Maternal preconception PFOS exposure of Drosophila melanogaster alters reproductive capacity, development, morphology and nutrient regulation

Abstract

Perfluorooctanesulfonic acid (PFOS) is a persistent synthetic surfactant widely detected in the environment. Developmental PFOS exposures are associated with low birth weight and chronic exposures increase risk for obesity and type 2 diabetes. As an obesogen, PFOS poses a major public health exposure risk and much remains to be understood about the critical windows of exposure and mechanisms impacted, especially during preconception. Here, we leverage evolutionarily conserved pathways and processes in the fruit fly Drosophila melanogaster (wild-type Canton-S and megalin-UAS RNAi transgenic fly lines) to investigate the window of maternal preconception exposure to PFOS on reproductive and developmental toxicity, and examine receptor (megalin)-mediated endocytosis of nutrients and PFOS into the oocyte as a potential mechanism. Preconception exposure to 2 ng PFOS/female resulted in an internal concentration of 0.081 ng/fly over two days post exposure, no mortality and reduced megalin transcription. The number of eggs laid 1-3 days post exposure was reduced and contained 0.018 ng PFOS/egg. Following heat shock, PFOS was significantly reduced in eggs from megalin-knockdown transgenic females. Cholesterol and triglycerides were increased in eggs laid immediately following PFOS exposure by non-heat shocked transgenic females whereas decreased cholesterol and increased protein levels were found in eggs laid by heat shocked transgenic females. Preconception exposure likewise increased cholesterol in early emerging wildtype F1 adults and also resulted in progeny with a substantial developmental delay, a reduction in adult weights, and altered transcription of Drosophila insulin-like peptide genes. These findings support an interaction between PFOS and megalin that interferes with normal nutrient transport during oocyte maturation and embryogenesis, which may be associated with later in life developmental delay and reduced weight.

Keywords: Development; Drosophila melanogaster; Nutrient regulation; Perfluorooctanesulfonic acid (PFOS); Preconception exposure; Reproduction.

Fig 1.

(A) Cumulative egg numbers from PFOS-treated females and acetone-treated control for 7 days (left) and statistical significance of the number of eggs compared to that from control females analyzed by two-way ANOVA (right; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001). (B) Survival rate of PFOS-treated females and control (left) and statistical significance of survival rate compared to control analyzed by two-way ANOVA (right). Error bars indicate standard deviation (SD).

Fig. 2.

(A) Cumulative egg numbers from 2 ng PFOS-treated females and acetone-treated control for 5 days; (B) Hatchability of the eggs collected for every 24 h from PFOS-treated females and acetone-treated control. Asterisks (***) indicate statistical differences using two-way ANOVA (P <0.001); (C) Decreased transcription levels of megalin following PFOS treatment with transcripti on levels of megalin compared to acetone-treated control flies every 24 h. An asterisk (*) indicat es statistical differences using two-way ANOVA (*, P<0.05; **, P<0.01; ***, P<0.001). Error bars indicate standard deviation (SD).

Fig. 3.

(A) Validation of megalin RNAi in transgenic flies. One group was heat shocked (HS) to induce RNAi of megalin. The other group (non-heat shocked; NHS) was used as control; (B) Cumulative egg numbers from HS females and NHS control for 5 days; (C) Hatchability of the eggs collected for every 24 h from HS females and NHS control. Asterisks (*) indicate statistical differences using two-way ANOVA (***, P<0.001; ****, P<0.0001). Error bars indicate standard deviation (SD).

Fig. 4.

Comparison of three major nutrients in the eggs of control (NHS) and megalin-knockdown (HS) in non-exposed and PFOS-exposed females. Measurements of (A) cholesterol, (B) protein, and (C) triglycerides were determined in samples collected over a 5 day period. The average difference (D) cholesterol, (E) protein, and (F) triglycerides in NHS-PFOS or HS-PFOS from the NHS non-exposed eggs were calculated at each day after maternal treatment. Bars represent mean ± SEM. *p<0.05 ** p<0.01, **p<0.001, Two Way ANOVA, Tukey’s post hoc. N = 15–21 pooled samples.

Fig. 5.

Preconception PFOS exposure delays development, decreases body weight and increases lifespan of early group F1 progeny: (A) Average time to adulthood for progeny of the groups indicated. n=20–26 mating pairs/treatment. D’Augostino & Pearson Normality test, Unpaired t-test *p<.05, ****p<.0001. (B) Weight of mixed gender adult flies 1–3 days post-eclosion. Weights are averaged for progeny of each mating pair, n=10–15 mating pairs/treatment, 16–25 flies/mating pair. D’Augostino & Pearson Normality test, Unpaired T-test *p<.05. Boxes indicate the 25th and 75th percentiles, horizontal bars indicate mean and error bars indicate min-max values. (C-F) Survival curves for early group female (C), early group male (D), late group female (E) and late group male (F) progeny of acetone control (black) and PFOS exposed (red) females. N= 6–10 vials of progeny/treatment group/sex. The comparison of survival curves (Kaplan-Meier method) was performed by using Log-rank (Mantel-Cox) test using GraphPad Prism (ver. 8, GraphPad Inc., San Diego, CA, USA).

Fig. 6.

Nutritional content of adult F1 progeny that developed from early laid eggs of CS mothers following either preconception PFOS or acetone exposure: (A) triglycerides, (B) cholesterol, (C) total sugar. Error bars indicate standard deviation (SD). N= 10–15 animals/treatment group. D’Augostino & Pearson Normality test, Mann-Whitney U test **p<0.01, ***p<0.001, ****p<0.000.

Fig. 7.

Preconception PFOS exposure alters Drosophila insulin-like peptide (dilp) expression levels in early group F1 progeny. Fold-change in expression of dilp 2 (A), 3 (B), 5 (C), 6 (D) and 8 (D) for female or male progeny from either preconception PFOS or acetone exposed mothers at 1 day and 14 days post-eclosion (see treatment legend). Fold-change in expression is calculated as 2^−ΔΔct using tubulin as a reference gene. Error bars indicate standard deviation (SD). N=8–10 animals/treatment. D’Augostino & Pearson Normality test, Mann-Whitney U test **p<0.01, ***p<0.001, ****p<0.000.

Fig. 8.

Detected amount of PFOS in maternal CS female flies (A) and their F1 eggs (B) following preconception exposure to PFOS. PFOS levels in eggs from non-heat shocked (NHS-megalin(+) or heat shocked (HS-megalin(−) transgenic female flies following preconception exposure to PFOS (C). Eggs were collected every 24 h from PFOS- treated female.