Current Biology 21, 1548–1551, September 27, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2011.08.009 Report Regulation of Sexual Plasticity in a that Produces Males, Females, and

Jyotiska Chaudhuri,1,2 Vikas Kache,1,2 , dauer formation is a facultative stage and occurs and Andre Pires-daSilva1,* when conditions are unfavorable for growth. 1Department of Biology, University of Texas at Arlington, To confirm the relationship between dauer formation and Arlington, TX 76019, USA hermaphroditic fate in Rhabditis sp. SB347, we isolated dauers by killing the nondauer worms with a 1% sodium dodecyl sulfate solution. We found that all dauers (n = 1,015) Summary developed into hermaphrodites. Under conditions when food is present, -fated larvae remain arrested in the The mechanisms by which new modes of reproduction dauer stage for about 24 hr before continuing development evolve remain important unsolved puzzles in evolutionary [3]. We also tracked the development of another set of larvae biology. Nematode worms are ideal for studying the evolu- to determine the fate of animals that do not pass through the tion of mating systems because the phylum includes both dauer stage. All animals tested that did not undergo dauer a large range of reproductive modes and large numbers of formation developed into females (n = 69). evolutionarily independent switches [1, 2]. Rhabditis sp. SB347, a nematode with sexual polymorphism, produces Hermaphrodite Development Can Be Induced males, females, and hermaphrodites [3]. To understand A Rhabditis sp. SB347 selfing hermaphrodite produces on how the transition between mating systems occurs, we char- average smaller proportions of hermaphrodite F1s within its acterized the mechanisms that regulate female versus first 15 hr of adulthood compared to the total proportion of hermaphrodite fate in Rhabditis sp. SB347. Hermaphrodites hermaphrodite F1s over several days in the presence of develop through an obligatory nonfeeding juvenile stage, cholesterol (p < 0.001, post hoc Mann-Whitney U test) (Fig- the dauer larva. Here we show that by suppressing dauer ure 2A). To test whether female-fated larvae can be redirected formation, Rhabditis sp. SB347 develops into females. to hermaphrodite development, larvae hatched from eggs laid Conversely, larvae that under optimal growth conditions within the first 15 hr of a selfing parent were cultured in media develop into females can be respecified toward hermaphro- lacking cholesterol. Cholesterol is an important precursor for ditic development if submitted to dauer-inducing condi- the dafachronic acid hormones, which inhibit dauer formation tions. These results are of significance to understanding in various distantly related nematodes [6–8]. Therefore, deple- the evolution of complex mating systems present in para- tion of cholesterol induces worms to become dauers. We sitic nematodes. found that a higher proportion of larvae developed into hermaphrodites in cholesterol-depleted medium and low Results food compared to larvae cultured in standard conditions (p < 0.001, post hoc Mann-Whitney U test) (Figure 2A). Rhabditis sp. SB347 is a sexually polymorphic nematode, con- Rhabditis sp. SB347 larvae that develop into hermaphro- sisting of hermaphrodites, females, and males [3]. Hermaphro- dites and females under standard conditions can be distin- dites are protandrous, with a small number (w300) of sperm guished by the mid-L1 stage by their different rates of gonad produced first that are stored for later use in fertilizing the development [3]: larvae with larger gonad primordia develop animal’s own oocytes. The somata of adult hermaphrodites to become females, whereas larvae with smaller gonads molt and females are practically identical (Figure 1A), whereas into dauer before continuing toward hermaphrodite develop- males have specialized behavior and morphological structures ment (see Figure S1 available online). Mid-L1 larvae that used for mating. Somatic determination is likely to be develop into females have gonads that are almost twice the mediated by an XX:XO mechanism, in which XX animals length of those in larvae that develop into hermaphrodites. develop into hermaphrodites and females, and XO animals When selecting worms with the dissecting microscope at 3 become males [4]. 80 magnification, 84% (n = 291) of the large-gonad (LG) In Rhabditis sp. SB347, the developmental switch that regu- larvae developed into females under standard culture condi- lates hermaphrodite versus female fate is poorly understood. tions (Figure 2B). To test whether LG larvae can go through Previous studies suggested that the postembryonic develop- the dauer stage, we selected mid-L1s and cultured them in ment of Rhabditis sp. SB347 differs between females and cholesterol-depleted media. Of the LG larvae, 74% (n = 61) hermaphrodites [3](Figure 1B). Developing females pass became dauers, indicating that larvae with the potential to through four feeding larval stages (L1–L4) before becoming re- become females are capable of undergoing dauer formation. producing adults. In contrast, developing hermaphrodites Importantly, most of the larvae with LGs became hermaphro- pass through an obligatory nonfeeding dauer larva stage dites if submitted to dauer-inducing conditions (Figure 2B). following their L2 stage. Dauer is a larval form that can with- Because dauers are highly dispersive and sometimes there stand long periods of stressful conditions, such as lack of is a discrepancy between the number of eggs and resultant food, exposure to hazardous chemicals, and extreme temper- adults, we only considered broods in which the difference atures [5]. In elegans and most terrestrial between eggs and adults was less than 10% (see Supple- mental Experimental Procedures). In conclusion, larvae that usually develop into females under standard conditions can 2These authors contributed equally to this work be redirected to hermaphrodite development when cultured *Correspondence: [email protected] in low food supply and in the absence of a cholesterol source. Sexual Plasticity in a Nematode 1549

