Evaluation of commercial aerosol insecticides for control of Aedes aegypti susceptible or resistant to pyrethroids.

OBJECTIVE: To evaluate indoor use of commercial aerosols for dengue vector mosquito control, and estimate the number of treatable houses per can. MATERIALS AND METHODS: Four aerosol products containing combinations of pyrethroids (two containing propoxur and one containing synergists too), were evaluated with mosquitoes in a room of a Tapachulastyle house. Eight cages containing 20 insecticide susceptible or resistant females were hung from tripods, another set was placed in sheltered areas of the room. From the entrance of the room, one of 4-9 concentrations was sprayed for each aerosol, leaving the mosquitoes for 30 min after sprayed. Mortality was recorded after 24 h and lethal concentrations were calculated. RESULTS: Aerosol A had the highest LC50, with 0.308 g for mosquitoes hanging from tripods and 0.453 g for sheltered mosquitoes; followed by aerosols C, D and B, with statistical differences between types of exposure. CONCLUSIONS: Aerosols B-D could spray 20-25 3-room houses (56 m3-room), killing all resistant mosquitoes. Aerosols may become a good tool for indoor mosquito control, if the optimal concentration and correct spray method are used.

Aerial release of Aedes aegypti male mosquitoes using an unmanned aerial vehicle: a novel control strategy.

OBJECTIVE: To development of a methodology for the chilling, handling, transport, and release of male Aedes aegypti mosquitoes, reared in insectary conditions to release in the field with unmanned vehicles to compete sexually with wild males in the field. MATERIALS AND METHODS: A population of Ae. aegypti from different areas in Tapachula, Chiapas, was used. Laboratory tests were conducted: Effect of temperature and cooling time on the knockdown, recovery of males, and copulatory success. RESULTS: The chilling temperature of 3 ± 1ºC for 30 min, was used as a knockdown temperature before handling, packing, transportation, and aerial release. The males subjected to the entire process, including the semi-field aerial release test, showed normal sexual behavior activity, obtaining 100% of females inseminated. CONCLUSION: These results present the feasibility of applying a new control methodology using unmanned aerial vehicle (UAV) as support for the sterile insect release technique (SIT), use of Wolbachia or both, in male Ae. aegypti, for the design of strategies to control their populations.

Resistance to Pyrethroids in the Malaria Vector Anopheles albimanus in Two Important Villages in the Soconusco Region of Chiapas, Mexico, 2022.

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.

Composition and abundance of anopheline species according to habitat diversity in Mexico / Composición y abundancia de especies de anofelinos según la diversidad de hábitats en México

Abstract: Objective: To determine the abundance and geographic distribution of the main malaria vectors, which are influenced by habitat characteristics and ecological factors that directly impact adult density and the dynamics of malaria transmission in Mexico. Materials and methods: Samples of larvae were collected from 19 states in Mexico. Each larval habitat was characterized in situ determining the following parameters: water depth, turbidity, percentage of vegetation cover, amount of detritus, presence of algae, light intensity, type of vegetation, amount of predators, habitat stability, altitude, and hydrologic type. Results: A total of 21 687 larvae corresponding to 13 anopheline species were obtained from 149 aquatic habitats. The most abundant species were Anopheles pseudopunctipennis (52.91%), An. albimanus (39.14%) and An. franciscanus (5.29%). The multiple logistic regression analysis showed a negative association between An. pseudopunctipennis and water turbidity (ß=-1.342; Wald=6.122; p=0.013) and the amount of detritus (ß=-2.206; Wald=3.642; p=0.050). While in An. albimanus, there was a significant positive association with water turbidity (ß=1.344; Wald=4.256; p=0.039), a negative correlation was found with the altitude (ß=-3.445; Wald=5.407; p=0.020). The highest mosquito species diversity index was found in Chiapas (Fisher's α=1.20) and the lowest diversity in Chihuahua (Fisher's α=0.26). The greatest richness was found in streams (n=11). Conclusions: The two most abundant species were: An. albimanus and An. pseudopunctipennis. Detailed knowledge of the distribution and characteristics of their larval habitats will be useful for the effective implementation of control strategies in Mexico.

