The effects of temperature stress and
ivermectin on the development time of yellow dung flies (Scathophaga stercoraria)
Warren Kunce
Degree project in biology, Master of science (2 years), 2012 Examensarbete i biologi 45 hp till masterexamen, 2012
Biology Education Centre, Uppsala University, and Department of Ecology, Swedish University of
Table of Contents
Abstract………...……....3
Introduction………...4
Methods………...5
Results………...8
Discussion……….10
Conclusion………...…….12
Acknowledgements………...…....13
References……….14
Abstract
In the face of global environmental change, wild animal populations are frequently subjected to multiple environmental stressors. While a population may remain healthy under the pressure of one stressor, it could be devastated under the negative influence of combined or synergistic effects of multiple stressors with different modes of action. The knowledge and techniques required to investigate the effects of stressors likely to be found together in nature, cross academic disciplines and are thus seldom brought together in the necessary
interdisciplinary approach. Using yellow dung flies (Scathophaga stercoraria; YDF) as a
model system I tested the synergistic effects of two anthropogenic stressors on the larval
development time in a common garden experiment. I used the common veterinary vermicide,
ivermectin (IV), known to have a detrimental effect on many species in the dung fauna as a
toxicological stressor. Since YDFs are known to be cold-tolerant and generally avoid high
temperatures, I also subjected them to a high temperature stress to simulate a climate change
stressor. A linear model fitted to the data reveals that larval development time, as expected for
ectotherms, increase with decreasing temperature while the IV contamination prolonged the
development period. In combination, the two stressors hasten development but not to the same
extent as temperature alone. The sexes reacted similarly to temperature stress and IV presence
when they were applied independently as solo stressors. However, when there was both a high
temperature stress and IV present there was a difference in response between the sexes.
Introduction
Ecotoxicology and risk assessment in particular, traditionally focus on the sources,
distribution, fate and effects of isolated chemicals within biological systems. Only recently has the discipline evolved to include the investigation of the chemical mixtures that more accurately reflect environmental conditions. However, there is still little known about the consequences of chemical pollution at the population level particularly in comparison with influence of other significant stressors such as over-exploitation, habitat loss or climate change (Cairns 2002).
There is mounting evidence that regional temperature increases are already affecting natural biological systems in the terrestrial ecosystem through poleward or elevational geographical habitat shifts, the timing of growth stages, migration alterations and changes in the abundance of specific species (IPCC 2007). Thus it is highly likely that wild populations are
encountering ecotoxicological stress in combination with rising temperatures. Yet, there is little information available on the effects of this combination of stressors. One study of the interactions between realistic levels of summer drought and a common contaminant of
agricultural soil (4-nonylphenol, NP) on the soil invertebrate, Folsomia candida, revealed that the exposure to NP significantly reduced the drought tolerance and reciprocally, the toxicity of NP during drought conditions more than doubled (Hojer et al. 2001). These synergistic results point to a need for a greater understanding of the effects of multiple anthropogenic stressors.
The vermicide, ivermectin (IV), is one of the most frequently studied veterinary
pharmaceuticals in ecotoxicology due to its persistence, toxicity and wide distribution (Floate et al., 2005). IV is used globally to treat cattle infected with parasites and residues of the drug are present in the dung for weeks or months after treatment (Floate 2008). The flies and beetles living and breeding in the dung are the most vulnerable populations to non-target effects (Rombke 2010). These invertebrate communities are not only responsible for the degradation of the dung and thus play a significant role in pasture hygiene (Floate 1998) but are also an important food supply for many bird and mammal species particularly during winter when food is scarce and also during the spring nesting period (McCracken 1993). The yellow dung fly (Scathophaga stercoraria; YDF) adults are also known to be an important food source for many bat species (McAney and Fairley 1989; Jones 1990 and Siel et al., 1991). Due to their prominent ecological role, wide distribution, short life-cycle, lack of obligate diapauses, sensitivity to dung contamination and the ease with which they can be reared in the lab, YDFs are often used as an indicator species for IV toxicity in dung dependent invertebrates (OECD 2008).
All invertebrates are sensitive to fluctuations in temperature. Since the increase in variability of daily temperature observed from 1977 to 2000 is due to an increase in warm extremes as opposed to a decrease in cold extremes (Klein Tank et al. 2002; Klein Tank and Können 2003) organisms sensitive to heat are likely to be experiencing temperature related stress.
YDFs are mainly distributed in cooler climates at either high latitudes or high altitudes.
