Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

Abstract

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

Figure 1

Vitellogenin Vtg2 mRNA levels following 72 hrs exposure of daphnids to various chemicals. Exposure concentrations are reported in Table 2. Data are reported as the mean and SEM (n=2, with each experimental unit consisting of 5 individuals) expression relative to concurrently evaluated controls which were set at 1.0 (horizontal line).

Figure 2

Relative expression of vitellogenin Vtg2 mRNA during continuous exposure of molt-synchronized daphnids to piperonyl butoxide (300 μg/L, panel A) and cyproterone acetate (2100 μg/L, panel B). Error bars represent the SEM (n=3). An asterisk denotes a significant (p<0.05) difference between the treatment (solid line, circles) and control (dashed line, squares) (Student’s t test).

Figure 3

Relative expression of selected mRNAs following 72 hrs exposure of daphnids to 20-hydroxyecdysone (481 μg/l, Panel A) or ponasterone A (232 μg/l, panel B). Data are reported as the mean and SEM (n=2, with each experimental unit consisting of 5 individuals) expression relative to concurrently evaluated controls which were set at 1.0 (horizontal line). An asterisk denotes a significant (p<0.05) difference between the treatment and control (Student’s t test).

Figure 4

Expression of vitellogenin Vtg2 mRNA levels during exposure to 481 μg/l 20-hydroxyecdysone. Values were normalized to concurrently evaluated control (untreated) animals. Error bars represent the SEM (n=3). An asterisk denotes a significant (p<0.05) reduction in Vtg2 level relative to levels at 0 hr (ANOVA, Tukey’s Multiple Comparison).

Figure 5

Relative expression of selected mRNAs following 72 hrs exposure of daphnids to the juvenoid hormone fenoxycarb (1000 μg/l)). Data are presented as the mean ± SEM (n=2, with each experimental unit consisting of 5 individuals) expression relative to concurrently evaluated controls which were set at 1.0 (horizontal line). An asterisk denotes a significant (p<0.05) difference between the treatment and control (Student’s t test).

Figure 6

Relative expression of selected mRNAs following 72 hrs exposure of daphnids to the juvenoid hormone methyl farnesoate (200 μg/l). Error bars represent the SEM (n=3–4, with each experimental unit consisting of 3 individuals). Data were normalized to concurrently maintained control (unexposed) daphnids. An asterisk denotes a significant (p<0.05) difference between the treatment and mRNA levels measured at 0 hr (ANOVA, Tukey’s Multiple Comparison).