Demonstration of RNAi Yeast Insecticide Activity in Semi-Field Larvicide and Attractive Targeted Sugar Bait Trials Conducted on Aedes and Culex Mosquitoes.

Eco-friendly new mosquito control innovations are critical for the ongoing success of global mosquito control programs. In this study, Sh.463_56.10R, a robust RNA interference (RNAi) yeast insecticide strain that is suitable for scaled fermentation, was evaluated under semi-field conditions. Inactivated and dried Sh.463_56.10R yeast induced significant mortality of field strain Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus larvae in semi-field larvicide trials conducted outdoors in St. Augustine, Trinidad, where 100% of the larvae were dead within 24 h. The yeast was also stably suspended in commercial bait and deployed as an active ingredient in miniature attractive targeted sugar bait (ATSB) station sachets. The yeast ATSB induced high levels of Aedes and Culex mosquito morbidity in semi-field trials conducted in Trinidad, West Indies, as well as in Bangkok, Thailand, in which the consumption of the yeast resulted in adult female mosquito death within 48 h, faster than what was observed in laboratory trials. These findings support the pursuit of large-scale field trials to further evaluate the Sh.463_56.10R insecticide, a member of a promising new class of species-specific RNAi insecticides that could help combat insecticide resistance and support effective mosquito control programs worldwide.

Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes.

G protein-coupled receptors (GPCRs), which regulate numerous intracellular signaling cascades that mediate many essential physiological processes, are attractive yet underexploited insecticide targets. RNA interference (RNAi) technology could facilitate the custom design of environmentally safe pesticides that target GPCRs in select target pests yet are not toxic to non-target species. This study investigates the hypothesis that an RNAi yeast insecticide designed to silence mosquito serotonin receptor 1 (5-HTR1) genes can kill mosquitoes without harming non-target arthropods. 5-HTR.426, a Saccharomyces cerevisiae strain that expresses an shRNA targeting a site specifically conserved in mosquito 5-HTR1 genes, was generated. The yeast can be heat-inactivated and delivered to mosquito larvae as ready-to-use tablets or to adult mosquitoes using attractive targeted sugar baits (ATSBs). The results of laboratory and outdoor semi-field trials demonstrated that consumption of 5-HTR.426 yeast results in highly significant mortality rates in Aedes, Anopheles, and Culex mosquito larvae and adults. Yeast consumption resulted in significant 5-HTR1 silencing and severe neural defects in the mosquito brain but was not found to be toxic to non-target arthropods. These results indicate that RNAi insecticide technology can facilitate selective targeting of GPCRs in intended pests without impacting GPCR activity in non-targeted organisms. In future studies, scaled production of yeast expressing the 5-HTR.426 RNAi insecticide could facilitate field trials to further evaluate this promising new mosquito control intervention.

Field trials reveal the complexities of deploying and evaluating the impacts of yeast-baited ovitraps on Aedes mosquito densities in Trinidad, West Indies.

The use of lure-and-kill, large-volume ovitraps to control Aedes aegypti and Aedes albopictus populations has shown promise across multiple designs that target gravid females (adulticidal) or larvae post-oviposition (larvicidal). Here we report on a pilot trial to deploy 10 L yeast-baited ovitraps at select sites in Curepe, Trinidad, West Indies during July to December, 2019. Oviposition rates among ovitraps placed in three Treatment sites were compared to a limited number of traps placed in three Control areas (no Aedes management performed), and three Vector areas (subjected to standard Ministry of Health, Insect Vector Control efforts). Our goal was to gain baseline information on efforts to saturate the Treatment sites with ovitraps within 20-25 m of each other and compare oviposition rates at these sites with background oviposition rates in Control and Vector Areas. Although yeast-baited ovitraps were highly attractive to gravid Aedes females, a primary limitation encountered within the Treatment sites was the inability to gain access to residential compounds for trap placement, primarily due to residents being absent during the day. This severely limited our intent to saturate these areas with ovitraps, indicating that future studies must include plans to account for these inaccessible zones during trap placement.

Assessment of Trinidad community stakeholder perspectives on the use of yeast interfering RNA-baited ovitraps for biorational control of Aedes mosquitoes.

