Effects of Interaction between Temperature Conditions and Copper

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Article pubs.acs.org/est

Effects of Interaction between Temperature Conditions and Copper Exposure on Immune Defense and Other Life-History Traits of the Blow Fly Protophormia terraenovae Mari Pölkki,* Katariina Kangassalo, and Markus J. Rantala Department of Biology, Section of Ecology, University of Turku, FIN-20014 Turku, Finland S Supporting Information *

ABSTRACT: Environmental pollution is considered one of the major threats to organisms. Direct effects of heavy metal pollution on various life-history traits are well recognized, while the effects of potential interactions between two distinct environmental conditions on different traits are poorly understood. Here, we have tested the effects of interactions between temperature conditions and heavy metal exposure on innate immunity and other life-history traits. Maggots of the blow fly Protophormia terraenovae were reared on either copper-contaminated or uncontaminated food, under three different temperature environments. Encapsulation response, body mass, and development time were measured for adult flies that were not directly exposed to copper. We found that the effects of copper exposure on immunity and other traits are temperaturedependent, suggesting that the ability to regulate toxic compounds in body tissues might depend on temperature conditions. Furthermore, we found that temperature has an effect on sex differences in immune defense. Males had an encapsulation response at higher temperatures stronger than that of females. Our results indicate that the effects of environmental conditions on different traits are much more intricate than what can be predicted. This is something that should be considered when conducting immunological experiments or comparing results of previous studies.

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INTRODUCTION Human-caused environmental pollution is a vast growing problem around the world. Concentrations of toxic compounds detected in the vicinity of industrial areas can be considerably high.1,2 Many pollutants, for instance, heavy metals, are transferred from soil to producers and eventually to other trophic levels.3−6 Furthermore, heavy metals have been found to accumulate in various plant, invertebrate, and vertebrate species.1,6 Additionally, exposure to heavy metals affects species composition and has a negative influence, for instance, on the functioning of ecosystems.2 Environmental conditions are generally considered as prominent factors that influence the development and overall condition of individuals. Most notably, environmental conditions experienced during early development have a strong impact on different adulthood traits.7−9 For insects and other ectothermic organisms, one of the most crucial abiotic environmental conditions is temperature. Dramatic changes in temperature are known to have an effect on survival and various traits. Temperature conditions experienced during early development have an influence on development time10−12 and adult size.11 In general, ectothermic organisms develop notably faster in warmer temperatures,10,13 until a certain limit. Negative effects appear when the temperature differs excessively from optimal conditions, which can elongate development time.11 However, there are differences between species and populations.10 Recently, a few studies have demonstrated that short- and long-term heat-shock © 2014 American Chemical Society

exposure has an impact on immune defense. In the study by Wojda and Jakubowicz,14 larvae of Galleria mellonella were found to have a better immune response when reared at a mild heat-shock temperature of 38 °C compared to the individuals raised at 28 °C. Similarly, after a short-term exposure to the lethal temperature of 43 °C, the larvae of G. mellonella showed stronger resistance against the fungus Beauveria bassiana.15 However, experiments considering the effects of temperature conditions on immune functions have been limited. Previously, various studies have reported that exposure to heavy metals has disadvantageous effects on different conditiondependent traits, for instance, development time, size, survival, longevity, and fecundity.16−20 Furthermore, some studies have revealed that heavy metals have an effect on the ability of individuals to resist pathogens.8,16,21−23 Individuals exposed to high concentrations of heavy metals are discovered to be more sensitive to both real pathogens21 and an artificial antigen,22,23 whereas smaller quantities have an intensifying effect on immune responses.8,23 Even though the direct effects of heavy metal pollution on the immune system and other life-history traits are fairly well known, the potential interactive effects of Received: Revised: Accepted: Published: 8793

August 28, 2013 June 3, 2014 June 13, 2014 June 13, 2014 dx.doi.org/10.1021/es501880b | Environ. Sci. Technol. 2014, 48, 8793−8799

