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. 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 F1 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 F10 generation. No copulation induction assistance was needed in subsequent generations. 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 (F1-F10). A mean insemination rate of 54.7 % (n = 5,907 females) ranging from 43.6 % (F2) to 66.6 % (F10) was recorded across nine generations (F2-F10). Free-mating was casually observed in the F8 generation, and subsequently confirmed in the F9 and F10 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 F14 generation without any noted changes in its productivity. By March 2015, the An. darlingi colony has been successfully reared to the F26 generation. 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.

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. 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. 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. 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.

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. 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. 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.

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

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.