Collection and DNA Detection of Dirofilaria immitis (Rhabditida Onchocercidae), Using a Novel Primer Set, in Wild-Caught Mosquitoes From Gainesville, FL.

Dirofilaria immitis, the causative agent of dog heartworm disease, is an important cause of canine morbidity and mortality, expensive to treat, and severe infections are often fatal. Much is known about the pathogen in the canine host, yet little is known on the basic ecology of the nematode in the mosquito vector. Thus, to evaluate the effectiveness of collection techniques on ability to capture dog heartworm-infected mosquitoes (Diptera Culicidae), we conducted a field study spanning 111 wk. Four methods were used: two aspirators types, sweep netting, and a CDC trap. All sites had canines present in either residential yards (n = 4) or dog kennel facilities (n = 3). Collected mosquitoes were sorted by site, trap, species, and date, then pooled into groups of up to 25 individuals. Mosquito head and thorax pools were extracted for DNA, that was screened using currently available protocols. These protocols were found unreliable; thus, we developed a novel qPCR primer and probe set. Using this method, the original samples were re-assayed and provided 494 positive pools. Approximately 10% of positive samples were confirmed by Sanger sequencing. Twenty-two mosquito species tested positive for dog heartworm DNA, including a new association with Wyeomyia mitchellii (Theobald). Although Aedes atlanticus (Dyar and Knab), Anopheles crucians Wiedemann, and Culiseta melanura (Coquillett) composed nearly 36% of the total collection, these species represented 42% of the qPCR positive pools. Infection rates within commonly collected mosquitoes ranged up to 2.5%, with more rarely collected species ranging up to 14%. The CDC trap was the most effective collection method at trapping infected mosquitoes.

Combining DL-Methionine and Bacillus thuringiensis Subspecies israelensis: Prospects for a Mosquito Larvicide.

Mosquito larvicides can reduce mosquito populations at the source, potentially decreasing biting rates and pathogen transmission. However, there is a growing need for mosquito larvicides that are environmentally sustainable. Bacillus thuringiensis subspecies israelensis (BTI) is a naturally occurring bacterium commonly used as a larvicide to manage mosquito populations. Methionine is an essential amino acid that has demonstrated toxic properties against larval mosquitoes in laboratory experiments, while having minimal effects on non-target organisms. The goal of this study was to evaluate the potential for a novel combination larvicide by testing for compatibility between these two active ingredients. We began by determining the lethal concentration values (LCs) of BTI and DL-methionine against Anopheles quadrimaculatus Say and Aedes aegypti Linnaeus (Diptera: Culicidae) larvae. These bioassays were conducted in glass jars and mortality was observed 48 h post-treatment. We found that while DL-methionine was more toxic to An. quadrimaculatus than Ae. aegypti, the opposite was true for BTI. Then, we used these LCs to conduct bioassays with a combination of BTI and DL-methionine to determine the relationship between the two active ingredients when used against An. quadrimaculatus and Ae. aegypti larvae. The findings of this study demonstrate that BTI and DL-methionine have the potential to be complementary due to their additive properties at higher concentrations and effect levels when tested against An. quadrimaculatus. However, an antagonistic relationship was detected at the concentrations tested with Ae. aegypti. These results are encouraging and imply that a DL-methionine or BTI/DL-methionine combination larvicide could be used in management of Anopheles species.