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Mapping the Genetic Relatedness of Outdoor-Biting Anopheles Mosquitoes in Zambia
by
, , , , and
Reneé L. M. N. Ali
1,2,*
,
Mary E. Gebhardt
1,2,
Limonty Simubali
3,
Kochelani Saili
3,
Westone Hamwata
4,
Hunter Chilusu
4,
Mbanga Muleba
4,
Conor J. McMeniman
1,2,
Anne C. Martin
2,5,
William J. Moss
2,5 and
Douglas E. Norris
1,2
1
The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA
2
The Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA
3
Macha Research Trust, Choma P.O. Box 630166, Zambia
4
National Health Research and Training Institute, Ndola P.O. Box 71769, Zambia
5
Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA
*
Author to whom correspondence should be addressed.
Insects 2025, 16(12), 1198; https://doi.org/10.3390/insects16121198
Submission received: 24 September 2025
/
Revised: 7 November 2025
/
Accepted: 14 November 2025
/
Published: 25 November 2025
(This article belongs to the Section Insect Molecular Biology and Genomics)
Simple Summary
Outdoor-biting Anopheles mosquitoes have been gaining attention due to their potential role in sustaining malaria transmission by avoiding indoor vector control interventions. The efficacy of mitigation efforts that primarily target indoor biting and resting mosquitoes may be undermined by these mosquitoes. The identification of these less studied mosquito taxa is challenging due to cryptic morphological features and the limited number of molecular reference sequences in databases. Advancements in sequencing technologies have led to a steady increase in the generation of mitochondrial genomes (mitogenomes). Mitogenomes have proved to be robust in resolving species identification, population structure and phylogenies in metazoans when compared to commonly used molecular barcodes. Our work highlights the use of mitochondrial genomes for understanding the genetic relatedness of the less-studied outdoor-biting anopheles with reference to the primary vectors of malaria. The datasets generated in this study can be used to improve interventions for malaria control and employ molecular diagnostics for accurate species identification.
Abstract
The zoophilic and exophilic traits of outdoor-biting Anopheles have led to this group largely being overlooked for their role in malaria transmission, despite several species now recognized as locally important in regions of sub-Saharan Africa. Given the current limitations with identification of these understudied species, it is crucial to accurately correlate morphological features to molecular data. Here, we produced high quality reference sequence data for representative understudied anopheline species to better understand the phylogenetic relationships between under- and well-studied vectors of malaria. For mitochondrial genome assembly, shallow shotgun sequencing was implemented on single mosquito specimens and phylogenetic analyses were performed on the concatenated protein coding genes of the mitogenomes using a Bayesian approach. This study generated 10 complete mitogenomes focusing on less studied taxa with an average length 15,380 bp and A-T content of 77.4% consistent with other anophelines containing 37 genes. Bayesian inference analysis yielded four main clades with molecular dating indicating that well-studied malaria vectors diverged from outdoor-biting species more than 63 million years ago. These findings support the taxonomic grouping of mosquitoes belonging to the Anopheles genus based on morphological characteristics and can provide molecular diagnostics for species identification enabling more precise and adept interventions for malaria control.
Keywords:
outdoor biting; mitochondrial genome; malaria; Anopheles; understudied
