The Chironomidae family, known as "non-biting midges" in their adult stage and "bloodworms" in their larval stage, consists of diverse dipteran insects inhabiting various global aquatic environments. Despite extensive global research, data on Chironomidae in the polluted Nyanza Gulf of Lake Victoria, Kenya, is scarce, and molecular identification methods have not been explored. This study aimed to quantify heavy metal concentrations in water, sediment, and insect samples and assess their impact on Chironomid species identified using mitochondrial DNA barcoding of the cytochrome oxidase subunit 1 (COI) gene. Analysis of Variance was used to determine if there were any statistically significant differences in heavy metal concentrations across different sample types or locations along the pollution gradient. Chironomids were collected from Nyanza Gulf, focusing on a pollution gradient. Results showed that concentrations of arsenic (As), lead (Pb), and cadmium (Cd) in insect, water, and sediment samples exceeded standard limits, while mercury (Hg) concentrations were within limits. Significant variations (p ≤ 0.05) in Pb levels were observed in water samples, and heavy metal concentrations in sediment samples varied significantly (p ≤ 0.05), with Pb showing the highest variation (p ≤ 0.0001). Insect samples exhibited significant differences (p ≤ 0.0001) in As and Hg contents. Genetic analysis identified two known species: Chironomus transvaalensis at the heavily polluted Kisumu station and Chironomus pseudothummi at the moderately polluted Kendu Bay and Homa Bay stations. Additionally, a unique Chironomus species was found on Ndere Island, a relatively clean site with restricted human activities. Sequence comparisons indicated proximity to global data but also highlighted the evolutionary significance and uniqueness of the identified species. This study demonstrated the potential use of genetic methods in determining Chironomid species diversity, community structure, and abundance in relation to heavy metal concentration. It suggests that heavy metal pollution may act as a selective pressure, driving the evolution of Chironomid species. The study recommends combining genetic approaches with other pollution sources for a comprehensive understanding of using this species in monitoring pollution
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