Journal of Insect Physiology 64 (2014) 1–6
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A blood-free protein meal supporting oogenesis in the Asian tiger mosquito, Aedes albopictus (Skuse) R. Jason Pitts ⇑ Department of Biological Sciences and Institute for Global Health, Vanderbilt University, 465 21st Ave. S., Nashville, TN 37232, USA
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Article history: Received 18 December 2013 Received in revised form 21 February 2014 Accepted 27 February 2014 Available online 6 March 2014 Keywords: Aedes albopictus Oogenesis Blood meal Blood-free formulation Mass rearing Hematophagy
a b s t r a c t Female mosquitoes require blood meals to complete oogenesis, or egg development. Current methods of maintaining laboratory colonies of mosquitoes generally rely on the use of whole blood to feed females. Blood feeding protocols require special handling techniques, impart numerous potential health hazards, involve significant costs, and are widely variable in terms of their success rates. In this study, a simple protein formulation was provided to Aedes albopictus using a membrane feeding system. Under the experimental conditions tested, females readily accepted the blood-free meal and produced eggs in greater numbers than cohort females that were fed with whole human blood. Moreover, fertility was comparable between treatments and survivorship of hatched larvae was equal among feedings. This implies that a readily available blood-free meal could be utilized in the laboratory rearing of this species. The elimination of blood handling, reduced cost, and consistency of blood-free meals would potentially be advantageous to mosquito rearing facilities generally, and in terms of scale, to mass rearing facilities specifically. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Females of anautogenous mosquito species require an ingested blood meal to complete ovarian follicle development, which remains in a suspended state (Clements, 1992). Various studies have concluded that protein is the major, if not exclusive, nutrient in the blood that is necessary for oogenesis (Clements, 1992). However, not all blood meals are equivalent and host species can affect female egg production. In some studies, Aedes aegypti (L.) females have produced fewer eggs after taking a human blood meal than after taking an animal blood meal (Chang and Judson, 1979; Greenberg, 1951). Fertility and fecundity were both higher in Culex pipiens quinquefasciatus (Say) that were fed chicken blood compared with bovine blood (Richards et al., 2012). In another study, oviposition rate was highest in Anopheles gambiae (Patton) after feeding on human or cow blood, but was lowest on chicken or dog blood (Lyimo et al., 2012). A low level of free isoleucine in human plasma relative to other species has been shown to account for this difference in fecundity, highlighting the importance of this essential amino acid in female mosquito oogenesis (Briegel, 1985; Chang and Judson, 1977; 1979; Greenberg, 1951). Moreover, proteins that are low in isoleucine content, like hemoglobins (human hemoglobin lacks isoleucine), fail to stimulate oogenesis ⇑ Tel.: +1 615 343 3718; fax: +1 615 936 0129. E-mail address: [email protected] http://dx.doi.org/10.1016/j.jinsphys.2014.02.012 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
while those that are high in isoleucine, such as globulins, support oogenesis (Spielman, 1971; Spielman and Wong, 1974). However, differences in fecundity between human blood and mouse blood feedings were negated in Ae. aegypti when sugar was withheld, and in this context females imbibed less fully, but more frequently on human blood than on mouse blood (Harrington et al., 2001). A recent study using radiolabeled tracers showed that free plasma isoleucine and protein-bound phenylalanine were incorporated at the highest rates into Ae. aegypti egg proteins compared to other essential amino acids, again demonstrating the importance of specific amino acids in oogenesis (Zhou and Miesfeld, 2009). Interestingly, Ae. aegypti were able to produce viable eggs when fed a meal consisting of only 12 amino acids, including isoleucine (Lea et al., 1956). Amino acids also act as signals for numerous metabolic processes in the mosquito midgut and stimulate a hormone-signaling cascade that initiates vitellogenesis in several culicine species (Uchida et al., 2001, 1998). While the gonotrophic requirement for blood protein is well established, blood-free meals for mosquito colony maintenance have not been widely tested for egg development or adopted for laboratory use. Successful oogenesis in Ae. aegypti can be supported by formulations made from protein sources like milk and egg powder; washed erythrocytes, supplemented with various proteins; and purified blood protein components, supplemented with isoleucine (Greenberg, 1951; Lea et al., 1956; Kogan, 1990). Absent from the published literature are studies that utilize blood
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meal substitutes to support egg production in anopheline species. One study specifically stated that An. gambiae fed inconsistently on an artificial meal and produced viable eggs in only 1 of 6 trials (Kogan, 1990). Another study described successful oviposition by An. quadrimaculatus resulting from artificial meals, but did not provide data regarding egg numbers or comparisons with controls (Lea et al., 1956). Few studies have provided detailed information about important parameters such as percentage of females fed on artificial meals, hatch rates of eggs, or survivorship to adult stage as compared to mosquitoes fed on live animals or whole blood delivered via artificial feeding apparatus. It thus appears that blood meal substitutes have the potential to be used successfully for laboratory mosquito colony rearing, but that more study is required in order to establish the utility of artificially delivered meals for laboratory colony maintenance in species other than Ae. aegypti. The current study was conducted to investigate the effects of a blood-free meal on fecundity of Ae. albopictus, an invasive species that is a competent vector for numerous arboviruses and has been implicated in several outbreaks of Chikungunya in various geographic locations within the past decade (Gratz, 2004; Pialoux et al., 2007; Paupy et al., 2009; Lambrechts et al., 2010). Ae. albopictus is a potential target for vector control programs that depend on the release of large numbers of sterile or genetically modified individuals (Benedict, 2003; Labbé et al., 2010; Alphey et al., 2011; Iturbe-Ormaetxe et al., 2011; Oliva et al., 2012). Such programs would depend upon consistent, high volume, cost effective, and pathogen free blood sources (Benedict et al., 2009). Here, a simple protein formulation supported significantly higher egg production in Ae. albopictus females compared with whole human blood and at a much lower cost. This outcome provides a framework for the development of blood-free formulations that will be useful for large-scale rearing of mosquitoes and potentially other hematophagous insects. 2. Materials and methods 2.1. Mosquito rearing Ae. albopictus eggs were initially obtained through the Malaria Reseacrh and Reference Reagent Resource Center (ALBOPICTUS F4, MRA-804). Eggs were submerged in an aqueous oak leaf infusion and placed in a vacuum chamber for 30 min to deoxygenate and stimulate hatching. Larvae were allowed to develop in clean tap water and fed ground koi fish food ad libitum. Pupae were collected by hand with an eyedropper and placed into clean water. Adults were allowed to emerge in cube-shaped cages measuring 30 cm3 (Megaview Science Co, Ltd.) and had access to a 10% sucrose solution ad libitum, except for 1 h prior to feeding experiments when females were separated from males and sucrose was withheld. All stages were reared at a temperature of 26 °C and 75% relative humidity (RH) in an upright incubator with a 12:12 light:dark cycle. 2.2. Feeding treatments Starved females were fed using a membrane delivery system (Hemotek, Ltd.) using 2 ml of whole human blood or artificial meal formulation, which was pipetted into a holding disc, sealed with stretched parafilm, and heated to 37 °C prior to delivery. An odor blend consisting of ammonium hydroxide, lactic acid, isovaleric acid, geranyl acetone, and butylamine, each at [ 5logM] concentrations in aqueous buffer was applied to parafilm membranes as a host-seeking stimulant. Whole human blood, collected from individual male subjects (blood type O ) and stored in lithium heparin was purchased from a biological supply company (BioChemed