During the peak summer temperatures they seek out cooler, forested areas. These patterns suggest a cold tolerant species sensitive to high temperatures (Ward and Simmons 1990;
Blankenhorn et al. 2001; Laugen et al. unpublished data).
YDFs are considered a suitable indicator species for estimating developmental toxicity in dung dependent Diptera because they are widespread across the globe, do not undergo obligate diapauses, are easy to rear in the lab, have a short life-cycle, have a prominent ecological role as both predators and prey and are very sensitive to chemical dung
contamination (OECD 2008). In fact, the registration of new veterinary compounds will soon require standardized bioassays using YDFs (Römbke et al. 2009). Even though YDFs can thrive on the dung of many large mammals such as sheep, horse, deer or wild boar, they are considered to be cow-dung specialists (Blackenhorn 2010).
Using YDFs as a model system, I tested the synergistic effects of IV and heat stress on larval survival and development time in a common garden experiment. Moreover, the experimental design allowed testing for sex differences in sensitivity to multiple environmental stress.
Methods
Dung Collection and Preparation
The dung was collected from a cattle farm in Mjölsta, Alunda, Sweden (60°0’48.92”N, 17°59’26.12”E) where the cows are known to have not been treated with pharmaceutical agents for at least 3 months prior to the collection, then homogenized with a cement mixer and stored in -80°C for a minimum of 2 weeks to kill any eggs/larvae of higher taxa that could be potential competitors.
A relevant environmental level of Ivermectin (IV) proven to have a slight negative impact on YDF emergence is 6.4μg / kg FW (Römbke et al. 2009). Due to the potency of Ivermectin powder (CAS 70288-86-7) compared to the low concentration desired for the experiment, first an IV stock solution was prepared by dissolving 4.22 mg IV powder in 100 ml of acetone. 5 ml of the stock solution were then diluted with 50 ml acetone giving a solution with a
concentration of 3.84 μg IV/ml. The 6.4μg / kg FW IV dung mixture was prepared by mixing 1 ml of solution 1 with 0.6 kg dung in 10 L buckets with a cement mixer. The control dung was prepared in separate 10 L buckets with the equivalent amount of acetone sans IV.
The dung in 10 L buckets was homogenized with a cement mixer and left to stand overnight
for the treatment solvent to evaporate prior to use. It was stirred for evenness before being
dispensed in 50 g allotments into 250 ml transparent plastic flasks. The flasks were plugged
with a permeable, cellulose acetate stopper and labeled IV (with ivermectin) or NO (control)
to specify the dung treatment.
Experiment Setup and Matings
In order to eliminate extraneous environmental effects from the field environment, the adult YDFs mated in this study were the third generation reared in the lab. The initial generation of flies was collected in September 2010 from Mjölsta, Alunda, Sweden (60°0’48.92”N,
17°59’26.12”E). After collection, the mating pairs were immediately transferred to polypropylene tubes (28.5 x 95mm) containing a filter paper smeared with dung and
transported to the lab. After mating, the males were removed and females allowed to lay eggs in the dung smear. After completed egg laying the female was removed and the eggs were divided into flasks (n= 10 – 15) of approximate 90 ml of homogenized cattle dung. Full- sibling matings were conducted for additional two generations to remove any non-genetic maternal effects (Mousseau & Fox 1998). The first two generations were reared in controlled temperature chambers at 20 °C, while the third generation was reared in 19 °C. The light/dark cycle was 16 h / 8 h for all generations. Larval development takes about three weeks under these conditions and after emergence, each adult fly was put in individual 250 ml flasks and given a water soaked sponge, sugar crystals and approximately 30 live Drosophila prey per week. While adults may survive on sugar and water live prey is necessary for gamete production (Foster 1967).
Figure 1. Experiment setup of matings and the distribution of eggs into the different treatment groups: NO indicating dung without ivermectin and IV indicating dung with ivermectin. The letters A and B indicate replicates of the same experimental treatment. Each male was crossed with 3 non-sibling females.
The third laboratory generation was used as parental generation for this experiment. 40 males from different families were each crossed with 3 non-sibling females as illustrated in Figure 1.
The matings were conducted in transparent polypropylene tubes (28.5 x 95mm). A filter paper with an even thin layer of IV free dung was placed inside the mating tube with the pair to be mated. The copulation commencement and conclusion as well as the time when the female started laying eggs were recorded in order to keep track of how well the chosen males and females were mating in case they should be re-mated or replaced due to lack of prowess or failure to lay. If the female did not lay eggs, she was mated again with the same male the following day with a fresh smear of dung. If she did not lay eggs after another 24 hours, she was replaced by another female. Each egg clutch was assigned a family designation indicating the identity of the sire and the dame.
