Estimating Reaction Norms for Predictive Population Parameters, Age Specific Mortality, And Mean Longevity in Temperature-Dependent Cohorts of Culex Quinquefasciatus Say (Diptera Culicidae)

354 Journal of Vector Ecology December2010

Estimating reaction norms for predictive population parameters, age specific mortality, and mean longevity in temperature-dependent cohorts of Culex

quinquefasciatus Say (Diptera: Culicidae)

Filiz Gunay, Bulent Alten, and Ergi Deniz Ozsoy

Hacettepe University, Science Faculty, Department of Biology, Evolutionary and Ecological Genetics Laboratory, 06800 Beytepe, Ankara, Turkey

Received 11 March 2010; Accepted 18 July 2010

ABSTRACT: Culex quinquefasciatus plays a major role in the transmission of important parasites and viruses throughout the world. Because temperature is an important limiting factor on growth and longevity of all mosquito species, estimating the reaction norms provides very important basic information for understanding both plasticity and individual variations of the population. In the present study, Cx. quinquefasciatus were maintained at five different constant temperatures (15, 20, 23, 27, and 30C) for two subsequent generations. Reproductive population parameters in blood-fed mated females and longevities of virgin and blood-fed mated adults reared at different temperatures were compared for the two generations. Longevity increased as temperature decreased within a range of 15 to 30C for the unmated adults, and 15 to 27C for the mated and blood-fed adults. Generation times were as long as 124.07 and 106.76 days for two subsequent generations reared at 15C, and the highest intrinsic rate of increase (rm) values were estimated at 0.22 and 0.18, respectively, from the cohorts reared at 27C. For survival rates, reproductive rates (R0), and rm values, 30C was found to be a critical temperature for this species. These cohorts produced the smallest amount of eggs (R0= 5.06), rm values decreasing across generations (from 0.11 to 0.06), and the survival rates from egg to adult were found to be insufficient (16.1 and 10.8%). Additionally, the rate of exponential increase with age and age specific mortalities (b) were calculated for the virgin cohorts. Age specific mortality rates increased as temperature decreased. The increase in mortality rates started to accelerate at 27C and was more pronounced at 30C, for both females and males. We estimated the coefficients of variation for the b values in which females have smaller coefficients than those of the males at all temperatures. Journal of Vector Ecology 35 (2): 354-362. 2010.

Keyword Index: Culex quinquefasciatus, temperature, variation, life table traits, longevity, age specific mortality.

INTRODUCTION

Life-history theory in evolutionary biology (Roff 1992, Stearns 1982) is a theory of fitness. The information regarding life history traits of organisms, from experiments carried out under laboratory conditions, could provide us with sound predictions of the expressions of their genetic potential, and thus could establish baselines for subsequent field studies. A fitness framework enables generalizations to be made not only for the species under study, but also for other species that could have been subjected to similar selective pressures (Nylin and Gotthard 1998).

The reaction norm is used to quantitatively model the dependence of fitness-related characters on environmental parameters such as ambient temperature and nutrition (Stearns 1982). Therefore, it defines phenotypic plasticity. As a generally accepted guideline, increased temperature results in higher growth rates, shorter development times and longevity, and smaller adult size in insects and other ectotherms (Sibly and Atkinson 1994, Li and Jackson 1996, Worthen 1996). Estimating the reaction norms in response to these components, such as temperature variations effecting distribution area of a given population, provides important basic information for understanding both plasticity and individual variations of the population.

The mosquito, Cx. quinquefasciatus Say 1823, plays a major role in the transmission of bancroftian filariasis, Chikungunya, West Nile, and St. Louis encephalitis world-wide. The species is widely distributed in the tropical and subtropical areas of the world from the Nearctic region to the end of the Palearctic region. It occurs in all climatic zones, and altitude does not appear to limit its distribution (Bhat 1975). Although waste water is a major larval habitat, this species can develop in virtually any type of aquatic habitat in the human environment. Females of Cx. quinquefasciatus generally feed indoors or outdoors on human blood. Because of these characteristics, Cx. quinquefasciatus is a good model organism to study predictive life traits in relation to reaction norms.