Resumen Objetivo: Determinar la abundancia y la distribución geográfica de los principales vectores de la malaria, las cuales están influenciadas por las características del hábitat y los factores ecológicos que afectan directamente la densidad de los adultos y la dinámica de la transmisión de la malaria en México. Material y métodos: Se obtuvieron muestras de larvas de 19 estados de México. Cada hábitat larvario se caracterizó in situ determinando los siguientes parámetros: profundidad del agua, turbidez, porcentaje de cobertura vegetal, cantidad de detritus, presencia de algas, intensidad de luz, tipo de vegetación, cantidad de depredadores, estabilidad del hábitat, altitud y tipo hidrológico. Resultados: Se identificaron un total de 21 687 larvas pertenecientes a 13 especies de anofelinos, de 149 hábitats acuáticos. Las tres especies más abundantes fueron Anopheles pseudopunctipennis (52.91%), An. albimanus (39.14%) y An. franciscanus (5.29%). El análisis de regresión logística múltiple mostró una asociación negativa para An. pseudopunctipennis y la turbidez del agua (ß=-1.342; Wald= 6.122; p=0.013) y la cantidad de detritus (ß=-2.206; Wald= 3.642; p=0.050). Para An. albimanus se encontró una asociación positiva significativa con la turbidez del agua (ß=1.344; Wald= 4.256; p=0.039) y una correlación negativa con la altitud (ß=-3.445; Wald=5.407; p=0.020). El índice de diversidad más alto se encontró en Chiapas (α de Fisher=1.20) y la diversidad más baja en Chihuahua (α de Fisher=0.26). La mayor riqueza se encontró en los arroyos (n=11). Conclusiones: Las dos especies más abundantes fueron An. albimanus y An. pseudopunctipennis. El conocimiento detallado de la distribución y características de sus hábitats larvales será útil para la implementación efectiva de las estrategias de control en México.

Composition and abundance of anopheline species according to habitat diversity in Mexico.

OBJECTIVE: To determine the abundance and geographic distribution of the main malaria vectors, which are influenced by habitat characteristics and ecological factors that directly impact adult density and the dynamics of malaria transmission in Mexico. MATERIALS AND METHODS: Samples of larvae were collected from 19 states in Mexico. Each larval habitat was characterized in situ determining the following parameters: water depth, turbidity, percentage of vegetation cover, amount of detritus, presence of algae, light intensity, type of vegetation, amount of predators, habitat stability, altitude, and hydrologic type. RESULTS: A total of 21 687 larvae corresponding to 13 anopheline species were obtained from 149 aquatic habitats. The most abundant species were Anopheles pseudopunctipennis (52.91%), An. albimanus (39.14%) and An. franciscanus (5.29%). The multiple logistic regression analysis showed a negative association between An. pseudopunctipennis and water turbidity (ß=-1.342; Wald=6.122; p=0.013) and the amount of detritus (ß=-2.206; Wald=3.642; p=0.050). While in An. albimanus, there was a significant positive association with water turbidity (ß=1.344; Wald=4.256; p=0.039), a negative correlation was found with the altitude (ß=-3.445; Wald=5.407; p =0.020). The highest mosquito species diversity index was found in Chiapas (Fisher's α=1.20) and the lowest diversity in Chihuahua (Fisher's α=0.26). The greatest richness was found in streams (n=11). CONCLUSIONS: The two most abundant species were: An. albimanus and An. pseudopunctipennis. Detailed knowledge of the distribution and characteristics of their larval habitats will be useful for the effective implementation of control strategies in Mexico.

OBJETIVO: Determinar la abundancia y la distribución geográfica de los principales vectores de la malaria, las cuales están influenciadas por las características del hábitat y los factores ecológicos que afectan directamente la densidad de los adultos y la dinámica de la transmisión de la malaria en México. MATERIAL Y MÉTODOS: Se obtuvieron muestras de larvas de 19 estados de México. Cada hábitat larvario se caracterizó in situ determinando los siguientes parámetros: profundidad del agua, turbidez, porcentaje de cobertura vegetal, cantidad de detritus, presencia de algas, intensidad de luz, tipo de vegetación, cantidad de depredadores, estabilidad del hábitat, altitud y tipo hidrológico. RESULTADOS: Se identificaron un total de 21 687 larvas pertenecientes a 13 especies de anofelinos, de 149 hábitats acuáticos. Las tres especies más abundantes fueron Anopheles pseudopunctipennis (52.91%), An. albimanus (39.14%) y An. franciscanus (5.29%). El análisis de regresión logística múltiple mostró una asociación negativa para An. pseudopunctipennis y la turbidez del agua (ß=-1.342; Wald= 6.122; p=0.013) y la cantidad de detritus (ß=-2.206; Wald= 3.642; p=0.050). Para An. albimanus se encontró una asociación positiva significativa con la turbidez del agua (ß=1.344; Wald= 4.256; p=0.039) y una correlación negativa con la altitud (ß=-3.445; Wald=5.407; p=0.020). El índice de diversidad más alto se encontró en Chiapas (α de Fisher=1.20) y la diversidad más baja en Chihuahua (α de Fisher=0.26). La mayor riqueza se encontró en los arroyos (n=11). CONCLUSIONES: Las dos especies más abundantes fueron An. albimanus y An. pseudopunctipennis. El conocimiento detallado de la distribución y características de sus hábitats larvales será útil para la implementación efectiva de las estrategias de control en México.