A B Hermaphrodite Rhabditis sp. SB347 Males/females

vulva Egg L1 L2 L3 L4 Adult oocytes egg Hermaphrodites Female Egg L1 L2d dauer L4 Adult

oocytes

vulva

Figure 1. Alternative Developmental Morphs in Rhabditis sp. SB347 (A) The germline of the adult hermaphrodite produces eggs by self-fertilization (top panel). The soma of the hermaphrodite is morphologically identical to that of the female (bottom panel). Scale bars represent 65 mm. (B) Females and males of Rhabditis sp. SB347 undergo four feeding larval stages. Hermaphrodites obligatorily pass through a nonfeeding dauer stage.

Larvae Can Be Redirected from Hermaphrodite to Female DA did not change their germline sex determination (data Development not shown). To confirm that the hormone is blocking dauer To test whether the dauer stage is necessary for hermaphro- formation in Rhabditis sp. SB347 as in other nematodes, we dite development in Rhabditis sp. SB347, we incubated L1 treated small-gonad L1s with 200 nM DA. Most animals larvae with the steroidal endocrine hormone D7-dafachronic (86%, n = 120) did not develop into dauers, indicating that acid (DA). DA acts as a ligand for the transcription factor DA inhibits dauer development. Together, these results indi- DAF-12, a biochemical interaction that is highly conserved in cate that by blocking dauer formation with DA, larvae that nematodes [7–9]. High levels of DA inhibit dauer formation usually would develop into hermaphrodites can be redirected and promote normal development through L1–L4, whereas to become females. low or absent levels of DA correlate with the induction of a da- uer stage [10–15]. Males Do Not Form Dauers We first sampled Rhabditis sp. SB347 L1 larvae, indepen- The sex of Rhabditis sp. SB347 is most likely determined by an dently of their gonad phenotype, from cultures maintained in XX:XO mechanism [4], in which hermaphrodites and females standard conditions. When incubated with 100 mM DA, very are XX and males are XO. It has been previously shown that few of the larvae (3%, n = 86) developed into hermaphrodites about 13% of the self-progeny of Rhabditis sp. SB347 compared to the control (75%, n = 146). To determine more hermaphrodites are of the male sex [3]. We confirmed these rigorously whether larvae specified for hermaphroditism can results by scoring total broods of virgin hermaphrodites, which be redirected toward female development, we isolated mid- resulted in a similar percentage (9%) of males in the F1 L1 larvae with small gonads and treated them with various progeny (n = 10 total broods) ([4]; this study). Interestingly, concentrations of DA. Larvae responded in a dosage-depen- outcrosses result in less than 2% male progeny. This low dent manner to DA, with a higher proportion of larvae devel- percentage of males after outcrossing is the consequence of oping into females when exposed to higher concentrations preferential fertilization of the male X-bearing sperm over the of the hormone (Figure 3). Postdauer animals treated with nullo sperm [4].

ABFigure 2. Proportion of Hermaphrodites Increases in Dauer- Inducing Conditions Hermaphr. Females (A) Total brood of hermaphrodite parents (total) results in more hermaphrodite than female progeny. Most larvae that 2630 376 351 291 401 hatch from eggs laid within the first 15 hr develop into 100- 100- females when cultured in standard conditions (15 hr +). In dauer-inducing conditions (15 hr 2), the proportion of larvae 80- 80- developing into hermaphrodites increases (p < 0.001, Mann- Whitney U test). Sample sizes are indicated above each column. 60- 60- (B) A higher proportion of larvae with large gonads (LG) develop into females when cultured in standard conditions (LG +) as opposed to cholesterol-free conditions (LG 2). - 40- 40 Sample sizes are indicated above each column. Color coding % each sexual morph % each sexual morph of the bars is as in (A). 20- 20- Data are represented as mean 6 standard error of the mean of ten replicates. See also Figure S1.

