The Effect of UV Radiation Absorption on The Reproductive Success of Enallagma carunculatum (Tule Bluet)

Alexandra Klimovitz

EEB 381 General Ecology, August 2014

The University of Michigan

ABSTRACT Human induced increases in DNA-damaging UV radiation due to ozone destruction have had many negative impacts on the reproductive success of native species. We investigated the effect of abdominal coloration on the clutch size produced by a female Enallagma carunculatum (Tule Bluet). In order to study this phenomenon we painted the abdomens of female tenerals white or black and after maturation observed the affect that UV radiation had on the ovulation rate. We hypothesized that individuals with dark abdomens would have more success in egg development due to increased UV radiation absorption. Our results were not consistent with this observation. We did not find a significant difference between abdominal treatments and clutch size. However, we did find that individuals with white abdominal treatments had a significantly higher mortality rate. Error from mortality rate and small sample size may have altered our data, thus further research is needed before conclusions are drawn. We suggest that future research investigate the effects that other environmental variables—such as weather and temperature—have on the reproductive success of E. carunculatum.

INTRODUCTION

It has become urgent to study the implications that ozone layer destruction and

global warming have on current species distribution and the reproductive success of these

species. Human induced increases in DNA-damaging UV radiation have been significant

in the last decade (McKenzie et al, 1999). These increases are most often cited as having

a negative impact on the ability of an organism to survive and reproduce successfully (Au

et al, 2002; Day et al, 1999; Grad and Williamson, 2001). However, there have been

instances reported in which small increases in UV radiation have positively impacted

species reproductive success (Kessler et al, 2008).

Certain invertebrate species such as damselflies have been shown to use the

reflection of UV light as an aid in reproduction (Silberglied & Taylor, 1978). By

observing how absorption rather than reflection affects egg development in female

damselflies, this study takes a novel approach to the potentially positive impact of

increasing UV radiation levels.

In order to study this phenomenon we painted the abdomens of female Enallagma

carunculatum (Tule Bluet) tenerals white or black, and after maturation observed the

affect that UV radiation had on the ovulation rate. We hypothesized that individuals with

dark abdomens would have more success in egg development due to increased UV

radiation absorption than individuals with lighter colored abdomens.

METHODS

Location

Our group of 4 undergraduate students conducted this study within residential

areas at The University of Michigan Biological Station (UMBS) along the shoreline of

South Fishtail Bay on Douglas Lake in Cheboygan Co., Michigan (45.559, -84.673).,

from June 10th, 2014 to August 6th, 2014.

Study Species

The damselfly E. carunculatum inhabits slow-moving rivers, lakes and open

ponds along the shores of the Great Lakes (Lam, 2004). The species is reproductively

active from mid-July to early September (Còrdoba-Aguilar, 2008).

Tenerals emerge from their aquatic nymph stage in late morning throughout the

summer (June - August) and are easily identified by their glossy and transparent bodies

(Lam, 2004).

Collecting/Processing Methods & Insectary Description

We collected newly emerged female E. carunculatum tenerals using nets in early

afternoon along the Pine Point Trail near the shoreline of Douglas Lake at UMBS. We

confirmed the species of each teneral, then sexed, and determined the age prior to

processing individuals. Collected individuals were put in small plasticine envelopes or

tupperware containers for transporting back to UMBS.

Processing took place behind Lakeside Lab at UMBS. We uniquely numbered

each individual on its wings using a felt tip pen. Then, using a small amount generic

paint, we coated the lower half of the abdomen. We painted random individuals white or

black in addition to an unpainted control group.

An insectary (Bioquip Inc., 1.8  ×  3.6  ×  1.8 m) was setup behind Lakeside Lab in a

sparsely wooded area approximately 10 meters from the shoreline of Douglas Lake. We

planted a variety of small leafy plants in buckets—with holes in the bottom to prevent

drowning—and placed them in the insectary to provide habitat and shelter for the

tenerals. After processing all marked tenerals were released into the insectary. During

later trials we used smaller generic mesh insectaries (0.3 x 0.3 x 0.3 m) due to high

mortality rates in the large insectary. In order to standardize the egg development time we

froze all tenerals three days following their capture.

Feeding

We fed the tenerals small flying insects collected with nets nearby the insectary

twice daily. Mesh bags filled with banana peels were hung inside and around the

insectary in order to attract Drosophila melanogaster to the area.

