Study site and sampling
Our team from the Ecology, Health, and Environment (ECOSEN) group, Université André Salifou, Zinder, embarked on a study of biodiversity of mosquitoes (Diptera: Culicidae) in Gayi (13.2402° N, 9.3499 °E), Wacha commune, Magaria Department, Zinder Region (Fig. 4) on 18 of August 2024. Gayi, a Sahelian settlement, characterised with seasonal malaria transmission is located 82 km southeast of Zinder, about 100 km from the border of Nigeria.
Map of Niger Republic showing Gayi locality, located south of Zinder region, near Nigerian border.
The project was approved by the Scientific Council of Université André Salifou, Zinder-Niger (Ref: N°000792 June 13, 2023).
The total population of Gayi was 32,709 inhabitants in 2024. Agriculture, livestock, and local trade constitute the primary livelihood activities, with flux of humans and animals between the locality and border towns in Nigeria. Female blood fed, unfed, and male, adult mosquitoes, resting indoor were collected from 19 houses, early in the morning (6:00–8:00 am) in Gayi using Improved Prokopack battery-operated aspirators (John. W. Hock, Florida, USA). During this month, Gayi had experienced a total of 1,927 reported malaria cases and an average monthly rainfall of 146.9 mm. Environmental conditions on the day of collection were: temperature of 29 °C, wind speeds of 2.14 km/h, and relative humidity of 64.06%. The mosquitoes were transported in temperature-regulated boxes and maintained on 10% sugar at 25 °C ± 2 and 75% relative humidity, before being sorted.
Morphological identification of mosquitoes genus
Mosquitoes were individually identified using morphological keys: for Anopheles gambiae complex30; for An. stephensi31, for Culex genera32 and for Aedes genera33.
Anopheles species identification to molecular levels
SINE 200 PCR identification of an. gambiae s.l. Complex mosquitoes
Genomic DNA (gDNA) was extracted from single legs each from 155 female adult An. gambiae s.l. mosquitoes using the LIVAK protocol34. For mosquitoes belonging to the Anopheles gambiae complex, species identification was performed using SINE200 PCR35.
COXI and ITS2 PCR identification of An.
stephensi
Legs from eight (8) female mosquitoes morphologically identified as An. stephensi were also gDNA-extracted. Universal primers LCO1490 and HC0219836 were utilised to amplify a fragment of the mitochondrial COXI gene using KAPA Taq DNA polymerase (KAPA Biosystems, Wilmington, MA, USA). The PCR reaction mix (15 µL) included 1 µL of gDNA, 1.5 µL of 10× Taq Buffer A, 0.4 mM (0.5 µL) each of forward and reverse primers, 1.25 mM (0.75 µL) MgCl2, 0.84 mM (0.5 µL) dNTP mix, and 0.2 µL KAPA Taq DNA polymerase (KAPA Biosystems, Wilmington, MA, USA), with the final volume adjusted using ddH2O. The thermal cycling conditions included an initial denaturation step of 2 min at 94 °C, followed by 35 cycles each of 30 s at 94 °C (denaturation), 30 s at 55 °C (primer annealing), and 30 s at 72 °C (extension). A final extension step of 10 min at 72 °C concluded the reactions. PCR products were resolved on a 1.5% agarose gel stained with peqGREEN, and visualised.
The same gDNA samples were also used for amplification of fragments of ITS2 of ribosomal DNA (rDNA) using size diagnostic PCR primers st-F and UD2-R, which amplify 438 bp An. stephensi–specific diagnostic amplicon, and primers 5.8 S-F and UD2-R which amplify a universal amplicon of varying sizes (> 600 bp) depending upon the length of ITS2 in a particular species37. Details of PCR mix and thermocycling conditions were as above. In addition, for both COXI and ITS2 amplifications gDNA from An. coluzzii, An. arabiensis, and An. gambiae s.s., Culex, and Aedes aegypti were include in the PCRs for comparison.
The PCR products were purified using the QIAquick® Gel Extraction Kit (QIAGEN, Hilden, Germany), ligated into the pJET1.2/blunt cloning vector (ThermoFisher Scientific, Waltham, MA, USA) and transformed into E. coli DH5α cells. Minipreps were prepared using the QIAprep® Spin Miniprep Kit (QIAGEN, Hilden, Germany) and sequenced using pJET1.2/blunt forward and reverse sequencing primers. Sequence trace files were manually examined using BioEdit version 7.7.138. Genetic diversity parameters, including the number of haplotypes (h), haplotype diversity (Hd), the number of polymorphic sites (S), and nucleotide diversity (π), were computed using DnaSP version 6.12.0339. Final sequences were used for BLASTn searches, in NCBI to retrieve previously published sequences from An. stephensi and other Anopheles mosquitoes sharing high similarities from GenBank. Multiple sequence alignments were carried out using CLC Sequence Viewer version 6.9 (http://www.clcbio.com/), and different haplotypes were compared by constructing maximum likelihood phylogenetic trees with MEGA XI40 using Kimura 3 parameter model, with 500 replicates bootstrapping. For outgroup, An. implexus (GQ165788) was used for COXI and An. sawadwongporni (JQ446420) for ITS2, respectively.
Estimation of entomological parameters of transmission
Entomological parameters were calculated, following the procedures outlined by the WHO41. Indoor resting density (IRD) was calculated from the number of Anopheles caught relative to the number of houses sampled. Human biting rate (HBR) was estimated for sampled rooms by dividing the number of mosquitoes captured by the number of occupants sleeping in the room.
To establish anthropophilic index, a set of blood fed females were dissected, heads/thoraces and abdomens used for gDNA extraction using DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol. The gDNA from 66 abdomens were utilised for PCR to detect source of blood. Human blood index was established from the proportion of Anopheles that have fed on humans relative to the total number of blood fed female Anopheles caught, using a cocktail PCR of Kent and Norris42.
A total of 122 gDNA from head/thoraces were tested for Plasmodium infection using nested PCR protocol targeting 18 S rRNA43. Parasite rate was calculated as percentage of mosquitoes with Plasmodium relative to the total number of the females tested. Entomological Inoculation Rate (EIR), which estimates the number of infectious bites per person per night, was calculated by multiplying the mean HBR by the parasite rate41.
