Embryonic gene expression among pollutant resistant and sensitive Fundulus heteroclitus populations

Abstract

Changes in gene expression, coupled with biochemical, physiological, and behavioral alterations, play a critical role in adaptation to environmental stress. Our goal was to explore ways natural populations may have adapted to local, polluted environments. We took advantage of natural populations of Fundulus heteroclitus, one of the few studied fish species in North America that has established resistant populations in highly contaminated urban estuaries. We analyzed morphology, physiology, and gene expression of developing F. heteroclitus embryos during late organogenesis (stage 31); these embryos were from both resistant and sensitive populations and were raised in a common, unpolluted environment. While cardiac heart rates show significant differences between embryos of parents from clean and heavily contaminated Superfund sites, time-to-stage, embryo morphology, and gene expression profile analyses do not differ significantly between untreated embryos from resistant and sensitive populations. Further evaluation that includes tissue-specific approaches in gene expression analysis and larger sample sizes may be necessary to highlight important phenotypes associated with mechanisms of sensitivity and resistance among natural F. heteroclitus embryo populations. Alternatively, population differences may be masked by developmental canalization, and biologically important differences between sensitive and resistant embryos may only manifest with exposure (e.g., be dependent on gene by environment interactions).

Figure 1. Loop Design for Embryo gene expression analysis

Loop consists of 4 biological replicates (embryo aRNA), each representing a family within a population, totaling 20 biological replicates (4 embryos × 5 populations). Two embryo RNAs are labeled with Cy3 and two with Cy5 within each population. Each arrow represents an array, and the direction of the arrow is from Cy3 to Cy5, so that two embryo RNAs from different populations labeled with different dyes are hybridized to the same array. M – Manteo, NC; E – Elizabeth River, VA; K – King’s Creek, VA; N – Newark Bay, NJ, S – Succotash, RI. Clear circles represent reference populations; gray circles represent polluted populations.

Figure 2. Embryo survival among five populations at stages 31 and 35

A. Stage 31 embryos. B. Stage 35 embryos. 1-way ANOVA (p<0.05) indicates significant differences among embryo population survival rates. Dunnett’s t-test indicates that Elizabeth River, VA and Succotash, RI embryos have significantly lower survival rates when compared to Newark Bay, NJ embryos.

Figure 3. Developmental delays among embryos from five populations

A. Stage 31 embryos. 1-way ANOVA (p<0.05) indicates significant differences among populations. Dunnett’s t-test indicates that Newark Bay, NJ and Succotash, RI embryos are significantly delayed when compared to King’s Creek, VA embryos. B. Stage 35 embryos. 1-way ANOVA (p<0.05) indicates significant differences among populations. Pairwise comparisons between populations revealed significant differences in the onset of developmental stage before hatching between King’s Creek, and Succotash, RI embryos (Bonferroni’s Multiple Comparisons test, p < 0.05; t = 3.13).

Figure 4. Heart rate (beats/min) differences during late organogenesis among five embryo populations

A. Stage 31 embryos. Two embryo populations from polluted sites (Elizabeth River, VA and Newark Bay, NJ) have significantly faster (p<0.05) heart rates when compared to embryos from clean reference sites. B. Stage 35 embryos. Heart rates differed significantly among 5 embryo populations at stage 35 (1-way ANOVA, p < 0.05). Bonferroni’s Multiple Comparisons Tests comparisons test between populations revealed that Elizabeth River embryos have significantly higher HRs then Manteo, NC (p < 0.01; t = 4.084), King’s Creek, NC (p < 0.001; t = 4.43), and Succotash, RI embryos p < 0.001; t = 4.78).

Figure 5. Embryo morphology among five populations during late organogenesis (stage 31)

A. Stage 31 embryos. B. Stage 35 embryos. Most embryos developed normally, and there were no significant differences in morphology (1-way ANOVA, p<0.05) among the three clean and two polluted embryo populations. Criteria used when scoring embryo morphology: 1 = normal; 2 = mildly-deformed; 3 = moderately-deformed; 4 = severely deformed; 5 = extremely deformed.