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Volume 47

Reciprocal effects of plasticity in defensive and mating-system phenotypes of freshwater snails.

Josh Auld, Department of Biological Sciences, University of Pittsburgh

Introduction and Background
Most organisms experience variation in biotic and abiotic environmental factors during ontogeny. To persist, they must survive and reproduce despite such environmental variability. Many organisms respond to environmental variation with conditional strategies that involve phenotypic changes based on environmental conditions (i.e. phenotypic plasticity). An understanding of how environmental variation affects the interaction between genotype and phenotype is fundamental to interpreting the process of evolution in natural systems. Many studies have documented how organisms respond to a single type of environmental variation, but in nature many environmental factors can vary simultaneously. Therefore, to understand the evolution of phenotypes in natural populations we have to understand the context in which phenotypes are expressed and how environmental factors interact to affect the expression and evolution of the phenotype.

I am examining the effects and consequences of environmental variation on phenotypes by examining two kinds of environmental factors that interact to affect the phenotype: predation risk and mate availability. These factors directly affect survival and reproduction and are therefore closely tied to fitness, making them excellent factors for a study of how interacting environmental factors affect a species' ecology and evolution. An increase in predation risk leads to the expression of inducible defenses that increase survival in the presence of a predator. However, inducible defenses frequently incur a cost of slower growth or decreased reproduction compared with the "no-predator" phenotype. Variation in mate availability plays a crucial role in determining mating systems and is a product of population size and structure. Responding to variation in mate availability can lead to patterns of inbreeding and outbreeding that deviate from random breeding. In nature, organisms will experience variation in predation risk and mate availability simultaneously.

Inducible defenses and mating systems are intimately linked because inducible defenses frequently involve adjustment of traits such as activity that also determine mating and reproductive success. Simultaneously, changes in the mating system can lead to inbreeding or outbreeding depression and therefore influence the development of an organism and the production of an inducible defense. Responding to variation in predation risk and mate availability frequently involves plastic adjustment of the same traits (e.g. age or size at first reproduction). Therefore, to understand the expression and evolution of defensive and mating-system phenotypes in nature we must examine their interaction. To explore how organisms respond to two independent sources of environmental variation, I am examining the effects of predator-induced plasticity on mating system expression and the implications of changes in the mating system on the production of an inducible defense.

Proposed research
In my grant application, I proposed research to address how the effects of plasticity in defensive and mating-system phenotypes interact in the hermaphroditic freshwater snail Physa acuta. Using a combination of laboratory and outdoor mesocosm experiments to decompose these reciprocal effects, I have made progress towards addressing two major hypotheses.

Hypothesis 1: Predator environments affect the mating system of a prey population by increasing the proportion of selfed progeny produced.
Predators can reduce prey density by thinning the prey population and induce changes in prey traits that affect the pool of available mates. If mates are available and inbreeding depression is high, individuals should mate and reproduce shortly after reaching sexual maturity. However, if mates are not available, individuals should wait a set amount of time before inbreeding to avoid the costs of inbreeding depression (Tsitrone et al., 2003). Concurrently, size-selective predation on small prey favours rapid growth and delayed reproduction whereas predation on large individuals favours slow growth and early reproduction (Stearns and Koella 1986). The combination of decreased mate availability due to predator thinning and delayed maturity (which should truncate the time delay before self-fertilization) induced by predators may result in an increase in the proportion of progeny produced by self-fertilization.

Hypothesis 2: Inbreeding impairs the production of prey defenses. I expect that with subsequent inbreeding, individual growth rate will decrease.
Thus, the development of morphological and life-history defensive phenotypes will be retarded in inbred individuals relative to outbred individuals due to their reduced growth potential. This decrease in plasticity should increase with subsequent inbreeding.

Research to date
Predators induce changes in the life-history traits of snails that likely affect the mating system. To examine how predators influence the mating system by 1) inducing changes in the timing of reproduction and 2) changing how individuals respond to mate availability, I experimentally examined how snails alter their life histories under combinations of predator presence and mate availability. I collected ten adult snails from a single, high-density (outcrossed) population and placed them in the lab for oviposition. Progeny from ten clutches were used in a full-factorial experimental combination of two predator treatments (no predator and caged crayfish) crossed with two mate-availability treatments (isolation and mate available three times a week for three hours at a time). These four treatments were replicated ten times for a total of 40 experimental units (1-L plastic tubs with weekly water changes and constant ad libitum food rations). I quantified growth rate, morphological changes in shell shape, age / size at first reproduction, progeny success (survival and growth rate), and fecundity.