Services), kept refrigerated at 4 °C for no longer than 1 week and used only one time per experiment. Blood meal substitute consisted of bovine serum albumin (fraction V; Research Products International Corp.) [100 or 200 mg/mL] dissolved in a phosphate buffered saline solution (NaCl [137 mM], Na2HPO4 [10 mM], KH2HPO4 [1.76 mM], KCl [2.68 mM], pH7.2) by heating briefly at 42 °C. Adenosine triphosphate (Sigma–Aldrich Co., LLC), was serially diluted in BSA solution from a 100 mM stock solution just prior to membrane feed at final concentrations of [1 mM], [0.1 mM], [10 lM], [1 lM] and [0 lM]. After feeding, females were provided 10% sucrose and were kept at 26 °C/75% RH for 72 h. Gravid females were placed in small cups containing 100 mL of distilled water with a partially submerged filter paper liner as a substrate for oviposition. For blood meal volumes, groups of unfed or fully fed females were cold anesthetized and weighed together on an electronic balance to the nearest milligram. Estimated mean mass was derived using the formula: total weight/# females. 2.3. Statistical treatment of data Ovaries were hand dissected from gravid females 72 h after feeding and teased apart using fine forceps. Stage V oocytes or oviposited eggs were placed under a dissecting microscope and counted with the aid of a handheld counter. Arithmetic mean, median, and standard deviation were determined for egg counting data. The Kruskal–Wallis analysis of variance was used to test for significant differences in distribution among all 3 groups. When significant differences were identified, the Mann–Whitney U statistic was calculated as a measure of differences in pairwise distributions. Interquartile range (IQR) was calculated by subtracting the highest value in quartile 3 (Q3) from the lowest value in quartile 1 (Q1). Whiskers were graphed as the highest or lowest data points falling within 1.5 * IQR of Q3 and Q1, respectively. Data points with values higher or lower than whiskers were plotted individually as outliers. The chi-square test was applied to the adult sex ratio data to determine significant deviation from the expected 1:1. 3. Results and discussion 3.1. Feeding responses Feeding responses of Ae. albopictus and subsequent oogenesis were compared separately using either whole human blood or a buffered formulation containing bovine serum albumin (BSA) as a protein source supplemented with adenosine triphosphate (ATP). Blood-free formulations induced qualitatively similar feeding responses at BSA concentrations of 200 mg/ml and 100 mg/ ml (BSA 200 and BSA100, respectively; Fig. 1). ATP was added to these formulations because it is a known phagostimulant in Ae. aegypti, where labral sensilla are sensitive to ATP and other soluble compounds (Galun et al., 1984, 1985; Werner-Reiss et al., 1999). To address the question of whether ATP was necessary for Ae. albopictus feeding, a series of trials was conducted in which the final ATP concentration in the BSA 200 meal ranged from a low of 0 mM to a high of 1 mM. A clear requirement for ATP was observed as no females fed on the BSA formulation lacking ATP (Table 1), although vigorous probing of the membrane was observed. The proportion of females that had fed on BSA improved with successively higher concentrations of ATP, reaching a maximum of 69% at 1 mM ATP, which outpaced the proportion feeding on whole human blood (Table 1). These observations strongly suggests that ATP is a phagostimulant for Ae. albopictus and may imply that this trait is a general feature of aedines. Additionally, groups of females were weighed immediately after feeding as a way of estimating ingested
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Fig. 1. Oogenesis in Ae. albopictus females fed with whole human blood or BSA meals. Left panels: females engorged with (A) human blood, (B) BSA [200 mg/mL], or (C) BSA [100 mg/mL]. Right panels: fully developed ovaries (stage V) dissected 72 h after feeding.
meal volumes. After feeding, individual females nearly doubled in mass to a calculated average of 3 mg as compared with unfed females (Table 1). Similar changes in mass were observed for blood fed or BSA fed females, suggesting that similar volumes of each meal were ingested, with the possible exception of the lowest concentration of ATP (Table 1). 3.2. Reproductive capacity Gonotrophic responses of Ae. albopictus females were characterized by quantification of egg production in gravid females. Initial studies quantified eggs that were oviposited as a measure of egg production. However, ovarian dissections of females that had recently oviposited revealed a high prevalence of partial, or skip oviposition, i.e. releasing only a portion of the fully developed eggs (Fig. 2; Supplemental Table 1), a phenomenon that has been observed previously for this species (Rozeboom et al., 1973; Trexler et al., 1998; Farjana and Tuno, 2013). This characteristic of gravid Ae. albopictus led to wide variations in observed egg counts, such that the actual total egg production per female would be obscured (Fig. 2). Consequently, total egg production was quantified as the sum of oviposited eggs plus stage V oocytes retained in the ovaries
Fig. 2. Skip oviposition in Ae. albopictus. y-Axis: stacked histograms showing number of eggs either oviposited (lighter shading) or retained in ovaries as stage V oocytes (darker shading) by individual females (x-axis) 72 h post-meal. (A) Whole human blood, (B) BSA [200 mg/ml], (C) BSA [100 mg/ml].
as observed upon ovarian dissection (Fig. 2). Females offered whole human blood or BSA at 2 different concentrations completed oogenesis within 72 h, with no discernable differences in timing. At least 5 trials of 5–10 females per trial were conducted to compare egg production of blood fed versus BSA fed individuals (Table 2). Egg counts for each trial were highly variable and the data were not normally distributed and had unequal variances, even when combined for all trials (Table 2; Supplemental Table 2). Various attempts to transform the data to allow parametric testing for differences in sample means were unsuccessful. Non-parametric analyses were therefore used to test for differences in egg production among groups. The results of a single trial are presented in Fig. 3A. In this example, females produced fewer eggs from BSA 100, but the ranked averages for blood and BSA 200 were not statistically different (Kruskal–Wallis H = 12.4, df = 2, p = 2.0 10 3; Fig. 3A). When data sets were combined and compared as a whole, all 3 groups differed significantly from one another (Kruskal–Wallis H = 47.4, df = 2, p = 5.1 10 11) and females produced the highest number
Table 1 Feeding Responses of Ae. albopictus females to BSA supplemented with ATP. BSA [200] + ATP
Proportion feeding Trials Mean weight mg n
Blood
1 mM
0.1 mM
10 lM
1 lM
None
75/127 (0.59) 8 3.17 63
63/91 (0.69) 5 2.93 56
46/103 (0.45) 7 3.22 41
43/99 (0.43) 6 2.91 35
12/89 (0.13) 4 2.67 12
0/99 (0.00) 6 1.70 41
Feeding frequency = # fully fed females/total # females. Numbers in parentheses = proportion of females taking a full meal. Trials = total number of independent replicates. Mean weight mg = average calculated mass per female in milligrams. n = Total number of fed females weighed.
