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.
When rearing mosquito vectors in the laboratory, researchers are often challenged by the method of feeding and by the source of blood that will effectively and efficiently facilitate predictability and success in terms of both colony maintenance and experimentation. Indeed, females of most species must take a vertebrate blood meal to acquire protein for egg production. In the development of a system for mass-rearing insects, efficiency and economics are of the utmost importance (Bailey et al. 1980). Anopheles dirus, An. cracens, An. minimus, An. sawadwongporni, and Aedes aegypti are colonized in our insectary at the Armed Forces Research Institute of Medical Sciences (AFRIMS) in Bangkok, Thailand. These mosquitoes are reared to support research on malaria and dengue fever, as well as to support repellent testing and pesticide resistance evaluations. Two techniques are commonly used for feeding female mosquitoes in the laboratory. One such technique, direct feeding, has several disadvantages, including the added expense and inconvenience associated with maintaining laboratory animals (Thomas et al. 1985). A more effective technique is feeding via artificial membranes. Such a method is low in cost, more standardized, and limits the involvement of animals (Cosgrove and Wood 1996, Benzon and Apperson 1987, O'Meara et al. 1993). Our insectary at AFRIMS uses membrane feeding techniques to rear mosquito colonies. There are several published studies on different aspects of mosquito membrane feeding (Samish et al. 1995, Robert 1998); however, there are no published reports comparing the relative impact of blood sources (i.e., human, sheep, guinea pig blood, hamster via direct feeding) on feeding, survival, fecundity, and egg hatch rates in laboratory colonies. Our insectary historically utilizes sheep blood to rear Aedes spp. and human blood for rearing Anopheles species because both are economical, locally acquired, and have provided adequate results in our past efforts to rear mosquitoes.
In this study, we test the hypothesis that blood source influences feeding success, female fecundity, egg hatching, and survival rates in five colonized mosquito species. Blood sources were selected because they are also available from our current blood provider, and they are comparable economically.
MATERIALS AND METHODS
An. dirus, An. cracens, An. minimus, An. sawadwongporni, and Ae. aegypti were reared in an established insectary located at AFRIMS in Bangkok, Thailand. Colonies were maintained and all experiments were carried out at a constant temperature of 25 ± 2º C and 80 ± 10% relative humidity. Eggs were submerged in 1.5 liters of distilled water in plastic trays (size 30×35×5 cm). After eggs hatched, ground fish food was added (0.1 g for 1st and 2nd instar larvae, 0.3 g for 3rd instar larvae, and 0.5 g for 4th instar larvae at 08:00 and 16:00 each day) to each tray for the successive two weeks until pupation of all larvae. Pupae were transferred to plastic containers with fine mesh netting at the top where healthy adults emerged and assumed resting positions on the side of the container. Weak adults that emerged and were unable to climb the container eventually drowned and were discarded with any unemerged pupae. Healthy emerged adults were provided with soaked cotton balls containing a 5% multivitamin solution (commercially available Multilim® children's vitamin syrup, containing 50% sucrose). The vitamin-saturated cotton balls were removed from the holding containers for 12 h prior to blood feeding and replaced with a water-soaked cotton ball. Six h prior to blood feeding, the water soaked cotton ball was removed.
A circulating water bath was set to 37° C and connected to feeding cups via tubing. Sausage casing membranes were stretched across the bottom of the feeders and secured with a rubber band. Cups containing mosquitoes were placed under the feeders, all the while ensuring that the bottom of each feeder was in contact with the mesh netting fitted to the top of the cup. Blood was added (1.5 ml) to the glass feeder well. After 30 min feeding, the number of mosquitoes that successfully fed was recorded and mosquitoes that did not feed or only partially fed were discarded. Mosquitoes were provided access to a vitamin solution immediately after blood feeding.
Origin of blood sources/hamsters
Our study utilized three blood sources (sheep, guinea pig, and human). Commercial defibrinated sheep blood and guinea pig blood were purchased from the National Laboratory Animal Centre, Mahidol University (NLAC-MU), Nakhon Pathom, Thailand. Commercial human blood was purchased from Interstate Blood Bank, Inc., Tennessee, U.S.A., and the Thai Red Cross Society, Thailand. Hamsters used in direct feeding were purchased from the National Laboratory Animal Centre, Mahidol University (NLAC-MU), Nakhon Pathom, Thailand. The animals were first anesthetized and placed on plastic containers.
