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.

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.

Alterations and Interchange of Morphometric Characters in Different Life Cycle Stages with Reference to Genomic Variations of Anopheles subpictus (Diptera; Culicidae) Sibling Species Complex in Sri Lanka.

The species complex of the mosquito Anopheles subpictus is designated by the sibling species A⁻D, depending on morphological characters of life cycle stages and variations in polytene chromosomes. However, morphological aberrations in the life cycle stages make the identification of sibling species uncertain and imprecise. The objective of the present study is to determine the suitability of morphological variations of sibling species and their genomic variations to identify the sibling species status of an An. subpictus population in Sri Lanka. Life cycle stages of larvae, pupal exuviae, and adults were examined for previously reported distinctive morphological features. Five nuclear and mitochondrial genome regions, including the Internal transcribed spacer 2 (ITS2) region, D3 region, white gene, cytochrome c oxidase I (COI), and Cytochrome b (Cyt-b), were sequenced and analyzed for variations. The eggs changed their distinct sibling morphological characters during metamorphosis (89.33%). The larvae, pupal exuviae, and adult stages showed deviation from their sibling characters by 26.10%, 19.71%, and 15.87%, respectively. However, all the species from the analysis shared two distinct sequence types for all regions, regardless of the morphological variations. In conclusion, the An. subpictus sibling species complex in Sri Lanka is not identifiable using morphological characters due to variations, and the genomic variations are independent from the morphological variations.