0 - - - -

0 - Total 15 hr + 15 hr - LG + LG - Current Biology Vol 21 No 18 1550

males (n = 10 total broods, 2,897 scored F1s). XO males result- ing from selfing parents are likely to be derived from chromo- somal nondisjunction, although further studies are required to confirm this [4]. The two types of nonmale Rhabditis sp. SB347 larvae can be distinguished shortly after hatching, indicating that speci- fication of both sexual morphs occurs during embryogenesis [3]. Under standard growth conditions, larvae with LGs usually develop into females, and the ones with small gonads develop into hermaphrodites. When larvae with LGs are cultured in an environment depleted in cholesterol, they undergo dauer formation and develop into hermaphrodites. Therefore, dauer formation seems to respecify female-fated larvae to develop into hermaphrodites. The link between dauer formation and sexual fate is also found in larvae with small gonads. These larvae usually undergo dauer formation and become hermaphrodites. When dauer formation in small- gonad larvae is blocked, they develop into females. It is not Figure 3. Dafachronic Acid Redirects from Hermaphrodite to Female Devel- clear how exactly the dauer pathway interacts with germline opment sex determination in Rhabditis sp. SB347. A correlation L1 larvae with small gonads were grown in the presence of ethanol (E, between expression of reproductive genes and dauer forma- control) and various concentrations of dafachronic acid (DA). Sample sizes tion has been recently established in C. elegans [21]. Postdauer are indicated above each column. Data are represented as mean 6 standard C. elegans hermaphrodites have a higher reproductive output error of the mean of seven to ten replicates. Differential mortality or dauer when compared to hermaphrodites that did not pass through migration does not account for differences in percentage of sexual morphs. the dauer stage [21]. Experimental evidence indicates that epigenetic changes occur in postdauer animals, resulting in the increased production of sperm. It will be interesting to From plates in overcrowded and starved conditions, all determine whether epigenetic changes triggered by dauer Rhabditis sp. SB347 dauers become hermaphrodites (n = formation in Rhabditis sp. SB347 underlie sperm production 1,015). One hypothesis for the lack of males developing into da- in XX animals, and thus hermaphrodite development. uers is that under these conditions, very few males are present. Although more ecological data are necessary to investigate However, visual inspection of the plates revealed that they the role of each sexual morph, the role of the Rhabditis sp. could be found in these cultures. An alternative hypothesis is SB347 dauers that later become hermaphrodites is probably that males cannot form dauers. To test this, we isolated early to disperse to new food sources. Dauers exhibit morpholog- larvae with morphological characteristics of animals that ical, physiological, and behavioral adaptations for dispersal develop into males (e.g., large B cell in the tail) and incubated [5]. By coupling dauer formation with hermaphrodite develop- them in cholesterol-depleted media. Consistent with the ment, Rhabditis sp. SB347 can colonize and reproduce in new previous experiments, none of the male larvae (n = 100) molted habitats in the absence of a mating partner. It is important to into the dauer stage. note that in C. elegans, which probably has a similar lifestyle, males have an enhanced capacity of undergoing dauer forma- tion relative to hermaphrodites [22, 23]. In contrast, Rhabditis Discussion sp. SB347 males do not seem to go through dauer. At this point, it is not clear why Rhabditis sp. SB347 males do not Rhabditis sp. SB347 as a Model for Evolution of Mating go through dauer, but the fact that hermaphrodites produce Systems about 10% males by self-fertilization may compensate for The presence of three sexual morphs in a population has been this. interpreted as an intermediate evolutionary step between gon- Sexual polymorphism may be the precursor of more ochorism (male/female) and hermaphroditism [16, 17]. Corrob- complex mating systems. In some species of parasitic orating the theoretical prediction that (males, females, nematodes, a hermaphroditic parasitic generation alternates and hermaphrodites) is evolutionarily unstable, C. elegans with a free-living, gonochoristic generation [24]. Similar to populations engineered to mimic a trioecious species revert Rhabditis sp. SB347, the hermaphrodite of parasites of the to androdioecy (males/hermaphrodites) within a few genera- genus Rhabdias obligatorily develops through the diapause tions [18, 19]. However, a direct comparison to C. elegans is stage. A second similarity is that most of the Rhabdias males difficult because sexual morph development in Rhabditis sp. are derived from selfing rather than from outcrossing [25]. SB347 is dependent on the timing of the production of the The amenability to genetic manipulation in laboratory cultures egg, the type of cross from which it is derived, and the avail- makes Rhabditis sp. SB347 a useful model to study the evolu- ability of a natural precursor for producing endocrine tion of complex mating systems such as those found in hormones. In contrast to C. elegans, in which a germline sex parasites. determination mutation transforms a hermaphrodite into a female [20], the Rhabditis sp. SB347 female phenotype does not seem to segregate as a simple Mendelian character. Supplemental Information The strain Rhabditis sp. SB347 has been inbred by selfing over Supplemental Information includes one figure and Supplemental Experi- ten generations, and the mean brood of a parental hermaphro- mental Procedures and can be found with this article online at doi:10. dite results in 46% hermaphrodites, 44% females, and 10% 1016/j.cub.2011.08.009. Sexual Plasticity in a Nematode 1551

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