Egg Division
The freshly laid eggs were counted through a binocular microscope and carefully transferred to wet pieces of filter paper, 7 eggs a piece. The time of the division of the egg clutch and the total number of eggs were recorded as well as which treatments and replicates received them.
T he eggs from each female were placed into 4 different treatment groups in order to assess the combined impact of IV and temperature stress on larval survival and development time.
The two temperatures in this scheme, 19 °C and 23 °C, were chosen because 19 °C is
considered a non-stressful temperature for YDFs whereas 23 °C is proven stressful in terms of lower survival (Laugen et al. unpublished data). The eggs were therefore divided into 4 flasks/groups with 2 replicates for each group if there were enough eggs: Temperatures 19 °C and 23 °C and within each temperature: without IV (NO A and NO B) and with IV (IV A and IV B) as illustrated in Figure 1 with 7 eggs per group. The minimum number of eggs used from a female was 28 (7 eggs per each treatment group) and the maximum was 56 (2 flasks of 7 eggs per each treatment group). After the first 28 eggs were distributed to each treatment group, if there were less than 56 eggs, the treatments that received the replicates were chosen by a randomization procedure. If a female laid less than 7 eggs in total, the eggs were
discarded and the male was mated with a replacement female.
The filter papers holding the eggs were then each placed into the specified treatment/replicate flask. The flasks were labeled with the family designation, treatment and replicate letter and then stored in climate chambers at 19 °C or 23 °C and a light/dark cycle of 16 h / 8 h. After 48 h, the number of hatched eggs were counted through a binocular microscope and recorded in order to differentiate between hatching success and larval survival.
Emergence
Adult flies usually emerge after a total pre-adult (egg + larval + pupal) development time between 17 and 80 days depending on the temperature with flies from the same family
emerging across several days (Blackenhorn 2010). To determine the larval development time,
the flasks were checked once a day before 10:00 AM for emerging flies beginning 18 days
after the first eggs were laid. The emergence day and sex were recorded for each fly. The data
is separated by sex since males are known to develop slower than females on average and
because there may be difference in stress tolerance between the sexes. Emerged flies were saved at -20 °C for future morphological measurements. The total number of emerged flies for each treatment group is available in Table 1.
Statistical Analysis
The data was checked for homogeneity of variances and normal distribution to satisfy the assumptions of analysis of variance. The half-sib design of this study implies that the
offspring of the same sire are more similar to each other than larvae of different sires, and the individual data points are therefore not independent. To ensure independent data points, the mean development time for all of the offspring of each sire in each treatment category was used as response variable. Sample sizes for each treatment category are given in Table 1.
Table 1. Number of sires and total number of offspring emerged per experimental treatment combination.
Treatment No. of Sires No. of Offspring Emerged
19 ° C 57 3340
19 ° C + IV 57 3366
23 ° C 56 3303
23 ° C + IV 56 3320
I fitted a general linear model to the data as follows
ݕ
ൌ ߤ ߙ
ߚ
ߛ
ߙ
ߚ
ߙ
ߛ
ߚ
ߛ
ߙ
ߚ
ߛ
߳
where the individual observation ݕ
is the sum of the grand mean ߤ, the effect of the ith level (low or high) of the temperature treatment (ߙ), the effect of the jth level (present or absent) of the IV treatment (ߚሻ and the effect of the kth category (male or female) of sex (ߛ) and the interactions between these variables (߳ሻ.To allow interpretation of main effects of treatments independently of interaction effects, the input variables were centered by subtracting the mean from all the values as outlined by Schielzeth (2010). The level of statistical significance is P = <0.5.
Results
The effects of temperature stress and IV on the development time of the YDFs is illustrated in
Figure 2 and parameter estimates from the statistical model in table 2. The mean development
time for the entire dataset regardless of sex or treatment was 21.67±0.05 days (Table 2). The
main effects of the statistical analysis (Table 2) are interpreted as follows. Males, on average
required an additional 1.62±0.10 days of development time than females. In the higher
temperature (23 °C) larvae developed on average 4.32±0.10 days faster compared to the lower temperature (19 °C). The IV treatment, on the other hand increased the development time by 2.85±0.10 days compared to those developing in the absence of IV.