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R. Jason Pitts / Journal of Insect Physiology 64 (2014) 1–6
Table 2 Aedes albopictus female egg production 72 h post feeding. Treatment
Mean St. Dev Median Range Trials n
Blood
BSA 200
BSA 100
83.7 19.8 84 18–124 7 67
92.2 33.6 96 7–162 6 58
57.0 23.6 59 5–97 5 55
Mean = average number of eggs produced per female. St. Dev = standard deviation. Median = median egg production. Trials = number of independent replicates. n = Total number of females in all trials.
of eggs after being fed BSA 200 (Table 2; Fig. 3B). This trend included a much higher maximum egg production for the BSA 200 fed females, where several individuals produced more than 130 eggs from a single meal (Table 2; Supplemental Table 2), a magnitude that has been reported previously for Ae. albopictus (Blackmore and Lord, 2000), but that was not achieved by any of the individuals that were fed whole blood or BSA 100 (Supplemental Table 2). Importantly, the mean numbers of eggs produced by blood fed (83.7) or BSA 200 fed (92.2) females in this study (Table 2) is very similar to published estimates of the average number of 50–60 ovarioles per ovary in Ae. albopictus when reared under optimal conditions (Yamany et al., 2012; Farjana and Tuno, 2013). Perhaps not coincidentally, the 200 mg/ml concentration of BSA is similar to the average protein content of whole human blood (Kogan, 1990), although the total isoleucine content of the BSA 200 meal in protein-bound form [80 lM] is higher than the plasma-free form reported in human blood plasma [40 lM] (Harrington et al., 2001). Thus the differences in egg production between whole human blood and BSA 200 observed in this study could potentially be accounted for by variable isoleucine content. If so, a higher concentration of BSA might be expected to elicit even greater egg production in Ae. albopictus females. A single trial was conducted with BSA at 400 mg/ml in which 16 fed females produced an average of 71.6 eggs (median = 81.5, range = 12– 109). Taken together, egg quantification data support the conclusion that near-maximum egg production was achieved in the BSA 200 feedings in this study. In addition it appears that most females
were indeed fully fed because blood meal volume has been positively correlated with total egg production in Ae. albopictus (Barnard and Xue, 2009). Future studies should be directed towards elucidating the effects artificial meals impose on total lifetime reproductive capacity by quantifying female longevity and egg production after multiple feedings. In the context of small scale laboratory rearing these issues may not be important as a single meal per generation is usually sufficient for production needs. However, mass rearing facilities may benefit from increased lifetime reproductive output if imparted by a non-blood formulation. 3.3. Survivorship of progeny Hatch rates and survivorship were determined for eggs oviposited by females that were fed with human blood or BSA 200 meals (Table 3). For the six trials reported here, hatch rates were highly variable, ranging from 27–86% in blood fed treatments and 25– 96% in BSA 200 fed treatments (Table 3). Although eggs from the BSA 200 trials hatched at a reduced rate overall, survivorship of hatched larvae during development was very high (>90%) in both treatments, with no apparent difference between BSA 200 or blood treatments (Table 3). Moreover, the numbers of adult males and females were approximately equal (Table 3) and the summed data were not significantly different than the expected 1:1 ratio in either treatment (blood fed: X2 = 1.575, df = 1, P = 0.2095; BSA fed: X2 = 3.429, df = 1, P = 0.0641). Numerous egg storage and hatching conditions were attempted, but the highest rates were obtained using eggs that were >2 weeks old, were free of mold, and were hatched using deoxygenated water (Hawley, 1988). Despite the overall challenge of hatching Ae. albopictus eggs, the results of several independent feeding experiments indicate that this species can be successfully reared without using animals or animal blood. During the course of this study, a colony of Ae. albopictus was successfully maintained for 6 consecutive generations using BSA 200 as the exclusive protein source for female oogenesis. 3.4. Health benefits and cost savings The use of an artificial meal and a membrane feeder system virtually eliminates the health hazards associated with handling human blood or animals (Askarian et al., 2011; Hill, 1999). Feeding mosquitoes using human volunteers or live animals, while practiced in many laboratories, is often unacceptable due to the potential infection of mosquitoes and exposure of individuals to
Fig. 3. Ae. albopictus egg production 72 h after feeding. y-Axes: total number of eggs (oviposited + stage V oocytes). (A) Graphical representation of a single trial: whole human blood (diamonds), BSA [200 mg/ml] (circles), or BSA [100 mg/ml] (triangles). Lower case letters indicate statistically different rank distributions as determined by and Mann–Whitney U tests (U = 32, p = 0.174 blood vs. BSA 200; U = 14, p = 0.007 blood vs. BSA 100; U = 9.5, p = 0.002 BSA 200 vs. BSA 100). (B) Box plot representation of compiled data. Boxes represent 1st and 3rd quartile intervals and whiskers represent ±1.5 * IQR. Horizontal bars represent median values. Lower case letters indicate statistically different rank distributions as determined by Mann–Whitney U tests (U = 1456, p = 0.016 blood vs. BSA 200; U = 727, p < 2 10 6 blood vs. BSA 100; U = 569.5, p < 2 10 6 BSA 200 vs. BSA 100).
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R. Jason Pitts / Journal of Insect Physiology 64 (2014) 1–6 Table 3 Aedes albopictus egg hatch rates and survivorship. # Larvaea
# Pupaeb
# Adultsc
# Males
# Females
Whole human blood 1 63 2 93 3 78 4 88 5 79 6 51 Sum 452
54 (0.86) 51 (0.55) 54 (0.69) 55 (0.63) 55 (0.70) 14 (0.27) 283 (0.63 ± 0.08)
54 (1.00) 50 (0.98) 51 (0.94) 52 (0.93) 43 (0.78) 13 (0.93) 263 (0.93 ± 0.03)
54 (1.00) 50 (0.98) 50 (0.93) 52 (0.93) 35 (0.64) 13 (0.93) 254 (0.90 ± 0.05)
32 26 19 19 15 6 117
22 24 31 33 20 7 137ns
BSA 200 1 2 3 4 5 6 Sum
49 (0.96) 26 (0.33) 44 (0.50) 21 (0.25) 22 (0.39) 23 (0.31) 185 (0.43 ± 0.10)
49 (1.00) 25 (0.96) 43 (0.98) 19 (0.90) 19 (0.86) 21 (0.91) 176 (0.95 ± 0.02)
46 (0.94) 24 (0.92) 43 (0.98) 19 (0.90) 19 (0.86) 17 (0.74) 168 (0.91 ± 0.03)
26 13 24 14 8 11 96
20 11 19 5 11 6 72ns
Female
# Eggs
51 79 88 83 56 74 431
# Eggs = number of eggs oviposited by an individual female. Columns 2–5 = numbers of living animals at each developmental stage (proportion). Columns 6 and 7 = numbers of eclosed adult males and females; ns – male:female ratio does not differ significantly from 1:1. Sum = total numbers in each column (proportion ± standard error of the mean). a Proportion of eggs hatched. b Proportion of larvae surviving to pupal stage. c Proportion of larvae surviving to adult stage.
