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.

Induction of ethylene inhibits development of soybean sudden death syndrome by inducing defense-related genes and reducing Fusarium virguliforme growth.

Ethylene is a gaseous hormone that regulates plant responses to biotic and abiotic stresses. To investigate the importance of ethylene in soybean resistance to Fusarium virguliforme (Fv), the causal agent of sudden death syndrome (SDS), soybean cultivars Williams 82 (SDS-susceptible) and MN1606 (SDS-resistant) were treated 24 h before and 24h after Fv inoculation with either ethephon (ethylene inducer), cobalt chloride (ethylene biosynthesis inhibitor), or 1-MCP (ethylene perception inhibitor). Inoculated plants were grown for 21 days at 24°C in the greenhouse and then evaluated for SDS severity and expression of soybean defense genes. In both cultivars, plants treated with ethephon showed lower SDS foliar severity compared to the other treatments, whereas those treated with cobalt chloride or 1-MCP showed the same or higher SDS foliar severity compared to the water-treated control. Ethephon application resulted in activation of genes involved in ethylene biosynthesis, such as ethylene synthase (ACS) and ethylene oxidase (ACO), and genes involved in soybean defense response, such as pathogenesis-related protein (PR), basic peroxidase (IPER), chalcone synthase (CHS), and defense-associated transcription factors. Cobalt chloride and 1-MCP treatments had little or no effect on the expression of these genes. In addition, ethephon had a direct inhibitory effect on in-vitro growth of Fv on PDA media. Our results suggest that ethephon application inhibits SDS development directly by slowing Fv growth and/or by inducing soybean ethylene signaling and the expression of defense related genes.