Evaluation of "Caserotek" a low cost and effective artificial blood-feeding device for mosquitoes.

Entomological research studies on mosquito vector biology, vector competence, insecticide resistance, dispersal, and survival (using mark-release-recapture techniques) often rely on laboratory-reared mosquito colonies to produce large numbers of consistently reared, aged, and sized mosquitoes. We developed a low-cost blood feeding apparatus that supports temperatures consistent with warm blooded animals, using commonly available materials found in low resource environments. We compare our system ("Caserotek") to Hemotek and glass/membrane feeding methods. Two experiments were conducted with Aedes aegypti (Linnaeus 1762) and one with Anopheles darlingi (Root 1926) (Diptera: Culicidae); 3 replicates were conducted for each experiment. Aedes aegypti female mosquitoes were provided chicken blood once per week for 30 min (Experiment #1) for 14 days or 1 hour (Experiment #2) for 21 days. Anopheles darlingi were fed once for 1 hour (Experiment #3). Blood-feeding rates, survival rates, and egg production were calculated across replicates. Caserotek had a significantly higher 30-min engorgement rate (91.1%) than Hemotek (47.7%), and the glass feeder (29.3%) whereas for 1-hour feeding, Hemotek had a significantly lower engorgement rate than either of the other two devices (78% versus 91%). Thirty-day survival was similar among the feeding devices, ranging from 86% to 99%. Mean egg production was highest for the Caserotek feeder (32 eggs per female) compared to the glass feeder and Hemotek device (21-22 eggs per female). Our new artificial feeding system had significantly higher blood feeding rates than for more expensive artificial systems and was equivalent to other fitness parameters. Caserotek only requires the ability to boil water to maintain blood temperatures using a Styrofoam liner. It can be easily scaled up to large production facilities and used under austere conditions.

Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos, Peru.

Over half the world's population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito-human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = -9.11) and 12.4% (95% CI 4.2%, ∞); z = -2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Antibody Responses Against Anopheles darlingi Immunogenic Peptides in Plasmodium Infected Humans.

Introduction: Malaria is still an important vector-borne disease in the New World tropics. Despite the recent decline in malaria due to Plasmodium falciparum infection in Africa, a rise in Plasmodium infections has been detected in several low malaria transmission areas in Latin America. One of the main obstacles in the battle against malaria is the lack of innovative tools to assess malaria transmission risk, and the behavioral plasticity of one of the main malaria vectors in Latin America, Anopheles darlingi. Methods: We used human IgG antibodies against mosquito salivary gland proteins as a measure of disease risk. Whole salivary gland antigen (SGA) from Anopheles darlingi mosquitoes was used as antigen in Western blot experiments, in which a ~65 kDa protein was visualized as the main immunogenic band and sent for sequencing by mass spectrometry. Apyrase and peroxidase peptides were designed and used as antigens in an ELISA-based test to measure human IgG antibody responses in people with different clinical presentations of malaria. Results: Liquid chromatography-mass spectrometry revealed 17 proteins contained in the ~65 kDa band, with an apyrase and a peroxidase as the two most abundant proteins. Detection of IgG antibodies against salivary antigens by ELISA revealed a significant higher antibody levels in people with malaria infection when compared to uninfected volunteers using the AnDar_Apy1 and AnDar_Apy2 peptides. We also detected a significant positive correlation between the anti-peptides IgG levels and antibodies against the Plasmodium vivax and P. falciparum antigens PvMSP1 and PfMSP1. Odd ratios suggest that people with higher IgG antibodies against the apyrase peptides were up to five times more likely to have a malaria infection. Conclusion: Antibodies against salivary peptides from An. darlingi salivary gland proteins may be used as biomarkers for malaria risk.

Feasibility of feeding Aedes aegypti mosquitoes on dengue virus-infected human volunteers for vector competence studies in Iquitos, Peru.

