- Open Access
Effects of inbreeding and genetic modification on Aedes aegypti larval competition and adult energy reserves
© Koenraadt et al; licensee BioMed Central Ltd. 2010
- Received: 19 July 2010
- Accepted: 6 October 2010
- Published: 6 October 2010
Genetic modification of mosquitoes offers a promising strategy for the prevention and control of mosquito-borne diseases. For such a strategy to be effective, it is critically important that engineered strains are competitive enough to serve their intended function in population replacement or reduction of wild mosquitoes in nature. Thus far, fitness evaluations of genetically modified strains have not addressed the effects of competition among the aquatic stages and its consequences for adult fitness. We therefore tested the competitive success of combinations of wild, inbred and transgenic (created in the inbred background) immature stages of the dengue vector Aedes aegypti in the presence of optimal and sub-optimal larval diets.
The wild strain of Ae. aegypti demonstrated greater performance (based on a composite index of survival, development rate and size) than the inbred strain, which in turn demonstrated greater performance than the genetically modified strain. Moreover, increasing competition through lowering the amount of diet available per larva affected fitness disproportionately: transgenic larvae had a reduced index of performance (95-119%) compared to inbred (50-88%) and wild type larvae (38-54%). In terms of teneral energy reserves (glycogen, lipid and sugar), adult wild type mosquitoes had more reserves directly available for flight, dispersal and basic metabolic functions than transgenic and inbred mosquitoes.
Our study provides a detailed assessment of inter- and intra-strain competition across aquatic stages of wild type, inbred, and transgenic mosquitoes and the impact of these conditions on adult energy reserves. Although it is not clear what competitive level is adequate for success of transgenic strains in nature, strong gene drive mechanisms are likely to be necessary in order to overcome competitive disadvantages in the larval stage that carryover to affect adult fitness.
- Genetically Modify
- Wild Strain
- Diet Level
- Transgenic Strain
- High Diet
The incidence of arthropod-borne diseases is increasing globally [1, 2]. Control of diseases such as malaria and dengue is complicated by the lack of effective vaccines  and new vector control strategies. Genetic modification of arthropods offers a promising strategy for the prevention and control of the diseases they transmit [4–7]. Currently, efforts are underway to develop and evaluate the potential of genetically sterile and disease-refractory Anopheles gambiae Giles and Aedes aegypti L. mosquitoes [8–10], vectors of malaria and dengue fever, respectively. The goal of this endeavor is to release genetically modified (GM) mosquitoes to either reduce population densities, or replace the wild population with a disease-refractory one [11, 12].
Of critical importance is the ability of released GM mosquitoes to survive, mate and pass on desirable genetic traits . GM mosquitoes will have greater success if genetic modification imparts low fitness costs . Fitness is a complex parameter that is impacted by survival and development time of immatures, mating success, adult survival, age of first reproduction and lifetime reproduction [15, 16]. Deleterious effects of transgenesis on mosquito fitness may be the result of insertional mutagenesis and/or added burden of the transgene product . Often transgenic insects are inbred to develop a strain that is homozygous for the insertion, further decreasing fitness. Previous studies have not examined the effects of transgene insertion versus the effects of mass-rearing which may lead to the fixation of recessive, fitness-reducing mutations.
An unresolved issue is how competition among the immature stages of wild and GM mosquitoes may affect population reduction or replacement. Irvin et al.  reported on larval development rate for transgenic versus a wild type laboratory strain of Ae. aegypti, but not at varying (sub-optimal) nutrition levels or with mixtures of transgenic and wild-type mosquitoes, as would eventually occur in nature. Furthermore, other studies have not considered the effects of larval competition and only provided insight into adult survival and fecundity [18, 19]. Another drawback of previous studies is that they compared GM strains with highly inbred wild-type strains (the 'outcross' strain).
Therefore, we evaluated the impact of inter- and intra-strain competition on the performance of three strains of Ae. aegypti: (1) a wild-type (second generation) strain collected from Mexico (referred to as 'Wild'), (2) Higgs' white eye (HWE), an inbred, white eye mutant strain (referred to as 'Inbred') and (3) a transgenic strain with a green fluorescent protein (GFP) insert (referred to as 'Transgenic'). The latter strain was created using the HWE genetic background. Because breeding sites of Ae. aegypti, such as water storage containers and tires, are often food-limited which may lead to density-dependent competition [20–22], we tested how the outcome of competition is altered under optimal and sub-optimal amounts of food. Finally, we investigated differences between wild, inbred and transgenic strains in terms of their energetic reserves upon emergence. These reserves are critical for key behaviors in adult life such as flight, dispersal and mating .