The effects of temperature on the development of Cx. quinquefasciatus have been reported previously (Shelton 1973, Suleman and Reisen 1979, Rayah and Groun 1983, Service 1986, Rueda et al. 1990, Mogi 1992). In this study, the effects of constant temperatures on life table traits of Cx. quinquefasciatus were determined. Longevity comparisons were also designed to reveal the reaction norms of the adults with respect to the different constant temperatures at which they developed. For this reason, as well as the life table mortality outcomes, virgin adult cages were prepared to exclude effects of mating and blood feeding on mortality.

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Age specific survivorship

yx = the number of females or males on each day, x.

Net reproductive rate per cohort

Age of mean cohort

reproduction

Intrinsic rate of increase

Maximum likelihood estimations of the rate of increase in age specific mortality (b parameter of the Gompertz function) were calculated in virgin Cx. quinquefasciatus cohorts. Variation in age specific mortality in response to temperature was defined in terms of a reaction norm after one generation of rearing.

MATERIALS AND METHODS

Maintenance of the colony in the laboratory The lab colony used in this study was previously reared

in France (ISEM) but originated in California in the 1980s. Egg rafts from the colony were transferred to the Ecological Research Laboratory at Hacettepe University (ESRL) in Ankara, Turkey, in 2005. The rearing and feeding of adults and larvae followed the methods of Kasap and Kasap (1983) with a temperature 271C, 605 RH%, and 14:10 h (L:D) photoperiod. Individuals used in the experiments were obtained from the same generation (F30) to avoid the effects of genetic differences between generations.

Maintenance of cohorts in the climate chambers Age-specific (horizontal) life tables were constructed

to determine whether temperature affects the basic demography of Cx. quinquefasciatus adults. Three replicates of 750 1st instar larvae were transferred into standard polyethylene cups (27x16x17 cm) that contained 1 liter of distilled water. The cups were placed in five climate chambers programmed at five different temperatures (15, 20, 23, 27, and 30C) and exposed to a 14:10 (L:D) photoperiod with a constant relative humidity of 60%. The larvae were fed each day with 0.04 g sinking Tetramin, which was spread evenly over the water surface. Pupal development and the number of pupae present were checked daily. All pupae were transferred to glass vials before eclosion. For the virgin adults, female (100 individuals) and male (100 individuals) cages were prepared separately, with each chamber containing 30% sucrose solution, for the estimation of the rate of exponential increase in mortality with age (the b parameter of the Gompertz function). In this experiment, females were not blood-fed. In another experiment, 100 females and 100 males were placed together in 20x20x20 cm cloth cages containing plastic cups with distilled water as oviposition sites. Females fed on quail blood every four days for 2 h. Experiments were replicated three times for each temperature regime. This procedure was followed for two subsequent generations.

Life tables and statistical data analysesHorizontal life table parameters were calculated from

daily records of mortality and fecundity of each cohort of Cx. quinquefasciatus. Life table attributes of adult mosquitoes, the calculation procedures, formulae used, and their rationale in the present study were according to Belen and Alten (2005) and summarized in Table 1.

Homogeneity of the data was tested with the Shapiro Wilk test and the predictive population parameters were compared using the non-parametric Kruskall-Wallis test.

xlx mx /R0

lxmx

lx= yx/yo

m is the mean number of female progeny produced by females of age x. The value of mx was calculated by mx=Exs where Ex is the mean number of eggs produced per female per age x, and s=the proportion of the offspring (eggs) that were females.

starting at x=1, the day of adult emergence.

l, m, w are as above, e is the base of natural logarithms, x is the age interval, lxmx is the product of the survivorship of each cohort female by its fecundity at an age x.

lxmxe-rmx

Table 1. Life table attributes used in this study and their rationale.

Age specific mortality parameters from the Gompertz function (Gompertz 1825), i.e., the baseline mortality rate (a) and the rate of exponential increase in mortality with age (b), were estimated with the virgins, per sex per treated temperature, by the method of Pletcher (1999). A likelihood X2 ratio test with 1 degree-of-freedom (only b was constrained) was performed to assess statistical significance.