pathogens or allergens (Samadi et al., 2013). Moreover, handling host animals can be hazardous in terms of potential bites, scratches, allergic reactions and needle punctures when injecting anesthetic agents (Gargiulo et al., 2012; National Research Council, 2011; Samadi et al., 2013). In addition to reduced exposure, another advantage of a blood meal substitute would be reduced costs associated with the use of biohazards, which include personal protective consumables, disposal fees, and personnel hours spent in training, handling and disposing of biohazards (Occupational Safety and Health Administration, 2011). Artificial meals can significantly lower the direct costs of mosquito rearing, reflected in reduced requirements for reagents, animal housing and related supplies. These savings would be most important to mass rearing facilities where the scale of blood feeding can be exceptionally large and frequent (Benedict et al., 2009). In this study, whole human blood was purchased for $35.00 per 10 ml, while BSA 200 was prepared for approximately $1.50 for the same volume, which is sufficient to feed >3000 Ae. albopictus females, where blood meal volumes range from 1 to 3ul per fully engorged female (Konishi and Yamanishi, 1984; Konishi, 1989; Barnard and Xue, 2009). In sum, the total costs of blood feeding with live animals will likely far exceed blood meal substitutes and specifically the BSA 200 formulation described here. In addition, the use of humans or animals in research labs generally requires approval from institutional review boards and/or animal care and use committees, necessitating extra administrative effort. The information in this study provides a starting point for the further development of blood-free formulations that will be useful for rearing Ae. albopictus. Given the numerous advantages that an artificial feeding system offers, serious consideration should be given to their use, particularly in the context of mass rearing for sterile or genetically modified mosquito release programs and, by extension, other hematophagous disease vectors. Acknowledgements Ae. albopictus eggs were obtained through the Malaria Reseacrh and Reference Reagent Resource Center (ALBOPICTUS F4, MRA804) as part of the Biodefense and Emerging Infections Research Resources Repository, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, deposited by Sandra Allan. The author acknowledges Dr. Laurence Zwiebel and the Dept. of Biological Sciences, Vanderbilt University, for the use of insectary space and equipment. The author also thanks David C. Rinker, Vanderbilt University, for critical reading of the manuscript and members of the Zwiebel Lab for helpful discussions. This work was supported by the International Atomic Energy Agency, Technical Contract No. 16985, to RJP. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2014. 02.012. References Alphey, N., Alphey, L., Bonsall, M.B., 2011. A model framework to estimate impact and cost of genetics-based sterile insect methods for dengue vector control. PLoS One 6, e25384. Askarian, M., Yadollahi, M., Kuochak, F., Danaei, M., Vakili, V., Momeni, M., 2011. Precautions for health care workers to avoid hepatitis B and C virus infection. Int. J. Occup. Environ. Med. 2, 191–198. Barnard, D., Xue, R.-D., 2009. Bloodmeal mass and oviparity mediate host avidity and DEET repellency in Aedes albopictus (Diptera: Culicidae). J. Med. Entomol. 46, 1235–1239. Benedict, M., 2003. The first releases of transgenic mosquitoes: an argument for the sterile insect technique. Trends Parasitol. 19, 349–355. Benedict, M.Q., Knols, B.G.J., Bossin, H.C., Howell, P.I., Mialhe, E., Caceres, C., Robinson, A.S., 2009. Colonisation and mass rearing: learning from others. Malar. J. 8 (Suppl 2), S4. Blackmore, M.S., Lord, C.C., 2000. The relationship between size and fecundity in Aedes albopictus. J. Vector Ecol. 25, 212–217. Briegel, H., 1985. Mosquito reproduction: incomplete utilization of the blood meal protein for oögenesis. J. Insect Physiol. 31, 15–21. Chang, Y., Judson, C., 1977. Role of isoleucine in differential egg production by mosquito Aedes aegypti Linnaeus (Diptera: Culicidae) following feeding on human or guinea pig blood. Comp. Biochem. Phys. A 57, 23–28. Chang, Y., Judson, C., 1979. Amino-acid composition of human and guinea-pig blood proteins, and ovarian proteins of the yellow-fever mosquito Aedes aegypti and their effects on the mosquito egg-production. Comp. Biochem. Phys. A 62, 753– 755. Clements, A., 1992. The Biology of Mosquitoes V1: Development, Nutrition and Reproduction. CABI Publishing, New York, NY. Farjana, T., Tuno, N., 2013. Multiple blood feeding and host-seeking behavior in Aedes aegypti and Aedes albopictus (Diptera: Culicidae). J. Med. Entomol. 50, 838–846.
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Occupational Safety and Health Administration, 2011. Laboratory Safety Guidance. US Department of Labor. Oliva, C.F., Jacquet, M., Gilles, J., Lempérière, G., Maquart, P.-O., Quilici, S., Schooneman, F., Vreysen, M.J.B., Boyer, S., 2012. The sterile insect technique for controlling populations of Aedes albopictus (Diptera: Culicidae) on Reunion Island: mating vigour of sterilized males. PLoS One 7, e49414. Paupy, C., Delatte, H., Bagny, L., Corbel, V., Fontenille, D., 2009. Aedes albopictus, an arbovirus vector: from the darkness to the light. Microbes Infect. 11, 1177– 1185. Pialoux, G., Gaüzère, B.-A., Jauréguiberry, S., Strobel, M., 2007. Chikungunya, an epidemic arbovirosis. Lancet Infect. Dis. 7, 319–327. Richards, S.L., Anderson, S.L., Yost, S.A., 2012. Effects of blood meal source on the reproduction of Culex pipiens quinquefasciatus (Diptera: Culicidae). J. Vector Ecol. 37, 1–7. Rozeboom, L.E., Rosen, L., Ikeda, J., 1973. Observations on oviposition by Aedes (S.) albopictus Skuse and A. (S.) polynesiensis Marks in nature. J. Med. Entomol. 10, 397–399. Samadi, S., Wouters, I.M., Heederik, D.J., 2013. A review of bio-aerosol exposures and associated health effects in veterinary practice. Ann. Agric. Environ. Med. 20, 206–221. Spielman, A., 1971. Bionomics of autogenous mosquitoes. Annu. Rev. Entomol. 16, 231–248. Spielman, A., Wong, J., 1974. Dietary factors stimulating oogenesis in Aedes aegypti. Biol. Bull. 147, 433–442. Trexler, J.D., Apperson, C.S., Schal, C., 1998. Laboratory and field evaluations of oviposition responses of Aedes albopictus and Aedes triseriatus (Diptera: Culicidae) to oak leaf infusions. J. Med. Entomol. 35, 967–976. Uchida, K., Oda, T., Matsuoka, H., Moribayashi, A., Ohmori, D., Eshita, Y., Fukunaga, A., 2001. Induction of oogenesis in mosquitoes (Diptera: Culicidae) by infusion of the hemocoel with amino acids. J. Med. Entomol. 38, 572–575. Uchida, K., Ohmorib, D., Eshita, Y., Oda, T., Kato, Y., Tomizawa, K., Moribayashi, A., Fukunaga, A., 1998. Ovarian development induced in decapitated female Culex pipiens pallens mosquitoes by infusion of physiological quantities of 20-hydroxyecdysone together with amino acids. J. Insect Physiol. 44, 525–528. Werner-Reiss, U., Galun, R., Crnjar, R., Liscia, A., 1999. Sensitivity of the mosquito Aedes aegypti (Culicidae) labral apical chemoreceptors to phagostimulants. J. Insect Physiol. 45, 629–636. Yamany, A.S., Mehlhorn, H., Adham, F.K., 2012. Yolk protein uptake in the oocyte of the Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae). Parasitol. Res. 111, 1315–1324. Zhou, G., Miesfeld, R., 2009. Differential utilization of blood meal amino acids in mosquitoes. OAIP, 1–12.