Dissecting Methods

We dissected between 5 hours and one day following the freezing of mature

individuals. After using a scissors to remove the head and wings of the frozen damselfly,

we used insect pins to secure the abdomen to a wax plate. We opened the abdomen using

fine-tipped forceps and insect pins. Using a Bausch and Lomb (0.7x - 3x) dissecting

microscope and light, we examined the contents of the abdomen and counted all eggs

found in each individual.

Statistical Analysis

We plotted means and standard deviation on a box plot. We used a one way

analysis of variance to analyze average egg counts of the different treatment types (black,

white, and control). We used a one-tailed t-test to test for differences between only black

and control treatments, then we tested for differences between only black and white. We

used a Chi-squared test for independence to analyze the mortality rates of each treatment

type.

RESULTS

The average number of eggs in each treatment found through dissection of mature

female damselflies was found to be highest in black-painted individuals, followed by

control individuals, and finally white-painted individuals ( black = 22.23, control = 13.83,

white = 12.5; Fig. 1). However, there was high variation from the average egg counts

(stdblack = 8.89, stdcontrol = 12.57, stdwhite = 11.54; Fig. 1). These differences in average

number of eggs in each treatment type were not found to be significant (F(2,28) = 2.302,

p >0.05; Fig. 1).

In a comparison of just black and control treatments we did not find significant

differences in clutch size (t = 1.091, p = 0.193; Table 1). However, in a comparison of

just black and white treatments we observed marginally significant differences in clutch

size (t = 1.5302, 0.1>p>0.05; Table 2). Additionally, we observed a significantly higher

mortality in white-treated individuals (70%, n = 10), versus black (25%, n = 12) or

control-treated individuals (0%, n = 6; χ² value = 18.52; d.f = 2, p ≤ 0.05; Fig. 2).

DISCUSSION

The data collected does not support our original prediction that a darker abdomen

promotes a larger clutch size. Our results indicate that UV radiation played a marginal—

if any—role in determining the ovulatory success of E. carunculatum. This study was

limited by a very small sample size with high variation, thus it is possible that future

research may find significance differences.

Another source of error stems from the high mortality rate we observed in

individuals with white-painted abdomens. We suggest that further research investigate

whether these deaths stem from a lack of UV radiation or error. We speculate that

differences in the reactions to chemicals in the paint may have contributed to increased

mortality. Black paint is colored using carbon, where as white paint is colored using

titanium dioxide (Woodford, 2013). Exposure to titanium dioxide has negative effects on

invertebrate species if ingested, suggesting that our method of treating individuals may

have caused mortality (Lovern and Klapper, 2009). Heavy rain during our trials may have

also contributed to higher than expected mortality rates, however we were unable to

quantify this effect.

To our knowledge this is the first study which investigated the effect of UV

radiation on E. carunculatum. One study by Beatty et al (2010) explored the implications

of small changes in weather and temperature on damselflies and also failed to find

significant results. A compilation of these results may show that damselflies are not

effected by climate change, however more studies would be needed to confirm this

pattern.

While our results were insignificant, they provide useful information on how

future research should approach the question. Climate change may have significant

impacts on invertebrate species such as E. carunculatum, and it is crucial to determine

whether these effects will be negative or positive.

FIGURES

Figure 1. Boxplot showing the average number of eggs for each treatment group: Figure does not show a significant difference in the number of eggs between treatment groups (ANOVA; F(2,28) = 2.302, p = 0.120, df = 28). Table 1. Results of a one-tailed t-test run to compare clutch size of control and black treatments. Figure does not show a significant difference between number of eggs found in each treatment.

Category Number Mean Standard Deviation

t-test value

P-value

Control 6 13.83 8.886 1.091 0.193

Black 10 21.10 14.138

     Control                      Black                  White  

Table 2. Results of a one-tailed t-test run to compare clutch size of white and black treatments. Figure shows a difference approaching significance (alpha less than 0.1) between number of eggs found in each treatment.

Category Number Mean Variance t-test value

P-value one-tail

White 10 2.156699 1.306 1.5302 0.0704

Black 13 2.8524 2.8525

Figure 2. Bar graph showing the percent mortality in each treatment type. Figure shows a significant difference between mortality of white (70%, n = 10), black (25%, n = 12), and control (0%, n = 6) treatments (χ² value = 18.52; d.f = 2, p ≤ 0.05).

0   10   20   30   40   50   60   70   80  

White  

Black  

Control  

%  Mortality  

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