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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.

ABSTRACT: 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.

SUBMITTER: Jayatunga DPW

PROVIDER: S-EPMC6163451 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

Jayatunga Dona P W DPW Harischandra Iresha N IN Chandrasekharan Naduviladath V NV de Silva Nissanka K NK

Insects 20180724 3

The species complex of the mosquito <i>Anopheles subpictus</i> 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 s ...[more]

PMID: 30042298

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Project description:BackgroundAnopheles subpictus s.l., an important malaria vector in Sri Lanka, is a complex of four morphologically identified sibling species A-D. Species A-D reportedly differ in bio-ecological traits that are important for vector control. We investigated possible variations that had not been reported previously, in the susceptibility to common insecticides and resistance mechanisms among the An. subpictus sibling species.MethodsAdult An. subpictus were collected from localities in four administrative districts in the dry zone of Sri Lanka. Single female isoprogeny lines were established and sibling species status determined according to reported egg morphology. World Health Organization's standard protocols were used for insecticide bioassays and biochemical assays to determine insecticide susceptibility and resistance mechanisms. Susceptibility of mosquitoes was tested against DDT (5%), malathion (4%), deltamethrin (0.05%) and λ-cyhalothrin (0.05%). Biochemical basis for resistance was determined through assaying for esterase, glutathione-S-transferase and monooxygenase activities and the insensitivity of acetycholinesterase (AChE) to propoxur inhibition.ResultsAll sibling species were highly resistant to DDT. However there were significant differences among the sibling species in their susceptibility to the other tested insecticides. Few species A could be collected for testing, and where testing was possible, species A tended to behave more similarly to species C and D than to B. Species B was more susceptible to all the tested insecticides than the other sibling species. This difference may be attributed to the predominance of species B in coastal areas where selection pressure due to indoor residual spraying of insecticides (IRS) was lower. However there were significant differences between the more inland species C and D mainly towards pyrethroids. Higher GST activities in species C and D might have contributed to their greater DDT resistance than species B. Malathion resistance in both species C and D may be caused by elevated GST activity and an altered insensitive target site in AChE. In addition, a carboxylesterase based malathion resistance mechanisms was also detected in species C and D. Elevated esterase levels in species C and D might have contributed to the low levels of pyrethroid resistance. However an absence of elevated activity of monooxygenases in species B, C and D indicates that monooxygenases are unlikely to be the cause of this partial resistance to pyrethroids.ConclusionsThe differences in insecticide susceptibility and insecticide resistance mechanism shown by An. subpictus sibling species are important considerations for developing the malaria control and eradication program in Sri Lanka. Similar studies on species complexes of other anopheline vectors of malaria are necessary for effective malaria control worldwide. The differential susceptibility findings are also consistent with most, if not all, morphologically identified An. subpictus species B in Sri Lanka belonging to the An. sundaicus complex. There is a need therefore to develop molecular techniques that can be used to differentiate morphologically similar anopheline species in field conditions for more effective vector control.

Project description:BackgroundAlthough 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).MethodsAdult 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).ResultsAnopheles 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.ConclusionsGene 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.

Project description:BackgroundGenus 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.MethodsCytochrome 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.ResultsAccording 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.ConclusionsThe 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.

Project description:BackgroundAnopheles subpictus sensu lato, a widespread malaria vector in Asia, is reportedly composed of four sibling species A - D. Mosquitoes morphologically identified as belonging to the Subpictus complex were collected from different locations near the east coast of Sri Lanka, and specific ribosomal DNA sequences determined to validate their taxonomic status.MethodsAnopheles subpictus s.l. larvae and blood-fed adults were collected from different locations in the Eastern province and their sibling species status was determined based on published morphological characteristics. DNA sequences of the D3 domain of 28 S ribosomal DNA (rDNA) and the internal transcribed spacer -2 (ITS-2) of mosquitoes morphologically identified as An. subpictus sibling species A, B, C and D were determined.ResultsPhylogenetic analysis based on D3 domain of rDNA resulted in two clades: one clade with mosquitoes identified as An. subpictus species A, C, D and some mosquitoes identified as species B, and another clade with a majority of mosquitoes identified as species B with D3 sequences that were identical to Anopheles sundaicus cytotype D. Analysis of ITS-2 sequences confirmed a close relationship between a majority of mosquitoes identified as An. subpictus B with members of the An. sundaicus complex and others identified as An. subpictus B with An. subpictus s.l.ConclusionsThe study suggests that published morphological characteristics are not specific enough to identify some members of the Subpictus complex, particularly species B. The sequences of the ITS-2 and D3 domain of rDNA suggest that a majority that were identified morphologically as An. subpictus species B in the east coast of Sri Lanka, and some identified elsewhere in SE Asia as An. subpictus s.l., are in fact members of the Sundaicus complex based on genetic similarity to An. sundaicus s.l. In view of the well-known ability of An. sundaicus s.l. to breed in brackish and fresh water and its proven ability to transmit malaria in coastal areas of many Southeast Asian countries, the present findings have significant implications for malaria control in Sri Lanka and neighbouring countries.

Dataset Information

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.

Publications

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

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