The effects of human and rhesus macaque blood meal sources on mosquito reproduction and adult survival under laboratory conditions.

Mass rearing of mosquitoes as required to fulfil research studies is a technically challenging endeavor. Blood meal source has been recognized as a key consideration in mass rearing of mosquitoes that affects colony health and fecundity. Four species of laboratory-colonized mosquitoes from the Department of Entomology, US Army Medical Directorate - Armed Forces Research Institute of Medical Sciences (USAMD-AFRIMS); Anopheles cracens, An. dirus, An. minimus and An. sawadwongporni were fed blood meals from human and rhesus macaque sources using an artificial membrane feeder. The effects of different blood meal sources were evaluated concerning blood-feeding, survival and reproduction (fecundity and hatching rates). Adult survival was monitored at days 7, 14 and 21 post blood-feeding. Although the mosquitoes fed on human blood exhibited higher rates of engorgement, there were no significant differences in blood-feeding rates in An. cracens (P = 0.08) and An. dirus (P = 0.91) between rhesus macaque and human blood sources. Twenty-one days post-feeding, no significant differences were observed in the survival rates of mosquitoes fed on human versus rhesus macaque blood. Except for An. dirus, which had better survival rates with human blood (97.5%) than after feeding on rhesus macaque blood (95.4%). All mosquito species fed on human blood produced significantly more eggs when compared to those fed on rhesus macaque blood. However, there was no statistical difference in hatching rates between blood sources, except for An. dirus, which had better hatching rates with human blood. These results indicate that human and rhesus macaque blood may be a viable alternative for maintaining Anopheles mosquitoes in colony.

Laboratory studies of oviposition site choice of Toxorhynchites splendens: Role of larval holding-rearing water and predator-prey interactions.

Oviposition preference by Toxorhynchites mosquito species is an important aspect of their biological control potential. When ovipositing, the females deposit individual eggs by hovering above the water, indicating that they may be capable of detecting chemical cues from above the water surface. These chemical cues may help the females to determine whether the water is suitable for their larvae. We investigated whether female Tx. splendens has the ability to discriminate against oviposition sites in habitats containing turbid larval/pupal rearing water and predator-prey treatments. In this study, the number of eggs in the test and control cups were compared to assess oviposition preference. According to the results, female Tx. splendens preferred ovipositing in cups with turbid immature water (prey absence) and predator-prey turbid water (prey and predator presences) over ovipositing in filtered water as a control. Results from the third experiment revealed that cups with alive and dead Ae. aegypti larvae proved strongly attractive to Tx. splendens females, while those from conspecific Toxorhynchites larvae caused oviposition deterrence when compared to other treatments in a dual choice test. Our results indicated that both turbid immature water and predator-prey turbid water can efficiently attract Tx. splendens females to deposit eggs. These findings will aid in the development of Toxorhynchites mosquito as a biocontrol agent for the control of Aedes spp. larvae.

Vector Competence and the Susceptibility of Anopheles pullus and Anopheles belenrae to Plasmodium vivax-Infected Blood From Thai Patients.

There are eight Anopheles spp. present in the Republic of Korea (ROK), including five members of the Anopheles Hyrcanus Group that cannot be identified using current morphological methods. The vector competence of only Anopheles sinensis s.s., An. lesteri, and An. kleini have been investigated. As the geographical distribution of Anopheles spp. varies in the ROK, determining the relative vector competence of the Anopheles spp. provides a basis for delineating malaria risks to Korean populations and U.S. military/civilian populations deployed to the ROK. Anopheles belenrae and An. pullus, collected from a malaria high-risk area in the ROK, were evaluated for vector competence of P. vivax. A total of 1,000 each of An. dirus (Thai strain), and Korean strains of An. pullus and An. belenrae were fed on P. vivax infected blood collected from Thai patients via artificial membrane feeding. The overall oocyst infection rates for An. dirus, An. pullus, and An. belenrae dissected on days 8-9 postfeed were 64.1, 12.0, and 11.6%, respectively. The overall sporozoite infection rates for those species dissected on days 14-15 postfeed were 84.5, 3.4, and 5.1% respectively. The salivary gland sporozoite indices for positive females with +4 (>1,000 sporozoites) were observed in An. dirus (72.8%), but not observed for either An. pullus or An. belenrae. Most sporozoite-positive An. pullus (83.3%) and An. belenrae (71.4%) females were observed with only +1 (1-10 sporozoites) salivary glands. These data indicate that both An. belenrae and An. pullus are very poor vectors of P. vivax.