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
A blood-free protein meal supporting oogenesis in the Asian tiger mosquito, Aedes albopictus (Skuse) R. Jason Pitts ⇑ Department of Biological Sciences and Institute for Global Health, Vanderbilt University, 465 21st Ave. S., Nashville, TN 37232, USA
a r t i c l e
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Article history: Received 18 December 2013 Received in revised form 21 February 2014 Accepted 27 February 2014 Available online 6 March 2014 Keywords: Aedes albopictus Oogenesis Blood meal Blood-free formulation Mass rearing Hematophagy
a b s t r a c t Female mosquitoes require blood meals to complete oogenesis, or egg development. Current methods of maintaining laboratory colonies of mosquitoes generally rely on the use of whole blood to feed females. Blood feeding protocols require special handling techniques, impart numerous potential health hazards, involve significant costs, and are widely variable in terms of their success rates. In this study, a simple protein formulation was provided to Aedes albopictus using a membrane feeding system. Under the experimental conditions tested, females readily accepted the blood-free meal and produced eggs in greater numbers than cohort females that were fed with whole human blood. Moreover, fertility was comparable between treatments and survivorship of hatched larvae was equal among feedings. This implies that a readily available blood-free meal could be utilized in the laboratory rearing of this species. The elimination of blood handling, reduced cost, and consistency of blood-free meals would potentially be advantageous to mosquito rearing facilities generally, and in terms of scale, to mass rearing facilities specifically. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Females of anautogenous mosquito species require an ingested blood meal to complete ovarian follicle development, which remains in a suspended state (Clements, 1992). Various studies have concluded that protein is the major, if not exclusive, nutrient in the blood that is necessary for oogenesis (Clements, 1992). However, not all blood meals are equivalent and host species can affect female egg production. In some studies, Aedes aegypti (L.) females have produced fewer eggs after taking a human blood meal than after taking an animal blood meal (Chang and Judson, 1979; Greenberg, 1951). Fertility and fecundity were both higher in Culex pipiens quinquefasciatus (Say) that were fed chicken blood compared with bovine blood (Richards et al., 2012). In another study, oviposition rate was highest in Anopheles gambiae (Patton) after feeding on human or cow blood, but was lowest on chicken or dog blood (Lyimo et al., 2012). A low level of free isoleucine in human plasma relative to other species has been shown to account for this difference in fecundity, highlighting the importance of this essential amino acid in female mosquito oogenesis (Briegel, 1985; Chang and Judson, 1977; 1979; Greenberg, 1951). Moreover, proteins that are low in isoleucine content, like hemoglobins (human hemoglobin lacks isoleucine), fail to stimulate oogenesis ⇑ Tel.: +1 615 343 3718; fax: +1 615 936 0129. E-mail address: [email protected] http://dx.doi.org/10.1016/j.jinsphys.2014.02.012 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
while those that are high in isoleucine, such as globulins, support oogenesis (Spielman, 1971; Spielman and Wong, 1974). However, differences in fecundity between human blood and mouse blood feedings were negated in Ae. aegypti when sugar was withheld, and in this context females imbibed less fully, but more frequently on human blood than on mouse blood (Harrington et al., 2001). A recent study using radiolabeled tracers showed that free plasma isoleucine and protein-bound phenylalanine were incorporated at the highest rates into Ae. aegypti egg proteins compared to other essential amino acids, again demonstrating the importance of specific amino acids in oogenesis (Zhou and Miesfeld, 2009). Interestingly, Ae. aegypti were able to produce viable eggs when fed a meal consisting of only 12 amino acids, including isoleucine (Lea et al., 1956). Amino acids also act as signals for numerous metabolic processes in the mosquito midgut and stimulate a hormone-signaling cascade that initiates vitellogenesis in several culicine species (Uchida et al., 2001, 1998). While the gonotrophic requirement for blood protein is well established, blood-free meals for mosquito colony maintenance have not been widely tested for egg development or adopted for laboratory use. Successful oogenesis in Ae. aegypti can be supported by formulations made from protein sources like milk and egg powder; washed erythrocytes, supplemented with various proteins; and purified blood protein components, supplemented with isoleucine (Greenberg, 1951; Lea et al., 1956; Kogan, 1990). Absent from the published literature are studies that utilize blood
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R. Jason Pitts / Journal of Insect Physiology 64 (2014) 1–6
meal substitutes to support egg production in anopheline species. One study specifically stated that An. gambiae fed inconsistently on an artificial meal and produced viable eggs in only 1 of 6 trials (Kogan, 1990). Another study described successful oviposition by An. quadrimaculatus resulting from artificial meals, but did not provide data regarding egg numbers or comparisons with controls (Lea et al., 1956). Few studies have provided detailed information about important parameters such as percentage of females fed on artificial meals, hatch rates of eggs, or survivorship to adult stage as compared to mosquitoes fed on live animals or whole blood delivered via artificial feeding apparatus. It thus appears that blood meal substitutes have the potential to be used successfully for laboratory mosquito colony rearing, but that more study is required in order to establish the utility of artificially delivered meals for laboratory colony maintenance in species other than Ae. aegypti. The current study was conducted to investigate the effects of a blood-free meal on fecundity of Ae. albopictus, an invasive species that is a competent vector for numerous arboviruses and has been implicated in several outbreaks of Chikungunya in various geographic locations within the past decade (Gratz, 2004; Pialoux et al., 2007; Paupy et al., 2009; Lambrechts et al., 2010). Ae. albopictus is a potential target for vector control programs that depend on the release of large numbers of sterile or genetically modified individuals (Benedict, 2003; Labbé et al., 2010; Alphey et al., 2011; Iturbe-Ormaetxe et al., 2011; Oliva et al., 2012). Such programs would depend upon consistent, high volume, cost effective, and pathogen free blood sources (Benedict et al., 2009). Here, a simple protein formulation supported significantly higher egg production in Ae. albopictus females compared with whole human blood and at a much lower cost. This outcome provides a framework for the development of blood-free formulations that will be useful for large-scale rearing of mosquitoes and potentially other hematophagous insects. 