RESULTS

The survival rates of the immature stages from egg to adult are shown in Table 2. The highest survival rates were from the cohorts at 23C and the lowest rates were seen at the highest temperature condition (30C) for both generations.

Separate adult life tables were constructed for each of the cohorts reared under different temperature regimes for two subsequent generations, and predictive population parameters (R0, Tc, rm) were also calculated. The results are compared in Table 3 for the first and second generations. While the net reproductive rate, R0 was lowest at the highest temperature for two subsequent generations, the highest value was calculated from the cohort reared at 23C while it decreased at the second generation. As a specific observation, at this temperature males lived longer than expected, which could mean the death caused by mating was lower. R0 value was found to be slightly higher (52.31) at 15C than at 27C (50.22), which is the laboratory

356 Journal of Vector Ecology December2010

optimum for the first generation, but it decreased to 37.23 for the second generation at the lowest temperature (15C). In spite of these differences, for both generations there was no discrepancy between the temperatures and R0 values statistically (p=0.1377, p=0.0581). At the lowest temperature, the generation time, Tc, was found to be almost three months (106.76 days) for the first, and four months (124.07 days), for the second generation. Tc showed a negative correlation with the temperature, except at 30C for both generations and at 23C for the second generation, in which the females produced fewer eggs than expected. There was a significant difference between temperatures and Tc values for the first (p=0.0102) and second (p=0.0193) generations. For generation times, the greatest significant difference was between 15 and 27C (p=0.0101, p=0.0127, respectively) for both generations among all cohorts.

Although the R0 values were greater at 15C than at 30C in the first generation, rm, the intrinsic rate of increase gave the opposite outcome. Another important point was that the rm value decreased in the course of one generation at 30C. The highest value was calculated from the cohorts at 27C, and the differences between the cohorts for both generations are statistically significant (p=0.0159; p=0.0164, respectively).

Interaction between longevity and temperature variation is shown in Table 5 and Figure 1 for mated (life table experiments) and unmated females and males of the second generation. For mated sexes, longevity was significantly affected by temperature and by sex, but there were no significant differences between replicates (Table 4).

While the longevity of Cx. quinquefasciatus was normally higher at lower temperatures, the mean longevity of both female and male adults was clearly low at extremely high temperatures (Table 5). Although there were no significant differences between the replicates, differences were obtained between females and males of the temperature-dependent cohorts (Table 5, Figure 1). The

males demonstrated a significantly shorter longevity than females at all temperatures (p

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respectively) except that of 23C which was not significant (X2= 0.1986, p= 0.6558). In contrast to the general trend of females that showed lower age specific mortality rates than males presented above, an opposite pattern was found for the adults at 27C. Mean longevity of the females and males at this temperature were very similar, unlike those from the other temperature regimes.

Figure 2 also shows the coefficient of variation (CV) of the b between the temperatures for each sex. Females had less variability than males at all temperature conditions. At 27C, the optimum temperature of the laboratory colony,

both females (0.286) and males (0.357) had smaller variation coefficients than those of the other temperature regimes.

DISCUSSION

Although there have been many studies on the effects of temperature variation on the pre-adult and adult stages of mosquitoes, few have collected data in the appropriate form and in sufficient detail to provide a distribution of values required for calculations in mathematical models or estimates for temperature-dependent development (Gomez et al. 1977, Suleman and Reisen 1979, Mogi 1992, Su and Mulla 2001). Our results showed Cx. quinquefasciatus is a good model organism to study the predictive life traits in relation to reaction norms of the temperature variation affecting populations.

All of the temperature regimes we used affected re-productive parameters. Even though the main colony was reared at 27C, the optimum temperature on the basis of fecundity was 23C for the first, and 20C for the second, generations. Nevertheless, there was no significant differ-ence between the cohorts for R0 values for both generations (p=0.1377, p=0.0581, respectively). As for the rm, the opti-mum temperature was 27C for the two consecutive gen-erations. It is well known that one of the most valuable ap-plications of the intrinsic rate of increase concept is in the delineation of the livable environment of a species (Vargas et al. 2000). Similar to the rm values calculated from differ-ent populations around the world (Walter and Hacker 1974, Gomez et al. 1977, Suleman and Reisen 1979), rm values in our study were also found to be positive under a wide range of temperature regimes (15, 20, 23, 27, and 30C), so

Table 4. General linear model for the effects of temperature, replicate and sex on longevity from life table experiments with mated + blood-fed adults of Cx. quinquefasciatus.