Determination of engorged females and survival rates post-feeding
On day 1, 50 five to seven-day-old females were placed into 8.5×8 cm paper cartons. Each container had one open end and a piece of nylon mesh was fitted over this open end to facilitate feeding. Mosquitoes were allowed to feed for 30 min either through the sausage casing membrane or via direct feeding as described previously. All fully-engorged mosquitoes were counted and recorded, while those that failed to engorge were removed and discarded. Survival rates of engorged females were recorded on days 1, 3, and 7 (Table 1). The sequence was repeated five times per blood source including direct feeding on hamsters.
Table 1. Experimental timeline for feeding, mating, and egg-laying.
|Day 0||Mosquitoes fed on blood|
|Day 1||Observed after blood feeding|
|Day 2||Artificial insemination method|
|Day 3||Observed 1 day after artificial insemination method /3 days post-feeding|
|Day 4||Separated engorged females individually into glass holding vials|
|Day 5||Added some water to glass holding vial|
|Day 6||Females remained in glass holding vial for 48 hrs|
|Day 7||Observed 48 hrs after female laid eggs/7 days post-feeding|
Fecundity and egg hatching rate
Two days after a 30-min period of blood feeding, five engorged females were selected by random from each species and mated manually using an artificial insemination method (Yang et al. 1963) (Table 1). Two of the anopheline species studied, An. dirus and An. sawadwongporni, will not mate under laboratory conditions and must be mated artificially. Both An. cracens and An. minimus, as well as Ae. aegypti, were allowed to mate naturally. Each blood-fed and mated female was held in an individual glass vial containing a piece of moist filter paper as an oviposition substrate. Numbers of eggs laid were recorded for each female up to 48 h post-feeding. All eggs of five engorged females per species were immersed in plastic rearing trays with water for hatching observation. Two days later, the number of newly hatched larvae was recorded.
Developmental period and survival rate of offspring from blood-fed adults
Larval and pupal periods were determined and time spent from first instar to pupation was recorded for all hatched larvae. Immediately after adult emergence, the number of adults were counted and compared with the number of eggs to compute the survival rate of offspring.
Data were subject to a one-way analysis of variance (ANOVA) and Duncan's multiple comparisons by SPSS for Windows (version 16.0).
Engorgement and adult post-feeding survival rates
The mean percentages of engorgement from feeding on guinea pig blood (97.7%) and human blood (97%) were significantly higher relative to hamster and sheep blood (83.0% and 89.3%, respectively) (P<0.05) (Table 2). By day 7 post-feeding, engorged females utilizing human blood had the highest survival rate (100%) relative to other blood sources, although not significantly higher than guinea pig blood. In contrast, the survival rate of engorged females post-feeding on sheep blood (67.2%) was significantly less than other blood sources (P<0.05) (Table 3).
Table 2. Mean percent engorged female mosquitoes fed various blood sources.
|Hamster blood by direct feeding||83.0 ± 4.4c||91.3 ± 6.0a||89.7 ± 5.7a||70.3 ± 6.0b||95.3 ± 3.5a|
|Guinea pig blood||97.7 ± 1.2a||82.3 ± 8.3ab||77.7 ± 13.5ab||64.0 ± 11.5bc||66.7 ± 16.6b|
|Human blood||97.0 ± 1.0a||71.7 ± 9.3b||70.7 ± 9.5b||48.7 ± 7.0c||67.0 ± 19.9b|
|Sheep blood||89.3 ± 3.1b||89.0 ± 7.9a||90.7 ± 6.1a||90.7 ± 7.0a||86.7 ± 8.5ab|
Table 3. Percent survival of engorged female mosquitoes after feeding on various blood sources at days 1, 3, and 7 post-feeding. All replicates were run at the same time using the same batches of blood.
