BACKGROUND: Decrease in malaria rates (e.g. incidence and cases) in Latin America maintains this region on track to achieve the goal of elimination. During the last 5 years, three countries have been certified as malaria free. However, the region fails to achieve the goal of 40% reduction on malaria rates and an increase of cases has been reported in some countries, including Ecuador. This scenario has been associated with multiple causes, such as decrease of funding to continue anti-malarial programmes and the development of insecticide resistance of the main malaria vectors. In Ecuador, official reports indicated phenotypic resistance in Aedes aegypti and Anopheles albimanus to deltamethrin and malathion, particularly in the coastal areas of Ecuador, however, information about the mechanisms of resistance have not been yet elucidated. This study aims to evaluate phenotypic response to deltamethrin and its relationship with kdr mutations in An. albimanus from two localities with different agricultural activities in southern coastal Ecuador.
METHODS: The CDC bottle assay was carried out to evaluate the phenotypic status of the mosquito\'s population. Sequencing the voltage gated sodium channel gene (VGSC) sought knockdown mutations (kdr) in codons 1010, 1013 and 1014 associated with resistance.
RESULTS: Phenotypic resistance was found in Santa Rosa (63.3%) and suspected resistance in Huaquillas (82.1%); with females presenting a higher median of knockdown rate (83.7%) than males (45.6%). No statistical differences were found between the distributions of knockdown rate for the two localities (p = 0.6048) which indicates no influence of agricultural activity. Although phenotypic resistance was confirmed, genetic analysis demonstrate that this resistance was not related with the kdr mechanism of the VGSC gene because no mutations were found in codons 1010 and 1013, while in codon 1014, 90.6% showed the susceptible sequence (TTG) and 7.3% ambiguous nucleotides (TKK and TYG).
CONCLUSIONS: These results highlighted the importance of continuous monitoring of resistance in malaria vectors in Ecuador, particularly in areas that have reported outbreaks during the last years. It is also important to elucidate the mechanism involved in the development of the resistance to PYs to propose alternative insecticides or strategies for vector control in areas where resistance is present.

Chiapas State comprises the largest malaria foci from Mexico, and 57% of the autochthonous cases in 2021, all with Plasmodium vivax infections, were reported in this State. Southern Chiapas is at constant risk of cases imported due to migratory human flow. Since chemical control of vector mosquitoes is the main entomological action implemented for the prevention and control of vector-borne diseases, this work aimed to investigate the susceptibility of Anopheles albimanus to insecticides. To this end, mosquitoes were collected in cattle in two villages in southern Chiapas in July-August 2022. Two methods were used to evaluate the susceptibility: the WHO tube bioassay and the CDC bottle bioassay. For the latter, diagnostic concentrations were calculated. The enzymatic resistance mechanisms were also analyzed. CDC diagnostic concentrations were obtained; 0.7 μg/mL deltamethrin, 12 μg/mL permethrin, 14.4 μg/mL malathion, and 2 μg/mL chlorpyrifos. Mosquitoes from Cosalapa and La Victoria were susceptible to organophosphates and to bendiocarb, but resistant to pyrethroids, with mortalities between 89% and 70% (WHO), and 88% and 78% (CDC), for deltamethrin and permethrin, respectively. High esterase levels are suggested as the resistance mechanism involved in the metabolism of pyrethroids in mosquitoes from both villages. Mosquitoes from La Victoria might also involve cytochrome P450. Therefore, organophosphates and carbamates are suggested to currently control An. albimanus. Its use might reduce the frequency of resistance genes to pyrethroids and vector abundance and may impede the transmission of malaria parasites.

IgG serology can be utilized to estimate exposure to Anopheline malaria vectors and the Plasmodium species they transmit. A multiplex bead-based assay simultaneously detected IgG to Anopheles albimanus salivary gland extract (SGE) and four Plasmodium falciparum antigens (CSP, LSA-1, PfAMA1, and PfMSP1) in 11,541 children enrolled at 350 schools across Haiti in 2016. Logistic regression estimated odds of an above-median anti-SGE IgG response adjusting for individual- and environmental-level covariates. Spatial analysis detected statistically significant clusters of schools with students having high anti-SGE IgG levels, and spatial interpolation estimated anti-SGE IgG levels in unsampled locations. Boys had 11% (95% CI: 0.81, 0.98) lower odds of high anti-SGE IgG compared to girls, and children seropositive for PfMSP1 had 53% (95% CI: 1.17, 2.00) higher odds compared to PfMSP1 seronegatives. Compared to the lowest elevation, quartiles 2-4 of higher elevation were associated with successively lower odds (0.81, 0.43, and 0.34, respectively) of high anti-SGE IgG. Seven significant clusters of schools were detected in Haiti, while spatially interpolated results provided a comprehensive picture of anti-SGE IgG levels in the study area. Exposure to malaria vectors by IgG serology with SGE is a proxy to approximate vector biting in children and identify risk factors for vector exposure.