Environmental Science & Technology

Article

libitum (Pirkka beef pâté) but otherwise maintained in a similar manner. Flies were allowed to acclimatize to the laboratory conditions for several generations before the experiments were conducted.8 Experimental Design and Treatments. For the experiment, eggs were harvested from the stocks and transferred randomly into six vials containing 50 g of either uncontaminated (control) or contaminated (treatment) food (beef pâté). Vials were then covered with foil. Contaminated food was supplemented with 200 μg of copper sulfate/g of larval food (Sigma-Aldrich, copper CuSO4, 10.00 g for a 1 L standard solution, diluted in deionized water), whereas uncontaminated food contained the same amount of distilled water. Vials were randomly placed into controlled temperature conditions at 23, 28, or 33 °C, and one contaminated vial and one uncontaminated vial were chosen for each temperature condition (approximately 200 eggs per vial). To avoid mortality-related selection, both the temperature conditions and copper concentration were preliminarily determined to have no significant effect on the survival of maggots or adult flies.8 Similar or even higher copper concentrations can be found near pollution sources2,38 that animals have to confront in nature. When the maggots were in the second instar, they were placed into 200 mL plastic vials containing 45 g of either contaminated or uncontaminated food (eight food treatment vials representing four uncontaminated and four contaminated vials within each of three temperature conditions, 24 rearing vials in total with 40 maggots each). Individuals were kept under the same food treatment and temperature conditions throughout their development. Vials were covered with foil and put into jars containing a 2 cm layer of sawdust at the bottom for pupation. Pupae were collected daily and placed individually into plastic vials. Hatched adults were checked and transferred once a day to cages (21 cm × 21 cm × 15 cm) containing dry powder food (see above) and fresh water ad libitum. Males and females were kept in separate cages to guarantee their virginity. Only adult flies were used in the immune and size assays. During the experiment, adult flies were kept under the same temperature conditions as the maggots (23, 28, or 33 °C). Immune Assay, Adult Size, and Development Time. Two days after eclosion, the encapsulation response of the adult flies was measured with nylon monofilament implants that were used as an artificial intruder to activate the immune system of the flies.39 The left side of the thorax was pierced by a sterile needle, and a 2 mm piece of nylon monofilament (0.18 mm in diameter) was inserted through the piercing. The flies’ immune system was allowed to activate and react with the foreign particle for 4 h before removal of the implant. Prior to implantation and removal of the implants, flies were anesthetized with carbon dioxide.8 To measure the encapsulation rate, monofilament implants were photographed from both sides and the average darkness (representing the formed capsule) was analyzed with ImageJ version 1.42 (see refs 8 and 39). Received values were then scaled so that highest darkness corresponds to the most intense encapsulation [an artificial unit (see refs 8 and 39)]. This method has been found to be a sufficient measure for testing individuals’ capacity to resist and encapsulate real pathogens.40,41 To obtain dry body mass, flies were dried at 60 °C for 24 h and weighed to the nearest 0.1 mg. To determine the development time, hatched adults were checked daily and the