Statistical model results of survival
Strain * Competitor
Statistical model results of development time
Statistical model results of pupal size
Strain * Competitor
Our study demonstrated lower survivorship and increased development time of the inbred and transgenic strain of Ae. aegypti larvae when compared to their wild counterpart. Clearly, this outcome of competition was mediated by the relative competitive strength of each strain as well as the amount of food available. First, the results of increased development time of Inbred and Transgenic larvae were most pronounced at high diet conditions for both males and females. Second, when competition for food was highest (i.e., at the low diet level), the wild-type strain was a superior competitor in terms of survival over the inbred strain, which in turn was superior over the transgenic strain (Figure 1-B). Because nutritional resources in the aquatic environment of Ae. aegypti are limited, such effects of competition for nutrients are likely to occur in nature as well [20–22].
Our experimental set-up with microcosms was based on earlier work that investigated the effects of density-dependent competition on the life-history of Ae. aegypti . In the field, a substantial diversity in container habitats can be found. These range from large, rain-filled water storage containers (~500 L) to very small containers such as ant-traps filled with tap water. The minimalist approach uses small numbers in small volumes , but still with comparable densities as encountered in the field . This experimental design allowed us to track larvae throughout their development and control for the amount of food available per larva.
It is possible that some of the observed effects may offset each other in nature. For example, lower survivorship could result in less frequent interactions with remaining larvae, which in turn could lead to larger, more fecund adults, and eventually result in no net effect on relative fitness. Using the index of performance that incorporates survival, development time and size-related fecundity, we found that the Wild strain had an overall competitive advantage in the larval stage. The Inbred strain was more fit than the Transgenic strain which in turn was less fit than the Wild strain, suggesting that genetic modification imparted a fitness cost on top of inbreeding costs.
The results on energetic reserves of the three strains suggest that inbreeding and genetic modification also affect the mosquito's metabolism. Such differences could be the result of poorer resource acquisition rate during the larval stage (e.g. amount of time spent feeding or efficiency of filter feeding). In general, glycogen reserves for the Wild strain were higher than for the Inbred and/or Transgenic strains. Lipid reserves showed a reverse trend for males, but not significantly so, whereas for females lipids showed a similar trend as glycogen levels. This suggests sex-specific variation in energetic budget as demonstrated for the malaria vector Anopheles gambiae . Because glycogen is an important stored energy source for mosquito flight and dispersal that can be rapidly utilized after eclosion, these differences could have a serious impact on mating and eventual reproductive success [23, 27].
Although the inbred and transgenic strains had a different origin than the wild strain (Puerto Rico versus Mexico), and intrinsic genetic differences could not be ruled out completely, our results on reduced competitive ability and altered metabolism are most likely the consequence of detrimental effects of both inbreeding and genetic modification. Ideally, transformation should be conducted on genetically diverse laboratory strains which could mitigate the impact of modification and inbreeding .
Although it is not clear just how competitive modified mosquitoes need to be compared with wild type mosquitoes in order to replace vector populations, the fitness effects observed in our study are likely to be relevant. First, the strong dependence on food level suggests that the outcome of competition is not fixed and thus fitness evaluations should not be performed only under 'ideal' laboratory conditions [17–19]. Next, our results support earlier statements that incorporation of strong gene drive mechanisms in the transgenic construct will be important . If they are not, a competitive disadvantage in the larval stage may lead eventually to the exclusion of the 'weaker' GM strain in nature  ultimately compromising control efforts. For a population reduction strategy based on RIDL or SIT, reducing the potential for density-dependent competition may negate the effectiveness of a mosquito control program, because less competition may result in increased wild type larval survival and potentially more fecund adult mosquitoes. This effect could thereby offset the initial positive effects of a reduced population , although the importance of density-dependent competition under field conditions remains unclear . Design of late-acting dominant lethal genetic systems may be promising in this regard as they carry a GM larval population through the density-dependent phase .
Future studies on competition and fitness of transgenic mosquitoes should address how the competitive ability of various transgenic strains is affected when exposed to different scenarios of density-dependent (e.g., container size and food availability) and density-independent factors (e.g., temperature). These studies should address both the fitness cost of inbreeding as well as genetic modification. In line with our results, other studies have shown that expression of genetic background strongly depends on environmental conditions (gene by environment interactions) [33, 34]. Similarly, there is an urgent need to evaluate transgenic mosquito lines under more realistic field conditions [13, 14] and across life stages.