| An. dirus ||Direct feeding||99.6||99.6||96.8||96.0 ± 0.8b|
| ||Guinea pig blood||100||100||98.3||98.3 ± 0.6a|
| ||Human blood||100||100||100||100.0 ± 0.0a|
| ||Sheep blood||70.1||98.9||97||67.2 ± 1.8c|
| An. cracens ||Direct feeding||99.2||99.3||99.3||97.8 ± 1.0d|
| ||Guinea pig blood||100||96.4||95||91.6 ± 7.0d|
| ||Human blood||96.6||89.2||97||90.3 ± 21.1d|
| ||Sheep blood||95.7||42||80||35.0 ± 27.0e|
| An. minimus ||Direct feeding||100||97.5||96.5||94.1 ± 0.3fg|
| ||Guinea pig blood||100||99||99.2||98.2 ± 1.1f|
| ||Human blood||100||96.8||91.1||88.2 ± 5.2g|
| ||Sheep blood||54.3||95.1||100||51.5 ± 7.0h|
| An. sawadwongporni ||Direct feeding||96.7||95||92.9||85.2 ± 1.4j|
| ||Guinea pig blood||100||92.2||96.3||88.6 ± 0.4j|
| ||Human blood||100||100||97.4||97.4 ± 2.3i|
| ||Sheep blood||60.6||83.8||93.7||46.7 ± 8.1k|
| Ae. aegypti ||Direct feeding||100||100||97.2||97.2 ± 1.6l|
| ||Guinea pig blood||100||100||99.3||99.3 ± 1.2l|
| ||Human blood||100||100||100||100.0 ± 0.0l|
| ||Sheep blood||84.2||100||100||84.2 ± 17.4l|
The mean percentages of engorged females feeding directly on hamster blood (91.3%) and on sheep blood via a membrane (89.0%) were higher than with either guinea pig blood (82.3%) or human blood (71.7%) (Table 2). The survival rate of engorged females feeding on sheep blood was lowest at 42.0% between days 1 and day 3 relative to other sources (Table 3). Overall survival post-feeding on sheep blood (35.0%) was significantly less than with guinea pig blood (91.6%), direct feeding on hamsters (97.8%), and human blood (90.3%) (P<0.05).
The mean percentages of engorged females after feeding on sheep blood, direct feeding on hamsters, guinea pig blood, and human blood were 90.7, 89.7, 77.7 and 70.7, respectively (Table 2). Survival declined rapidly on the first day (54.3%) post-feeding with sheep blood. Moreover, overall survival post-feeding on sheep blood (51.5%) was significantly less than with guinea pig blood (98.2%), direct feeding on hamsters (94.1%), and human blood (88.2%) (P<0.05) (Table 3).
The mean percentage of engorged females after feeding on sheep blood was significantly higher (90.7%) relative to the other blood sources (Table 2). Additionally, there was a marked drop in survival at day 1 (60.6%). Also, the overall survival rate of engorged females that fed on sheep blood (46.7%) was significantly less than with human blood (97.4%), guinea pig blood (88.6%), and direct feeding on hamsters (85.2%) (P<0.05) (Table 3).
The mean percentage of engorged females from direct feeding (95.3%) was higher than the other blood sources; sheep blood (86.7%), human blood (67.0%), and guinea pig blood (66.7%) (Table 2). At 7 days post-feeding, the survival rate with human blood was 100%. Guinea pig blood, direct feeding on hamsters, and sheep blood were showing survival rates at 99.3, 97.2, and 84.2, respectively, although there were no statistically significant differences among the blood sources (P>0.05) (Table 3). Presumably, survival rate is not affected by the blood source for this particular species.
Fecundity and egg hatching rate
For An. dirus, the mean number of eggs laid by direct feeding on a hamster (219 eggs ±6.5) was not significantly different from on guinea pig blood (204.3 eggs ±23.8). However, the number was higher than what was observed with human blood (151.3 eggs ±3.2) and sheep blood (95.0 eggs ±42.9) (Table 4). The hatching rate was significantly lower with sheep blood (19.5%) relative to the other blood sources (Table 4).