Immunological priming in insects is defined as a previous contact with non-virulent pathogens, which induces protection after a second virulent infection. The mechanism of this process is not well understood. We have observed midgut DNA synthesis (endoreplication) in Plasmodium berghei exposure mosquitoes (primed) and after the immune challenge, which could be an essential component of the priming response in the mosquito. Endoreplication requires cell cycle components re-direction to make multiple DNA copies. Therefore, it is fundamental to understand the role of cell cycle components in priming. Here, we analyzed the expression of the cyclins A, B, E, and AurkA, and the endoreplication components NOTCH and HNT in the mosquito Anopheles albimanus; after priming with non-infective Plasmodium berghei and challenged with an infective P. berghei. The overexpression of cell cycle elements occurred seven days after priming with a quick reduction 24 h after the challenge. Hnt and NOTCH overexpression occurred 24 h after priming. Antimicrobial peptide cecropin is quickly overexpressed after 24 h in primed mosquitoes, then is downregulated at day seven and overexpressed again after parasite challenge. We also found that DNA synthesis occurs in cells with different nuclear sizes, suggesting a change in midgut epithelial dynamics after Plasmodium exposure. Inhibition of DNA synthesis via cisplatin revealed that DNA synthesis is required for priming to limit Plasmodium infection. Our results indicate the importance of cell cycle components on DNA synthesis and Notch pathway during priming response in An. albimanus mosquitoes.

Pyrethroid resistance in the malaria vector Anopheles albimanus presents an obstacle to malaria elimination in the Americas. Here, An. albimanus CYP6P5 (the most overexpressed P450 in a Peruvian population) was functionally characterized. Recombinant CYP6P5 metabolized the type II pyrethroids, deltamethrin and α-cypermethrin with comparable affinities (KM of 3.3 μM ± 0.4 and 3.6 μM ± 0.5, respectively), but exhibited a 2.7-fold higher catalytic rate for α-cypermethrin (kcat of 6.02 min-1 ± 0.2) versus deltamethrin (2.68 min-1 ± 0.09). Time-course assays revealed progressive depletion of the above pyrethroids with production of four HPLC-detectable metabolites. Low depletion was obtained with type I pyrethroid, permethrin. Transgenic expression in Drosophila melanogaster demonstrated that overexpression of CYP6P5 alone conferred type II pyrethroid resistance, with only 16% and 55.3% mortalities in flies exposed to 0.25% α-cypermethrin and 0.15% deltamethrin, respectively. Synergist bioassays using P450 inhibitor piperonylbutoxide significantly recovered susceptibility (mortality = 73.6%, p < 0.001) in synergized flies exposed to 4% piperonylbutoxide, plus 0.25% α-cypermethrin, compared to non-synergized flies (mortality = 4.9%). Moderate resistance was also observed towards 4% DDT. These findings established the preeminent role of CYP6P5 in metabolic resistance in An. albimanus, highlighting challenges associated with deployment of insecticide-based control tools in the Americas.

It is important to identify repellents that can provide reliable protection from arthropod biting and prevent arthropod-borne diseases, such as malaria. In the present study, the spatial repellent activity and toxicity of two novel pyridinyl amides (1 and 2) were evaluated against Anopheles albimanus, Anopheles quadrimaculatus, and Anopheles gambiae. In vapor repellency bioassays, compound 2 was generally more effective than DEET and 2-undecanone, while compound 1 was about as active as these standards. Overall, transfluthrin was the most active compound for inducing anopheline mosquito repellency, knockdown, and lethality. Although they were not the most active repellents, the two experimental amides produced the largest electroantennographic responses in female antennae. They also displayed modest toxicity to anopheline mosquitoes. Significant synergism of repellency was observed for the mixture of a pyrethroid-derived acid and the repellent 2-undecanone against anopheline mosquitoes, similar to that observed previously in Aedes aegypti. Overall, this study provides insight for further synthesis of alternative amide compounds for use as spatial treatments.

BACKGROUND: The STECLA strain of Anopheles albimanus 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 (4-day LC50 of 1468 ng/ml) than all other Anopheles species tested to-date (LC50 values range from 7 to 56 ng/ml). The ability of An. 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 An. albimanus is a primary vector.
METHODS: To investigate this, host-seeking An. albimanus were captured in northern Belize and used in membrane feeding bioassays of ivermectin, employing the same methods as described earlier with the STECLA strain.
RESULTS: Field-collected An. albimanus in Belize were 55 times more susceptible to ingested ivermectin than were the STECLA reference strain. Oral susceptibility to ivermectin in wild An. albimanus from Belize (4-day LC50 of 26 ng/ml) was equivalent to that of other Anopheles species tested.
CONCLUSIONS: Contrary to initial assessments using a highly inbred strain of mosquito, laboratory studies using a field population indicate that ivermectin treatment of livestock could reduce An. albimanus populations in areas of Central America and the Caribbean where malaria transmission may occur. Toxicity screening of ivermectin and other systemic parasiticides for malaria control should examine wild populations of the vector species being targeted.

BACKGROUND: Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings.
METHODS: Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2-5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed.
RESULTS: Results showed location-associated heterogeneity in both F1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche.
CONCLUSIONS: These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F1 An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

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.

In invertebrates, \"immunological priming\" is considered as the ability to acquire a protective (adaptive) immune response against a pathogen due to previous exposure to the same organism. To date, the mechanism by which this type of adaptive immune response originates in insects is not well understood. In the Anopheles albimanus - Plasmodium berghei model, a DNA synthesis that probably indicates an endoreplication process during priming induction has been evidenced. This work aimed to know the transcriptomic profile in the midguts of An. albimanus after priming induction. Our analysis indicates the participation of regulatory elements of the cell cycle in the immunological priming and points out the importance of the cell cycle regulation in the mosquito midgut.