2. Materials and methods 2.1. Mosquito rearing Ae. albopictus eggs were initially obtained through the Malaria Reseacrh and Reference Reagent Resource Center (ALBOPICTUS F4, MRA-804). Eggs were submerged in an aqueous oak leaf infusion and placed in a vacuum chamber for 30 min to deoxygenate and stimulate hatching. Larvae were allowed to develop in clean tap water and fed ground koi fish food ad libitum. Pupae were collected by hand with an eyedropper and placed into clean water. Adults were allowed to emerge in cube-shaped cages measuring 30 cm3 (Megaview Science Co, Ltd.) and had access to a 10% sucrose solution ad libitum, except for 1 h prior to feeding experiments when females were separated from males and sucrose was withheld. All stages were reared at a temperature of 26 °C and 75% relative humidity (RH) in an upright incubator with a 12:12 light:dark cycle. 2.2. Feeding treatments Starved females were fed using a membrane delivery system (Hemotek, Ltd.) using 2 ml of whole human blood or artificial meal formulation, which was pipetted into a holding disc, sealed with stretched parafilm, and heated to 37 °C prior to delivery. An odor blend consisting of ammonium hydroxide, lactic acid, isovaleric acid, geranyl acetone, and butylamine, each at [ 5logM] concentrations in aqueous buffer was applied to parafilm membranes as a host-seeking stimulant. Whole human blood, collected from individual male subjects (blood type O ) and stored in lithium heparin was purchased from a biological supply company (BioChemed
Services), kept refrigerated at 4 °C for no longer than 1 week and used only one time per experiment. Blood meal substitute consisted of bovine serum albumin (fraction V; Research Products International Corp.) [100 or 200 mg/mL] dissolved in a phosphate buffered saline solution (NaCl [137 mM], Na2HPO4 [10 mM], KH2HPO4 [1.76 mM], KCl [2.68 mM], pH7.2) by heating briefly at 42 °C. Adenosine triphosphate (Sigma–Aldrich Co., LLC), was serially diluted in BSA solution from a 100 mM stock solution just prior to membrane feed at final concentrations of [1 mM], [0.1 mM], [10 lM], [1 lM] and [0 lM]. After feeding, females were provided 10% sucrose and were kept at 26 °C/75% RH for 72 h. Gravid females were placed in small cups containing 100 mL of distilled water with a partially submerged filter paper liner as a substrate for oviposition. For blood meal volumes, groups of unfed or fully fed females were cold anesthetized and weighed together on an electronic balance to the nearest milligram. Estimated mean mass was derived using the formula: total weight/# females. 2.3. Statistical treatment of data Ovaries were hand dissected from gravid females 72 h after feeding and teased apart using fine forceps. Stage V oocytes or oviposited eggs were placed under a dissecting microscope and counted with the aid of a handheld counter. Arithmetic mean, median, and standard deviation were determined for egg counting data. The Kruskal–Wallis analysis of variance was used to test for significant differences in distribution among all 3 groups. When significant differences were identified, the Mann–Whitney U statistic was calculated as a measure of differences in pairwise distributions. Interquartile range (IQR) was calculated by subtracting the highest value in quartile 3 (Q3) from the lowest value in quartile 1 (Q1). Whiskers were graphed as the highest or lowest data points falling within 1.5 * IQR of Q3 and Q1, respectively. Data points with values higher or lower than whiskers were plotted individually as outliers. The chi-square test was applied to the adult sex ratio data to determine significant deviation from the expected 1:1. 3. Results and discussion 3.1. Feeding responses Feeding responses of Ae. albopictus and subsequent oogenesis were compared separately using either whole human blood or a buffered formulation containing bovine serum albumin (BSA) as a protein source supplemented with adenosine triphosphate (ATP). Blood-free formulations induced qualitatively similar feeding responses at BSA concentrations of 200 mg/ml and 100 mg/ ml (BSA 200 and BSA100, respectively; Fig. 1). ATP was added to these formulations because it is a known phagostimulant in Ae. aegypti, where labral sensilla are sensitive to ATP and other soluble compounds (Galun et al., 1984, 1985; Werner-Reiss et al., 1999). To address the question of whether ATP was necessary for Ae. albopictus feeding, a series of trials was conducted in which the final ATP concentration in the BSA 200 meal ranged from a low of 0 mM to a high of 1 mM. A clear requirement for ATP was observed as no females fed on the BSA formulation lacking ATP (Table 1), although vigorous probing of the membrane was observed. The proportion of females that had fed on BSA improved with successively higher concentrations of ATP, reaching a maximum of 69% at 1 mM ATP, which outpaced the proportion feeding on whole human blood (Table 1). These observations strongly suggests that ATP is a phagostimulant for Ae. albopictus and may imply that this trait is a general feature of aedines. Additionally, groups of females were weighed immediately after feeding as a way of estimating ingested
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R. Jason Pitts / Journal of Insect Physiology 64 (2014) 1–6
Fig. 1. Oogenesis in Ae. albopictus females fed with whole human blood or BSA meals. Left panels: females engorged with (A) human blood, (B) BSA [200 mg/mL], or (C) BSA [100 mg/mL]. Right panels: fully developed ovaries (stage V) dissected 72 h after feeding.
meal volumes. After feeding, individual females nearly doubled in mass to a calculated average of 3 mg as compared with unfed females (Table 1). Similar changes in mass were observed for blood fed or BSA fed females, suggesting that similar volumes of each meal were ingested, with the possible exception of the lowest concentration of ATP (Table 1). 3.2. Reproductive capacity Gonotrophic responses of Ae. albopictus females were characterized by quantification of egg production in gravid females. Initial studies quantified eggs that were oviposited as a measure of egg production. However, ovarian dissections of females that had recently oviposited revealed a high prevalence of partial, or skip oviposition, i.e. releasing only a portion of the fully developed eggs (Fig. 2; Supplemental Table 1), a phenomenon that has been observed previously for this species (Rozeboom et al., 1973; Trexler et al., 1998; Farjana and Tuno, 2013). This characteristic of gravid Ae. albopictus led to wide variations in observed egg counts, such that the actual total egg production per female would be obscured (Fig. 2). Consequently, total egg production was quantified as the sum of oviposited eggs plus stage V oocytes retained in the ovaries
Fig. 2. Skip oviposition in Ae. albopictus. y-Axis: stacked histograms showing number of eggs either oviposited (lighter shading) or retained in ovaries as stage V oocytes (darker shading) by individual females (x-axis) 72 h post-meal. (A) Whole human blood, (B) BSA [200 mg/ml], (C) BSA [100 mg/ml].