MS d.f. F PReplicate 899.0 2 1.3 0.267

Sex 527,975.0 1 775.9

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Figure 1. Box and Whisker plots of longevity (+1 SE) for mated + blood-fed and virgin adults of Cx. quinquefasciatus (a), survival curves of mated and blood-fed females and males (b), virgin females and males (c) at five different constant temperature (15C, 20C, 23C, 27C and 30C). Data were pooled across replicates (fm: female, m: male).

A

B

C

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we can conclude that Cx. quinquefasciatus may increase its population size under all these temperature conditions but with some differences. This is one explanation for why the species has the ability to disperse widely in nature.

Minimal and maximal temperature thresholds for embryonic development of Cx. quinquefasciatus are 13 and 39C, respectively. The hatching rate varies directly with temperature, up to 32C, after which eclosion rates drop gradually (Rayah and Groun 1983). We found that at 15C, this species could complete its life cycle with a positive rm and a very long generation time (124.07 days) (Table 3). Suleman and Reisen (1979) have shown that this species could overwinter in Pakistan, in a gonoactive state, with females emerging during late autumn, persisting through the cold winter months and into early spring. Overwintering strategies of this nature coupled with a high degree of human and bird feeding seemed to indicate that this species could provide an overwintering mechanism for West Nile Virus and perhaps for other disease agents (Suleman and Reisen 1979). Regarding the variety of the viruses transmitted by this species (Jozan et al. 2003, Kilpatrick et al. 2007, Peterson et al. 2008), our results suggest that Cx. quinquefasciatus can overwinter in cooler areas than Pakistan (Lanciotti et al. 1999, Epstein 2000, Githeko et al. 2000, Kilpatrick et al. 2004, Hayes et al. 2005).

According to McCann et al. (2009), better larval conditions result in females capable of greater reproductive output. In the present study, the most suitable condition in terms of survival rates for the immature stages was found to be 23C (Table 2). In the second generation at 23C, we observed unexpected results. We were expecting to find a greater R0 value at 23C, and indeed obtained it for the first generation (Table 3), but in contrast, the R0 value was found to be lower, and the Tc was longer, for the second generation at the same temperature regime. One of the factors that can

affect the observed decrease in R0 and rm is the production of offspring at an older age (Futuyma 1998). Since we detected a delay at the second generation, this factor may be responsible for the decrease in these parameters and consequently in the increase of mean longevity of the adults, eventually in Tc. Similar results were found at 30C temperature regime for both generations. These unexpected values of R0 and rm may also be the result of high mortality among the cohort females that lived a maximum of 51 days and started to reproduce at the age of 21, while the females that lived at the optimum temperature laid eggs on the seventh day of emergence (not shown). Even though at the coldest temperature rm values were found to be smaller than those of the hottest for both generations, rm increased with temperature increases for both generations, but it was significantly decreased at 30C (Table 3). Changes in the rm matters strongly for understanding the fitness and permanency of the cohorts (Tatar 2001, Simsek et al. 2005). These findings suggest that extremely warm temperatures could be more detrimental, and possibly more limiting, than cold temperatures for this species. For life span, the specific patterns were mostly as expected and thus in agreement with the general notion of pronounced differences between different rearing temperatures (Tatar 2001). Like many of the previous studies, the pattern of increasing life span of cohorts at low temperatures was evident in both mated and unmated cohorts as shown previously (Su and Mulla 2001, Norry and Loeschcke 2002, Gilles et al. 2005, Kasap and Alten 2006, Aytekin et al. 2009, Karl and Fisher 2009). This frequent pattern is related to increasing metabolic rates with increasing temperatures in ectotherms, with higher metabolic rates in turn being correlated with shorter life spans (Karl and Fisher 2009). Life table experiments at the highest temperature (30C) for mated and blood-fed adults (Table 5) yielded a higher corresponding adult

Figure 2. Exponential increase curves in age specific mortalities (b values x 10-2) and coefficients of variation (CV) at five different constant temperatures in Cx. quinquefasciatus.