As this was a fully factorial experiment, the higher order interactions indicate whether the development time in one experimental group depends on the value of one or more of the other groups. Thus the parameter estimates of higher-order interactions are not straight forward to interpret and thus the group averages are illustrated in Fig. 2 for interpretation. The interaction between temperature and IV is not statistically significant indicating that the change in
development time between the two temperatures is independent of the IV treatment. The sexes reacted similarly to treatments when they were applied as solo stressors as indicated by the non-significant two-way interactions sex x temperature and sex x IV. However, as
indicated by the significant three-way interaction (sex x temperature x IV), when temperature was high and IV present there was a difference in response between the sexes.
Within each treatment category, the females develop quicker than the males. The high temperature (23 °C) without IV decreased the average larval development time by 3.77 days for males and 4.47 days for females (Fig 2, Table 2). IV exposure alone, on the other hand, increased the larval development time by 3.13 days for females and 2.93 days for males (Fig 2, Table 2). Combined, the high temperature stress and IV contamination decreased the development time by 1.41 days for females and 1.52 days for males (Fig. 2, Table 2).
Table 2. Parameter estimates from a linear model of larval development time (days) in yellow dung flies (Scathophaga stercoraria) from a factorial laboratory experiment with two experimental treatments; temperature 19 °C and 23 °C and the veterinary vermicide, ivermectin (IV) present at a concentration of 6.4μg / kg FW or absent (See Statistical Analysis p. 5). To allow interpretation of main effects of treatments independently of interaction effects, the input variables were centered by subtracting the mean from all the values. Because the explanatory variables are categorical, the statistical analysis uses one of the levels as a baseline and only returns parameter estimates for the remaining level, indicating the deviation of the focal group from the baseline group (Schielzeth 2010). The intercept is the grand mean development time regardless of sex and treatments.
Abbreviations: males (M), high temperature (H) and present IV (P).
Estimate±SE t-value P-value ___________________________________________________________________________
Intercept 21.67±0.05 428.51 <0.001
Sex(M) 1.62±0.10 15.97 <0.001
Temperature(H) –4.32±0.10 –42.67 <0.001
IV (P) 2.85±0.10 28.13 <0.001
Temp(H) x IV(P) –0.37±0.20 –1.84 0.067
Sex(M):Temp(H) –0.28±0.20 –1.41 0.161
Sex(M):IV(P) 0.21±0.20 1.05 0.296
Sex(M):Temp(H):IV(P) 0.84±0.40 2.08 0.039
___________________________________________________________________________
Figure 2. The average larval development time (days, mean ± SD) in male (triangles) and female (circles) yellow dung flies (Scathophaga stercoraria) from a factorial laboratory experiment with two experimental treatments; temperature 19 (low) and 23 (high) °C and the veterinary vermicide, ivermectin (IV) absent (solid line) or present (dashed line).
Discussion
As expected for ectotherms, increased temperature decreases the observed development time
while the IV exposure prolonged it. Together the two treatments hastened development but
not to the same extent as temperature alone. The development time for males and females
reacts similar to an increase in temperature and IV presence when they are applied as solo
treatments. However, when both stressors are present simultaneously, the IV increases
development time more in males than in females
I chose a sublethal IV concentration according to (Römbke and Floate, et al. 2009) and while my chosen non-stressful temperature was one degree lower than in their study (20 ±2 °C vs.
19 °C), the results are still comparable. The observed effects of 6.4 μg/kg FW IV on the development time of YDF larvae at 20 ±2 °C for several studies have been summarized (Römbke and Floate, et al. 2009) and are similar to the results of this study for IV-exposed flies that developed in 19 °C.
Development and growth rates in ectotherms increase with increasing temperature (Angilletta 2009). While there is evidence for genetic variation in development and growth rates both between and within populations (Angilletta 2009) a substantial amount of variation in development and growth rates is environmentally driven. In other words, if ectotherms are subjected to higher temperatures, their development and growth rates increase. However, because of the difference in limiting physiological mechanisms between development (mainly cell division) and growth (mainly through protein synthesis), development rate has a tendency to increase faster with temperature than growth rate (van der Have and de Jong 1996).
Conversely, as temperature decreases, development rate slows down faster than growth rate.
This means that at a given development stage (such a as metamorphosis or eclosion) individuals raised in higher temperatures will be smaller than individuals raised in lower temperatures.