Table 4. Mean comparison of the number of eggs laid and percent egg hatch for each blood source.
|Direct feeding||219.7 ± 6.5a||88.6 ± 1.5a||122.3 ± 13.0a||85.5 ± 12.5a||93.0 ± 13.0a||74.9 ± 8.3a||133.0 ± 53.4a||88.9 ± 3.2a||95.0 ± 13.1a||92.5 ± 6.5a|
|Guinea pig blood||204.3 ± 23.8a||79.3 ± 1.1a||159.7 ± 17.9a||96.9 ± 2.6a||59.0 ± 7.8b||52.41 ± 7.7a||109.7 ± 29.1a||82.8 ± 6.2a||61.7 ± 13.6b||74.4 ± 22.5a|
|Human blood||151.3 ± 3.2b||87.2 ± 2.4a||157.3 ± 40.7a||82.9 ± 9.2a||50.7 ± 11.7b||50.8 ± 8.1a||92.0 ± 28.6a||87.7 ± 6.7a||101.0 ± 21.0a||90.8 ± 9.2a|
|Sheep blood||95.0 ± 42.9c||19.5 ± 20.7b||72.0 ± 16.5b||64.0 ± 1.7b||0c||NA||0b||NA||42.3 ± 14.0b||90.2 ± 14.0a|
For An. cracens, the mean number of eggs laid and hatching rate associated with a sheep blood diet (72 eggs ±16.5, 64.0%) was significantly less than what was observed with guinea pig blood (159.7 eggs ±17.9, 96.9%), human blood (157.3 eggs ±40.7, 82.9%), and direct feeding on a hamster (122.3 eggs ±13.0, 85.5%) (Table 4).
No An. minimus fed on sheep blood and therefore egg-laying/hatching rates could not be computed. Meanwhile, the number of eggs laid and hatching rates by direct feeding on hamsters (93 eggs ±13.0, 74.9%) were greater than those observed for guinea pig blood (59.0 eggs ±7.8, 52.4%) and human blood (50.7 eggs ±11.7, 50.8%) (Table 4).
No An. sawadwongporni fed on sheep blood and therefore egg-laying/hatching rates could not be computed (Table 4). However, there were no significant differences among the mean number of eggs laid and eggs hatched due to feeding on guinea pig blood (109.7 eggs ±29.1, 82.8%), human blood (92.0 eggs ±28.6, 87.7%), and direct feeding on hamsters (133.0 eggs ±53.4, 88.9%).
The mean number of eggs laid by Ae. aegypti that fed on sheep blood (42.3 eggs ±14.0) was significantly lower than that of human blood (101.0 eggs ±21.0), direct feeding on hamster blood (95.0 eggs ±13.1), and guinea pig blood (61.7 eggs ±13.6) (Table 4). No significant differences were observed among the mean percentages of hatched eggs associated with each blood source (P>0.05).
Developmental period and survival rate of offspring
The mean developmental time from egg to adult of offspring from An. dirus parents fed on one of four blood sources was 10.3 days (human blood), 12.0 days (hamster), 12.3 days (guinea pig), and 13.7 days (sheep blood) (Table 5). Mean developmental time to adult after parents were fed on sheep blood was significantly longer relative to the developmental times of other blood sources (P<0.05). The survival rate of offspring originating from adults fed with sheep blood (19.2%) was significantly less than that observed with other blood sources: hamster blood (84.8%), human blood (80.1%), and guinea pig blood (71.4%) (Table 6).
Table 5. Mean comparison of developmental times of Anopheles spp and Ae. aegypti progeny by species and blood source.
| An. dirus ||Direct feeding||1.0 ± 0.0||3.0 ± 0.0||2.3 ± 0.6||2.0 ± 0.0||1.7 ± 0.6||2.0 ± 0.0||12.0 ± 1.0de|
| ||Guinea pig blood||1.0 ± 0.0||3.0 ± 0.0||2.3 ± 0.6||2.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||12.3 ± 0.6de|
| ||Human blood||1.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||1.3 ± 0.4||2.0 ± 0.0||2.0 ± 0.0||10.3 ± 0.4d|