Colonization and Maintenance of Anopheles belenrae and Anopheles pullus From the Republic Of Korea.

The Anopheles Hyrcanus Group in the Republic of Korea (ROK) consists of 5 morphologically indistinct species that can only be identified with certainty by polymerase chain reaction (PCR). A total of 86 bloodfed Anopheles spp. were collected from a cow barn located in the village of Tongilchon near the demilitarized zone in the ROK on June 13, 2016, and sent to the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, where they were identified to species by PCR. The 1st shipment contained 15 An. belenrae and 37 An. pullus females that were used to start the colonies. Parent females that oviposited were identified by PCR for colonization. A higher proportion of F1-F4 females of An. belenrae than An. pullus bloodfed when provided both blood meals on human arms and using a membrane feeding system with human blood. Following blood meals, the females were forced mated for colony maintenance. The mean numbers of eggs oviposited per female for An. belenrae was 127.7 ± 19.3 and for An. pullus was 136 ± 23.6. On average, at 25°C (±2°C) An. belenrae and An. pullus took 15.1 and 16.1 days to develop from egg to adult, respectively. A 2nd group of bloodfed Anopheles spp. was collected at the same location in the ROK on June 24, 2017. This group contained 13 An. belenrae and 27 An. pullus. Similarly, eggs were obtained and adults identified by PCR and then reared to adults and subsequent generations forced mated to members of each of the existing colonies to increase genetic diversity. The colonies were established to evaluate their susceptibility to vector vivax malaria, which is essential to better understand the epidemiology of malaria transmission in Korea. This is the 1st report of colonization of both An. belenrae and An. pullus.

Sugar and Multivitamin Diet Effects on The Longevity and Mating Capacity of Laboratory-Reared Male Anopheline Mosquitoes.

Successful mating by male mosquitoes is dependent on several factors, with sugar feeding being particularly important. The effect of ingested vitamins on adult male mosquitoes is poorly understood. This laboratory study used 3 anopheline species, Anopheles campestris, An. dirus, and An. sawadwongporni, to study the effect of sugar and vitamins on male longevity, copulation, and fecundity. Males were fed 1 of 5 diets containing different combinations of sugar and vitamins: 10% glucose, 10% sucrose, 10% multivitamin syrup, 10% multivitamin syrup + 10% glucose, and 10% multivitamin syrup + 10% sucrose. The longevity of males was measured for a period of 15 days. Forced mating was used to simulate copulation, and fecundity was measured by counting the number of eggs oviposited and the hatch rate of larvae. The longevity of An. campestris and An. dirus was greatest when fed a diet of 10% multivitamin syrup + 10% glucose, and the longevity of An. sawadwongporni was greatest when fed a diet of 10% multivitamin syrup + 10% sucrose. The 1st mating routinely produced the most viable eggs when males were mated with several females. The diet of 10% multivitamin syrup + 10% sucrose produced numerically greater egg production and larval emergence for all 3 species, although this was not always statistically significant due to variability and small sample size. These results indicate that the addition of multivitamin syrup to sucrose may produce healthier and more fit male anophelines. This has potential implications for increasing insectary operations and improving the fitness of laboratory-reared male mosquitoes that will be released for mosquito and disease-pathogen control studies.