as observed upon ovarian dissection (Fig. 2). Females offered whole human blood or BSA at 2 different concentrations completed oogenesis within 72 h, with no discernable differences in timing. At least 5 trials of 5–10 females per trial were conducted to compare egg production of blood fed versus BSA fed individuals (Table 2). Egg counts for each trial were highly variable and the data were not normally distributed and had unequal variances, even when combined for all trials (Table 2; Supplemental Table 2). Various attempts to transform the data to allow parametric testing for differences in sample means were unsuccessful. Non-parametric analyses were therefore used to test for differences in egg production among groups. The results of a single trial are presented in Fig. 3A. In this example, females produced fewer eggs from BSA 100, but the ranked averages for blood and BSA 200 were not statistically different (Kruskal–Wallis H = 12.4, df = 2, p = 2.0 10 3; Fig. 3A). When data sets were combined and compared as a whole, all 3 groups differed significantly from one another (Kruskal–Wallis H = 47.4, df = 2, p = 5.1 10 11) and females produced the highest number
Table 1 Feeding Responses of Ae. albopictus females to BSA supplemented with ATP. BSA [200] + ATP
Proportion feeding Trials Mean weight mg n
Blood
1 mM
0.1 mM
10 lM
1 lM
None
75/127 (0.59) 8 3.17 63
63/91 (0.69) 5 2.93 56
46/103 (0.45) 7 3.22 41
43/99 (0.43) 6 2.91 35
12/89 (0.13) 4 2.67 12
0/99 (0.00) 6 1.70 41
Feeding frequency = # fully fed females/total # females. Numbers in parentheses = proportion of females taking a full meal. Trials = total number of independent replicates. Mean weight mg = average calculated mass per female in milligrams. n = Total number of fed females weighed.
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R. Jason Pitts / Journal of Insect Physiology 64 (2014) 1–6
Table 2 Aedes albopictus female egg production 72 h post feeding. Treatment
Mean St. Dev Median Range Trials n
Blood
BSA 200
BSA 100
83.7 19.8 84 18–124 7 67
92.2 33.6 96 7–162 6 58
57.0 23.6 59 5–97 5 55
Mean = average number of eggs produced per female. St. Dev = standard deviation. Median = median egg production. Trials = number of independent replicates. n = Total number of females in all trials.
of eggs after being fed BSA 200 (Table 2; Fig. 3B). This trend included a much higher maximum egg production for the BSA 200 fed females, where several individuals produced more than 130 eggs from a single meal (Table 2; Supplemental Table 2), a magnitude that has been reported previously for Ae. albopictus (Blackmore and Lord, 2000), but that was not achieved by any of the individuals that were fed whole blood or BSA 100 (Supplemental Table 2). Importantly, the mean numbers of eggs produced by blood fed (83.7) or BSA 200 fed (92.2) females in this study (Table 2) is very similar to published estimates of the average number of 50–60 ovarioles per ovary in Ae. albopictus when reared under optimal conditions (Yamany et al., 2012; Farjana and Tuno, 2013). Perhaps not coincidentally, the 200 mg/ml concentration of BSA is similar to the average protein content of whole human blood (Kogan, 1990), although the total isoleucine content of the BSA 200 meal in protein-bound form [80 lM] is higher than the plasma-free form reported in human blood plasma [40 lM] (Harrington et al., 2001). Thus the differences in egg production between whole human blood and BSA 200 observed in this study could potentially be accounted for by variable isoleucine content. If so, a higher concentration of BSA might be expected to elicit even greater egg production in Ae. albopictus females. A single trial was conducted with BSA at 400 mg/ml in which 16 fed females produced an average of 71.6 eggs (median = 81.5, range = 12– 109). Taken together, egg quantification data support the conclusion that near-maximum egg production was achieved in the BSA 200 feedings in this study. In addition it appears that most females
were indeed fully fed because blood meal volume has been positively correlated with total egg production in Ae. albopictus (Barnard and Xue, 2009). Future studies should be directed towards elucidating the effects artificial meals impose on total lifetime reproductive capacity by quantifying female longevity and egg production after multiple feedings. In the context of small scale laboratory rearing these issues may not be important as a single meal per generation is usually sufficient for production needs. However, mass rearing facilities may benefit from increased lifetime reproductive output if imparted by a non-blood formulation. 3.3. Survivorship of progeny Hatch rates and survivorship were determined for eggs oviposited by females that were fed with human blood or BSA 200 meals (Table 3). For the six trials reported here, hatch rates were highly variable, ranging from 27–86% in blood fed treatments and 25– 96% in BSA 200 fed treatments (Table 3). Although eggs from the BSA 200 trials hatched at a reduced rate overall, survivorship of hatched larvae during development was very high (>90%) in both treatments, with no apparent difference between BSA 200 or blood treatments (Table 3). Moreover, the numbers of adult males and females were approximately equal (Table 3) and the summed data were not significantly different than the expected 1:1 ratio in either treatment (blood fed: X2 = 1.575, df = 1, P = 0.2095; BSA fed: X2 = 3.429, df = 1, P = 0.0641). Numerous egg storage and hatching conditions were attempted, but the highest rates were obtained using eggs that were >2 weeks old, were free of mold, and were hatched using deoxygenated water (Hawley, 1988). Despite the overall challenge of hatching Ae. albopictus eggs, the results of several independent feeding experiments indicate that this species can be successfully reared without using animals or animal blood. During the course of this study, a colony of Ae. albopictus was successfully maintained for 6 consecutive generations using BSA 200 as the exclusive protein source for female oogenesis. 3.4. Health benefits and cost savings The use of an artificial meal and a membrane feeder system virtually eliminates the health hazards associated with handling human blood or animals (Askarian et al., 2011; Hill, 1999). Feeding mosquitoes using human volunteers or live animals, while practiced in many laboratories, is often unacceptable due to the potential infection of mosquitoes and exposure of individuals to
Fig. 3. Ae. albopictus egg production 72 h after feeding. y-Axes: total number of eggs (oviposited + stage V oocytes). (A) Graphical representation of a single trial: whole human blood (diamonds), BSA [200 mg/ml] (circles), or BSA [100 mg/ml] (triangles). Lower case letters indicate statistically different rank distributions as determined by and Mann–Whitney U tests (U = 32, p = 0.174 blood vs. BSA 200; U = 14, p = 0.007 blood vs. BSA 100; U = 9.5, p = 0.002 BSA 200 vs. BSA 100). (B) Box plot representation of compiled data. Boxes represent 1st and 3rd quartile intervals and whiskers represent ±1.5 * IQR. Horizontal bars represent median values. Lower case letters indicate statistically different rank distributions as determined by Mann–Whitney U tests (U = 1456, p = 0.016 blood vs. BSA 200; U = 727, p < 2 10 6 blood vs. BSA 100; U = 569.5, p < 2 10 6 BSA 200 vs. BSA 100).
