Anopheles ecology, genetics and malaria transmission in northern Cambodia

Abstract

In the Greater Mekong Subregion, malaria cases have significantly decreased but little is known about the vectors or mechanisms responsible for residual malaria transmission. We analysed a total of 3920 Anopheles mosquitoes collected during the rainy and dry seasons from four ecological settings in Cambodia (villages, forested areas near villages, rubber tree plantations and forest sites). Using odor-baited traps, 81% of the total samples across all sites were collected in cow baited traps, although 67% of the samples attracted by human baited traps were collected in forest sites. Overall, 20% of collected Anopheles were active during the day, with increased day biting during the dry season. 3131 samples were identified morphologically as 14 different species, and a subset was also identified by DNA amplicon sequencing allowing determination of 29 Anopheles species. The investigation of well characterized insecticide mutations (ace-1, kdr, and rdl genes) indicated that individuals carried mutations associated with response to all the different classes of insecticides. There also appeared to be a non-random association between mosquito species and insecticide resistance with Anopheles peditaeniatus exhibiting nearly fixed mutations. Molecular screening for Plasmodium sp. presence indicated that 3.6% of collected Anopheles were positive, most for P. vivax followed by P. falciparum. These results highlight some of the key mechanisms driving residual human malaria transmission in Cambodia, and illustrate the importance of diverse collection methods, sites and seasons to avoid bias and better characterize Anopheles mosquito ecology in Southeast Asia.

Figure 1

Map of the mosquito collection sites in Kaev Seima district, Mondulkiri, Cambodia. Land use map from Landsat satellite imagery retrieved from SERVIR Mekong (https://landcovermapping.org/en/). Map created in QGIS version 3.14.0 Pi (https://www.qgis.org/en/site/). Collection sites: F = forest site, V = village, FNV = forest area near the village, P = rubber tree plantation.

Figure 2

Average number of Anopheles females collected per trap per hour in the different collection sites in the HBNTs and the CBNTs.

Figure 3

Day biting rate (proportion of Anopheles females collected between 6am and 6 pm) of mosquitoes captured in the different collection sites and seasons in (a) HBNTs and (b) CBNTs. The day biting rate was significantly higher during the dry season compared to the rainy season in both kind of traps (HBNTS χ² = 19.08, df = 1, p < 0.001 and CBNTS χ² = 115.53, df = 1, p < 0.0001). Data show average proportion ± 95% confidence intervals.

Figure 4

Malaria parasite prevalence in mosquitoes captured across different collection sites and seasons in (a) HBNTs and (b) CBNTs. Data show average proportion ± 95% confidence intervals. Numbers below the bars indicate the total number of mosquitoes. Zero in the graph indicates no infected mosquitoes. Malaria parasite prevalence was significantly higher during the dry season compared to the rainy season in the HBNTS (χ² = 8.57, df = 1, p = 0.003) whereas the opposite was observed in the CBNTS (χ² = 5.44, df = 1, p = 0.02). In the CBNTS, malaria parasite prevalence was significantly affected by daytime (χ² = 8.13, df = 1, p = 0.004) and the interaction between collection sites and seasons (χ² = 6.94, df = 2, p = 0.03).

Figure 5

Proportions of the molecularly identified samples in the CBNTS and HBNTS.

Figure 6

Insecticide resistance mutations are very frequent in Anopheles peditaeniatus. All A. peditaeniatus (n = 108) in the molecularly typed set of 844 are depicted here sorted by their collection location and within collection site by their insecticide resistance genotypes. Each row of three boxes represents genotype data from a single A. peditaeniatus individual. Out of 108 A. peditaeniatus, 106 carried the G119S mutation (3 heterozygotes, 103 homozygotes), 78 carried the L1014F mutation (22 heterozygotes, 56 homozygotes), and 104 carried the A296S mutation (12 heterozygotes, 92 homozygotes). Forty nine percent (53/108) of individuals have homozygous resistant genotypes for all 3 loci and an additional 23% (25/108) carry at least one resistant allele for each of the 3 insecticide loci typed. Of the remaining 28% of individuals (30/108), 28 carried the homozygous susceptible allele at the L1014F kdr mutation (2 individuals with no available sequence), 17 were homozygous for the resistant allele at both ace-1 and rdl, another 7 individuals were homozygous for ace G119S and heterozygous for rdl A296S. A single individual was homozygous for the rdl resistant mutation and heterozygous for the ace-1 mutation. Five individuals carried a resistance mutation at only one of the 3 loci tested, 3 were ace-1 mutation homozygotes, 1 was heterozygous for rdl resistance and 1 was homozygous for rdl resistance with no sequence available for ace-1 resistance.

Figure 7

Geographic distribution of insecticide resistance mutations in A. peditaeniatus. Allele frequencies for ace-1 G1119S (top), kdr L1014F (middle) and rdl A196S (bottom) are shown as a function of sampling location. For kdr there is a detectable geographic pattern with the Forest site 2 exhibiting significantly (p = 0.03) higher susceptible allele frequency than all other sites combined, while Forest near Village 2 (p = 0.04) and 3 (p = 0.02) exhibit significantly higher resistant allele frequencies than all other sites combined. A similar pattern is observed for rdl, but here the total number of susceptible alleles does not provide the power to detect significant differences. For locations: F = Forest, FNV = Forest Near Village, P = Plantation, and V = Village. Only those 8 sites with at least 5 A. peditaeniatus (10 alleles) are shown here. Resistant (res) and susceptible (sus) alleles are colored according to the legends, with resistant alleles in the darker shade.

Figure 8

(a) Percentage of each land cover category per collection site determined by a 2 km buffer zone and based on Landsat imagery (Landsat 4–8, 30 m resolution, 2017). Land cover classes defined as: cropland (land with herbaceaous and shrubby crops with harvesting and bare soil periods), evergreen broadleaf (land dominated by trees > 60% canopy cover with a tree above 5 m, evergreen broadleaf trees make up > 60% of the total tree cover), forest (land spanning more than 0.5 hectares with trees higher than 5 m and a canopy cover of more than 10%), mixed forests (land with > 60% tree canopy cover, tree height is greater than 5 m, and the forest composition is mixed such that no single forest type makes up > 60% of the total tree cover), plantation forests (land cultivated with perennial crops that reach heights above 5 m and occupy the land for long periods). The table is indicating patch density (PD), edge density (ED), Shannon’s diversity index (SHDI), and Simpson’s diversity index (SIDI) for each buffer zone. (b) Percentage of each mosquito species (molecular identification) sampled in each collection site. Sample sizes are given in parentheses.