Competition experiments were carried out with combinations of three strains of Ae. aegypti: (1) wild-type (Wild); larvae were second generation offspring of field collected pupae from Tapachula, Mexico (14° 54'N, 92° 15'W); (2) Higgs' white eye (HWE), an eye-pigment deficient variant of the Puerto Rican Rexville D strain  as the result of a spontaneous mutation (S. Higgs, pers. comm.; ) and (3) enhanced green fluorescent protein (EGFP), a HWE strain in which the GFP gene has been inserted through germ-line transformation using the piggyBac transposable element (A.A. James, pers. comm.; ). All strains were maintained in separate environmental chambers set at a temperature and humidity comparable to the origin of the Wild strain (Tapachula, Mexico): 27°C, 80% RH and a photoperiod of 12:12 L:D. The Inbred (HWE) and Transgenic (GFP) strains were kept at 28°C and 80% RH prior to shipment to our laboratory facilities. Experiments were executed in another environmental chamber with the same temperature, humidity and light settings. Standard rearing conditions for all strains used 200 larvae per tray (27 × 20 × 8 cm) filled with 1 L of tap water. We added 30, 60, 90 and 90 mg of food to the rearing trays on days 0, 1, 3 and 5, respectively (1.65 mg/larva). Food consisted of a 1:1 ratio of lactalbumin: brewers yeast mixture.
We followed the approach of Agnew et al.  to study the effects of larval competition in Ae. aegypti. Wells of 12-well cell culture plates (2.2 cm diameter, Corning Incorporated Life Sciences, Lowell, MA) were filled with five ml tap water. Water was added daily to account for evaporation. Newly hatched first-instar larvae (~ 4 h old) were introduced in the following six combinations at high and low diet regimens: (1) 4 Wild larvae, (2) 4 Transgenic larvae, (3) 4 Inbred larvae, (4) 2 Wild plus 2 Transgenic larvae, (5) 2 Wild plus 2 Inbred larvae and (6) 2 Transgenic plus 2 Inbred larvae. We added 0.15, 0.3, 0.6 and 0.6 mg of food per larva on days 0, 1, 3 and 5, respectively, for the high diet regimen (total 1.65 mg/larva). This diet amount was found to be optimal for development of large body size mosquitoes in previous experiments in our laboratory. The low diet consisted of half the amount of the high diet regimen (total 0.825 mg/larva). Mortality was recorded daily and, over the course of the study, the amount of food added to each well was adjusted to the number of larvae remaining. Thirty replicates per treatment were carried out.
Time to pupation was recorded, as well as sex of pupae . Size of pupae was measured as an indicator of adult body size as described by Koenraadt . Briefly, after moving the pupa to its lateral side, cephalothorax length was measured as the distance between the anterior point of the median keel and the ventral tip of the pupal wing sheath . The relationship could be expressed as y = 1.110x + 0.014 and y = 0.974x + 0.119 for females and males, respectively, whereby x = cephalothorax length (mm) and y = adult wing length (mm). GFP expression in the eyes of pupae was visualized using a Stemi 2000-C stereomicroscope (Carl Zeiss MicroImaging Inc., Thornwood, NY) equipped with an Endura Bright Royal Blue (450 nm) LED-light (Opto Technology, Inc., Wheeling, IL) and a yellow barrier filter (Edmund Optics Inc., Barrington, NJ). The experiments were terminated when all larvae had died or pupated.
To test for differences in nutritional reserves of teneral adults emerging from the single species treatment, we determined glycogen, lipid and sugar content using previously published protocols [40–42]. Body size dependent variation in nutrient quantities were controlled for by expressing nutritional reserves per mm pupal height.
