Building on a recent study showing that gonad structures form in asexually reproducing individuals of the annelid Pristina leidyi [32], in this study we aimed to characterize gonad plasticity and to investigate gonad regeneration in this species. We identified both extrinsic and intrinsic factors that influence the presence and size of gonad structures and found that gonads in P. leidyi can develop by morphallactic regeneration in previously non-gonadal segments. Furthermore, although gonad size remains relatively small in all asexual individuals, we identified a sexually mature individual from our laboratory cultures indicating that this laboratory strain of P. leidyi retains the potential to become fully sexual. Together, our findings demonstrate that gonads in P. leidyi are highly plastic with respect to their presence, their size, and their location. We find that, as a group, clitellate annelids can form gonads in at least four different contexts, making this group a particularly useful one for investigating the developmental, physiological, and molecular mechanisms involved in gonad formation.
Nutritional status, individual age, and parental age all strongly influence gonad size in asexual P. leidyi
Although cultures of P. leidyi reproduce clonally, and thus little if any genetic variation is expected among individuals, we noted very high variation in the size of developing gonads among individuals in our cultures. Our experiments showed that gonad size is extremely sensitive to both extrinsic and intrinsic factors, specifically feeding level, individual age, and parental age (Figs. 1, 2). The functional consequence of differences in gonad size is not yet known, but a possible explanation is that this variation correlates with individuals being closer to or farther from sexual maturity. Testing this idea would require knowing the factors triggering sexual maturation, such that individuals with gonads of different sizes could be assayed for how readily or quickly they become fully sexual. Although such factors are not yet known, precluding testing this idea at this time, the identification of a sexually mature individual from our cultures indicates that sexual maturity can indeed be reached in this species even after extended periods of exclusively asexual reproduction (Fig. 4).
Starvation-induced gonad regression is known to occur in many animal groups, including several annelids [6, 9, 21, 41, 42]. When starved, P. leidyi individuals stop fissioning and their gonads shrink dramatically, becoming undetectable (by either in situ hybridization or histological staining) by 3–4 weeks without food. We estimate that if gonads are present at all, they must be composed of only a few cells at most and either do not express the piwi homolog we investigated, PRIle-piwi1, or do so at extremely low levels, below detectability by in situ hybridization. However, when starved worms are refed, they redevelop detectable gonads with strong PRIle-piwi1 expression within just a few days. Cells within the gonads of well-fed worms are positive for EdU labeling, indicating that gonad growth occurs, at least in part, by cell proliferation within the gonad. In refed worms, PRIle-piwi1 expression in the gonad area appears to arise de novo, since starved worms do not express detectable levels of PRIle-piwi1 anywhere in the body. What the cellular source is of the reestablished gonads remains an important question to address. It is possible that a few gonad cells or stem cells persist at the gonad site and regrow the gonads when feeding is restored, or that all gonad cells disappear under starvation but, when feeding is restored, source cells are induced and/or migrate from some other location to reestablish the gonad. Recent studies in P. leidyi have documented migration of cells using live imaging during both regeneration [43] and fission [32], and a study in another clitellate annelid has inferred migration of germline precursor cells from snapshot data during regeneration [17]; the cell migration hypothesis for gonad establishment is thus a viable one worth investigating further. Whether the source cells of reestablished gonads express PRIle-piwi1 at very low levels or not at all, and whether they express PRIle-piwi1 protein, should also be investigated in future studies.
Since many animals do not produce mature gonads until they reach a certain size or age, we had expected that, if age effects were present in P. leidyi, older worms would be the ones to have larger gonads (on the assumption that they are closer to maturation). However, we found the exact opposite: The largest gonads are found in the youngest worms (newly fissioned posterior worms) (Fig. 2). Specifically, we found that young fission products start off with large gonads and these gonads shrink dramatically over the next few weeks. Several scenarios could account for gonad shrinkage as individuals age. Shrinkage could be mediated by environmental cues. For example, the default state of young individuals (young fission products) may be to become sexually mature (and thus to have large gonads) but then, in response to cues from the environment (which in the laboratory favor only asexual reproduction), they may invest resources preferentially into other processes rather than maintaining large gonads. The cause of shrinkage could also be due to changes in the individual’s energy budget, which is known to be dynamically allocated in this species [38]. For example, young worms might start off with the greatest energy budget, having received considerable investment from their (anterior) parent, and thus having the most energy available to invest into gonads; once these animals need to sustain themselves by feeding and invest energy into fission products themselves, energy may be better allocated into these processes rather than in maintaining large gonads. Gonad shrinkage could also result from a general decrease in body function, namely senescence, and/or a depletion of germline cells. If, for example, the cells that make the gonads of a fission product must come from the (anterior) parent worm’s gonads, successive rounds of fission might eventually deplete the anterior worm’s germline cells. Testing these hypotheses and investigating the ultimate and proximate causes of gonad shrinkage (and growth) will help elucidate mechanisms of germline plasticity.
Our study also demonstrated a strong parental effect on asexually produced offspring (Fig. 2). The offspring (posterior zooids) of young parents (anterior zooids) had significantly larger gonads than the offspring of older parents. Therefore, just as is found in sexually produced offspring of many species [44–48], and also in offspring produced via parthenogenesis [49], features of asexually produced offspring can be strongly influenced by the age of the parent. The cause(s) of this parental effect could include scenarios similar to those described above for individual-level age effects. Whether the parental effect we identified in P. leidyi is in some way indicative of senescence of the anterior zooid remains to be determined. Evidence of senescence has been demonstrated in several asexual invertebrates (e.g., colonial tunicates, bryozoans, hydroids in [50]), including another fissioning naidid (Paranais litoralis) [51], so this is a viable possibility.
