Multigenerational study of life history traits, bioaccumulation, and molecular responses of Pseudodiaptomus annandalei to cadmium.

Metal pollution provide a substantial challenge for environmental health. This study investigated the multigeneration effects of cadmium on populations of the copepod species Pseudodiaptomus annandalei, exposed to a sublethal concentration, 40 µg/L of cadmium (Cd), over 10 generations. At the end of each generation, copepod individuals were collected to estimate fecundity, bioaccumulation, and real time qPCR quantification of selected differentially expressed genes to evaluate Cd effects and sex-specific responses of copepods across multiple generations. Our results revealed a sex-specific accumulation of Cd integrating 10 successive generations. The concentration of Cd was significantly higher (p < 0.05) in males than in females. We also observed a generational increase in Cd accumulation. Fecundity increased, with the exception of the first generation, possibly as a compensation for a decrease of copepod population size under Cd exposure. Protein expression of copepods exposed to Cd occurred in a sex-specific manner. Hemerythrin was mostly up-regulated in both copepod sexes exposed to Cd with males having the highest expression levels, while heat shock protein 70 was mostly up-regulated in males and down-regulated in female copepods, both exposed to Cd. Although copepods are known to develop adaptive mechanisms to tolerate toxic chemicals, continuous exposure to metals could lead to the bioaccumulation of metals in their offspring through maternal transfer and direct uptake from the medium over several generations. As a consequence, increased metal concentrations in copepods could result in physiological damage, reducing their fitness, and possibly compromise copepod population structures. This study showed that mortality, life history traits and molecular responses of a copepod species provided important toxicological endpoints and bio-markers for environmental risk assessments. Environmental pressure resulting from continuous exposure to persistent pollutants like Cd, could have evolutionary significance. The tendency for copepods to selectively adapt to a toxic environment through modifications, could increase their chance of survival over a long term.

Differential gene expression profile of male and female copepods in response to cadmium exposure.

In this study, the whole transcriptome and sex-specific differential gene expression of the copepod Pseudodiaptomus annandalei exposed to cadmium (Cd) were investigated. P. annandalei were exposed to 40 µg/L Cd from the naupliar stage to male and female adults. High-throughput transcriptome sequencing (RNA-seq) was performed with copepod samples using an Illumina Hiseq™ 2000 platform. TransDecoder analysis found 32,625 putative open reading frame contigs. At p-values of <0.001, a total of 4756 differentially expressed genes (DEGs) (2216 up-regulated and 2540 down-regulated genes) were found in male copepods. Whereas a total of 2879 DEGs (2007 up-regulated and 872 down-regulated genes) were found in female copepods. A few selected cellular stress response genes, involved in xenobiotic metabolism, energy metabolism, growth, and development as a result of Cd exposure in the copepods were discussed. The study showed that most of these processes were changed in a sex-specific manner, accounting for the different sensitivities of male and female copepods. Results suggest and reinforce that sex is an important factor to be considered in ecotoxicogenomics.

Bioaccumulation of metals in calanoid copepods by oral intake.

We demonstrated that oral intake of water by two calanoid copepods, Pseudodiaptomus annandalei and Eurytemora affinis takes place and has implications for their ecotoxicology. In the first experiment, copepods were exposed to a dyed medium, which allowed us to visually examine the possibility of water uptake by the copepod. We observed that both copepod species were taking in water orally and evacuated dye at different speeds. This exposure left concentrated dye in the guts of the copepods indicating adsorption into the gut epithelium. This was further demonstrated by exposing both copepod species independently to dissolved metals (Cd,17 µg/L; Cu,13.8 µg/L; Ni, 29.3 µg/L) and to dietary metals (Cd,18.8 µg/g; Cu, 35.3 µg/g; Ni, 32.5 µg/g). The results showed that although the concentration of dissolved metals they were exposed to were lower than those of the dietary metals, nevertheless, uptake of metals by both copepod species from the dissolved phase alone was substantially higher than from dietary exposure. This provides clear evidence to support our hypothesis that higher metal body burden observed in copepods exposed to dissolved metals than in those exposed to dietary metals is an implication of oral intake of water. P. annandalei showed higher excretion rate of metals when exposed to dissolved metals than E. affinis. However, the excretion rate of metals from both copepod species exposed to dietary metal was similar. We conclude here that both copepod species take in water orally. Our study further showed that metal uptake depends on the exposure routes and the uptake and excretion rates are dependent on the type of metals, amounts and the species.