Plasmodium vivax CSP-Pvs25 variants from southern Mexico produce distinct patterns of infectivity for Anopheles albimanus versus An. pseudopunctipennis, in each case independent of geographical origin.

BACKGROUND: The susceptibility of Anopheles albimanus and An. pseudopunctipennis to local Plasmodium vivax has been associated in southern Mexico with two ookinete surface proteins (Pvs25/28) polymorphism. Perhaps parasite population selection (i.e. adaptation to local vectors) contributes to this phenomenon. It is also possible that certain molecular interactions exist between P. vivax and each mosquito species independently of geographical origin. This study aimed to explore the susceptibility of An. albimanus and An. pseudopunctipennis (collected from different geographical sites) to P. vivax cspVk/Pvs25-130 haplotypes from southern Mexico. RESULTS: Of the 120 P. vivax-infected blood samples used to simultaneously feed An. albimanus and An. pseudopunctipennis mosquitoes originating from various geographical sites, 80 produced at least one infected mosquito species. Three parasite haplotypes were identified in infected blood: Vk210/Pvs25-A (12.5%), Vk210/Pvs25-B (20%) and Vk247/Pvs25-B (67.5%). Two parameters (the proportion of infected mosquitoes and number of oocysts/mosquito) showed a similar pattern for each mosquito species (independently of geographical origin). For An. albimanus mosquitoes (from the Pacific coast, Mexican gulf and Lacandon Forest lowlands), these two parameters were higher in specimens infected with P. vivax Vk210/Pvs25-A versus Vk210/Pvs25-B or Vk247/Pvs25-B (P < 0.001). For An. pseudopunctipennis mosquitoes (from the Pacific coast, northeast Mexico and east Guatemala foothills), the same two parameters were higher in specimens infected with Vk247/Pvs25-B or Vk210/Pvs25-B versus Vk210/Pvs25-A (P < 0.001). Higher infection rates were caused by Vk247/Pvs25-B than Vk210/Pvs25-B parasites in An. pseudopunctipennis (P = 0.011) and An. albimanus (P = 0.001). The greatest parasitaemia, gametocytaemia and microgamete formation was observed in Vk247/Pvs25-B infected blood, and each of these parameters correlated with each other and with the number of oocysts in An. pseudopunctipennis from the sympatric colony. CONCLUSIONS: Plasmodium vivax Vk247/Pvs25-B infections were the most prevalent, likely due to the higher parasitaemia produced in the susceptible vector (especially An. pseudopunctipennis). The analysis of mosquito-parasite interactions indicate that An. pseudopunctipennis and An. albimanus each have a unique pattern of transmitting genetic variants of P. vivax, and this is not dependent on geographical origin. The present findings highlight the importance of parasite genotyping to understand transmission dynamics and vectorial participation.

Ookinete-Specific Genes and 18S SSU rRNA Evidenced in Plasmodium vivax Selection and Adaptation by Sympatric Vectors.

