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Morphological deformities and levels of fluctuating asymmetry in organisms are caused by several factors, such as developmental instabilities, environmental stress, and genetic factors 1. The structural development of mouthpart morphology, in chironomids, for instance, is connected with the organisms' exposure to anthropogenic environmental pollutants 2. Therefore, the method has frequently been tested and proposed as a suitable biomonitoring tool for the presence of toxicants in water bodies 3, 4. However, one difficulty in interpreting the outcomes of such studies is to distinguish the effects of external environmental stress and internal genetic stress, as may be caused by increasing frequencies of recessive deleterious mutations in a homozygous state through inbreeding. High levels of inbreeding have phenotypic effects (inbreeding depression) in most studied naturally outcrossing organisms, such as decreased reproductive fitness or morphological deformities 5. For instance, the detrimental effects of inbreeding on fitness and the resulting implications for toxicological bioassays have been shown for laboratory strains of the ecotoxicological model organism Chironomus riparius6, 7. However, no study has yet examined the potential impact of inbreeding on mouthpart deformities, despite its importance for sound interpretations of biomonitoring approaches using this parameter. To test the impact of low genetic diversity and inbreeding on the level of mouthpart deformities in chironomids, we used a collection of ethanol-fixed larvae of the species from a large-scale multigenerational study, which was conducted with one highly inbred and one outcrossed C. riparius strain 8. Both strains were exposed to a sublethal concentration of the antifouling agent tributyltin (TBT). We analyzed the proportions of commonly investigated mouthpart deformities and compared them with levels of genetic diversity and life history. We aimed to answer two specific questions: (1) Do inbred C. riparius cultures show significantly enhanced levels of mouthpart deformities compared with outcrossed strains? (2) Are mouthpart deformities a suitable biomarker for the indication of detrimental effects of a toxic pollutant (TBT) in a sublethal concentration?
MATERIALS AND METHODS
A multigeneration experiment was performed with two C. riparius strains. One strain was obtained from crossbreeding of 11 C. riparius cultures from different laboratories (GEN+) 6, 8. This strain was shown to have a similar level of genetic diversity compared with diverse field populations. We considered this strain as outcrossed. In contrast, a second strain (GEN-) was used in parallel. The GEN- is a highly inbred laboratory culture that was kept in the laboratory for several years without any genetic refreshing from field populations or other laboratory strains 9.
Both GEN- and GEN+ were exposed under standardized laboratory conditions for 10 consecutive generations under moderate TBT stress (nominal TBT concentration of 80 µg as Sn/kg sediment dry wt). A solvent control was run in parallel. A detailed description of the experimental procedure can be found in Vogt et al. 8 and Nowak et al. 9. Besides recording life-history parameters, levels of genetic diversity were monitored as average heterozygosity levels across five variable microsatellite loci 10 over the course of the experiment 8.
Head capsule analysis
Before microscopic preparation, the ethanol-fixed head capsules were separated mechanically from the rest of the larval exuviae and stored in Roti-histol (Carl Roth GmbH) overnight for dewaxing and dehydration. For preparation, the head capsules were mounted with the ventral side up on a glass slide in Roti-Histokit (Carl Roth GmbH), covered with a glass coverslip, and squeezed gently. The menta were evaluated visually with a light microscope, using 40-fold magnification (Zeiss Axiostar plus, Germany). As deformities, missing teeth, extra teeth, and Köhn gaps were counted 4, 11–13. During observations, special care was taken to distinguish deformities from physical wearing. The ratio of individuals with deformed mouthparts to the total number of examined individuals was calculated 14. Additionally, the number of head capsules with a certain deformation of type and extent was set in relation to the total number of examined individuals. Head capsule deformities were analyzed for generations 1, 2, 3, 5, and 10 for GEN+ and GEN-. The number of total analyzed head capsules per generation and population varied between 9 and 49 because of differences in mortality and capsule recovery rates.
Statistical analysis of the data was performed with the software programme GraphPad Prism, Version 4.03 (GraphPad Software), for Windows and R Version 2.10 (http://www.r-project.org/). Differences in the occurrence of mouthpart deformities were analyzed using generalized mixed models with logit link and binomial error. Generation, treatment, and genetic variation were added as fixed effects to the model. Because repeated observations were analyzed within independent replicates, replicate (nested within treatment and genetic variation level) was added as random effect. Tests of the fixed effects were based on likelihood ratio tests.
