DEVELOPMENT, LIFE HISTORY Genetic Variance and Genotype-by-Environment Interaction of Immune Response in Aedes aegypti (Diptera: Culicidae) MIGUEL MORENO-GARCIA,1,2 �� HUMBERTO LANZ-MENDOZA,2 AND ALEX CORDOBA-AGUILAR1�� Departamento de Ecolog��a �� Evolutiva, Instituto de Ecolog��a, �� Universidad Nacional Autonoma �� de Mexico, �� Circuito Exterior s/n, Apdo. Postal 70-275, Mexico, �� D. F. 04510, Mexico�� J. Med. Entomol. 47(2): 111��120 (2010) DOI: 10.1603/ME08267 ABSTRACT Immune response can be negatively affected by resource limitation, so it is expected that organisms evolve strategies to minimize the impact of this environmental outcome. Phenotypic plasticity in immune response could represent a genetic response to face such situations. We inves- tigated the effects of high and low quality and quantity of food at the larval stage on two important immune components, phenoloxidase activity (PO) and nitric oxide production (NO) measured in adults of the Dengue vector, Aedesaegypti. We reared families to determine the magnitude and pattern of expression of genetic variance, environmental variance and genotype-by-environment interaction (GEI). In addition, we quanti��ed whether there were differences in plastic immune responses in both sexes. Our results indicated additive variance for PO and NO, but rearing environment did not produce differences among individuals. For NO and PO in males, there were large differences among families in plasticity, as indicated by the different slopes produced by each reaction norm. Therefore, there is additive genetic variation in plasticity for NO production and PO activity. One possible interpre- tation of these results is that different genotypes may be favored to ��ght pathogens under the different food quality situations. Males and females showed similar overall GEI strategies but there were differences in PO and NO. Males showed a phenotypic correlation between PO and NO, but we did not ��nd genetic correlations between immune parameters in both sexes. KEY WORDS immune response, quantitative genetics, phenotypic plasticity, Aedes aegypti Immune response is a trait closely linked to survival and reproduction (Schmid-Hempel 2003, Schulen- berg et al. 2009). Despite the fact that investment to immunity is adaptive, immune response is strongly impacted by environmental conditions. This occurs in situations of environmental heterogeneity (such as variation in food abundance), in which an overall ��tness decrement for a given genotype is observed (e.g., Leclaire and Brandl 1994, Fellowes et al. 1998, Metcalf and Monagham 2001, Siva-Jothy and Thomp- son 2002). In mosquitoes, for example, larvae that have been reared in crowded or undernourished condi- tions, give rise to adults with a weak cellular encap- sulation (Suwanchaichinda and Paskewitz 1998). On the same line of research, it has been also found that when mosquito adults are stressed with food shortage, encapsulation immune response decreases (Chun et al. 1995, Schwartz and Koella 2002). Therefore, the organisms are expected to deal with the problem of maximizing ��tness in changing or stressful environ- ments. A genetic background that expresses changes in phenotype expression could have a selective advan- tage in a given environment (Zhivotovsky et al. 1996). In some cases, a single genotype can have the ability to produce distinct phenotypes when exposed to dif- ferent environments, a phenomenon known as phe- notypic plasticity (Roff 1997, Nylin and Gotthard 1998, Schlichting and Pigliucci, 1998). Phenotypic plasticity is shown by many traits and immune response is not an exception (Fordyce 2006). In fact, such immune plasticity could be used as an indicator of the strategies that an organism has followed when dealing with en- vironmental heterogeneity. Paradoxically, given the recent explosion in ecological and evolutionary stud- ies of immunity, only a handful of studies have ap- proached immune response using a phenotypic plas- ticity perspective (but see Barnes and Siva-Jothy 2000, Mucklow and Ebert 2003, Cotter et al. 2004a, Lazzaro et al. 2008, McKean et al. 2008). To have a better understanding of the evolution of immune responses, more studies of phenotypic plasticity are therefore needed. To afford the problem of whether plastic immune responses could have an impact in population evolu- 1 Departamento de Ecolog����a Evolutiva, Instituto de Ecolog����a, Univer- sidad Nacional Autonoma �� de Mexico, �� Circuito Exterior s/n, Apdo. Postal 70-275, Mexico, �� D. F. 04510, Mexico �� (e-mail: miguelmoga2000@ yahoo.com.mx). 2 Centro de Investigaciones Sobre Enfermedades Infecciosas, In- stituto Nacional de Salud Publica, �� Avda. Universidad 655. Col. Sta. Mar����a Ahuacatitlan, �� 62100 Cuernavaca, Morelos, Mexico. �� 0022-2585/10/0111��0120$04.00/0 2010 Entomological Society of America