In the southern Pacific coast of Chiapas, Mexico (SM), the two most abundant vector species, Nyssorhynchus albimanus and Anopheles pseudopunctipennis, were susceptible to different Plasmodium vivax Pvs25/28 haplotypes. To broaden our understanding of the existing P. vivax in the area, genes encoding proteins relevant for ookinete development and the 18S rRNA were studied. P. vivax infectivity (percentage of infected mosquitoes and oocyst numbers) was evaluated by simultaneously feeding infected blood samples from patients to Ny. albimanus and An. pseudopunctipennis female mosquitoes. Three infectivity patterns were identified: one group of parasites were more infective to An. pseudopunctipennis than to Ny. albimanus, another group was more infective to Ny. albimanus, while a third group infected both vectors similarly. In 29 parasite isolates, the molecular variations of ookinete-specific genes and the 18S rRNA-type S were analyzed. Using concatenated sequences, phylogenetic trees, and Structure analysis, parasite clustering within SM isolates and between these and those from other geographical origins were investigated. A ML phylogenetic tree resolved two parasite lineages: PvSM-A and PvSM-B. They were associated to a different 18S rRNA variant. PvSM-A parasites had 18S rRNA variant rV2 and correspond to parasites causing high oocyst infection in Ny. albimanus. A new ML tree and Structure analysis, both comprising global sequences, showed PvSM-A clustered with Latin American parasites. Meanwhile, all isolates of PvSM-B had 18S rRNA variant rV1 and remained as unique genetic cluster comprising two subgroups: PvSM-Ba, producing high infection in An. pseudopunctipennis, and PvSM-Bb, causing similar oocyst infection in both vector species. PvSM-A parasites were genetically similar to parasites from South America. Meanwhile, PvSM-B were exclusive to southern Mexico and share ancestry with Asian parasites. The results suggest that these lineages evolved separately, likely by geographic and vector restriction.

Larval habitat characterization of Anopheles darlingi from its northernmost geographical distribution in Chiapas, Mexico.

BACKGROUND: Anopheles darlingi is considered the most efficient malaria vector in the Neotropical region. In Mexico, its role as an incriminated vector of Plasmodium has not been confirmed in the Lacandon forest. Similarly, knowledge about bionomic and larval ecology is scarce. The study aim was to identify and describe the larval habitats of An. darlingi in Chiapas, México. METHODS: Standard larval collections were performed in the Lacandon forest region and in the Soconusco region of southern Chiapas from January 2010 to April 2014, including dry and rainy seasons. Mean larval density of An. darlingi was estimated according to hydrological types, and associations between the presence of An. darlingi and environmental factors including ecological parameters and geographic positions were statistically analysed. RESULTS: One hundred and twelve aquatic habitats were analysed, 80 from the Lacandon forest region and 32 from the Soconusco region; 94.64% of these sites presented anopheline larvae. In total, 10,977 larvae belonging to 11 Anopheles species were collected. The 19 (out of 112) larval habitats positive to An. darlingi were: rain puddles (26.32%), ground pools (21.05%), ponds (15.79%), ditches (15.79%), river margins (10.53%) and streams (10.53%). Overall, the average (±SD) larval density was 6.60 ± 2.41 larvae per dip. Multiple logistic regression analysis showed that temporary habitats, green algae presence and stagnant water were associated with An. darlingi larval presence. The positive habitats were found in the Lacandon forest region during the rainy season (May-September). No specimens were found in the Soconusco region of the coastal plain of Chiapas. CONCLUSION: The mosquito An. darlingi larval habitats were found in different hydrological types. The habitat stability, presence of algae and water current were the main factors for An. darlingi larval occurrence. The information on the characteristics of the larval habitats of An. darlingi will be useful in sustainable programmes for malaria control in the Lacandon forest region, Chiapas.

Establishment of a free-mating, long-standing and highly productive laboratory colony of Anopheles darlingi from the Peruvian Amazon.

BACKGROUND: Anopheles darlingi is the main malaria vector in the Amazon region and is among the most efficient malaria vectors worldwide. However, due to the lack of a well-established laboratory colony, key control-relevant aspects of the bionomics, behaviour, genetics, and vector-parasite relationships of An. darlingi remain unknown. Here, biological parameters that had been successful in initiating other Anopheles colonies were optimized and improved for An. darlingi, with the aim of establish a free-mating, stable, and highly productive laboratory colony. METHODS: Wild An. darlingi adult females were field collected from Zungarococha, Loreto Department, Peru (03°49'32.40″S, 73°21'00.08″W), and taken to the NAMRU-6 Insectary in Iquitos where F(1) offspring were produced and reared. Natural copulation was successfully induced in F1 adults under a thermoperiod of 30 ± 1 °C during the day and 25 ± 1 °C at night, and with a 30-min LED light stimulation period at dusk. Oviposition success was enhanced using egg-laying containers with a dark-coloured surface. Larval feeding regimes were standardized for optimal larval development. Optimized copulation induction methods were used to facilitate mating in An. darlingi until the F(10) generation. No copulation induction assistance was needed in subsequent generations. RESULTS: In 19 generations, the An. darlingi colony produced a total of 763,775 eggs; 441,124 larvae; 248,041 pupae; and 231,591 adults. A mean of 0.56 sexual encounters/female/cage (n = 36 cages) was recorded across the first ten generations (F(1)-F(10)). A mean insemination rate of 54.7 % (n = 5,907 females) ranging from 43.6 % (F(2)) to 66.6 % (F(10)) was recorded across nine generations (F(2)-F(10)). Free-mating was casually observed in the F(8) generation, and subsequently confirmed in the F(9) and F(10) generations; comparable insemination rates and egg laying between stimulated (51.6 %, 12.9 eggs/female), and non-stimulated (52.3 %, 11.2 eggs/female) females were recorded. The time from egg to adult development ranged from 10 to 20 days. Moreover, the colony was relocated to a new laboratory within Iquitos in the F(14) generation without any noted changes in its productivity. By March 2015, the An. darlingi colony has been successfully reared to the F(26) generation. CONCLUSIONS: This constitutes the first report of a free-mating, highly productive, and long-standing An. darlingi laboratory colony established through natural copulation induction, which will support critical malaria research. This rearing methodology may be a transferable, cost-effective alternative to labour-intensive forced mating practices widely used in maintaining other Anopheles colonies.