Gonads can develop in a variety of post-embryonic contexts in P. leidyi and in annelids more broadly
Findings from this and other studies suggest that gonads can be established in a variety of post-embryonic contexts in P. leidyi and in clitellate annelids more broadly (Fig. 5) [17, 27, 32, 52]. Gonads can be induced by feeding, can form by morphallaxis following amputation, can form during paratomic fission, and can develop by epimorphic regeneration. We found that the first three of these can all occur in P. leidyi (this study; [32]), indicating that this species has a particularly broad repertoire for establishing gonads post-embryonically; the last of these appears to not be possible in P. leidyi but has been described in several other annelids [17, 27, 52].
Findings from this study indicate that the presence of detectable gonads in P. leidyi is sensitive to feeding regime, with individuals losing detectable gonads when starved and regrowing gonads within days after refeeding (Fig. 5a). A strong effect of feeding on gonads has also been demonstrated in other annelids, such as the annelid Enchytraeus japonensis in which sexualization is dependent on feeding [23]. Food availability is known to influence gonad development and maintenance in a number of animals; for example, in macrostomid flatworms, gonads of starved adults shrink to below detectability [41, 42], similar to our findings in P. leidyi, and in insects, starved larvae form fewer ovarioles [53] and adults can resorb oocytes [9]. We therefore expect that feeding-dependent gonad formation may be widespread among annelids.
Our study also confirms that gonads can form by morphallaxis of previously non-gonadal segments following amputation in P. leidyi (Fig. 5b). In this species, a maximum of four segments can regenerate anteriorly; if more than four are amputated anteriorly, the normal morphology of the animal is reestablished by tissue remodeling of the remaining original segments. In this study, following the removal of seven anterior segments, we found that gonads are established in segments that were formerly non-gonadal segments (segments 9 and 10) when these segments acquire the body position typical of gonadal segments (segments 6 and 7) (Fig. 3). Gonad formation by morphallaxis has been documented in another clitellate annelid [17], but otherwise remains poorly studied. Given that morphallaxis of somatic body features is common in annelids [54], the possibility that gonad morphallaxis occurs more broadly among annelids should be further investigated.
In P. leidyi, the process of paratomic fission involves the novel formation of the six anterior-most segments, including the septum between segments 5 and 6 and the septum between segments 6 and 7; it is on these two newly formed septa (the two posterior-most septa formed within the new head) that P. leidyi forms gonad structures during fission (Fig. 5c). Thus, P. leidyi paratomy involves formation of gonads from newly developed tissues, similar to gonad formation during epimorphic regeneration (Fig. 5d). Paratomy in P. leidyi is thought to have evolved as a modification of the epimorphic regeneration process [34, 55]; thus, it is possible that the similarity between gonad formation during paratomy and that during epimorphic regeneration (present in other annelids, though not in P. leidyi) is due to the shared ancestry of these processes. Paratomy has evolved a number of times within annelids, and investigating gonad formation in additional species representing independent origins of paratomy will be needed to determine how widespread this mechanism of gonad development is in annelids.
Finally, in some annelids, though not P. leidyi, gonads form in segments that regenerate by epimorphosis (Fig. 5d). In P. leidyi, epimorphic regeneration of gonads does not occur because the maximum number of segments that can regenerate in this species (four) is less than the segment number in which gonads normally form (segments 6 and 7); thus, the gonadal segments are not regenerated epimorphically (but instead by morphallaxis of old segments). P. leidyi appears to be unusual in this regard, however. More typically, in clitellates, the gonadal segments, or at least the septa on which gonads will form, can regenerate; indeed, these are often the posterior-most structures that can be regenerated anteriorly [27, 54, 56, 57]. In this regard, an interesting case involves a fissioning species of Enchytraeus, in which individuals that develop from zygotes form gonads in segments 10 and 11, but in which a maximum of seven new segments can regenerate anteriorly. Individuals produced by fragmentation/regeneration have gonads shifted anteriorly with respect to embryonically derived individuals; gonads of these asexually produced individuals occur on the posterior-most regenerated septa, namely segments 7 and 8, instead of in segments 10 and 11 [17, 52]. Regenerating gonads through epimorphosis, that is, on newly developed septa, thus appears to be an ancestral mechanism of gonad regeneration among clitellates, and one that is retained in certain asexually reproducing species, though not P. leidyi.
Importantly, across these four post-embryonic contexts (feeding, morphallaxis, paratomy, and epimorphosis), gonads form in different types of segments and with different cell type associations (Fig. 5). When starved worms are refed, gonads form in original segments that previously possessed gonads; during morphallaxis, gonads form in original segments that were previously non-gonadal; and during both paratomic fission and epimorphic regeneration, gonads form in newly formed segments. Several studies in annelids, including a prior study in P. leidyi, have suggested that isolated piwi-positive cells may be involved in germline development in annelids [17, 18, 32, 58]. In particular, our previous work in P. leidyi suggested that a population of piwi-positive cells found along the ventral nerve cord appears to be migratory and involved in development of gonads during fission [32], and a study in the clitellate Enchytraeus similarly inferred that gonad formation is associated with migration of piwi-positive cells [17]. However, in this study, we found no evidence that gonad formation is associated with the presence of piwi-positive presumptive germ cells during refeeding and morphallaxis. Together, these differences across post-embryonic contexts suggest the possibility that gonads may form by different post-embryonic processes in annelids, a possibility that should be investigated in future studies.