Mosquito breeding water parameters are important determinants for Microsporidia MB in the aquatic stages of Anopheles species

Abstract

Background: Microsporidia MB disrupts Plasmodium development in Anopheles mosquitoes, making it a possible biocontrol tool for malaria. As a tool for vector/disease control, its ecological distribution and the factors that determine their occurrence must be defined. We investigated the frequency of Microsporidia MB in Anopheles mosquitoes across selected sites in northern and southern Ghana, as well as the physicochemical parameters of mosquito breeding water that are associated with the occurrence of the fungus, by fitting regression models.

Methods: A non-column extraction method was used to extract DNA from the abdomens of 4255 adult Anopheles mosquitoes that emerged from larvae and pupae collected between August and October of 2021 and 2022. Detection of Microsporidia MB was achieved using quantitative PCR (qPCR), while mosquito species were molecularly identified using short interspersed nuclear elements (SINE), restriction fragment length polymorphism (RFLP) methods, and the ANOSPP algorithm.

Results: Overall Microsporidia MB distribution was 2.2% (92/4255). Male mosquitoes exhibited a higher frequency of infections and had a predicted probability of infection that was 85% higher than that of females. Sites in Ghana's Savannah zone had the highest Microsporidia MB distribution (68.5%). Biochemical oxygen demand in mosquito breeding water was estimated to be positively associated with and significantly predicts Microsporidia MB in mosquitoes with an accuracy of 94%. Increasing ammonium ion concentrations reduced the chances of finding Microsporidia MB-positive mosquitoes. According to our data, all Anopheles mosquitoes, including minor species such as An. squamosus, An. pretoriensis and An. rufipes, had equal probability of Microsporidia MB infection.

Conclusions: These results provide preliminary information on micro-ecological factors that potentially support the sustainability of Microsporidia MB infection in mosquitoes during their aquatic life stages. It will be important, therefore, to explore the impact of strategies for larval source management on these factors to ensure that the symbiont's persistence during the host's aquatic stages may not be adversely affected should it be used as an integrated approach for mosquito/disease control.

Keywords: Anopheles mosquitoes; Microsporidia MB; Mosquito breeding site; Physicochemical parameters.

Fig. 1

Map of Ghana showing towns where mosquito breeding sites were inspected for larval and pupal collections. The map was generated with ArcGIS software using GPS co-ordinates that were obtained with the KoboCollect app

Fig. 2

Frequencies of Anopheles mosquitoes across study sites. Each bar represents the total percentage of mosquitoes from the collection attributed to a specified site. Stacked bars are colored according to species and show the percentage of Anopheles spp. contributed by each site. The study sites are arranged from the northernmost to the southernmost regions (Fig. 1)

Fig. 3

Microsporidia MB distribution among mosquito species, study sites and mosquito sex. A Pie chart of the percentage of mosquito species found to be infected. B Distribution of the total Microsporidia MB abundance according to site and mosquito sex

Fig. 4

Assessment of the selected model for explaining Microsporidia MB status based on study site and mosquito sex. A Receiver operating characteristic (ROC) curve showing area under the curve (AUC), which depicts the accuracy of the model. B The predicted probabilities of detecting Microsporidia MB infection in mosquitoes from sampled sites, showing that the odds for Microsporidia MB positivity status are higher in males across all sites

Fig. 5

Boxplots of estimated water parameters in the study sites showing the distribution of the physicochemical parameters among breeding sites at each study area. The horizontal line depicts the median value. P-values were calculated with the Kruskal-Wallis test to compare the median among study sites for each parameter. Significant P-values are shown in bold. BOD, Biochemical oxygen demand; DO, dissolved oxygen; NH4-N, ammonium-nitrogen; NO3-N, nitrate-nitrogen; PO4-P, phosphate-phosphorus; SAL, salinity; SO4, sulfate; TURB, turbidity

Fig. 6

Results from the best selected model that explains probability of detecting Microsporidia MB-infected mosquitoes based on physicochemical parameters of the mosquito breeding water. A Principal coordinates analysis (PCA) biplot of the correlation between Microsporidia MB and the quantitative parameters. Arrows show the direction of each variable in relation to the principal components (PC), and the lengths of the arrows represent the strength of the contribution of the variable to the PC. Arrows close together are positively correlated with each other, while those opposite each other are negatively correlated. Variables at a 90º angle have no correlation. B Receiver Operating Characteristic (ROC) curve shows the area under the curve (AUC). C Predicted probabilities of detecting Microsporidia MB infection in mosquitoes from sampled sites based on arbitrary BOD and NH4-N. BOD, Biochemical oxygen demand; DO, dissolved oxygen; NH4-N, ammonium-nitrogen; NO3-N, nitrate-nitrogen; PO4-P, phosphate-phosphorus; SO4, sulfate