Sequence variation of ookinete surface proteins Pvs25 and Pvs28 of Plasmodium vivax isolates from Southern Mexico and their association to local anophelines infectivity.

The polymorphism of Pvs25 and Pvs28 ookinete surface proteins, their association to circumsporozoite protein repeat (CSPr) genotypes (Vk210 and Vk247) and their infectivity to local Anopheles albimanus and Anopheles pseudopunctipennis were investigated in Plasmodium vivax-infected blood samples obtained from patients in Southern Mexico. The pvs25 and pvs28 complete genes were amplified, cloned and sequenced; and the CSPr genotype was determined by PCR amplification and hybridization. The amino acid Pvs25 and Pvs28 polymorphisms were mapped to their corresponding protein structure. Infected blood samples were simultaneously provided through artificial feeders to both mosquito species; the ratio of infected mosquitoes and oocyst numbers were recorded. The polymorphism of pvs25 and pvs28 was limited to few nucleotide positions, and produced three haplotypes: type A/A parasites presented Pvs25 and Pvs28 amino acid sequences identical to that of Sal I reference strain; parasites type B1 presented a mutation 130 Ile-->Thr in Pvs25, while type B2 presented 87 Gln-->Lys/130 Ile-->Thr in the same molecule. Both types B1 and B2 parasites presented changes in Pvs28 at 87 Asn-->Asp, 110 Tyr-->Asn and five GSGGE/D repeat sequences between the fourth EGF-like domain and the GPI. Most P. vivaxparasites from the coastal plains and the overlapping region were Pvs25/28 A/A, CSPrVk210 and were infective only to An. albimanus (p< or =0.0001). Parasites originating in foothills were Pvs25/28 type B1/B or B2/B and CSPrVk210 or Vk247, and were more infective to An. pseudopunctipennis than to An. albimanus (p< or =0.001). These results and the analysis of Pvs25/28 from other parts of the world indicated that non-synonymous variations in these proteins occur in amino acid residues exposed on the surface of the proteins, and are likely to interact with midgut mosquito ligands. We hypothesize that these molecules have been shaped by co-evolutionary adaptations of parasites to their susceptible vectors.

Plasmodium berghei ookinetes induce nitric oxide production in Anopheles pseudopunctipennis midguts cultured in vitro.

The Anopheles pseudopunctipennis nitric oxide synthase gene (ApNOS) was identified and its partial sequence showed high homology with NOS from A. stephensi, A. gambiae (putative sequence), and Drosophila melanogaster. ApNOS was mainly expressed in male and female adult mosquitoes and was induced by a blood meal. Nitric oxide (NO) was produced by in vitro-cultured mosquito midguts inoculated by enema with Plasmodium berghei ookinetes, Saccharomyces cerevisiae, Gram-positive bacteria (Micrococcus luteus), but not with Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli or Serratia marcescens). Dihydroxyphenylalanine (L-DOPA) oxidation induced the generation of NO in midguts in vitro, and hydrogen peroxide generated during its oxidation induced ApNOS expression. P. berghei ookinetes exposed in vitro to L-DOPA and sodium nitroprusside (a NO generator) were killed. These observations demonstrate that reactive oxygen and nitrogen intermediates constitute a part of the cytotoxic arsenal employed by Anopheles mosquitoes against microbial pathogens and Plasmodium ookinetes.