlorodiphenyltrichloroethane and Malathion. Its preimaginal development in wells and water tanks was susceptible to predation by the larvivorous guppy fish Poecilia reticulata. The arrival, establishment, and spread of An. stephensi in northern Sri Lanka are analyzed in relation to anthropogenic factors that favor its range expansion. The implications of the findings for global public health challenges posed by malaria and other mosquito-borne diseases are discussed.

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.