Date of Award

January 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Jefferson A. Vaughan

Abstract

Malaria continues to be one of the most severe public health concerns worldwide. The malaria parasite, Plasmodium, is transmitted from person to person through the mosquito vector Anopheles. Vector control is a crucial element of anti-malarial campaigns and works best when there is thorough knowledge about an Anopheles vector’s biology and behavior in a given region. To fully eradicate malaria, new vector control strategies must be developed that exploit the behaviors of different vector species. One promising strategy to target vectors that primarily bite outdoors and display generalist feeding behaviors (zoophagic) is the use of veterinary systemic parasiticides. Veterinary parasiticides are drugs used in companion animals and livestock to kill endo- and/or ecto-parasites. By treating animals with systemic parasiticides, the mosquitoes feeding on these animals will be killed or sterilized, reducing the vector population and eliminate residual malaria transmission.The lethality and sublethal effects of seven veterinary systemic parasiticides (ivermectin, eprinomectin, moxidectin, abamectin, fipronil, fluralaner, and afoxolaner) were evaluated in two laboratory strains of zoophagic Anopheles vectors: Anopheles albimanus STECLA and An. stephensi STE2. One compound, ivermectin, evaluated for its effects on a wild population of An. albimanus (An. albimanus BELIZE). First, these compounds were fed to mosquitoes in vitro, using membrane feeders and mortality and reproductive capacities were examined. An. albimanus STECLA was highly tolerant of ivermectin (LC50 1468 ng/ml) and was most susceptible to fipronil (23 ng/ml). An. stephensi STE2 was most susceptible to ivermectin (LC50: 7 ng/ml). An. albimanus BELIZE was much more susceptible to ivermectin compared to An. albimanus STECLA (26.4 ng/ml). Ivermectin, fipronil, and afoxolaner significantly decreased reproductive capacity in treated An. stephensi STE2 and An. albimanus STECLA. In vivo tests using mice were also used to determine if systemic parasiticide vector control would be feasible in a live organism. Ivermectin reduced survival in both An. stephensi (χ2=87.1, p<0.0001) and An. albimanus (χ2=12.5, p=0.0004) that fed one day after mice were treated. Reproduction capacity also decreased in An. stephensi feeding on mice one day post treatment (t=8.18, p<0.0001). However, when fed three days after mouse treatment, only An. stephensi was still susceptible to ivermectin (χ2=16.8, p<0.0001). Neither species had significantly reduced fecundity when fed on mice three days post treatment. Mice were also treated with either injectable or topical fipronil and mortality was quantified for mosquitoes feeding on the mice for up to 11 days post treatment. Fipronil significantly decreased survival in both species feeding on treated mice for up to 11 days post treatment. By day 11 post treatment, mortality decreased in both species feeding on mice injected with fipronil. By contrast, topically applied fipronil was still extremely toxic to both species. Ivermectin and fipronil were tested in cattle to determine if systemic parasiticides would work in a tropical environment. In Belize, three heifers were treated with topical fipronil, one cow was treated with an injectable form of ivermectin, and two left untreated. Mortality, blood meal digestion, and ovarian development were recorded for mosquitoes feeding 2-, 5-, 7- and 14- days post treatment. Both ivermectin and fipronil decreased survival in wild An. albimanus fed on the treated animals for up to 7 days post treatment. By day 14 post treatment, ivermectin was no longer lethal to mosquitoes. Fipronil was still signifcantly lethal to mosquitoes on day 14 post treatment, but the effect had waned. Both compounds reduced ovarian development in surviving mosquitoes, but only ivermectin reduced blood meal digestion. To test how ivermectin distributes in the body and the effect of formulation on drug effectiveness, calves were treated with either a topical or injectable form of ivermectin. An. stephensi STE2 were fed on different body postions on calves and mortality, blood meal digestion, and ovarian development were recorded for 2, 5, 9, 14 and 23 days post treatment. Injectable and topical ivermectin both reduced survival until day 14, no matter where they fed on the body. Only mosquitoes fed on topically treated calves near the site of drug application experienced a reduction in survival. Mosquitoes fed on calves injected with ivermectin experienced a significant delay in blood meal digestion until after day 14 post treatment. Mosquitoes fed on calves treated with topical ivermectin experienced a significant delay in blood meal digestion until day 14 post treatment, and a slight delay in blood meal digestion on day 23 post treatment. Ovarian development was also hindered in both treatments until day 14. On day 23 post treatment, mosquitoes fed on calves injected with ivermectin had no significant delay in ovarian development, while those fed on calves treated with topical ivermectin still had a significant problem developing fully ovaries. Anopheles albimanus STECLA had the highest tolerance to ivermectin of any Anopheles species tested to date. To explore why, three possible mechanisms of resistance were examined. The first, midgut absorption was tested using parenteral injections of ivermetin. This bypassed the midgut and allowed ivermectin to directly interact with its target receptor. Ivermectin was significantly more toxic to An. albimanus STECLA when injected (LC50: 188 ng/ml) than when ingested (LC50: 1468 ng/ml). Next, an potential increase in cytochrome P450, an enzyme responsible for breaking down ivermectin, was tested by examining An. albimanus STECLA resistance to another compound broken down by cytochrome P450, permethrin. An. albimanus STECLA had no resistance to permethrin, ruling out this mechanism or resistance. Finally, conformation changes in the target of ivermectin, the glutmate-gated chloride channels could be responsible for ivermectin tolerance. The sequences of several Aedes and Anopheles species were analyzed using phylogenetic methods to examine the glutamate-gated choride channel gene for selection. Seven exons were analyzed and an phylogenetic tree was constructed. The gene was highly conserved across the mosquito species, except on exon seven. While positive selection on this exon may influence ivermectin tolerance, it is not conclusive. In conclusion, veterinary systemic parasiticides are effective against two species of Anopheles mosquitoes. However, mosquitoes vary in their suceptibility to different compounds. Before implementing systemic parasiticides for vector control, local vector species must be evaluated for their susceptibility to the intended drug. The pilot trial provides proof-of-concept supporting this method of vector control. This study also is a cautionary tale that ivermectin resistance in mosquitoes can develop, and the mechanisms should be further examined.

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