Effect of irrigation on Anopheles vector larval ecology and insecticide resistance status and mechanisms in mixed crop irrigation in Homa Bay County, Western Kenya

Abstract

Malaria is a debilitating parasitic disease that causes high economic and health burden especially in sub- Saharan Africa. The control and elimination of malaria is greatly jeopardized by drug resistance and increasing insecticide resistance witnessed in different regions with high vector population. The main aim of this study was to determine the effect of irrigation on malaria vector larval ecology, larval survivorship and insecticide resistance in irrigated and non-irrigated areas of Homa Bay County, Western Kenya. Larval sampling was conducted throughout the dry and wet seasons of 2018 and 2019 to assess the availability and productivity of vector aquatic stages in temporary, semi-permanent, and permanent habitats. The productivity of female adult vectors from various habitat types per week was determined using emergent traps. In 2019, monthly larval abundance measurements and predator experiments were also undertaken to assess the densities of mosquito aquatic stages throughout the year and the survivorship of Anopheles arabiensis larvae and habitat productivity in four permanent habitat types respectively. Furthermore, the duration of habitat stability was compared between cohorts of selected habitats followed for a year in irrigated and non-irrigated eco-systems. Water samples were collected from malaria vector larval positive and negative aquatic habitats in the irrigated and non- irrigated sites. Bacteria were cultured from these samples, and the colonies identified using Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). To validate and further identify the bacterial species present in the water samples, DNA was extracted from these bacterial cultures, polymerase chain reaction (PCR) and sequencing were performed. Finally, the metabolite composition of larval positive and negative habitats was examined (total polyphenols, free radical scavenging activity, mineral analysis, chlorophyll and carotenoid, crude protein, fatty acid and lipid/ oil, and sugar contents). In addition, the status and mechanism of insecticide resistance in malaria vectors in irrigated and non-irrigated areas and the contribution of public health interventions and agriculture to insecticide resistance was investigated. Overall, An. gambiae complex was the most predominant vector (95.2%), with An. arabiensis (a sibling species) as most common (98.3%) in both irrigated and non-irrigated areas. The irrigated areas had more habitat variety (74.8%) and Anopheles larvae (72.3%) than the non-irrigated areas. For Anopheles and Culex, single species infestation rate in non-irrigated areas was greater than in irrigated areas. Furthermore, larval densities decreased significantly with age in both irrigated and non-irrigated zones. During the seasonal sample period, temporary habitats were the most productive in both irrigated and non- irrigated areas. The number of semi-permanent and permanent habitats was significantly different in irrigated and non-irrigated areas during monthly dynamics sampling. In the predator experiment, fish was the most efficient predator of all examined predators. Culex larval density dropped as predator density increased. The mortality rate from larva to pupa was above 97% for An. arabiensis and 100% for An. funestus. The highest larval stage survival rate was between larval stages I and II, while the lowest was between larval stages IV and pupa. Life tables for each developmental stage revealed substantial mortality rates, particularly at larval stages II and III. Bacillus was the only genera identified from larval sources in the non-irrigated zone using MALDI-TOF MS. Shigella was the dominant genera in the irrigated region, while Escherichia coli was the most prevalent species. Bacillus was found in 65% of the sequenced isolates. Brevibacillus brevis, Bacillus subtilis, and Exiguobacterium profundum were isolated and classified as larvicidal isolates, together with Bacillus mojavensis, Bacillus tequilensis, Bacilus stercoris, and Brevibacillus agri. Compared to non-irrigated sites, irrigated areas with larvae had lower crude fat and crude protein content. Larvae were present and absent in non-irrigated areas with comparable protein levels. The presence of larvae was seen in both irrigated and non-irrigated areas with high total chlorophyll content. Sites with larvae in both irrigated and non-irrigated locations showed higher sugar concentrations than sites without larvae; however, when compared to identical sites in irrigated areas, non-irrigated sites with larvae had higher sugar concentrations. Furthermore, significant amounts of Manganese, Calcium, and Copper were detected in larvae-infested sites in both irrigated and non-irrigated areas. Pyrethroids were the most common chemical class of pesticides used for crop and animal protection, with very high coverage of LLINs impregnated with pyrethroids and IRS impregnated with organophosphate insecticides. Larvae were susceptible to various dilutions of Diazol and Thunder, resulting in 96% - 100% mortality. Milraz, on the other hand, caused 23.2% and 11.2% larval mortality in irrigated and non-irrigated areas, respectively, at 1:10,000 dilutions, while no mortality was seen at 1:50,000 dilutions in both irrigated and non-irrigated areas. An. arabiensis was the sole species tested in irrigated areas and the main species in non-irrigated areas, with the rest being An. gambiae sensu stricto. In 2018, susceptibility to deltamethrin and malathion was reported in irrigated areas while phenotypic resistance to deltamethrin with susceptibility to malathion was observed in the non-irrigated areas. However, in 2019, phenotypic resistance was observed against deltamethrin in both areas. In 2019, suceptility to DDT and PBO- deltamnethrin was observed in both populations. There were observed low frequencies of L1014F with mutation frequencies ranging from 1% to 16%, and nearly no alteration in the ACE-1 gene. There were higher oxidase and β-Esterase levels in mosquitoes from the irrigated and non- irrigated sites. However, the enzyme levels of oxidase, GST and esterase were not statistically different between the irrigated and non- irrigated area. Malaria breeding sites in Homa Bay have increased as a result of environmental modification. Irrigation has promoted habitat stability, potentially stabilizing malaria breeding and transmission throughout the year, making larval source management a serious problem as a supplementary strategy of malaria control. The presence of B. brevis, B. subtilis, and E. profundum may have altered larval availability, and the existence of related clustered isolates B. mojavensis, B. tequilensis, B. stercoris, and Brevibacillus agri allows for further study as possible larvicidal or adulticidal agents. A high concentration of fatty acids, chlorophyll, sucrose, and manganese may have an effect on larval production. The absence of insecticide reistance in immature vectors to commonly used pesticides in the area is encouraging as this might not be the origin of insecticide resistance observed in the area. However, the widespread use of pyrethroids in agriculture and public health may have contributed to the fast spread of insecticide resistance. The susceptibility of these malaria vectors to organophosphates and PBO synergists in pyrethroids suggests that IRS and ITN-based vector control strategies have a bright future.