Akilah T. M. Stewart

There have only been few studies utilizing next generation sequencing technologies for assessing microbial communities of domestic water in the Caribbean. In the current study, Illumina sequencing of the V4 region of 16S rRNA gene amplicons was used to profile the bacteriome of domestic water utilized by residents of Carriacou, Grenada. Analysis of 184 water samples resulted in a total of 11,472 amplicon sequence variants (ASVs) that were dominated by Proteobacteria (52.2%) and Bacteroidota (15.5%). The families Comamonadaceae and Pedosphaeraceae were the most abundant in harvested rainwater stored in both cisterns and tanks. Nostocaceae and Gemmataceae dominated in protected borehole samples and Methylophilaceae and Gallionellaceae in open wells. Genera of potential public health importance were detected at varying prevalence levels, including Acinetobacter and Pseudomonas in rainwater stored in both cisterns and tanks. Amplicons of Escherichia-Shigella were detected in 12.6% of all samples. The detection of non-cultured bacteria via 16S rRNA gene sequencing cannot be used as a specific measure of risk. However, this study shows the occurrence of potential microbial hazards in domestic water in Carriacou that require further investigation. This is the first detailed study to profile the bacteriome of domestic water on a Caribbean island, specifically focusing on harvested rainwater from a rural community across two seasons. Further studies including the use of qPCR for increased data resolution are recommended for assessing risks to inhabitants to guide decisions on managing the microbiological quality of domestic water locally.

Spotted wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), are invasive vinegar flies of East Asian origin that are an increasingly global threat to the small fruit industry. It is essential that new classes of eco-friendly insecticides and cost-effective strategies for SWD control are developed. Here, we describe the preparation of a strain of RNA interference (RNAi) Saccharomyces cerevisiae expressing shRNA that specifically targets the SWD RNA-binding Fox protein 1 (Rbfox1) gene. The yeast effectively silences the SWD Rbfox1 gene, resulting in significant loss of fly neural activity. Laboratory trials demonstrated that the RNAi yeast can be mixed with soda, which functions as SWD attractive targeted sugar bait (ATSB) that can be delivered in a soda bottle feeder. The ATSB, mixed with yeast that was heat-killed prior to suspension in the ATSB, resulted in 92 ± 1% mortality of SWD flies that consumed it, yet had no impact on non-target dipterans. Rbfox.687 yeast delivered in ATSB feeders may one day be a useful component of integrated SWD control programs.

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.

The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention.

This study investigated the sources and microbiological quality of domestic water used by rural communities of Speightstown, Barbados; Carriacou, Grenada; and Nariva, Trinidad. The primary water source was harvested rainwater in Carriacou, and the public water supply for Nariva and Speightstown. Secondary water supplies of the communities came from untreated sources including rainwater, wells, boreholes and springs. E. coli was detected at higher frequencies in water from Carriacou (41.3%) and Nariva (47.4%) than Speightstown (3.6%). Generally, more untreated samples of rainwater (44.6%) and surface/ground water (58.3%) were E. coli positive than treated water obtained from the public supply (9.9%). These findings demonstrate the increased risk to residents in rural communities of the Caribbean who utilize untreated rainwater and environmental sources such as springs and wells. These results demonstrate the usefulness of traditional methods such as the compartment bag test in determining the microbiological quality of domestic water in resource-challenged rural communities of the Caribbean.

The Caribbean region experiences significant water supply challenges, especially for 30% of the population who live in rural areas. Improved water resource management in this region would enhance water availability for its population, but this requires reliable quantitative data to guide relevant policy decisions. The Water Poverty Index (WPI) and its components can be valuable sources of such data but there has not been any recent calculation of this index for countries or communities in the Caribbean. In this study, the WPI was determined for three rural communities in the southern Caribbean: Carriacou, Grenada; Nariva, Trinidad and Tobago; and Speightstown, Barbados. Using data collected from 606 surveys of households as well as published statistics, the overall WPI scores were calculated to be 65/100 for Carriacou, 65/100 for Nariva and 70/100 for Speightstown, which all fell in the ‘medium-low’ and ‘low’ water poverty scale respectively. Resource and Use components had the lowest scores and thus require urgent attention. There were no statistically significant differences across the major components, which demonstrates the common water-related challenges in these Small Island Developing States, despite geographical diversity. This research will aid in defining national water resource management policies in rural communities of the Caribbean.