Food quality effects on copepod growth and development: implications for bioassays in ecotoxicological testing.

We evaluated effects of six algal species in 25 combinations on growth and reproduction of the harpacticoid copepod Nitocra spinipes. In the first lifecycle test, Rhodomonas salina, Phaeodactylum tricornutum, and Dunaliella tertiolecta were used. The results showed that R. salina was the best food, whereas P. tricornutum (0% development success) and D. tertiolecta (41.7% malformations) were poor food items. In the second lifecycle test, a mixture of R. salina, Tetraselmis suecica, and Thalassiosira weisflogii (selected from screening tests) was tested together with a mono-diet of R. salina. Also in this test, copepods fed R. salina performed better (i.e. had higher survival and reproductive success) compared with the other treatment. We conclude that R. salina is appropriate to use as food in toxicity testing with N. spinipes, whereas some of the algae commonly used as feed in ecotoxicological tests with other copepods had detrimental effects on the development, reproduction, and survival of N. spinipes.

Expression of ecdysteroids and cytochrome P450 enzymes during lipid turnover and reproduction in Calanus finmarchicus (Crustacea: Copepoda).

The marine copepod Calanus finmarchicus is the most abundant zooplankton species in the northern regions of the Atlantic Ocean and the Barents Sea. Very little is known about molecular regulation of hormone metabolism, moulting and reproduction in copepods. To investigate these processes, we sampled adult male and female copepods (females at three distinct reproductive stages) and copepodites stage five (CV) from the culture at SINTEF/NTNU Sealab. Copepods were individually photographed, analyzed biometrically (body size, length and lipid storage size) and for ecdysteroid concentrations. In addition, we analyzed copepods for gene expression of three putative cytochrome P450 enzymes possibly involved in ecdysteroid regulation: CYP301A1, CYP305A1 and CYP330A1. The CV group exhibited the highest ecdysteroid concentrations and the largest lipid storage size, and a significant positive correlation was found between these parameters. Also, two of the P450 enzymes (CYP305A1 and CYP330A1) were more highly expressed at CV than at the adult stage, suggesting that these P450 enzymes are involved in ecdysteroid synthesis and lipid storage regulation. The expression of CYP330A1 was higher in newly moulted females than in females that had produced eggs. In addition, we observed that ecdysteroid concentrations were higher in females with large egg sacs, suggesting that ecdysteroids may be involved in egg maturation and reproduction. The CYP301A1 was more highly expressed in males and post-spawning females, and may be involved in ecdysteroid degradation since these groups also exhibited the lowest ecdysteroid concentrations.

Integrating individual ecdysteroid content and growth-related stressor endpoints to assess toxicity in a benthic harpacticoid copepod.

Anthropogenic chemicals released into the environment may have so-called endocrine disruptive effects. For instance, innumerous observations on subtle alterations of fish reproductive systems have been published in the scientific literature during the last decades. At the same time, the evidence is scarce regarding similar effects in crustaceans, which is probably related to the limited understanding of basic crustacean endocrine systems and pathways, rather than absence of endocrine disruption within the crustacean subphylum. This knowledge gap is particularly evident in micro-crustacean species, which are frequently used in environmental risk assessment of chemicals, and adequate tools for identification of potential endocrine disrupters are missing in current chemicals regulation. The main objective of the current study was therefore to utilize an enzyme immunoassay to establish a stable protocol for analysis of individual ecdysteroid levels in the benthic harpacticoid copepod Nitocra spinipes, a species which has been used in ecotoxicological investigations for more than 30 years. Further, to assess the usefulness of the individual ecdysteroid level as a stressor endpoint, it was integrated with a growth-related stressor endpoint battery, i.e. individual RNA content, mean development times, and growth rate, by exposing individual N. spinipes to the insecticide and known ecdysteroid antagonist lindane at 25-400 microg L(-1). The results showed that the ecdysteroid levels were significantly different from the control (71 pg individual(-1)) in the 100 microg L(-1) treatment (124 pg individual(-1)). Although the ecdysteroid levels were not significantly different from the control in the 25-50 microg L(-1) treatments (83-93 pg individual(-1)), these results still show a clear concentration-related trend. In the 200 microg L(-1) treatment, the ecdysteroid content was the highest (165 pg individual(-1)), however not significantly different from the control due to high variation. The 400 microg L(-1) treatment resulted in lowered ecdysteroid levels (107 pg individual(-1)), indicating a profound lindane-induced stress, which was confirmed by high mortality in the same treatment (79%). For all other endpoints there were clear concentration-related effects of lindane, with development time and growth rate as the most sensitive endpoints. In conclusion, this study presents for the first time a tool for identification of endocrine alterations in N. spinipes. By using the established enzyme immunoassay protocol, we obtained individual ecdysteroid levels that integrated well with the growth-related stressor endpoints previously used on this species.