The amount of thermal stress experienced by an organism under a certain thermal regime depends on the thermal performance curve of the organism (Angilletta 2009).The
performance of an organism is often more precipitous at temperatures above the optimal range than below it (Scholwalter 2009).When faster development results in a less fit organism due to trade-offs with other fitness traits such as survival or larger body size it can be considered stressful. Additionally, while shorter development time can be beneficial in time-constrained environments such as high-latitudes (Laugen et al 2003) or ephemeral habitats (Lind et al 2008) it may incur a cost if metamorphosing or enclosing with a smaller size. For instance, larger females can often produce more or larger eggs than smaller females and larger males are often more successful in competing for females (Blankenhorn 2009). Increased
development rate may also trade off against survival or developmental stability (Blankenhorn 1997). Moreover, there is evidence that male YDFs raised in 23 °C are less successful in sperm competition experiments than males raised in 15 °C (Laugen et al unpublished data) and that females raised in 23 °C are less willing to mate compared to their counterparts raised in 19 °C (Laugen et al unpublished data).
This study supports previous findings that IV alone prolongs development time (Römbke 2009). IV has also been shown to reduce body mass implying retarded larval growth (Id.).
Since IV is retarding growth and higher temperatures are speeding up the development, it is
possible that the combination could result in a greater body mass reduction than IV alone. The
different responses between the sexes when exposed to both stressors could be related to
sexual dimorphism, since males are, on average, larger than females. The next step in this
study is to analyze the morphology of both the male and female flies from each treatment
group to determine if there are, indeed, synergistic effects on the size morphology and to look
for further clues to the differing response between the sexes.
In addition to larval development, this study also collected data on larval survival (i.e. the proportion of eggs laid to emerged adults). An elucidation of potential synergistic effects of IV and high temperature on survival is imperative to drawing definite conclusions,
particularly on population dynamics. The future completion of the analysis of this data will likely provide further significant insights.
There is some evidence that raising YDFs in a constant temperature in the laboratory as opposed to fluctuations in temperature in the field environment is a stress that could have implications on development itself (Blankenhorn 1999). Further investigation into the importance of temperature fluctuations should be conducted to improve the robustness of laboratory studies of development.
Significant reductions in body size, fitness and fecundity as a result of climate change and chemical induced stress could influence the population dynamics and therefore also the ecological niche, as well as natural selection in the wild. The experimental design of the present study allows for future quantitative genetics analysis of the results. Such an analysis will shed light on the evolutionary impact of these stressors by teasing out the environmental effects on variation from the parental effects.
Conclusion
Larval development in YDFs is influenced differently by the combination of IV and high temperature stress than by these stressors in isolation with a difference in the effects
according to sex. Further investigation into the impact of temperature fluctuations on larval development would improve the robustness of laboratory studies on dung fly development.
Analysis of the larval survival data from this study will provide insight into the possible synergistic effects of temperature stress and IV exposure on YDF survival. Future
morphological analysis may provide insight on the potential impact on population dynamics by evaluating possible synergistic effects on body size, fitness and differing responses
between the sexes. A future quantitative genetics analysis could shed light on the evolutionary impact of these stressors by teasing out the environmental effects on variation from the
parental effects.
Acknowledgements
It is a pleasure to thank those who made this thesis possible. I would like to gratefully acknowledge Dr. Ane T. Laugen for contributing her time, funding and teaching into supervising this work. In addition to my gratitude for all that I learned under her guidance, I am also deeply thankful for her support both academic and emotional during my recovery from surgery. Financing was provided by the Marie Curie European Reintegration Grant
"SUPAFLY" (Grant agreement PERG05-GA-2009-247995) and research support funding from the U it of Population Ecology, Dept of Ecology, SLU, both awarded to Dr. Ane T.
Laugen.
Dr. Beatrice Lindgren is to be thanked for her insights during the planning process and her invaluable camaraderie in the lab. Many aspects of this project would not have been possible without her or the greatly appreciated assistance of Berrit Kiehl, Ling Shen, Ellen
Salomonsson, Rishu Vallabhu, Simon Leivo, Karin Lundin and Carin Eriksson. For his detailed and thoughtful feedback on the manuscript, I thank Jan Örberg.
Lastly, I would like to thank my family for their love and support. Despite the geographical
distance, my father’s love, encouragement and confidence in me was never lacking during the
entirety of my studies. Sofia managed to deftly keep my priorities in perspective; Miguel was
there to pick me up whenever I fell and Simon was my beloved companion.
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