Oral Susceptibility to Ivermectin is Over Fifty Times Greater in a Wild Population of Anopheles Albimanus Mosquitoes from Belize than the STECLA Laboratory Reference Strain of A. Albimanus

Background: The STECLA strain of Anopheles albimanus Wiedemann has been in continuous colony for many years and is the reference strain on which genomic studies for the species are based. Recently, the STECLA strain was demonstrated to be much less susceptible to ivermectin ingested in a blood meal (LC 50 of 1468 ng/ml) than all other Anopheles species tested to-date (LC 50 values range from 7 – 56 ng/ml). The ability of A. albimanus to survive ingestion of ivermectin at concentrations far beyond that typically found in the blood of ivermectin-treated people or livestock ( i.e ., 30 – 70 ng/ml) could invalidate the use of ivermectin as a malaria vector control strategy in areas where A. albimanus is a primary vector. Methods: To investigate this, we captured host-seeking A. albimanus in northern Belize and conducted membrane feeding bioassays of ivermectin, using the same methods as described earlier with the STECLA strain. Results: Field-collected A. albimanus in Belize were 55 times more susceptible to ingested ivermectin than were the STECLA reference strain. Oral susceptible to ivermectin in wild A. albimanus from Belize (LC 50 of 26 ng/ml) was equivalent to that of other Anopheles species tested. Conclusion: Contrary to our initial assessment using a highly inbred laboratory strain of mosquito, we show that ivermectin treatment of livestock could reduce A. albimanus populations in areas of Central America and the Caribbean where malaria transmission may occur. Future toxicity screening of ivermectin and other systemic parasiticides for malaria control should consider examining wild populations of the vector species being targeted.

Heat Shock Causes Lower Plasmodium Infection Rates in Anopheles albimanus

The immune response of Anopheles mosquitoes to Plasmodium invasion has been extensively studied and shown to be mediated mainly by the nitric oxide synthase (NOS), dual oxidase (DUOX), phenoloxidase (PO), and antimicrobial peptides activity. Here, we studied the correlation between a heat shock insult, transcription of immune response genes, and subsequent susceptibility to Plasmodium berghei infection in Anopheles albimanus. We found that transcript levels of many immune genes were drastically affected by the thermal stress, either positively or negatively. Furthermore, the transcription of genes associated with modifications of nucleic acid methylation was affected, suggesting an increment in both DNA and RNA methylation. The heat shock increased PO and NOS activity in the hemolymph, as well as the transcription of several immune genes. As consequence, we observed that heat shock increased the resistance of mosquitoes to Plasmodium invasion. The data provided here could help the understanding of infection transmission under the ever more common heat waves.

Dietary and Plasmodium challenge effects on the cuticular hydrocarbon profile of Anopheles albimanus

The cuticular hydrocarbon (CHC) profile reflects the insects’ physiological states. These include age, sex, reproductive stage, and gravidity. Environmental factors such as diet, relative humidity or exposure to insecticides also affect the CHC composition in mosquitoes. In this work, the CHC profile was analyzed in two Anopheles albimanus phenotypes with different degrees of susceptibility to Plasmodium, the susceptible-White and resistant-Brown phenotypes, in response to the two dietary regimes of mosquitoes: a carbon-rich diet (sugar) and a protein-rich diet (blood) alone or containing Plasmodium ookinetes. The CHCs were analyzed by gas chromatography coupled to mass spectrometry or flame ionization detection, identifying 19 CHCs with chain lengths ranging from 20 to 37 carbons. Qualitative and quantitative changes in CHCs composition were dependent on diet, a parasite challenge, and, to a lesser extent, the phenotype. Blood-feeding caused up to a 40% reduction in the total CHC content compared to sugar-feeding. If blood contained ookinetes, further changes in the CHC profile were observed depending on the Plasmodium susceptibility of the phenotypes. Higher infection prevalence caused greater changes in the CHC profile. These dietary and infection-associated modifications in the CHCs could have multiple effects on mosquito fitness, impacts on disease transmission, and tolerance to insecticides.