Acute and chronic toxicity of cadmium on the copepod Pseudodiaptomus annandalei: A life history traits approach.

We determined the effect of acute and chronic toxicity of cadmium (Cd) on Pseudodiaptomus annandalei, according to their developmental and reproductive stages. Firstly, to estimate the 50% lethal concentration (LC50), acute exposure of nauplii and copepodids to 20, 40, 80, 150, and 300 µg/L of Cd was tested, and the effects of 5 µg/L and 40 µg/L of Cd on copepod developmental rate was done. Female lifespan and number of nauplii produced were compared. Secondly, one generation of copepod was exposed to dissolved (WCd) and dietary (DCd) Cd, and sex-specific Cd uptake and population density were estimated. 96 h LC50 was 40 µg/L Cd for nauplii and 120 µg/L Cd for copepodids. Duration of copepod development was 3.5 days and 5.5 days longer than the control when exposed to 5 µg/L and 40 µg/L of Cd, respectively. Female lifespan in both treatments were 9 and 8 days shorter than in the control, respectively. Total number of nauplii produced per female lifespan was 440 (control), 450 (5 µg/L Cd), and 365 (40 µg/L Cd). Cd uptake in copepods increased from nauplii to adults when exposed to dietary Cd and decreased when exposed to dissolved Cd. Dietary uptake of Cd was significantly higher in males than in females and Cd uptake from water was higher in males than in females, but not statistically significant. The total population of copepods were significantly affected by Cd. The toxic effects of Cd in copepods appear to depend on developmental stage, sex, duration and uptake route.

Differences in lethal response between male and female calanoid copepods and life cycle traits to cadmium toxicity.

This study determined the effect of cadmium (Cd) toxicity comparatively on two copepods, Eurytemora affinis (Poppe 1880) from a temperate region (Seine Estuary, France) and Pseudodiaptomus annandalei (Sewell 1919) from a subtropical region (Danshuei Estuary, Taiwan), according to their sex and reproductive stages. In addition, the effect of Cd to their life cycle traits was quantified. In the first experiment, both copepod sexes were exposed to 40, 80, 150, 220, and 360 µg/L of Cd and a control cultured in salinity 15, except that the temperature was 18 °C for E. affinis and 26 °C for P. annandalei. This allowed calculating median lethal concentration (LC50) of Cd after 96 h. This was 120.6 µg/L Cd for P. annandalei males which were almost twice as sensitive as P. annandalei females (LC50 = 239.5 µg/L Cd). For E. affinis females, the LC50 was 90.04 µg/L Cd, reflecting a 1.4 times higher sensitivity of females than of males (LC50 = 127.75 µg/L Cd). The males of both species were similarly sensitive; however, the E. affinis females were 2.7 times more sensitive than the P. annandalei females. We also compared the sensitivity of ovigerous females (OVF) and non-ovigerous females (NOF) of both species to Cd. Mortality was higher in NOF than in OVF of both copepod species in both the control and the 40 µg/L Cd treatment. Finally, the total population, fecundity and female morphology of both copepod species were estimated after exposing one generation cycle (nauplius to adult) to 40 µg/L Cd (for E. affinis) and 160 µg/L Cd (for P. annandalei). A significant decrease in cohort production, survival and clutch size but no significant difference in the prosome length of both copepod species exposed to Cd were detected. The ratio of OVF:NOF was high in both copepod species exposed to Cd. Cd toxicity did not significantly affect the M:F sex ratio and % OVF of E. affinis. However, the effect of Cd toxicity in P. annandalei was significant in the M:F sex ratio and was in favor of females and their reproductive activities due to an increase in % OVF. Moreover, there was a significant decrease in total production of P. annandalei due to high mortality in their nauplii and copepodid developmental stages. Toxicity to Cd appears to be affected by multiple factors including sex, reproductive life stage and species. The ecological implication of Cd toxicity on E. affinis and P. annandalei copepod ecology is more related to a skewed sex ratio, low egg production, reduced hatchability and reduced survival that affects the recruitment potential of the copepod nauplii resulting in a decreasing copepod population. Mortality, reproduction and population growth of model species may provide important bio-indicators for environmental risk assessment.