BACKGROUND: Transmission of dengue virus (DENV) from humans to mosquitoes represents a critical component of dengue epidemiology. Examinations of this process have generally been hampered by a lack of methods that adequately represent natural acquisition of DENV by mosquitoes from humans. In this study, we assessed artificial and natural blood feeding methods based on rates of DENV infection and dissemination within mosquitoes for use in a field-based epidemiological cohort study in Iquitos, Peru. METHODOLOGY/PRINCIPAL FINDINGS: Our study was implemented, stepwise, between 2011 and 2015. Participants who were 5 years and older with 5 or fewer days of fever were enrolled from ongoing clinic- and neighborhood-based studies on dengue in Iquitos. Wild type, laboratory-reared Aedes aegypti were fed directly on febrile individuals or on blood collected from participants that was either untreated or treated with EDTA. Mosquitoes were tested after approximately 14 days of extrinsic incubation for DENV infection and dissemination. A total of 58 participants, with viremias ranging from 1.3 × 10(2) to 2.9 × 10(6) focus-forming units per mL of serum, participated in one or more feeding methods. DENV infection and dissemination rates were not significantly different following direct and indirect-EDTA feeding; however, they were significantly lower for mosquitoes that fed indirectly on blood with no additive. Relative to direct feeding, infection rates showed greater variation following indirect-EDTA than indirect-no additive feeding. Dissemination rates were similar across all feeding methods. No differences were detected in DENV infection or dissemination rates in mosquitoes fed directly on participants with different dengue illness severity. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates the feasibility of using direct and indirect feeding methods for field-based studies on vector competence. Direct mosquito feeding is preferable in terms of logistical ease, biosecurity, and reliability.

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi.

BACKGROUND: The mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America. METHODS: To determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 µg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated. RESULTS: IVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions. CONCLUSION: In conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

Ivermectin susceptibility, sporontocidal effect, and inhibition of time to re-feed in the Amazonian malaria vector Anopheles darlingi.

BACKGROUND: Outdoor malaria transmission hinders malaria elimination efforts in the Amazon region and novel vector control tools are needed. Ivermectin mass drug administration (MDA) to humans kills wild Anopheles, targets outdoor-feeding vectors, and can suppress malaria parasite transmission. Laboratory investigations were performed to determine ivermectin susceptibility, sporontocidal effect and inhibition of time to re-feed for the primary Amazonian malaria vector, Anopheles darlingi. METHODS: To assess ivermectin susceptibility, various concentrations of ivermectin were mixed in human blood and fed to An. darlingi. Mosquito survival was monitored daily for 7 days and a non-linear mixed effects model with Probit analysis was used to calculate lethal concentrations of ivermectin that killed 50% (LC50), 25% (LC25) and 5% (LC5) of mosquitoes. To examine ivermectin sporonticidal effect, Plasmodium vivax blood samples were collected from malaria patients and offered to mosquitoes without or with ivermectin at the LC50, LC25 or LC5. To assess ivermectin inhibition of mosquito time to re-feed, concentrations of ivermectin predicted to occur after a single oral dose of 200 µg/kg ivermectin were fed to An. darlingi. Every day for 12 days thereafter, individual mosquitoes were given the opportunity to re-feed on a volunteer. Any mosquitoes that re-blood fed or died were removed from the study. RESULTS: Ivermectin significantly reduced An. darlingi survivorship: 7-day-LC50 = 43.2 ng/ml [37.5, 48.6], -LC25 = 27.8 ng/ml [20.4, 32.9] and -LC5 = 14.8 ng/ml [7.9, 20.2]. Ivermectin compound was sporontocidal to P. vivax in An. darlingi at the LC50 and LC25 concentrations reducing prevalence by 22.6 and 17.1%, respectively, but not at the LC5. Oocyst intensity was not altered at any concentration. Ivermectin significantly delayed time to re-feed at the 4-h (48.7 ng/ml) and 12-h (26.9 ng/ml) concentrations but not 36-h (10.6 ng/ml) or 60-h (6.3 ng/ml). CONCLUSIONS: Ivermectin is lethal to An. darlingi, modestly inhibits sporogony of P. vivax, and delays time to re-feed at concentrations found in humans up to 12 h post drug ingestion. The LC50 value suggests that a higher than standard dose (400-µg/kg) is necessary to target An. darlingi. These results suggest that ivermectin MDA has potential in the Amazon region to aid malaria elimination efforts.

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.