RESULTS AND DISCUSSION
The rate of mouthpart deformities varied considerably among strains and generations during the multigeneration study (Fig. 1A). The GEN- showed higher rates of deformities compared with GEN+ (9.8–2.2% across all generations). This difference was greatest in the first generations, with mean deformity rates peaking at 24% (TBT treatment) and 32% (control). Interestingly, deformity rates decreased steeply after the first generation in both GEN- strains and remained in the range of GEN +, but with slightly higher (but not significant) values in most generations (Gen 3, 5, and 10). This observation can be explained by an increase in genetic diversity starting in the second generation of both treatments. Although microsatellite analysis indicated no allelic variation in the first generation in GEN-, at least some variability was detected in the second generation (Fig. 1B) and all further generations, except for generations 3 and 4 for the TBT treatment, which is likely attributable to an insufficient resolution provided by the five microsatellite markers. The increase in genetic variation, which is observed after the second generation of the Gen-SC strain, is most probably attributed to the appearance of previously undetected alleles within the first generations in the GEN- strains. This needs careful inspection, because it seems to contradict the predictions of genetic drift. The detection of higher rates of genetic variation (measured as expected heterozygosity) strongly indicates reduced inbreeding, which is confirmed by reduced inbreeding depression as documented by increased population fitness in the course of the study 9. In contrast to GEN-, the degree of mouthpart deformities in the outcrossed GEN+ strains remained on a relatively constant low level (Fig. 1A). This observation is in good agreement with constantly high levels of genetic diversity (Fig. 1B) and no signs of inbreeding depression in this strain 9.
In the present study, levels of mouthpart deformities of C. riparius were impacted by inbreeding rather than TBT exposure. Likelihood ratio tests of the occurrence of mouthpart deformities in relation to TBT exposure, genetic variation, generation, and all two-way interactions revealed no evidence for the impact of TBT on mouthpart deformity rates (Table 1). In contrast, significant effects were observed for the impact of the factors generation and strain (GEN- vs GEN + ), confirming the observation that inbreeding was the dominant factor explaining rates of mouthpart deformities in the present study. Although this observation might question the sound application of mouthpart deformities as an indicator of pollution, one has to note that inbreeding-induced deformity rates disappeared with slightly increasing levels of genetic diversity. Because very high inbreeding rates are rare in wild populations, and population sizes as well as gene-flow rates are usually high in Chironomus15, that inbreeding will impact rates of mouthpart deformities in natural populations of the investigated species seem highly unlikely. The lack of TBT impact on mouthpart deformities confirms the observation of overall low effects of this environmentally relevant exposure concentration on C. riparius16, 17. In summary, the present study cannot confirm the suitability of mouthpart deformity assessments in indicating sublethal TBT exposure from reared populations. Because strong inbreeding may severely increase deformity rates in this species, this factor has to be considered in laboratory studies, but it might not be relevant in field investigations. For routine ecotoxicological testing these results clearly indicate that a control group with the same genetic diversity is mandatory. The used animals should be derived from the same laboratory breeding stock to make the outcome of the experiments valuable. Furthermore, the results from pure laboratory studies should be discussed very carefully, because a translation to the more complex field situation is nearly impossible. Field populations of C. riparius have a higher degree of genetic diversity because of crossbreeding and therefore a lower sensitivity to head capsule deformities induced by stressors.
Table 1. Likelihood ratio tests of the occurrence of mouthpart deformities in relation to tributyltin (TBT) exposure, genetic variation, generation, and all two-way interactionsa
In all analyses, independent replicate was added as random effect. GEN = inbreeding level; TBT = TBT exposure.
GEN × TBT
GEN × generation
TBT × generation
The help of S. Well and S. Schmidt for collecting the head capsules is much appreciated. The present study was financed by the BW-Plus program “Programm Lebensgrundlage Umwelt und ihre Sicherung” of the federal state Baden-Württemberg (contract number BWR 22018). Additional funding came from the research funding programme “LOEWE–Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz” of Hesse's Ministry of Higher Education, Research, and the Arts.