Dengue, Zika, chikungunya and yellow fever viruses continue to be a major public health burden. Aedes mosquitoes, the primary vectors responsible for transmitting these viral pathogens, continue to flourish due to local challenges in vector control management. Yeast interfering RNA-baited larval lethal ovitraps are being developed as a novel biorational control tool for Aedes mosquitoes. This intervention circumvents increasing issues with insecticide resistance and poses no known threat to non-target organisms. In an effort to create public awareness of this alternative vector control strategy, gain stakeholder feedback regarding product design and acceptance of the new intervention, and build capacity for its potential integration into existing mosquito control programs, this investigation pursued community stakeholder engagement activities, which were undertaken in Trinidad and Tobago. Three forms of assessment, including paper surveys, community forums, and household interviews, were used with the goal of evaluating local community stakeholders' knowledge of mosquitoes, vector control practices, and perceptions of the new technology. These activities facilitated evaluation of the hypothesis that the ovitraps would be broadly accepted by community stakeholders as a means of biorational control for Aedes mosquitoes. A comparison of the types of stakeholder input communicated through use of the three assessment tools highlighted the utility and merit of using each tool for assessing new global health interventions. Most study participants reported a general willingness to purchase an ovitrap on condition that it would be affordable and safe for human health and the environment. Stakeholders provided valuable input on product design, distribution, and operation. A need for educational campaigns that provide a mechanism for educating stakeholders about vector ecology and management was highlighted. The results of the investigation, which are likely applicable to many other Caribbean nations and other countries with heavy arboviral disease burdens, were supportive of supplementation of existing vector control strategies through the use of the yeast RNAi-based ovitraps.

Community acceptance of yeast interfering RNA larvicide technology for control of Aedes mosquitoes in Trinidad.

RNA interference (RNAi), a technique used to investigate gene function in insects and other organisms, is attracting attention as a potential new technology for mosquito control. Saccharomyces cerevisiae (baker's yeast) was recently engineered to produce interfering RNA molecules that silence genes required for mosquito survival, but which do not correspond to genes in humans or other non-target organisms. The resulting yeast pesticides, which facilitate cost-effective production and delivery of interfering RNA to mosquito larvae that eat the yeast, effectively kill mosquitoes in laboratory and semi-field trials. In preparation for field evaluation of larvicides in Trinidad, a Caribbean island with endemic diseases resulting from pathogens transmitted by Aedes mosquitoes, adult residents living in the prospective trial site communities of Curepe, St. Augustine, and Tamana were engaged. Open community forums and paper surveys were used to assess the potential acceptability, societal desirability, and sustainability of yeast interfering RNA larvicides. These assessments revealed that Trinidadians have good working knowledge of mosquitoes and mosquito-borne illnesses. A majority of the respondents practiced some method of larval mosquito control and agreed that they would use a new larvicide if it were proven to be safe and effective. During the community engagement forums, participants were educated about mosquito biology, mosquito-borne diseases, and the new yeast larvicides. When invited to provide feedback, engagement forum attendees were strongly supportive of the new technology, raised few concerns, and provided helpful advice regarding optimal larvicide formulations, insecticide application, operational approaches for using the larvicides, and pricing. The results of these studies suggest that the participants are supportive of the potential use of yeast interfering RNA larvicides in Trinidad and that the communities assessed in this investigation represent viable field sites.

Identification of Host Blood Meals of Mosquitoes (Diptera: Culicidae) Collected at the Aripo Savannas Scientific Reserve in Trinidad, West Indies.

Surveillance for blood-fed female mosquitoes was performed between August 2015 and February 2016 at sites along the periphery of the Aripo Savannas Environmentally Reserve (ASSR) located in northeastern Trinidad, West Indies. We collected engorged female mosquitoes representing 13 species. DNA extractions from dissected abdomens were subjected to PCR amplification with three primer pairs targeting the mitochondrial cytochrome oxidase I and cytochrome b gene sequences. High-quality sequence information and host identification were obtained for 42 specimens representing eight mosquito species with at least one primer combination. A broad range of vertebrates including humans were identified, but the majority were nonhuman mammals, both domestic and wild. Domestic dogs were the most common host and may represent potential sentinel species for monitoring local enzootic arbovirus activity in Trinidad. Culex declarator Dyer and Knab and Culex nigripalpus Theobald were the most common blood-fed mosquito species comprising 79.1% of the total number identified. These species obtained blood meals from birds, nonhuman mammals, and human hosts, and therefore pose significant risks as potential bridge vectors for epizootic arbovirus transmission in the ASSR area as well as other sylvan areas in Trinidad. These data represent the first such results for Trinidad.

COH-SR4 reduces body weight, improves glycemic control and prevents hepatic steatosis in high fat diet-induced obese mice.

