Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia

Abstract

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI's nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

Keywords: Anopheles; Zambia; malaria; mosquito; phylogenetics; residual transmission.

FIGURE 1 |

NCBI Nucleotide database search results for each species name. Well-studied An. gambiae complex sibling species and An. funestus s.s. in blue. Understudied species in orange.

FIGURE 2 |

Collection site map. Each purple dot represents a household in Nchelenge. The public domain map, CleanTopo2 (43) was used for the base map on left two panels. World imagery from ArcGIS® software by Esri was used for the base map for the right panel.

FIGURE 3 |

(A) Phylogenetic tree based on COI sequences. (B) Tree based on ITS2 sequences. Trees include both well-defined sequences from this study (green or red node termini outlined in black) and from NCBI (green or red node termini without black outline), as well as sequences without a proper species identification (gray from this study and black from NCBI).

FIGURE 4 |

(A) Phylogenetic tree based on COI sequences of An. funestus group samples. (B) Tree based on ITS2 sequences. Trees include both well-defined sequences from this study (green node termini outlined in black) and from NCBI (green node termini without black outline).

FIGURE 5 |

(A) Phylogenetic tree based on COI sequences of An. squamosus and An. sp. 15 group samples. (B) Tree based on ITS2 sequences. Trees include both well-defined sequences from this study (green node termini outlined in black) and from NCBI (green node termini without black outline) as well as sequences without a proper species identification (gray from this study and black from NCBI).

FIGURE 6 |

(A) Phylogenetic tree based on COI sequences of An. coustani group samples. (B) Tree based on ITS2 sequences. Trees include both well-defined sequences from this study (red node termini outlined in black) and from NCBI (green or red node termini without black outline), as well as sequences without a proper species identification (black from NCBI).

FIGURE 7 |

(A) Phylogenetic tree based on COI sequences of An. sp. 6 samples. (B) Tree based on ITS2 sequences. Trees include both well-defined sequences from NCBI (green or red node termini without black outline) as well as sequences without a proper species identification (gray from this study and black from NCBI).

FIGURE 8 |

(A) Phylogenetic tree based on COI sequences of An. sp. 11 samples. (B) Tree based on ITS2 sequences. Trees include sequences without a proper species identification (gray from this study and black from NCBI).

FIGURE 9 |

(A) Phylogenetic tree based on COI sequences of other Anopheles samples. (B) Tree based on ITS2 sequences. Trees include both well-defined sequences from this study (green node termini outlined in black) and from NCBI (green or red node termini without black outline), as well as sequences without a proper species identification (gray from this study and black from NCBI).