No Slide Title

Hatch Plasticity in Ochlerotatus triseriatus in

Response to Hatch Stimulation Frequency

Christopher J. Vitek, Camilo Khatchikian, Todd P. Livdahl

Biology Department, Clark University, 950 Main St., Worcester, MA 01610

Introduction

Phenotypic plasticity is a mechanism organisms may use to avoid habitat disturbances and threats. Previous plasticity studies of container-breeding mosquitoes have examined the effect of habitat drying on the metamorphosis of Ochlerotatus triseriatus (Juliano et al, 1994), as well as the various effects that environmental influences may have on the hatch rate (Mogi, 1976; Edgerly et al, 1992; Livdahl et al, 1984). We chose to examine the effects of cues that may predict habitat risk where the cue itself is not a threat to the organism. One such cue could be the frequency of hatching stimulation for a container breeding mosquito. During plentiful rainfall conditions, eggs are stimulated more frequently, while under possible drought conditions less frequent stimulation would occur. Our previous work has indicated that Aedes albopictus, the Asian Tiger Mosquito, does show a plastic response to the frequency of hatch stimulation (Vitek et al, 2003). Eggs that are stimulated less frequently show less hatch delay than eggs that are stimulated more frequently. Here, we examine the hatch response of another container breeding mosquito, Ochlerotatus triseriatus. We tested a variety of populations collected from areas in the northeastern USA that have a range of natural frequencies of rainfall, as well differences in mean annual temperature and mean annual precipitation. We expected that any exhibited plasticity should be directly related to the natural habitat.

Methods

In the summer of 2002, eggs were collected from 7 locations in northeastern USA (see figure 1). These sites were chosen based on records of precipitation collected from National Weather Service stations (see table 1). These data were collected over the span of 15 to 54 years (depending on the site), and consisted of the number of days per month with a minimum of .01 inches of precipitation. Seven sites were chosen, including 4 sites at the upper end of the distribution and 3 sites with relatively low rainfall frequency. Eggs were collected after one month and brought into the laboratory. They were stored at a constant temperature (23° C) and at a humidity of 90% or greater, in a 16:8 h light:dark cycle. Half of the eggs were hatched and the larvae reared to adulthood. These adults were force mated to produce an F1 generation and stored under identical conditions as the parental eggs. Experimental eggs were drawn from both the wild-collected eggs and the F1 generation of laboratory eggs. Eggs were divided into batches of 50 and then subjected to one of two levels of a stimulation frequency treatment (3 or 7 days between stimuli). Hatch stimuli consisted of submerging the eggs into a .5g/l nutrient broth mixture for 24 hours. At the end of the stimulation period, hatched larvae were removed and recorded and the eggs were removed and placed back into storage until the next stimulation. This process was repeated for 10 stimuli for all egg batches. At the conclusion of the experiment, unhatched eggs were dissected to determined viability. Non-viable eggs were removed from the analysis to determine an accurate hatch percentage.

For all analyses conducted, egg batches that had 100% hatch after the first stimulus was excluded as the eggs were not exposed to differing treatments. The initial analysis tested the hatch rates during the first stimulus to determine if there was any difference between the two treatments. A secondary analysis was conducted comparing batches that had no subsequent eggs hatch to batches that had eggs hatch after the first stimulus. A third analysis compared the hatch rates for egg batches that did have any eggs hatching after the first stimulus. For this analysis, a 50% critical stimulus number was estimated using logistic regression. This number would be the hatch stimulus at which 50% or more of the remaining eggs after the first stimulus had hatched. For all analyses, a variety of environmental influences were tested including latitude, rainfall frequency, mean annual precipitation, and mean annual temperature.

Results

The initial analysis was to test for treatment effects and egg type (wild or force mated) and population differences in hatch rate on the first stimulus (see figure 2). Ideally, treatment would have no effect on the hatch rate at the first stimulus because the egg batches were not subjected to different treatments at that time. There was no significant influence of treatment on the hatch rates in the first stimulus (F = 2.7343, p = .1025). Egg type (wild collected or laboratory force mated) did have a significant influence (see table 2) with a p value of <.0001. Mean annual precipitation also had a significant positive influence on the first hatch rate (see figure 3).

Figure 4. The interaction effect between treatment and rainfall frequency. More frequent rainfall results in a greater degree of plasticity between high and low frequency stimulation.

Table 1. Summary of the sites

Figure 1. The seven selected sites from which eggs were collected. Sites were chosen based on the frequency of rainfall during the summer months, three sites with a low natural frequency (indicated by a red marker) and four sites with a high natural frequency (indicated by a blue marker). The eighth site (Binghamton) was excluded from this study.