In the absence of predators, snails that were isolated delayed reproduction compared to snails with partners. However, in the presence of predators, snails with and without mates delayed reproduction (Fig. 1). With crayfish, there was no additional waiting time prior to self-fertilizing. Thus, when predators are present, there is a reduced probability of locating a mate prior to initiating reproduction. Hence, individuals are more likely to self-fertilize prior to finding a mate in the presence of a predator than in the absence of a predator. I am currently testing this hypothesis further by using microsatellite allele frequency data to evaluate how the genetic mating system changes in the presence / absence of predators.
Figure 1. The effects predator presence and sexual partner availability on age and size at first reproduction, growth rate, and the number of eggs produced in the first week of reproduction. Data are means + 1 s.e.m.

To evaluate the prediction that inbreeding impairs the production of prey defenses, I am conducting the same experiment after several generations of self-fertilization or outcrossing in the lab. I expect that with subsequent inbreeding, growth rate and fecundity will decrease. Also, I am measuring the same morphological and life-history traits for inbred and outbred progeny to determine if inbreeding affects the plasticity of snail traits.
It is important to be able to compare snails produced through self-fertilization to an "outcrossed" control line of snails in order to control for any inadvertent selection that occurs under lab conditions and to control for any general environmental affects that differ between generations. In my original attempt at this (August - December 2005), I obtained snails for the second-generation experiment from my first-generation-experiment snails. It was obvious that one generation of self-fertilization had detrimental consequences on fitness (Fig. 2).
Figure 2. The effects of sexual partner availability and mating system on the number of eggs laid and the proportion of eggs hatching. These data are averaged across predator treatment. All data are means + 1 s.e.m

Selfed snails laid 36% fewer eggs than outcrossed snails and 15% more of their eggs did not hatch. Thus, self-fertilization impairs fitness and also impairs the production of predator-induced shell morphology (data not shown). However, despite these obvious differences between the first-generation traits and the drastic reduction in fitness associated with self-fertilization, my outcrossing control lines were intermediate in many measures between the generation-1 snails and the generation-2 snails produced through self-fertilization. This suggests that my outcrossing treatments were occasionally self-fertilizing. To correct for this and obtain higher-quality results, I have implemented a new protocol for outcrossing snails. In this protocol, "outcrossing" lines are given consistent access to a mate (marked with red paint), which is changed everyday for one month. In this way, outcrossing parents potentially have access to 30 different mates from which they can obtain allosperm (individuals of this species can store sperm for ~3 months; Wethington and Dillon 1991). This new protocol is having the desired effect of more accurately mimicking an outcrossing-selfing comparison. I am currently completing the generation-1 experiment and approximately 1 month into the generation-2 experiment. This change in protocol will allow a better comparison.

Conclusions and Significance
Life persists because organisms survive and reproduce despite environmental variation. To understand how organisms accomplish this, we need information on how individuals integrate their phenotypes to simultaneously survive predation, find mates, and maximize reproduction. This investigation into the interactions between plasticity in defensive and mating-system phenotypes will improve understanding of how inducible defenses are expressed in nature (in the context of the mating system) and how the mating system is affected by biotic interactions (such as predation risk).

The funding from the Malacological Society of London has been integral to the completion of this phase of the work, and I am extremely grateful for the financial assistance.

Literature Cited
Stearns, SC and JC Koella. 1986. The evolution of phenotypic plasticity in life-history traits:
  predictions of reaction norms for age and size at maturity. Evolution 40: 893-913.
Tsitrone, A, S Duperron, and P David. 2003. Delayed selfing as an optimal mating strategy
  in preferentially outcrossing species: theoretical analysis of the optimal age at first reproduction in relation to mate availability. Am. Nat. 162: 318-331
Wethington, AR, and RT Dillon. 1991. Sperm storage and evidence for multiple insemination
  in a natural population of the freshwater snail, Physa. Am. Malacol. Bull. 9:99-102.