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                                                                            Volume 5, March 2009 [Table of Contents]

The Effects of Ethanol Exposure on the Development of Caenorhabditis elegans

Alexandra Charron, Jeffrey Fox, and Jaymie Honold

Department of Biology, Lake Forest College, Lake Forest, Illinois 60045

Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Summary

Prenatal exposure to ethanol results in a wide range of birth defects in humans classified as Fetal Alcohol Spectrum Disorders (FASD). As a result, the effect of ethanol on fetal development has been highly studied in many vertebrate and mammalian models. Recently, Caenorhabditis elegans has been introduced as an ideal model organism for studying the effects of ethanol on development. In this experiment, we exposed C. elegans to three different concentrations of ethanol. We hypothesized that increasing ethanol concentrations would result in fewer offspring that were smaller in size. Our results were inconclusive in the quantity of offspring produced, but we found that worms developing in the presence of ethanol were significantly smaller than those that were not. From these observations, we concluded that exposure to ethanol during development inhibited growth. Future experiments include developing a more precise experimental procedure and increasing the concentrations of ethanol used.

Introduction

Abnormal human fetal development has long been associated with women consuming alcohol (ethanol) during pregnancy.  The resulting offspring frequently present what has come to be classified as Fetal Alcohol Spectrum Disorder (FASD) – colloquially known as Fetal Alcohol Syndrome (Davis, et al., 2008).  FASD is characterized by physical and neurological abnormalities.  These abnormalities result in observable behavioral symptoms, learning disorders, and irregular immune system function (Davis, et al., 2008).

To further understand the teratogenic effects alcohol may have on human development, studies on vertebrate models (mouse, rat, guinea pig, chick, sheep, pig-tail macaque) have been extensively conducted over the past several decades (Davis, et al., 2008).  Evidence from these experiments has consistently demonstrated the role ethanol likely plays in interfering with neural crest cell survival and migration (Ahlgren, et al., 2002).  During vertebrate embryonic development, neural crest cells migrate anteriorly along the developing head, ultimately differentiating into associated facial and neural tissues (Ahlgren, et al., 2002).  Sonic hedgehog (Shh) signaling has been identified as a prominent pathway directing this neural crest cell migration and is therefore largely responsible for producing normal head, brain, and facial structures (Ahlgren, et al., 2002).  In mouse models and humans with Shh mutations, extensive neural crest cell apoptosis has been observed, which result in phenotypic abnormalities coinciding with those demonstrated in FASD patients (Ahlgren, et al., 2002).  Experimentally administering antibodies blocking Shh signaling in chick embryos also produces phenotypes observed in humans with FASD and mouse, chick, and quail embryos exposed to ethanol (Ahlgren, et al., 2002).  When chick embryos are treated with ethanol, depressed levels of mRNAs involved in the Shh signaling cascades are detected (Ahlgren, et al., 2002).  Administering exogenous Shh to the ethanol treated chick models revives normal gene activity and mRNA concentrations (Ahlgren, et al., 2002).  Subsequently, interfering with Shh signaling appears to be the mechanism by which ethanol promotes neural crest cell apoptosis and abnormal embryonic development.

While data from these experiments has supported the observations of humans with FASD, vertebrate models make examining microbiological components difficult (Davis, et al., 2008; Wolf, et al., 2003).  Recently, in order to further observe the teratogenic effects of ethanol on this level, invertebrate models such as C. elegans and Drosophilia melanogaster have been proposed as alternate models for experimentation (Davis, et al., 2008; Wolf, et al., 2003; Ahlgren, et al., 2002; Kwon, et al., 2003).  These models have previously been used with success to investigate the microbiological components and pathways involved in drug and nicotine dependence, supporting their use in the investigation of alcohol’s teratogenic effects (Davis, et al., 2008; Wolf, et al., 2003; Feng, et al., 2006; Kwon, et al., 2003; Dhawan, et al., 1999; Davies, et al., 2003; Davies, et al., 2004; Ahlgren, et al., 2002).  Justified by these past successes, recent experiments have been conducted with ethanol and C. elegans.  The results of these studies have provided insight into ethanol’s physiological impact on C. elegans, many of which are postulated to have conserved mechanisms of action responsible for mammalian responses to ethanol (Davies, et al., 2003; Feng, et al., 2006; Kwon, et al., 2003).  More specifically, the results of Kwon, et al. (2003) found that 230 genes in C. elegans were altered in their expression when the nematodes were treated with ethanol, many of which are known homologs to genes found in humans (Kwon, et al., 2003).  Furthermore, Davis, et al. (2008) observed that ethanol inhibited normal C. elegans larval development, growth, onset of reproductive maturity, and negatively affected longevity and the ability to produce normal progeny in normal quantity (Davis, et al., 2008).  Many of these results correlate with observations of phenotypes seen in humans with FASD.  The potential of the C. elegans model in researching ethanol’s impacts on development – including human development – has yet to be fully realized.  With the C. elegans genome and normal embryonic developmental fates mapped in their entirety, this nematode appears to be an ideal model to study (Kwon, et al., 2003). 