Evidence of population genetic effects of long-term exposure to contaminated sediments-a multi-endpoint study with copepods.

In the environment, pollution generally acts over long time scales and exerts exposure of multiple toxicants on the organisms living there. Recent findings show that pollution can alter the genetics of populations. However, few of these studies have focused on long-term exposure of mixtures of substances. The relatively short generation time (ca. 4-5 weeks in sediments) of the harpacticoid copepod Attheyella crassa makes it suitable for multigenerational exposure studies. Here, A. crassa copepods were exposed for 60 and 120 days to naturally contaminated sediments (i.e., Svindersviken and Trosa; each in a concentration series including 50% contaminated sediment mixed with 50% control sediment and 100% contaminated sediment), and for 120 days to control sediment spiked with copper. We assayed changes in F(ST) (fixation index), which indicates if there is any population subdivision (i.e., structure) between the samples, expected heterozygosity, percent polymorphic loci, as well as abundance. There was a significant decrease in total abundance after 60 days in both of the 100% naturally contaminated sediments. This abundance bottleneck recovered in the Trosa treatment after 120 days but not in the Svindersviken treatment. After 120 days, there were fewer males in the 100% naturally contaminated sediments compared to the control, possibly caused by smaller size of males resulting in higher surface: body volume ratio in contact with toxic chemicals. In the copper treatment there was a significant decrease in genetic diversity after 120 days, although abundance remained unchanged. Neither of the naturally contaminated sediments (50 and 100%) affected genetic diversity after 120 days but they all had high within treatment F(ST) values, with highest F(ST) in both 100% treatments. This indicates differentiation between the replicates and seems to be a consequence of multi-toxicant exposure, which likely caused selective mortality against highly sensitive genotypes. We further assayed two growth-related measures, i.e., RNA content and cephalothorax length, but none of these endpoints differed between any of the treatments and the control. In conclusion, the results of the present study support the hypothesis that toxicant exposure can reduce genetic diversity and cause population differentiation. Loss of genetic diversity is of great concern since it implies reduced adaptive potential of populations in the face of future environmental change.

Application of growth-related sublethal endpoints in ecotoxicological assessments using a harpacticoid copepod.

In ecotoxicology, there is an increasing demand for sensitive sublethal endpoints. The primary aim of the present study was therefore to evaluate the relative sensitivity and usefulness of four sublethal endpoints - development time, body length, RNA content and growth rate - in the harpacticoid copepod Nitocra spinipes, using the reference molecule Simvastatin. Development time decreased significantly at low sublethal concentrations of Simvastatin (p < 0.001; F = 13.249; 0.16-1.6 microgL(-1)), while RNA content and body length increased significantly at 0.16 microgL(-1) (p < 0.001; F = 6.13) and 1.6 microgL(-1) (p < 0.01; F = 2.365), respectively. The growth rate increased significantly at 0.16-5 microgL(-1) (p<0.01-0.001). Hence, significant responses of growth-related traits were observed already at 0.16 microgL(-1), which is about 5,000 times lower than the acute toxicity (96 h-LC(50): 810 microgL(-1)). These results show that all assayed endpoints are very sensitive and indicate that current ecotoxicity testing used for environmental protection activities may underestimate the risk for harpacticoid copepods and most likely for other small invertebrates, when relying exclusively on acute toxicity measurements.