| ||Sheep blood||1.0 ± 0.0||3.0 ± 0.0||3.0 ± 0.0||2.0 ± 0.0||2.7 ± 0.6||2.0 ± 0.0||13.7 ± 0.6e|
| An. cracens ||Direct feeding||1.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||1.0 ± 0.0||2.3 ± 0.6||2.0 ± 0.0||11.3 ± 0.6c|
| ||Guinea pig blood||2.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||1.0 ± 0.0||2.3 ± 0.6||2.0 ± 0.0||11.3 ± 0.6c|
| ||Human blood||2.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||1.0 ± 0.0||2.3 ± 0.6||2.0 ± 0.0||11.3 ± 0.6c|
| ||Sheep blood||1.0 ± 0.0||4.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||2.7 ± 0.6||2.0 ± 0.0||13.7 ± 0.6b|
| An. minimus ||Direct feeding||2.0 ± 0.0||6.3 ± 0.6||5.7 ± 0.6||3.0 ± 0.0||2.7 ± 0.6||2.7 ± 0.6||22.3 ± 1.2f|
| ||Guinea pig blood||1.0 ± 0.0||6.0 ± 0.0||7.0 ± 0.0||2.3 ± 0.6||3.7 ± 0.6||2.3 ± 0.6||22.7 ± 1.2f|
| ||Human blood||2.0 ± 0.0||5.7 ± 0.6||5.3 ± 0.6||3.3 ± 0.6||3.0 ± 0.0||2.3 ± 0.6||21.7 ± 0.6f|
| ||Sheep blood||0||0||0||0||0||0||0.0g|
| An. sawadwongporni ||Direct feeding||3.0 ± 0.0||2.0 ± 0.0||3.0 ± 0.0||2.0 ± 0.0||3.0 ± 0.0||2.3 ± 0.6||15.3 ± 0.6h|
| ||Guinea pig blood||3.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||3.0 ± 1.7||2.7 ± 0.6||2.7 ± 0.6||15.3 ± 0.6h|
| ||Human blood||3.0 ± 0.0||2.0 ± 0.0||2.3 ± 0.6||2.0 ± 0.0||2.7 ± 0.6||2.3 ± 0.6||14.7 ± 0.6h|
| ||Sheep blood||0||0||0||0||0||0||0.0i|
| Ae. aegypti ||Direct feeding||2.0 ± 0.0||2.0 ± 0.0||1.0 ± 0.0||1.0 ± 0.6||2.7 ± 0.6||2.0 ± 0.0||10.7 ± 0.6a|
| ||Guinea pig blood||2.0 ± 0.0||1.7 ± 0.6||1.0 ± 0.0||1.3 ± 0.3||2.3 ± 0.6||2.3 ± 0.6||10.7 ± 0.6a|
| ||Human blood||2.0 ± 0.0||2.0 ± 0.0||1.0 ± 0.0||1.0 ± 0.0||2.7 ± 0.6||2.3 ± 0.6||11.0 ± 1.0a|
| ||Sheep blood||2.0 ± 0.0||2.0 ± 0.0||2.0 ± 0.0||1.0 ± 0.0||2.7 ± 0.6||2.0 ± 0.0||11.7 ± 0.6a|
Table 6. Mean percent offspring survival from egg to adult for each species and blood source.
|Direct feeding||84.8 ± 0.2a||93.9 ± 2.8a||70.8 ± 6.1a||80.7 ± 4.0a||84.5 ± 10.a|
|Guinea pig blood||71.4 ± 3.5a||85.3 ± 12.8a||46.9 ± 17.8b||63.9 ± 8.5b||75.8 ± 18.1a|
|Human blood||80.1 ± 8.8a||87.3 ± 12.5a||46.7 ± 7.9b||79.1 ± 7.8a||85.9 ± 11.2a|
|Sheep blood||19.2 ± 20.2b||63.5 ± 8.4b||NA||NA||67.7 ± 23.1a|
The mean developmental time for offspring from An. cracens fed sheep blood mimicked that observed for An. dirus (13.7 days) (Table 5). This result proved to be significantly longer than the 11.3 days observed for offspring from adults fed guinea pig, human, or hamster blood. No significant differences were found among the survival rates of offspring as a result of adult feeding on hamster (93.9%), guinea pig blood (87.3%), or human blood (85.3%) (Table 6). However, the survival rate of offspring from sheep-fed parents was significantly lower than what was observed with the other blood sources (63.5%) (P<0.05).
Mean developmental times for offspring from An. minimus fed guinea pig blood, direct feeding on hamster blood, or human blood were 22.7, 22.3, and 21.7 days, respectively (Table 5). In addition, no engorged females survived to lay eggs after feeding on sheep blood. Meanwhile, there was a 70.8% mean survival rate of offspring feeding on hamster blood which proved to be significantly higher than the 46.9% observed with guinea pig blood and 46.7% with human blood (Table 6).