heavy metal exposure and other environmental conditions on immunity are poorly understood. The immune defense systems of invertebrates and vertebrates share some homologous structural elements.24−28 The main difference is that invertebrates’ immune defense is based principally on innate immunity.24 This is one reason why insects are suitable model systems for immunological studies. Insects’ innate immune system can be divided into two distinct branches: cellular and humoral responses.29,30 Of these, the humoral defense system produces a variety of different kinds of circulating secretions, antimicrobial peptides (AMPs), which are used as an ancillary defense.29−31 The cellular process is based mainly on encapsulation response, nodulation, and phagocytosis, whose primary function is to destroy multicellular intruders.29,30 Of these, the primary defense mechanism against multicellular intruders29,32 and viruses33 is encapsulation response. Briefly, the complex encapsulation process starts from the recognition of the foreign particle, after which the novel intruder is isolated and destroyed by encapsulating it with multiple layers of hemocytes.29,32 However, to the best of our knowledge, studies testing the impact of two distinct environmental conditions on immune defense have been scarce, although there have been some recent multiple-stressor studies.34,35 The main purpose of these experiments was to study the impact of heavy metal exposure and temperature conditions on the innate immune system, size, and development time. More specifically, the aim was to test the potential interactive effects of different temperature conditions and heavy metal exposure on innate immunity and other traits of males and females. Possible sex differences in immunity and other traits under two distinct environmental conditions are not well-known. Here we measured the immune response by assessing the encapsulation rate against an artificial intruder, using the blow fly Protophormia terraenovae (Diptera: Calliphoridae; RobineauDesvoidy, 1830) as a model. Copper (Cu) was used to demonstrate the effects of heavy metal exposure on different traits. Copper is required for several metabolic processes as an important trace element, which is why it is thought to be more easily regulated in tissues than nonessential heavy metals.36,37 Even though copper is vitally needed, exposure to high concentrations is disadvantageous.17 The concentration used here was sublethal and selected to correspond to dosages found in polluted areas2,38 (see also Materials and Methods). For the experiment, maggots of P. terraenovae were reared under three different temperature environments with food supplemented with copper or uncontaminated food. The encapsulation response against nylon monofilament, development time, and dry body mass were measured for adult flies. Our findings show that (1) the strength of the encapsulation response in uncontaminated or contaminated groups is temperaturedependent and (2) males and females differ in their immune responses depending on temperature conditions.

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MATERIALS AND METHODS Stock Population. Individuals used in this experiment were obtained from a wild originated laboratory stock population (based on more than 600 flies captured in Turku in 2009). Adult flies were kept in several large (70 L) stock cages at a constant temperature of 24 ± 1 °C under constant light and were sustained with dry powder food (1:1:1 semolina/sugar/ baby’s milk formula mixture, containing altogether 10% dry yeast) and water ad libitum. Maggots were fed cat food ad 8794

dx.doi.org/10.1021/es501880b | Environ. Sci. Technol. 2014, 48, 8793−8799

Environmental Science & Technology

Article

time that elapsed between oviposition and adult eclosion was used to define the development time (in days). Statistics. The normality of the data was estimated using the Kolmogorov−Smirnov test and alternatively from the residuals. The equality of variances was checked using Levene’s test. In case variances were not equal (body mass), we reduced the significance level to P ≤ 0.01 to control the reliability of the P values. Variables that differed from the assumptions required for normality (body mass) were transformed (square-root transformation). If normality assumptions were not achieved, the variable was analyzed with nonparametric tests instead [development time (see below)]. Otherwise, univariate analysis of variance was used to discover the differences between treatment groups (encapsulation response and body mass). In the final analysis of variance (ANOVA) model, copper treatment (uncontaminated vs contaminated), temperature condition (23, 28, and 33 °C), and sex were used as fixed factors. Body mass or encapsulation response was used as a dependent variable. “Rearing vial” was a nonsignificant factor and was excluded from the final ANOVA model. The effect of rearing vial was analyzed using continuous vial number as a random factor. Pairwise comparisons between groups were conducted by using contrasts (body mass). Sidak post hoc tests were conducted separately for each of the sexes (encapsulation response). Sequential Bonferroni corrections were applied to correct the P values for multiple tests. Development time was analyzed using Cox regression, in which temperature condition, exposure to copper, and sex were used as categorical covariates. Kaplan−Meier survival analysis was used to compare the differences in development time between treatment groups. Because the number of different treatment groups was high, a reduced significance level was used (sequential Bonferroni corrections) in multiple comparisons to attain reliable P values in the final results of the Log Rank (Mantel-Cox). All statistical tests were conducted using IBM SPSS Statistics 21 for WINDOWS.

Table 1. Summary of Analysis of Variance of the Encapsulation Response Conducted Separately for Males and Femalesa df

a

temperature exposure interaction error

2 1 2 316

temperature exposure interaction error

2 1 2 374

type III SS

MS

Females 4794.643 2397.322 675.309 675.309 242.074 121.037 98610.649 312.059 Males 8051.799 4025.90 4747.772 4747.772 913.600 456.800 114196.947 305.339

F

P

7.682 2.164 0.388

0.001 0.142 0.679

13.185 15.549 1.496