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longevity. Likewise, Rueda et al. (1990) found the same decrease in lifespan in both Cx. quinquefasciatus and Aedes aegypti from 15 to 27C, but an increase at 30 and 34C. Interestingly, this trend was not determined in unmated cohorts in our study. On the other hand, Shelton (1973) showed that in the immature stages of Cx. quinquefasciatus, longevity increased while the temperature decreased in the range from 20 to 26C, but at 32C, longevity increased. Because even the immature stages show this response to the highest temperature, it might be expected that unmated adults would show the same reaction. We estimate that this could have occurred with the effect of mating as a specific trend. Perhaps there was a trade-off between the rise of temperature and the decrease of longevity in which, after some specific temperature is reached, some buffering physiology of the organism may be overturning the negative relationship between the temperature increase and the longevity. It is thus possible that some overall metabolic and genome expression changes could be linked to the mating status of aging flies at higher temperatures, as mating accelerates aging in general.

The longevity of a vector population is an extremely important facet of its vectorial potential. Longevity patterns in Cx. quinquefasciatus varied between sexes, with females living longer than males, as are found in many other animals (Smith and Warner 1989, Nuzhdin et al. 1997). Males generally appear to sacrifice viability for enhanced sexual performance, whereas females may benefit by investing more in immunity and longevity (Rolf 2002). As a rule, large animals tend to live longer than small animals (Lindstedt and Calder 1976, Speakman 2005), which may also have an impact on the pattern found in Cx. quinquefasciatus, as adult females are larger than males at all constant temperatures (data not shown). Sex differences were affected by temperature. While males and females showed similar life spans at the higher temperature, females lived much longer than males at the lower temperatures. This suggests that females may have an enhanced heat tolerance compared to males, which has already been shown in Drosophila (Sorensen et al. 2001, Jensen et al. 2007). Mating and blood feeding have important effects on adult survival (Yuval 2006), and that it is shown once again in this study. In what may be somewhat aberrant results at 30C, virgin adults did not live as long as those that were mated and blood fed, suggesting that temperature stress combined with blood deprivation and the inability to find mates caused an enhanced death rate.

We have excluded the effects of mating and blood feeding and examined the longevity of the virgin individuals to observe the reaction norm in response to temperature variation. Using the Gompertz function, we estimated a, initial mortality rate, and b, exponential rate of increase with age (Pletcher 1999). In general, total vectorial capacity is more sensitive to changes in b than a, which is understandable because b is exponentially, whereas a is multiplicatively, related to overall mortality. As overall mortality increases, the potential for mosquito populations to transmit pathogens declines (Styer et al. 2007).

Supporting our findings, Cx. quinquefasciatus adults have more vectorial importance at relatively low temperatures (15 and 20C). Tests indicated that with increasing temperature, the exponential rate of increase in mortality increased as we expected. Females at higher temperatures (27 and 30C) showed considerably higher b values than at lower temperatures. The reaction norm is a basic tool of evolutionary analysis that quantifies the relationship between environmental parameters and functional characters, including reproduction and longevity (Phelan and Rose 2006). In our study, the reaction norm relating the exponential rate of increase in age specific mortality to ambient temperature was fairly steep (Figure 2), especially for males at 30C. In addition, except at the middle temperature of 23C, there was a significant difference on b values at every constant temperature for adults. Variation coefficients of the b parameter suggest that the population is strongly adapted to 27C and females give more stable reactions than males.

Temperature may also affect the vectorial capacity and changes in the population densities of species (Olejnicek and Gelbic 2000). We conclude that temperature is a crucial factor in the evolution and ecology of Cx. quinquefasciatus. As climate changes could cause some unexpected effects on the dispersal of disease (Epstein 2000), our results show that this species is also a good candidate in this respect across its distribution range.

Acknowledgments

We thank Dr. Utku Perktas and Murat Ylmaz for their help and support. This study is part of an MSc thesis submitted to Hacettepe University.

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