Study area
Mosquitoes were collected from 14 districts in 12 administrative regions across Tanzania mainland (Figure 1). Tanzania has a broadly tropical climate, with four primary climatic zones: the hot and humid coastal plain (i.e., Pwani, Tanga, Lindi and Mtwara), the semi-arid central plateau (i.e., Dodoma, Kigoma, Katavi and Rukwa), the high rainfall lake regions (i.e., Kagera and Mwanza), and the cooler highlands (i.e., Morogoro and Ruvuma). On the Tanzanian coast and offshore islands, temperatures typically fluctuate between 27°C and 29°C. In the central, northern, and western regions, temperatures vary between 20°C and 30°C. The extended rainy season spans from March to May, while the shorter rainy season extends from October to early December, with dry season lasting from June to September. Overall, annual rainfall ranges from 550 mm in the central areas to 3690 mm in certain parts of the southwestern highlands 47. In most of these districts, majority of the rural households are subsistence farmers 48,49. Malaria prevalence in children under the age of 5 differs significantly in the study area, with the highest in the regions in the north-western (i.e., Kagera and Kigoma) and south-eastern regions (i.e., Mtwara and Lindi) to less than 1% in the central region (i.e., Dodoma) 50.
The data collection sites are shown in Figure 1. Specific districts were: Misenyi in Kagera, Kakonko and Kibondo in Kigoma, Chamwino in Dodoma, Ulanga and Kilombero in Morogoro, Tunduru in Ruvuma, Bagamoyo in Pwani, Nkasi in Rukwa, Tanganyika in Katavi, Misungwi in Mwanza, Mtama in Lindi, Mahurunga in Mtwara and Muheza in Tanga. These collection sites represent diverse geographical regions, including the hot and humid coastal plain (i.e., Bagamoyo, Muheza, Mtama and Mahurunga), the semi-arid central plateau (i.e., Chamwino, Kakonko, Kibondo, Tanganyika and Nkasi), the high rainfall lake regions (i.e., Misenyi and Misungwi), and the cooler highlands (i.e., Kilombero, Ulanga and Tunduru).
Figure 1: Map of Tanzania showing the regions where Anopheles funestus mosquitoes were collected.
Mosquito collection and processing
Mosquito collections were completed between December 2018 and December 2022. Whereas multiple mosquito species were collected, only An. funestus s.l. are used in this analysis. In each of the districts, at least two houses were selected upon consent from the household heads and used for the collection of adult mosquitoes. Centers for Disease Control and Prevention (CDC) light traps 51 and Prokopack aspirators 52 were used to sample indoor host-seeking and resting mosquitoes respectively. The sampling methods captured other mosquito species, thus mosquitoes were morphologically sorted to the species level and females of An. funestus individually packed in an Eppendorf tube with 80% ethanol. In addition, in some locations such as Dodoma, Tanga and Morogoro regions, larval collections were conducted using standard larval dippers 53. The collected larva were reared to adults as previously described 54, then also sorted by taxa as above.
Extraction of Genomic DNA
Genomic DNA was extracted from the heads and thoraces of collected mosquitoes using DNAzol method 55. Bead ruptor 96 well plate homogenizer (OMNI international, Kennesaw, GA, USA) was used for homogenization and the resulted DNA pellets were eluted in 50 µl of Tris-EDTA buffer.
Identification of the sibling species in the An. funestus group and detection of Plasmodium spp infections.
A cocktail of species-specific primers for the identification of the sibling species in the An. funestus group was used, as previously described by Koekemoer et al. 56; with a slight adaptation to include a primer for Anopheles rivulorum-like (Table 1) in the cocktail 42. A nested PCR assay was used for the detection of the Plasmodium spp., of which the first round of the PCR included universal forward and reverse primers for 18S rDNA Plasmodium spp. (Table 2) regardless of species; followed by a second round using the amplicon from the first round as DNA template. Species-specific primers for Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax and Plasmodium malariae were used in the second round (Table 2).
Further analysis of the ITS2 region in non-amplified Anopheles funestus samples to investigate polymorphisms
Ten samples underwent cloning and sequencing, employing the following primers: ITS2A: 5’ TGT GAA CTG CAG GAC ACA T 3’; (Forward) ITS2B: 5’ TAT GCT TAA ATT CAG GGG GT 3’ (Reverse). The PCR reaction mixture, conditions and procedures for the thermal cycling and electrophoresis were similar to those described earlier. The amplicons (approximately 840 base pairs) were excised from the gel and cleaned using Wizard® SV Gel and PCR Clean-Up System (Catalogue number: #A9281, Promega). The purified product was cloned using a plasmid vector pJET1.2/blunt (CloneJET PCR Cloning Kit, Catalogue number: #K1231, Thermo Scientific). The resulting recombinant plasmid DNA was isolated and purified (QIAprep Spin Miniprep Kit, Catalogue number #27106, Qiagen) and sent for sequencing. Sequencing of the recombinant plasmid DNA was carried out using the reverse PJET1.2 primer (5’- AAGAACATCGATTTTCCATGGCAG-3’). Plasmid primer regions trimming, sequence alignment, and analysis were performed using SeaView software 57.
Data Analysis
The data collected from the field included the number of traps used, the number of collection days, and the mosquitoes captured per trap, facilitating the calculation of trap nights (defined as the product of the number of traps and collection days). The annual entomological inoculation rate (EIR) was determined by multiplying human biting rates and Plasmodium sporozoite prevalence, then adjusted for 365 days 31. A coefficient of 0.68 was used for conversion of the sampling efficiency of the CDC light trap relative to human landing catch (HLC) 31,46. Mosquitoes collected using Prokopack aspirators and from larval collections were excluded from the EIR calculation, as these methods do not accurately reflect host-seeking behavior or the potential for infectivity. SeaView software 57 was used for the alignment and analysis of the ITS2 sequences of the non-amplified samples.
Table 1: Primers for PCR detection of Anopheles funestus group sibling species.
Primer orientation
|
Primer sequence
|
Sibling species
|
Size of the PCR-product (bp)
|
Universal forward
|
TGTGAACTGCAGGACACAT
|
-
|
-
|
FUN reverse
|
GCATCGATGGGTTAATCATG
|
An. funestus s.s.
|
505
|
VAN reverse
|
TGTCGACTTGGTAGCCGAAC
|
An. vaneedeni
|
587
|
RIV reverse
|
CAAGCCGTTCGACCCTGATT
|
An. rivulorum
|
411
|
PAR reverse
|
TGCGGTCCCAAGCTAGGTTC
|
An. parensis
|
252
|
LEES reverse
|
TACACGGGCGCCATGTAGTT
|
An. leesoni
|
146
|
RIVLIKE reverse
|
CCGCCTCCCGTGGAGTGGGGG
|
An. rivulorum-like
|
313
|
Table 2: Primers for a nested PCR detection of Plasmodium infection and species.
PCR detection
|
Primer orientation
|
Primer sequence
|
Size of the PCR-product (bp)
|
Plasmodium infection detection
|
Forward
|
AGTGTGTATCAATCGAGTTTC
|
783 - 821
|
Reverse
|
GACGGTATCTGATCGTCTTC
|
783 - 821
|
Plasmodium species-specific detection
|
Forward (Universal)
|
CTATCAGCTTTTGATGTTAG
|
-
|
P. falciparum reverse
|
GTTCCCCTAGAATAGTTACA
|
344
|
P. vivax reverse
|
AAGGACTTCCAAGCC
|
457
|
P. ovale reverse
|
CCAATTACAAAACCATG
|
202
|
P. malariae reverse
|
TCCAATTGCCTTCTG
|
241
|