Malaria risk stratification in Colombia 2010 to 2019

Background Heterogeneity and focalization are the most common epidemiological characteristics of endemic countries in the Americas, where malaria transmission is moderate and low. During malaria elimination, the first step is to perform a risk stratification exercise to prioritize interventions. This study aimed to identify malaria risk strata in the ecoepidemiological regions of Colombia. Methods This was a descriptive and retrospective study using cumulative malaria cases in 1,122 municipalities of Colombia from 2010 to 2019. To identify the strata, the criteria proposed by PAHO were adapted. To classify the receptive areas (strata 2, 3, and 4) and nonreceptive areas (stratum 1), 1,600 m above sea level, ecotypes, main malaria vector presence, Plasmodium species prevalence and occurrence of malaria cases were used. The area occupied by the receptive municipalities, the cumulative burden, and the at-risk population in the regions were calculated. Results Ninety-one percent of the Colombian territory is receptive to the transmission of malaria and includes 749 municipalities with 9,734,271 (9,514,243–9,954,299) million at-risk inhabitants. Stratum 4 accounted for 96.7% of the malaria burden, and cases were concentrated primarily in the Pacific and Uraba-Bajo Cauca-Sinu-San Jorge regions. Plasmodium vivax predominates in most of the receptive municipalities, except in the municipalities of the Pacific region, where P. falciparum predominates. Anopheles albimanus, An. nuneztovari s.l., and An. darlingi were the main vectors in receptive areas. Conclusions In Colombia, 91.2% of the territory is receptive to the transmission of malaria and is characterized by being both heterogeneous and focused. Stratum 4 contains the greatest burden of disease, with a relatively greater proportion of municipalities with a predominance of P. vivax. However, there is a low proportion of municipalities with P. falciparum mainly in the Pacific region. These findings suggest that the latter be prioritized within the malaria elimination plan in Colombia.

Dietary and Plasmodium challenge effects on cuticular hydrocarbons in Anopheles albimanus

The cuticular hydrocarbon (CHC) profile reflects the insect’s physiological states. These states include age, sex, reproductive stage, or gravidity. Environmental factors such as diet, relative humidity, or exposure to insecticides also affect the CHCs composition in mosquitoes. In this work, the CHC profile was analyzed in two Anopheles albimanus phenotypes with different degrees of susceptibility to Plasmodium: the susceptible-White and resistant-Brown phenotypes. The effects of the CHC profile were considered under a carbon-rich diet (sugar), a protein-rich diet (blood), and an infectious challenge (blood containing Plasmodium berghei ookinetes). The CHCs were analyzed by gas chromatography coupled with either mass spectrometry or flame ionization detection, identifying 19 CHCs with chain lengths ranging from 20 to 37 carbons. The qualitative and quantitative changes observed in the CHCs composition were dependent on the diet and parasite challenge, and independent of the phenotype. The exception was the challenged condition, where significant differences between the phenotypes were observed in Z-12 pentacosane, hexacosane 9-octyl, methyl-nonacosane, and methyl-hentriacontane. Since the lipid metabolism in Anopheles mosquitoes has been shown crucial for Plasmodium development, the changes in the CHC profiles associated with infection could have multiple effects on mosquito fitness and impacts on disease transmission.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds

The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

Oral Delivery of dsRNA Targeting a Female-Biased Flight Muscle Actin Impairs Flight in the Malaria Vector, Anopheles Albimanus.

BackgroundDespite the progress to eliminate malaria in Central America, focalized transmission persists, and insecticide resistance is on the rise in the primary vector, Anopheles albimanus. Many of the new control methods being developed depend on the release of a large number of male mosquitoes that must be sorted prior to release. However, An. albimanus manual pupal-sex-sorting is not feasible, and therefore, we explored the use of RNA interference (RNAi) targeting genes with a sex-biased expression for female elimination. Here, we evaluated the effect of feeding larvae with dsRNA for a female-biased orthologue of the flight muscle actin gene.ResultsTwo sex-biased actin forms were identified in An. albimanus. Gene expression analysis showed a >40-fold higher expression of the AALB015469 transcript in female pupae (p = 0.0048) and adults (p = 0.0078) when compared to males. Tissue-specific analysis also suggests this female-biased actin can be an orthologue of the flight muscle actin of Aedes aegypti. At the same time, the AALB015481 transcript showed a >40-fold higher expression in male pupae and adults when compared to females, with no detectable expression in flight muscle. The potential effects of oral-induced RNAi for the female-biased actin were evaluated. Larvae were fed a diet containing either dsRNA for the female-biased actin 3'-UTR alone or for the UTR with an adjoining portion of the C-terminal coding region. A significant number of flightless females resulted from feedings with 3' UTR alone (10.50 ± 5.92 %, p < 0.05) or with the coding region (6.00 ± 2.16 %, p < 0.01). Treatment with the 3' UTR alone resulted in a significant number of flightless males (8.25 ± 3.10 %, p < 0.01). Both diets produced significant mortality in both female and male adults (p < 0.0001). ConclusionsFeeding of An. albimanus larvae with dsRNA targeting the female-biased flight muscle actin orthologue impairs flight in both sexes and affects the overall survival of female and male mosquitoes. Providing dsRNA in the larval diet shows promise as a method for screening other differentially expressed genes as potential targets for female elimination in mosquito breeding facilities.