The relationship between wing length, blood meal volume, and fecundity for seven colonies of Anopheles species housed at the Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

Established colonies of Anopheles campestris, Anopheles cracens, Anopheles dirus, Anopheles kleini, Anopheles minimus, Anopheles sawadwongporni, and Anopheles sinensis are maintained at the Armed Forces Research Institute of Medical Sciences (AFRIMS). Females were provided blood meals on human blood containing citrate as an anticoagulant using an artificial membrane feeder. The mean wing length, used as an estimate of body size, for each species was compared to blood-feeding duration (time), blood meal volume, and numbers of eggs oviposited. Except for An. campestris and An. cracens, there were significant interspecies differences in wing length. The mean blood meal volumes (mm(3)) of An. kleini and An. sinensis were significantly higher than the other 5 species. For all species, the ratios of unfed females weights/blood meal volumes were similar (range: 0.76-0.88), except for An. kleini (1.08) and An. cracens (0.52), that were significantly higher and lower, respectively. Adult females were allowed to feed undisturbed for 1, 3, and 5min intervals before blood feeding was interrupted. Except for An. campestris and An. sawadwongporni, the number of eggs oviposited were significantly higher for females that fed for 3min when compared to those that only fed for 1min. This information is critical to better understand the biology of colonized Anopheles spp. and their role in the transmission of malaria parasites as they relate to the relative size of adult females, mean volumes of blood of engorged females for each of the anopheline species, and the effect of blood feeding duration on specific blood meal volumes and fecundity.

Factors influencing the feeding response of laboratory-reared Aedes aegypti.

We evaluated the effects of membrane surface area (cm2), female density, and container/cage size on feeding response in laboratory-reared Aedes aegypti. Female density did not affect feeding rates at low surface areas, but higher density did significantly increase feeding as surface area increased. Females in large, cloth cages fed less compared to those in large, plastic cups. The rate of feeding was higher when using live, anesthetized mice versus a membrane feeding system. The AFRIMS Insectary will continue to look for innovative ways to improve our membrane feeding system.

Colonization and maintenance of Anopheles kleini and Anopheles sinensis from the Republic of Korea.

Colonization of members of the Anopheles Hyrcanus Group is required to facilitate a better understanding of their roles in the epidemiology, vector competence, transmission potential, and maintenance of vivax malaria in the Republic of Korea (ROK). A total of 158 resting blood-fed wild caught Anopheles species were collected on August 26, 2011 at Tongilchon, near the demilitarized zone, in the ROK and shipped on August 28, 2011 to the Entomology Department, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand. Four of the 5 species of the Anopheles Hyrcanus Group (Anopheles kleini, An. sinensis, An. pullus, and An. belenrae) were identified by polymerase chain reaction. Eggs were obtained from parent populations, and F, progeny provided blood meals using membrane feeding techniques. The mean numbers of eggs/female for An. sinensis and An. kleini were 157 and 146, respectively. Membrane blood feeding rates for An. sinensis (90.6%) and An. kleini (89.0%) were similar for F1-F30 generations. Colonies of An. kleini and An. sinensis were established by force mating techniques and maintained by the Insectary Section, Entomology Department, AFRIMS, to date. This is the 1st report of successful laboratory colonization of An. kleini collected from a vivax malaria high-risk area in the ROK.

Maintenance of mosquito vectors: effects of blood source on feeding, survival, fecundity, and egg hatching rates.

Artificial membrane-feeding techniques have replaced direct feeding on animals for the maintenance of malaria and arbovirus vectors in many laboratories. Membrane feeding facilitates controlled experimentation of pathogen transmission during mosquito feeding. Sheep blood is commonly used due to its availability and low cost. We evaluated the impact of blood source (human, guinea pig, sheep, and hamster via direct feeding) on feeding rates, adult survival, fecundity, hatching rates, and developmental times for five species of laboratory-colonized mosquitoes (Anopheles dirus, An. cracens, An. minimus, An. sawadwongporni, and Ae. aegypti). We found that feeding rates differ among blood sources within mosquito species. Survival, fecundity, and hatching rates were lower in all Anopheles species and Ae. aegypti after membrane feeding on sheep blood. Survival rates seven days post-feeding on sheep blood were significantly lower (P<0.05) for An. dirus (84.2%), An. minimus (67.2%), An. sawadwongporni (51.5%), and An. cracens (35.5%) relative to other blood sources. An. minimus and An. sawadwongporni laid no eggs by seven days post-feeding with sheep blood, while An. dirus and An. cracens produced significantly fewer numbers of eggs and demonstrated significantly lower hatching rates relative to what was observed with the other blood sources. These findings support the conclusion that sheep blood is not a suitable blood source for laboratory rearing of Anopheles spp.