Obesity is a chronic metabolic disorder caused by imbalance between energy intake and expenditure, and is one of the principal causative factors in the development of metabolic syndrome, diabetes and cancer. COH-SR4 ("SR4") is a novel investigational compound that has anti-cancer and anti-adipogenic properties. In this study, the effects of SR4 on metabolic alterations in high fat diet (HFD)-induced obese C57BL/J6 mice were investigated. Oral feeding of SR4 (5 mg/kg body weight.) in HFD mice for 6 weeks significantly reduced body weight, prevented hyperlipidemia and improved glycemic control without affecting food intake. These changes were associated with marked decreases in epididymal fat mass, adipocyte hypertrophy, increased plasma adiponectin and reduced leptin levels. SR4 treatment also decreased liver triglycerides, prevented hepatic steatosis, and normalized liver enzymes. Western blots demonstrated increased AMPK activation in liver and adipose tissues of SR4-treated HFD obese mice, while gene analyses by real time PCR showed COH-SR4 significantly suppressed the mRNA expression of lipogenic genes such as sterol regulatory element binding protein-1c (Srebf1), acetyl-Coenzyme A carboxylase (Acaca), peroxisome proliferator-activated receptor gamma (Pparg), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr), as well as gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) in the liver of obese mice. In vitro, SR4 activates AMPK independent of upstream kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). Together, these data suggest that SR4, a novel AMPK activator, may be a promising therapeutic compound for treatment of obesity, fatty liver disease, and related metabolic disorders.

Renoprotective and lipid-lowering effects of LR compounds, novel advanced glycation end product inhibitors, in streptozotocin-induced diabetic rats.

The accelerated formation of advanced glycation/lipoxidation end products (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several natural and synthetic compounds have been proposed and advanced as inhibitors of AGE/ALE formation. We examined the effects of two new AGE/ALE inhibitors, LR-9 and LR-74, on the prevention of early renal disease and dyslipidemia in streptozotocin (STZ)-induced diabetic rats. Diabetic rats were treated with either LR-9 or LR-74 for 32 weeks. Progression of renal disease was evaluated by measurements of urinary albumin and plasma creatinine concentrations. AGE-induced chemical modification of the tail tendon collagen and levels of Nepsilon-(carboxymethyl)- and (carboxyethyl)- lysines (CML and CEL) in skin collagen were measured. AGE/ALE levels in kidneys were determined by immunohistochemistry. Plasma lipids and their lipid hydroperoxide concentrations were also determined. Treatment of either LR-9 or LR-74 significantly inhibited the increase in albuminuria, plasma creatinine, hyperlipidemia, and plasma lipid peroxidation in diabetic rats without any effects on hyperglycemia. Both compounds also reduced CML-AGE accumulation in kidney glomeruli and tubules, AGE-linked fluorescence and cross-linking of tail collagen, and levels of CML and CEL in skin collagen. These results suggest that both LR compounds can inhibit the progression of renal disease and also prevent dyslipidemia in experimental diabetes. These compounds may have an additional beneficial effect as an antioxidant against lipid peroxidation, and thus may provide alternative therapeutic options for the treatment of various diabetic macrovascular complications.

Prevention of early renal disease, dyslipidaemia and lipid peroxidation in STZ-diabetic rats by LR-9 and LR-74, novel AGE inhibitors.

BACKGROUND: Increased formation of advanced glycation/lipoxidation endproducts (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several compounds have been developed as inhibitors of AGE/ALE formation. We examined the effects of two new AGE/ALE inhibitors, LR-9 and LR-74, on the development of early renal disease and lipid metabolism in streptozotocin (STZ)-induced diabetic rats. METHODS: Diabetic Sprague-Dawley rats were treated with either of the LR compounds for 32 weeks. Progression of renal disease was evaluated by measurements of urinary albumin and plasma creatinine concentrations. AGE/ALE and nitrotyrosine levels in kidneys were determined by immunohistochemistry. AGE-induced chemical modification of the tail tendon collagen and levels of Nepsilon-(carboxymethyl) and (carboxyethyl)- lysines (CML and CEL) in skin collagen were measured. Plasma lipids and their lipid hydroperoxide concentrations were also determined. In vitro, both compounds were tested for inhibiting lipid peroxidation reactions. RESULTS: Treatment of either LR compounds significantly inhibited the increase in albuminuria, creatinaemia, hyperlipidaemia and lipid peroxidation in diabetic rats without any effect on hyperglycaemia. Both compounds also reduced CML-AGE and nitrotyrosine accumulation in kidney glomeruli and tubules, AGE-linked fluorescence and cross-linking of tail collagen, and levels of CML and CEL in skin collagen. In vitro, LR compounds inhibited the oxidation of human low-density lipoprotein (LDL). CONCLUSION: Both compounds can inhibit the progression of renal disease and also prevent dyslipidaemia in type-1 diabetic animals. These compounds may have an additional beneficial effect as an antioxidant against lipid peroxidation, and thus may provide alternative therapeutic options for the treatment of various diabetic macrovascular complications.