The mean developmental time of offspring after female An. sawadwongporni fed on hamster blood and human blood was 15.3 days for both blood sources (Table 5). The period from egg to adult for human blood was 14.7 days. There were no significant differences among mean developmental times associated with human blood, hamster blood, and guinea pig blood (P >0.05). Mean survival rates of offspring fed hamster blood (80.7%) and human blood (79.1%) were significantly higher than those observed for guinea pig blood (63.9%) (Table 6).
Mean developmental times associated with hamster blood and guinea pig blood for Ae. aegypti were 10.7 days. Those associated with human blood and sheep blood proved to be longer in duration (11.7 days). No significant differences in mean developmental times were found among blood sources (P>0.05) (Table 5). The mean survival rates of offspring from parents fed human blood, hamster blood, guinea pig, and sheep blood were 85.9, 84.6, 75.8, and 67.7, respectively, with no significant differences observed (Table 6).
Our results show that the blood meal source impacts feeding rates, adult survival, fecundity, hatching rates, and developmental times for five species of mosquitoes (An. dirus, An. cracens, An. minimus, An. sawadwongporni, and Ae. aegypti). Sheep blood had a significant, negative impact on the survival rates of An. dirus, An. cracens, and An. minimus. The mean number of eggs laid was reduced relative to the other blood sources when An. dirus, An. cracens, and Ae. aegypti adults were fed sheep blood. Sheep blood did not induce the production of eggs for An. sawadwongporni and An. minimus. Previous studies have in fact shown that mosquito fecundity is affected by the following factors: mosquito species (Briegel 1990), body size (Blackmore and Lord 2000), host (Taylor and Hurd 2001), size of the bloodmeal (Roitberg and Gordon 2005) and amino acids from erythrocytes (Hurd 2003). Furthermore, the offspring of An. dirus fed with sheep blood survived for a shorter period of time relative to what was shown with the other blood sources.
The results show that blood meal sources impact feeding rates, survivorship, and fecundity, and are consistent with those of other studies. Nayar and Sauerman (1975) studied the effects of nutrition from a blood meal on survival and calorie intake in mosquitoes when females were fed a meal of chicken blood ad libitum. Efficiency of blood ingested for survival was highest in Ae. aegypti (84.5 hr/cal.), followed by Ae. taeniorhynchus (68.5 hr/cal.), and Ae. sollicitans, Anopheles quadrimaculatus and Psorophora confinnis (44.0 to 48.4 hr/cal.). Xue (2008) reported that the survival of female Ae. albopictus that were fed on blood of two different hosts (double meal; 70.2%) was higher than the females fed on one host (single meal; 55.5%) and produces significantly more eggs. Kweka et al. (2010) investigated the feeding and mortality rates of Cx. quinquefasciatus, Ae. aegypti and An. arabiensis against three different hosts (rabbits, guinea pigs, and mice). Mosquito mortality was 54.9% for those introduced to mice as a host, 34.3% for the guinea pig, and 10.8% for those introduced to the rabbit. Survivorship in mosquitoes is related to many factors including temperature and available nutrition (Clements 1992). Also, human blood was the most ideal for oviposition rate (79.2%), while chicken and pig blood were the least favorable (46.8 and 48.1%, respectively). In addition, Richards et al. (2012) reported the impact of blood meal source on feeding and reproduction in Cx. quinquefasciatus when mosquitoes were fed on different blood meal sources (chicken blood and bovine blood). Fecundity and fertility of Cx. quinquefasciatus were greater in mosquitoes fed chicken blood than the other blood source. Offspring of parents reared under low food availability produced more eggs than the offspring from parents reared under high food conditions. Harre et al. (2001) found no differences in feeding success, mortality rates, egg numbers, and egg viability when sand flies (Phlebotomus papatasi) were fed on blood from different mammals (human, horse, cow, pig, dog, rabbit, guinea pig, and hamster). Our study demonstrates that sheep blood has a negative impact on adult survival, egg-laying, and hatching rates of Anopheles mosquitoes. Future studies will elucidate some of the mechanisms for these findings in terms of the chemical and physical composition of sheep blood or digestion processes.
Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition. Funding for this project was partially provided by the U.S. Army Medical and Material Command. The views expressed in this paper are those of the authors and do not represent the position and/or reflect the official policy of the Department of the Army, the Department of Defense or the U.S. Government. We also wish to express our gratitude to COL Chaiyaphruk Pilakasiri for reviewing and comments on the manuscript.