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R. Jason Pitts / Journal of Insect Physiology 64 (2014) 1–6 Table 3 Aedes albopictus egg hatch rates and survivorship. # Larvaea
# Pupaeb
# Adultsc
# Males
# Females
Whole human blood 1 63 2 93 3 78 4 88 5 79 6 51 Sum 452
54 (0.86) 51 (0.55) 54 (0.69) 55 (0.63) 55 (0.70) 14 (0.27) 283 (0.63 ± 0.08)
54 (1.00) 50 (0.98) 51 (0.94) 52 (0.93) 43 (0.78) 13 (0.93) 263 (0.93 ± 0.03)
54 (1.00) 50 (0.98) 50 (0.93) 52 (0.93) 35 (0.64) 13 (0.93) 254 (0.90 ± 0.05)
32 26 19 19 15 6 117
22 24 31 33 20 7 137ns
BSA 200 1 2 3 4 5 6 Sum
49 (0.96) 26 (0.33) 44 (0.50) 21 (0.25) 22 (0.39) 23 (0.31) 185 (0.43 ± 0.10)
49 (1.00) 25 (0.96) 43 (0.98) 19 (0.90) 19 (0.86) 21 (0.91) 176 (0.95 ± 0.02)
46 (0.94) 24 (0.92) 43 (0.98) 19 (0.90) 19 (0.86) 17 (0.74) 168 (0.91 ± 0.03)
26 13 24 14 8 11 96
20 11 19 5 11 6 72ns
Female
# Eggs
51 79 88 83 56 74 431
# Eggs = number of eggs oviposited by an individual female. Columns 2–5 = numbers of living animals at each developmental stage (proportion). Columns 6 and 7 = numbers of eclosed adult males and females; ns – male:female ratio does not differ significantly from 1:1. Sum = total numbers in each column (proportion ± standard error of the mean). a Proportion of eggs hatched. b Proportion of larvae surviving to pupal stage. c Proportion of larvae surviving to adult stage.
pathogens or allergens (Samadi et al., 2013). Moreover, handling host animals can be hazardous in terms of potential bites, scratches, allergic reactions and needle punctures when injecting anesthetic agents (Gargiulo et al., 2012; National Research Council, 2011; Samadi et al., 2013). In addition to reduced exposure, another advantage of a blood meal substitute would be reduced costs associated with the use of biohazards, which include personal protective consumables, disposal fees, and personnel hours spent in training, handling and disposing of biohazards (Occupational Safety and Health Administration, 2011). Artificial meals can significantly lower the direct costs of mosquito rearing, reflected in reduced requirements for reagents, animal housing and related supplies. These savings would be most important to mass rearing facilities where the scale of blood feeding can be exceptionally large and frequent (Benedict et al., 2009). In this study, whole human blood was purchased for $35.00 per 10 ml, while BSA 200 was prepared for approximately $1.50 for the same volume, which is sufficient to feed >3000 Ae. albopictus females, where blood meal volumes range from 1 to 3ul per fully engorged female (Konishi and Yamanishi, 1984; Konishi, 1989; Barnard and Xue, 2009). In sum, the total costs of blood feeding with live animals will likely far exceed blood meal substitutes and specifically the BSA 200 formulation described here. In addition, the use of humans or animals in research labs generally requires approval from institutional review boards and/or animal care and use committees, necessitating extra administrative effort. The information in this study provides a starting point for the further development of blood-free formulations that will be useful for rearing Ae. albopictus. Given the numerous advantages that an artificial feeding system offers, serious consideration should be given to their use, particularly in the context of mass rearing for sterile or genetically modified mosquito release programs and, by extension, other hematophagous disease vectors. Acknowledgements Ae. albopictus eggs were obtained through the Malaria Reseacrh and Reference Reagent Resource Center (ALBOPICTUS F4, MRA804) as part of the Biodefense and Emerging Infections Research Resources Repository, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, deposited by Sandra Allan. The author acknowledges Dr. Laurence Zwiebel and the Dept. of Biological Sciences, Vanderbilt University, for the use of insectary space and equipment. The author also thanks David C. Rinker, Vanderbilt University, for critical reading of the manuscript and members of the Zwiebel Lab for helpful discussions. This work was supported by the International Atomic Energy Agency, Technical Contract No. 16985, to RJP. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2014. 02.012. References Alphey, N., Alphey, L., Bonsall, M.B., 2011. A model framework to estimate impact and cost of genetics-based sterile insect methods for dengue vector control. PLoS One 6, e25384. Askarian, M., Yadollahi, M., Kuochak, F., Danaei, M., Vakili, V., Momeni, M., 2011. Precautions for health care workers to avoid hepatitis B and C virus infection. Int. J. Occup. Environ. Med. 2, 191–198. Barnard, D., Xue, R.-D., 2009. Bloodmeal mass and oviparity mediate host avidity and DEET repellency in Aedes albopictus (Diptera: Culicidae). J. Med. Entomol. 46, 1235–1239. Benedict, M., 2003. The first releases of transgenic mosquitoes: an argument for the sterile insect technique. Trends Parasitol. 19, 349–355. Benedict, M.Q., Knols, B.G.J., Bossin, H.C., Howell, P.I., Mialhe, E., Caceres, C., Robinson, A.S., 2009. Colonisation and mass rearing: learning from others. Malar. J. 8 (Suppl 2), S4. Blackmore, M.S., Lord, C.C., 2000. The relationship between size and fecundity in Aedes albopictus. J. Vector Ecol. 25, 212–217. Briegel, H., 1985. Mosquito reproduction: incomplete utilization of the blood meal protein for oögenesis. J. Insect Physiol. 31, 15–21. Chang, Y., Judson, C., 1977. Role of isoleucine in differential egg production by mosquito Aedes aegypti Linnaeus (Diptera: Culicidae) following feeding on human or guinea pig blood. Comp. Biochem. Phys. A 57, 23–28. Chang, Y., Judson, C., 1979. Amino-acid composition of human and guinea-pig blood proteins, and ovarian proteins of the yellow-fever mosquito Aedes aegypti and their effects on the mosquito egg-production. Comp. Biochem. Phys. A 62, 753– 755. Clements, A., 1992. The Biology of Mosquitoes V1: Development, Nutrition and Reproduction. CABI Publishing, New York, NY. Farjana, T., Tuno, N., 2013. Multiple blood feeding and host-seeking behavior in Aedes aegypti and Aedes albopictus (Diptera: Culicidae). J. Med. Entomol. 50, 838–846.
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