tion, it is necessary to evaluate if different environ- ments produce different phenotypes and if genotypes respond differently to these environments (genotype- by-environment interaction GEI, which represents genetic variation for phenotypic plasticity). It has been proposed that GEI can allow populations to evolve to an optimum phenotypic mean in different environments, promoting adaptation to heteroge- neous environments (Via and Lande 1985). As a result, the amount of genetic variance, as assessed by the slope of reaction norms (i.e., a graphical description of the GEI), may be a strong determinant of the popu- lation ��tness (Fry 1996). Notwithstanding, a lack of variation may lead to a failure to respond adaptively to environmental changes. There are theoretical advan- tages of using this approach. For example, phenotypic plasticity could explain the maintenance of genetic variance recently found in different immune compo- nents (e.g., see Ryder and Siva-Jothy 2001, Simmons and Roberts 2005, Cotter et al. 2004b, Rolff et al. 2005, Schwarzenbach et al. 2005, Fellowes et al. 1998). The fact that plasticity uncouples the phenotype from ge- notype and thus releases the gene pool from the im- mediate impact of natural or sexual selection (Stearns 1992), will lead to slow depletion of genetic variance. However, some reaction norms will not produce the optimal mean trait value in a given environment (Schlichting and Pigliucci 1998, Roff 1997), slowing down the rate of adaptation for immediate genera- tions. The conventional approach to see how a trait re- sponds to selective pressures is to analyze the trait in question using quantitative genetics by partitioning and estimating variances and covariances into causal components (Falconer and Mackay 1996). In this study we have explored the environmental heteroge- neity affecting immune components in the mosquito Aedes aegypti Linnaeus (Diptera: Culicidae), the prin- cipal vector of Dengue and Yellow Fever virus. We varied levels of food quality and quantity in the larval stage, and as response variables two immune markers were measured in the adult: the basal levels of phe- noloxidase (PO) activity and nitric oxide (NO) pro- duction. PO is an oxidoreductase enzyme used in in- sect cellular and humoral response such as cuticle melanization, wound repair, cytotoxin production, and melanotic encapsulation (Soderhall �� �� and Cerenius 1998). NO is a highly reactive and unstable free radical gas that inhibits protein catalytic activity and produces protein and harming effects on pathogens�� DNA (Riv- ero 2006). Recent studies have shown that PO activity is costly to produce (reviewed by Kanost and Gorman 2008). Although there is no direct evidence for such costs for NO, their physiological pathways strongly indicate that it is potentially costly (Rivero 2006, Car- ton et al. 2008). We evaluated genetic variation, phe- notypic plasticity and tested for GEIs of PO and NO using a split family design. We predicted the existence of genetic differences and environmentally sensitive production of alternative phenotypes by given geno- types on these immune markers and a negative effect because of limitation in food quality: mosquito adults whose larval stage had poor quality food would show decreased basal PO activity and NO production while the opposite would be found for mosquito adults whose larval stage had access to high quality food. Reproductive strategies could promote differences in the pattern of investment to immune defense by each sex. Males are expected to increase their ��tness by reducing their investment to immune defense while investing resources to reproductive effort (Zuk and McKean 1996, Sadd et al. 2006). Meanwhile, in females natural selection would favor an increase in resource investment to immunity, under the assump- tion that increased longevity could enhance ��tness via egg production (Rolff 2002). How this presumable sexual dimorphism translates into plastic response dif- ferences in both sexes has been little explored. Mc- Kean and Nunney (2005) found sex speci��c plastic responses in relation to the availability of limiting resources. Given this background information, we thus evaluated if males and females express different strategies in their plastic responses in PO activity, NO production, and GEIs strategies. Materials and Methods Mosquitoes were obtained from an insectary at the Instituto Nacional de Salud Publica, �� Cuernavaca, Mex- ico. The stock population had been held in this place for at least 130 generations. The colony was main- tained with a protocol that minimizes inbreeding: fairly high number of individuals per generation (over 2,000 individuals per generation) and random mating. During the study, the colony was kept on a 12L:12D cycle at 25��28 C. Experimental Design. For PO activity and NO pro- duction, we had 44 families by randomly choosing 44 mated and blood fed females. We then split their F1 hatched larvae into one of two rearing environments that differed in food quality and quantity: the high quality food (HQF) environment was based on rat chow, yeast extract and lactoalbumin hydrolisate (1: 1:1 mix 25 g/200 ml), while the low quality food (LQF) environment was based on rat chow only (25 g/200 ml). Preliminary observations have shown these dietary requirements are effective enough to change the individual condition, as re��ected by adult size (S. Hernandez-Martinez personal communication), life- span, and egg clutch and size (see Nasci 1986, Packer and Corbet 1989). Larvae were fed according to the schedule shown in Table 1. Each family of larvae was reared in plastic glasses containing 100 ml of water. Adult females and males of 3 d postemergence from these larvae were used to obtain PO and NO readings. To minimize degradation of molecules, individuals were collected and frozen at 70 C until they were processed. Given the ��nal large number of individuals and with the aim to avoid sample degradation, mac- eration, supernatant collection and readings were only done for the exact number of tests supported in the microwell plate (80 wells for test samples plus 16 for standard reference curves, see below) that can be processed and quanti��ed in a single day. A group of 112 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 2