Our analyses focused on detecting differences in survival to the pupal stage, development time and pupal size that were inherent to the strain (strain effect), how each strain affected survival, development and size of the other larvae in the same wells (competitor effect), and how these effects changed at optimal and sub-optimal diet levels. Random plate and well effects were not found to be significant in our initial model development; consequently, they were removed from further analysis. Survival data were analyzed with the binomial logistic regression procedure (JMP 7.0, Cary, NC, USA). Analyses of development time and pupal size were performed with standard least squares models (JMP 7.0, Cary, NC, USA). Significant main effects in all models without significant interactions were indicative of a 'ranking' in strain and competitor effects. In that case, post-hoc contrasts were specified to test the hypothesis that the Wild strain was 'stronger' than the Inbred strain (e.g., higher survivorship or larger size), that the Inbred strain was stronger than the Transgenic strain and that the Wild strain was stronger than the Transgenic strain. Similarly, we tested the hypothesis that the Wild or Inbred strain as a competitor had a greater negative impact on survival, development time or pupal size than the Transgenic strain. Significant interactions between strain and competitor were indicative of intra-strain versus inter-strain differences, i.e. survival, development time and pupal size for larvae reared with their own strain were different than when reared with any of the two other strains. This hypothesis was analyzed by specifying post-hoc contrasts, and significance was evaluated by using Tukey-HSD tests or by correcting significance levels using the Bonferroni correction.
N 0 represents the initial number of females (assumed to be 50% of the starting number in our experiments); A x is the number of adult females produced at time x of the experiment; represents the size of the emerging female and is a direct proxy for fecundity. For the present study we used female pupal size because of its strong correlation with female adult size . Indices were calculated for each cohort, whereby cohort was defined as all females from one treatment combination. After calculating the indices, we set the value of the Wild-Wild combination at 1 as the reference group. We did this separately for the high diet and the low diet experiment. All other calculated performance index values were then adjusted so that values >1 would indicate better performance and values <1 would indicate poorer performance than the Wild larvae in the presence of larvae of their own strain.
Finally, strain differences in glycogen, lipid and sugar content per unit body size were evaluated for the adults that emerged from the single species experiments. Standard least square regression procedures were used for this purpose. Post-hoc tests were based on least square differences (LSD).
This work was funded by a grant from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative.
We thank Dr. Janine Ramsey and Guillermo Bond (Centro Regional de Investigaciones en Salud Publica, Tapachula, Mexico) for collecting pupae for the low generation Wild colony. James Booth is acknowledged for statistical advice. We thank Fred Gould, Erik Poelman and Michelle Helinski for their comments on an earlier draft of this manuscript.
- Harrus S, Baneth G: Drivers for the emergence and re-emergence of vector-borne protozoal and bacterial diseases. Int J Parasitol. 2005, 35: 1309-1318. 10.1016/j.ijpara.2005.06.005.View ArticlePubMedGoogle Scholar
- Gratz NG: Emerging and resurging vector-borne diseases. Annu Rev Entomol. 1999, 44: 51-75. 10.1146/annurev.ento.44.1.51.View ArticlePubMedGoogle Scholar
- Monath TP: Dengue and yellow fever - Challenges for the developments and use of vaccines. New Engl J Med. 2007, 357: 2222-2225. 10.1056/NEJMp0707161.View ArticlePubMedGoogle Scholar
- Allen ML, Scholl PJ: Quality of transgenic laboratory strains of Cochliomyia hominivorax (Diptera: Calliphoridae). Journal of Economic Entomology. 2005, 98: 2301-2306. 10.1603/0022-0493-98.6.2301.View ArticlePubMedGoogle Scholar