Our aims were to conduct an experiment using this model to test two hypotheses:  (1) the quantity of progeny will negatively correlate with increasing alcohol concentrations and (2) the progeny will be smaller with increasing alcohol concentrations.  Based on the results of Davis, et al. (2008) and Wolf, et al. (2003), we expected to observe evidence in support of our hypotheses as well as associated behavioral symptoms that increased in severity with exposure to increasing ethanol concentrations.

Results

24 hours

The worms were observed 24 hours after being placed on the ethanol plates. Plate set-up is depicted in Table 1. The worms on the control plates had normal movement, two of the worms on control plate A (CPA) were gravid and one worm on control plate B (CPB) is obviously gravid. At 0.32%, the adult worms appeared to be moving very slowly on both plates and stopped frequently. None of them appeared to be gravid. On plate 3 (P3), at 0.16% ethanol, the worms had slightly inhibited movement, and two appeared swollen with eggs. On plate 4 (P4), at 0.16% ethanol, the worms appeared normal and none were gravid. At 0.08% ethanol, movement was normal in all worms. On plate 5 (P5), one appeared gravid and on plate 6 (P6), all three worms appeared gravid.

72 hours

The worms were observed 72 hours after being transferred to ethanol plates. The CPA and CPB had adult worms that exhibited normal movement (Table 2). P1, at 0.32% ethanol, had approximately 150 adult worms and over 400 worms that were half the size or less. The adult worms had abnormally slow movement while the small worms appeared to move normally. P2 showed a similar trend but there were also many eggs that had not hatched yet (Table 2). P3 was also similar to P1 and P2, but P4 differed in that the adult worms outnumbered the small worms (Table 2). P5, at 0.08% ethanol, had similar quantities of worms as P1-4, but the adult worms showed normal movement. P6 had fewer worms, and hundreds of unhatched eggs.

96 hours

The worms were observed after 96 hours. CPA and CPB contained mostly large adult worms with approximately half as many small worms (Table 3, Figure 1A). P1 and P2 (0.32% ethanol), showed hundreds of small worms and slightly less large worms than the control plates (Table 3, Figure 1C, 1D). The small worms were approximately five grid units long while the normal adults were about nine grid units long (Figure 2). The normal adult worms were estimated to be 0.5 grid units wide and the small worms were about 0.25 grid units wide (Figure 2). P3 and P4 (0.16% ethanol) had similar quantities of large adult worms as P1 and P2, but fewer smaller worms (Table 3, Figure 1E, 1F). P5 and P6 (0.08% ethanol) had similar quantities of large adult worms, fewer smaller worms than P1-4 but still more than the control plates (Table 3, Figure 1G, 1H).

Table 1. The plates were created with specific ethanol concentrations absorbed into the agar.

Table 2. Observations after 72 hours. The number of adult and small worms were counted as well as the number of eggs that had not hatched yet.

Table 3. Observations after 96 hours. More small worms were visible on ethanol plates than controls as shown by the ratio of adult/small worms.

Figure 1. C. elegans photographs at 96 hours; 40x magnification. A) CPA; mostly adult worms were visible, few small worms. B) CPB; mostly adult worms visible, few small worms. C) P1; at 0.32%, many more small worms visible than in controls. D) P2; at 0.32%, abundance of small worms. E) P3; at 0.16%, more small worms than controls but less than P1 and P2. F) P4; at 0.16%, more small worms than controls but less than P1 and P2. G) P5; at 0.08%, worm quantities appear similar to control. H) P6; at 0.08%, slightly more small worms than control groups but less than P1-4.