Figure 2. The hatch percentages of the first stimulus for each site and treatment (either high or low frequency of stimulation), with standard error bars.

Table 2. Summary of ANOVA results testing the hatch rate during the first stimulus against egg type, treatment, rainfall frequency, and Mean Annual Precipitation. Treatment is not significant, although both egg type (wild versus laboratory eggs) and Mean Annual Precipitation are significant.

Summary

These results seem to indicate that hatch plasticity is used by Ochlerotatus triseriatus in response to stimulation frequency. In addition, the plasticity response does seem to vary as a result of the natural habitat. The two forms of plasticity analyzed (hatching versus no hatching after the first hatch stimulus and the hatch rate after the first stimulus) both show significant interactions between the treatment and environmental factors including latitude, mean annual precipitation, and rainfall frequency. This indicates that plasticity responses may vary in the wild based on the natural conditions and that indirect cues such as rainfall frequency may be used as predictors of habitat stability. One possible source of error in this experiment is small sample size. Limited success with force mating reduced the number of laboratory reared eggs available and forced the inclusion of wild collected eggs. In addition a large number of the egg batches had a 100% hatch rate on the first stimulus, limiting the number of replicates for each treatment that were exposed to later stimuli.

Table 5. Summary of ANOVA results. Treatment interacted significantly with mean annual precipitation and rainfall frequency.

Literature Cited

Edgerly, J.S., Marvier, M.A. 1992. To Hatch of Not to Hatch? Egg Hatch Response to Larval Density and to Larval Contact in a Treehole Mosquito. Ecological Entomology, 17, 28-32.

Juliano, S.A., Stroffregen, T.L. 1994. Effects of Habitat Drying on Size at and Time to Metamorphosis in the Tree Hole Mosquito Aedes triseriatus. Oecologia 97, 369-376.

Livdahl, T.P., Koenekoop, R.K., Futterweit, S.G. 1984. The Complex Hatching Response of Aedes Eggs to Larval Density. Ecological Entomology, 9, 437-442.

Mota, M. 1976. Variation in Oviposition, Hatching Rate, and Setal Morphology in Laboratory Strains of Aedes Albopictus. Mosquito News, 42, 196-201.

Vitek, C.J., Livdahl, T.P. 2003. Phenotypic Plasticity in Hatch Rate and Development Rate of Aedes albopictus. American Mosquito Control Association Annual Conference. Minnesota, MN, USA. March 2003.

A copy of this poster can be found at http://posters.vitekweb.com/

The author can be contacted at cvitek@clarku.edu

Figure 3. 1st hatch fraction graphed against Mean Annual Precipitation, with egg type indicated. There is no interaction, but each influence is significant.

Egg batches were then tested for a hatch response after the first stimulus. They were categorized as either “non responders” (replicates with viable eggs remaining that failed to hatch) or “responders” (replicates with viable egg remaining, at least some of which did hatch). Egg type (wild versus laboratory collected eggs) was tested initially, along with environmental factors, to determine if conditions these eggs were exposed to in the wild resulted in the eggs not responding to subsequent hatch stimuli. ANOVA results indicate that egg type did not play a role in the possible response or non response of eggs to subsequent hatch stimulation (see table 3). A second analysis, incorporating the treatment factor, was then conducted, and indicated that treatment interacted with latitude to produce a significant influence on the egg’s response to subsequent hatch stimulation (the treatment*mean annual temperature interaction was almost significant). Rainfall frequency was also significant (see table 4), but this would not be a plasticity response because there is no interaction with treatment.

The 50% critical stimulus number was estimated using a logistic regression analysis for each egg batch that had any hatch after the first stimulus. This critical stimulus number represents the estimated stimulus at which 50% of the remaining eggs would hatch. Using this predicted stimulus, environmental factors and treatment (high or low frequency stimulation) were analyzed with ANOVA. The environmental factors were mean annual precipitation, mean annual temperature, latitude, and rainfall frequency. Treatment interacted significantly with both rainfall frequency and mean annual precipitation (see table 5). Figures 4 and 5 show this interaction effect, with regression lines drawn for both of the two treatments.

Table 3. ANOVA results testing egg type as a factor for responsive or non responsive egg hatch after the 1st stimulus

Table 4. ANOVA results testing the responders and non responders with treatment and environmental factors.

Figure 5. The interaction effect between treatment and mean annual precipitation. Greater precipitation results in a greater degree of plasticity between high and low frequency stimulation.