- Atkinson PW, Michel K: What's buzzing? Mosquito genomics and transgenic mosquitoes. Genesis - The Journal of Genetics and Development. 2002, 32: 42-48.Google Scholar
- Beard CB, Dotson EM, Pennington PM, Eichler S, Cordon-Rosales C, Durvasula RV: Bacterial symbiosis and paratransgenic control of vector-borne Chagas disease. Int J Parasitol. 2001, 31: 621-627. 10.1016/S0020-7519(01)00165-5.View ArticlePubMedGoogle Scholar
- Scott MJ, Heinrich JC, Li XL: Progress towards the development of a transgenic strain of the Australian sheep blowfly (Lucilia cuprina) suitable for a male-only sterile release program. Insect Biochem Mol Biol. 2004, 34: 185-192. 10.1016/j.ibmb.2003.11.001.View ArticlePubMedGoogle Scholar
- Franz AWE, Sanchez-Vargas I, Adelman ZN, Blair CD, Beaty BJ, James AA, Olson KE: Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proc Natl Acad Sci. 2006, 103: 4198-4203. 10.1073/pnas.0600479103.PubMed CentralView ArticlePubMedGoogle Scholar
- Riehle MA, Srinivasan P, Moreira CK, Jacobs-Lorena M: Towards genetic manipulation of wild mosquito populations to combat malaria: advances and challenges. Journal of Experimental Biology. 2003, 206: 3809-3816. 10.1242/jeb.00609.View ArticlePubMedGoogle Scholar
- Fu GL, Lees RS, Nimmo D, Aw D, Jin L, Gray P, Berendonk TU, White-Cooper H, Scaife S, Phuc HK: Female-specific flightless phenotype for mosquito control. Proc Natl Acad Sci. 2010, 107: 4550-4554. 10.1073/pnas.1000251107.PubMed CentralView ArticlePubMedGoogle Scholar
- Benedict MQ, Robinson AS: The first releases of transgenic mosquitoes: an argument for the sterile insect technique. Trends Parasitol. 2003, 19: 349-355. 10.1016/S1471-4922(03)00144-2.View ArticlePubMedGoogle Scholar
- Gould F, Schliekelman P: Population genetics of autocidal control and strain replacement. Annu Rev Entomol. 2004, 49: 193-217. 10.1146/annurev.ento.49.061802.123344.View ArticlePubMedGoogle Scholar
- Scott TW, Takken W, Knols BGJ, Boete C: The ecology of genetically modified mosquitoes. Science. 2002, 298: 117-119. 10.1126/science.298.5591.117.View ArticlePubMedGoogle Scholar
- Marrelli MT, Moreira CK, Kelly D, Alphey L, Jacobs-Lorena M: Mosquito transgenesis: what is the fitness cost?. Trends Parasitol. 2006, 22: 197-202. 10.1016/j.pt.2006.03.004.View ArticlePubMedGoogle Scholar
- Scott TW, Rasgon JL, Black WC, Gould F: Fitness studies: developing a consensus methodology. Bridging laboratory and field research for genetic control of disease vectors. Edited by: Knols BGJ, Louis C. 2006, Dordrecht, The Netherlands: Kluwer Academic Publishers, 11: 210-Bogers RJ (Series Editor)Google Scholar
- Begon M, Harper JL, Townsend CR: Ecology - Individuals, populations and communities. 1990, Boston, Oxford, London, Edinburgh, Melbourne: Blackwell Scientific Publications, 2Google Scholar
- Irvin N, Hoddle MS, O'Brochta DA, Carey B, Atkinson PW: Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes. Proc Natl Acad Sci. 2004, 101: 891-896. 10.1073/pnas.0305511101.PubMed CentralView ArticlePubMedGoogle Scholar
- Catteruccia F, Godfray HC, Crisanti A: Impact of genetic manipulation on the fitness of Anopheles stephensi mosquitoes. Science. 2003, 299: 1225-1227. 10.1126/science.1081453.View ArticlePubMedGoogle Scholar
- Moreira LA, Wang J, Collins FH, Jacobs-Lorena M: Fitness of anopheline mosquitoes expressing transgenes that inhibit Plasmodium development. Genetics. 2004, 166: 1337-1341. 10.1534/genetics.166.3.1337.PubMed CentralView ArticlePubMedGoogle Scholar
- Dye C: Competition amongst larval Aedes aegypti: the role of interference. Ecol Entomol. 1984, 9: 355-357. 10.1111/j.1365-2311.1984.tb00859.x.View ArticleGoogle Scholar
- Gilpin ME, McClelland GAH: Systems analysis of the yellow fever mosquito Aedes aegypti. Fortschritte der Zoologie. 1979, 25: 355-388.PubMedGoogle Scholar
- Southwood TR, Murdie G, Yasuno M, Tonn RJ, Reader PM: Studies on the life budget of Aedes aegypti in Wat Samphaya, Bangkok, Thailand. Bull World Health Org. 1972, 46: 211-226.PubMed CentralPubMedGoogle Scholar
- Briegel H, Knusel I, Timmermann SE: Aedes aegypti: size, reserves, survival, and flight potential. J Vector Ecol. 2001, 26: 21-31.PubMedGoogle Scholar
