Disturbances in reproduction and expression of steroidogenic enzymes in aquatic invertebrates exposed to components of the herbicide Roundup

Abstract

Exposure of organisms to environmental contaminants is a growing concern. We have investigated the effects of the individual active ingredients of the herbicide Roundup (glyphosate and diquat dibromide [DD]) since Roundup causes alterations in reproduction, mortality, and development in the aquatic snail Lymnaea palustris. Snails chronically treated with elevated but ecologically relevant levels of DD exhibit reduction in fecundity (p < 0.05), while fecundity in glyphosatetreated snails is comparable to or exceeds control levels. To investigate a possible mechanism for the reproductive disturbance, we monitored levels of steroid acute regulatory (StAR) protein in whole snails and observed a correlation in StAR protein decrease with treatment with Roundup, glyphosate, or DD. We detect StAR in organs where steroid biosynthesis occurs (ovotestis, brain, kidney); StAR protein is reduced following chronic exposure to Roundup, glyphosate, or DD (p < 0.01). Estradiol and testosterone concentrations in hemolymph were measured by enzyme-linked immunosorbent assay following 3-week exposure of snails to 3.5 mg/L glyphosate or 140 mg/L DD. Testosterone levels decrease in DD-treated groups (p < 0.05); a trend of lower testosterone is also observed in glyphosate-treated groups (p > 0.05). Estradiol concentration is greater than or equal to control levels in glyphosate, but decreased in DD (p < 0.05). Because of its role in the conversion of testosterone to estradiol, we monitored abundance of aromatase and observed a reduction (p < 0.05) in DD-treated snails (consistent with the drop in fecundity and estradiol levels) and a comparable level to control in glyphosate-treated snails (consistent with their high fecundity and estradiol levels). Although the toxicity of commercially-available Roundup to aquatic animals may have many contributing factors including its inactive surfactant, the constituent of Roundup associated with the greatest reproductive disturbances and observed developmental abnormalities of offspring is DD. This study details the analysis of particular herbicide constituents and their effect on specific targets in the reproductive pathway.

Sex steroids and steroidogenesis-related genes in the sea cucumber, Holothuria scabra and their potential role in gonad maturation

“…The HscStAR gene has also been reported in the giant freshwater prawn M. rosenbergii 23 , the aquatic snail Lymnaea palustris 54 , and the Yesso scallop Mizuhopecten yessoensis 10 . Its presence is a key indicator for a core function in steroid synthesis through cholesterol conversion.…”

Cellular effects of diquat dibromide exposure: Interference with Wnt signaling and cytoskeletal development

“…Normal seminal vesicles of control snails were dissected and compared to those of snails chronically exposed to DD for 3 weeks (Figure 4(a)). It has been reported that DD reduces fecundity and steroid sex hormone levels, in part through a significant reduction in steroid acute regulatory protein (StAR) expression 2 ; however, the increased seminal vesicle size and sperm count (Figure 4(b) and (d)) suggest that sperm release and/or maturation are compromised as well. Sperm collected from snails chronically exposed to 5X or 10X DD exhibit greater density, erratic motility, and tendency to form twisted cable-like structures (Figure 4(b)) in contrast to the smoother sinusoidal movements of control sperm (Figure 4(c); Online Supplemental Data 2).…”

“…Aberrant motility appeared to increase with increased density and therefore increased DD concentration. Impaired StAR expression and hormonal regulation, 2 hypertrophy of the seminal vesicles as a possible consequence of DD-induced disruption in Wnt signaling, and increased ROS formation are all factors that likely play a role in producing the observed irregularities in sperm abundance, viability, and motility, though the exact mechanism is unclear; these may all contribute to the severely impaired fecundity of snails exposed to DD.…”

“…The effects of DD on nontarget organisms have been minimally studied, though previous studies indicate that herbicidal runoff into surface waters poses a risk to the fecundity, development, and viability of organisms that come into direct contact with DD. 1,2 DD is known to initiate the formation of reactive oxygen species (ROS) in plant tissues, leading to lipoperoxidation and apoptotic cell death. 3,4 While ROS are essential during animal development and regeneration 5 and serve as second messengers in several signaling pathways, 6 overproduction of ROS is implicated in a number of diseases and comprises a stress response to toxins.…”

“…DD has also been demonstrated to negatively affect fecundity in L. palustris adults. 2 While DD may indirectly decrease fecundity by altering abundance of steroidogenic enzymes in these hermaphroditic organisms, it is possible that DD may also directly interfere with reproduction by acting at the cellular level in sperm production, maturation, and/or motility. Wnt/Ca 2+ signaling, specifically mediated by Wnt 11 and Wnt 5a, 16 is required for sperm maturation and motility, 17 which are aberrant in sperm extracted from DD-treated snails.…”