- Agnew P, Hide M, Sidobre C, Michalakis Y: A minimalist approach to the effects of density-dependent competition on insect life-history traits. Ecol Entomol. 2002, 27: 396-402. 10.1046/j.1365-2311.2002.00430.x.View ArticleGoogle Scholar
- Koenraadt CJM, Jones JW, Sithiprasasna R, Scott TW: Standardizing container classification for immature Aedes aegypti surveillance in Kamphaeng Phet, Thailand. J Med Entomol. 2007, 44: 938-944. 10.1603/0022-2585(2007)44[938:SCCFIA]2.0.CO;2.View ArticlePubMedGoogle Scholar
- Huho BJ, Ng'habi KR, Killeen GF, Nkwengulila G, Knols BGJ, Ferguson HM: Nature beats nurture: a case study of the physiological fitness of free-living and laboratory-reared male Anopheles gambiae s. l. J Exp Biol. 2007, 210: 2939-2947. 10.1242/jeb.005033.View ArticlePubMedGoogle Scholar
- Yuval B, Hollidayhanson ML, Washino RK: Energy budget of swarming male mosquitos. Ecol Entomol. 1994, 19: 74-78. 10.1111/j.1365-2311.1994.tb00392.x.View ArticleGoogle Scholar
- James AA: Gene drive systems in mosquitoes: rules of the road. Trends Parasitol. 2005, 21: 64-67. 10.1016/j.pt.2004.11.004.View ArticlePubMedGoogle Scholar
- Grover JP: Resource competition. 1997, London: Chapman & Hall, 1View ArticleGoogle Scholar
- Atkinson MP, Su Z, Alphey N, Alphey LS, Coleman PG, Wein LM: Analyzing the control of mosquito-borne diseases by a dominant lethal genetic system. Proc Natl Acad Sci. 2007, 104: 9540-9545. 10.1073/pnas.0610685104.PubMed CentralView ArticlePubMedGoogle Scholar
- Legros M, Lloyd AL, Huang Y, Gould F: Density-dependent intraspecific competition in the larval stage of Aedes aegypti (Diptera: Culicidae): revisiting the current paradigm. J Med Entomol. 2009, 46: 409-419. 10.1603/033.046.0301.PubMed CentralView ArticlePubMedGoogle Scholar
- Phuc HK, Andreasen MH, Burton RS, Vass C, Epton MJ, Pape G, Fu GL, Condon KC, Scaife S, Donnelly CA: Late-acting dominant lethal genetic systems and mosquito control. BMC Biology. 2007, 5: 11-10.1186/1741-7007-5-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Devlin RH, D'Andrade M, Uh M, Biagi CA: Population effects of growth hormone transgenic coho salmon depend on food availability and genotype by environment interactions. Proc Natl Acad Sci. 2004, 101: 9303-9308. 10.1073/pnas.0400023101.PubMed CentralView ArticlePubMedGoogle Scholar
- Sundström LF, Lõhmus M, Tymchuk WE, Devlin RH: Gene-environment interactions influence ecological consequences of transgenic animals. Proc Natl Acad Sci. 2007, 104: 3889-3894. 10.1073/pnas.0608767104.PubMed CentralView ArticlePubMedGoogle Scholar
- Miller BR, Mitchell CJ: Genetic selection of a flavivirus-refractory strain of the Yellow Fever mosquito Aedes aegypti. Am J Trop Med Hyg. 1991, 45: 399-407.PubMedGoogle Scholar
- Adelman ZN, Jasinskiene N, Vally KJM, Peek C, Travanty EA, Olson KE, Brown SE, Stephens JL, Knudson DL, Coates CJ, James AA: Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti. Transgenic Research. 2004, 13: 411-425. 10.1007/s11248-004-6067-2.View ArticlePubMedGoogle Scholar
- Christophers SK: Aedes aegypti (L.) - the yellow fever mosquito. 1960, London: Cambridge University PressGoogle Scholar
- Koenraadt CJM: Pupal dimensions as predictors of adult size in fitness studies of Aedes aegypti (Diptera : Culicidae). J Med Entomol. 2008, 45: 331-336. 10.1603/0022-2585(2008)45[331:PDAPOA]2.0.CO;2.View ArticlePubMedGoogle Scholar
- Knight KL: A mosquito taxonomic glossary VII. The pupa. Mosquito Systematics Newsletter. 1971, 3: 42-65.Google Scholar
- Van Handel E: Rapid determination of glycogen and sugars in mosquitos. J Am Mosq Control Assoc. 1985, 1: 299-301.PubMedGoogle Scholar
- Van Handel E: Rapid determination of total lipids in mosquitos. J Am Mosq Control Assoc. 1985, 1: 302-304.PubMedGoogle Scholar
- Harrington LC, Edman JD, Scott TW: Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood?. J Med Entomol. 2001, 38: 411-422. 10.1603/0022-2585-38.3.411.View ArticlePubMedGoogle Scholar
- Livdahl TP, Sugihara G: Non-linear interactions of populations and the importance of estimating per-capita rates of change. Journal of Animal Ecology. 1984, 53: 573-580. 10.2307/4535.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.