Vasa, Piwi, and Pl10 Expression during Sexual Maturation and Asexual Reproduction in the Annelid Pristina longiseta

Naidids are tiny, transparent freshwater oligochaetes, which are well known for their ability to propagate asexually. Despite the fact that sexually mature individuals and cocoons with embryos are sometimes found in nature, in long-period laboratory cultures, worms reproduce agametically only. In this paper, we showed, for the first time, the expression of Vasa, Piwi, and Pl10 homologs in mature Pristina longiseta worms with well-developed reproductive system structures and germ cells. Although the animals have been propagated asexually by paratomic fission for over 20 years in our lab, some individuals become sexualized under standard conditions for our laboratory culture and demonstrate various stages of maturation. The fully matured animals developed a complete set of sexual apparatus including spermatheca, atrium, seminal vesicles, and ovisac. They also had a clitellum and were able to form cocoons. The cues for the initiation of sexual maturation are still unknown for P. longiseta; nevertheless, our data suggest that the laboratory strain of P. longiseta maintains the ability to become fully sexually mature and to establish germline products even after a long period of agametic reproduction. On the other hand, many of the sexualized worms formed a fission zone and continued to reproduce asexually. Thus, in this species, the processes of asexual reproduction and sexual maturation do not preclude each other, and Vasa, Piwi, and Pl10 homologs are expressed in both somatic and germline tissue including the posterior growth zone, fission zone, nervous system, germline cells, and gametes.


Introduction
Reproduction of metazoans occurs mainly by the sexual mode. It is carried out by special types of cells, namely germ cells, or gametes, which are most often formed in permanent or temporary gonads [1,2]. In case of hermaphroditism, the same animal develops both types of germ cells, the male and the female ones. In addition to conspicuous germline cells, the somatic part of the reproductive system may have a complex structure, to ensure both cross-fertilization and embryonic development. Thus, during sexual reproduction, being inherent in virtually all animals, each individual develops various organs necessary for the formation of germ cells to ensure the fertilization process. However, many animals are capable of asexual reproduction [3][4][5][6]. In this case, special germ cells and gonads are not required. Instead, reproduction relies on the separation of a body fragment from the maternal organism on the background of somatic cell proliferation. Agametic reproduction allows rapid expansion within distinct ecological niches, as well as effective multiplication In this study, we have shown that asexually reproducing individuals of naidid Pristina longiseta were able to establish germ cells and become fully sexually mature. We also have discovered that asexual propagation and germ cell/gonadal development/maturation are not mutually exclusive. We have identified five GMP genes, homologs of Vasa (Plovasa), Piwi (Plo-piwi1, Plo-piwiA, and Plo-piwi2), and Pl10 (Plo-pl10), and characterized their expression using whole-mount in situ hybridization. During asexual reproduction and sexual maturation, Vasa, Piwi, and Pl10 homologs are differentially expressed in both somatic and germline tissue including the posterior growth zone, fission zone, nervous system, germline cells, and gametes.

Animal Material and Fixation
The laboratory culture of Pristina longiseta was maintained using specimens originally found in a pond in the park of the Biological Institute of Saint-Petersburg State University (Russia) in 1999. Animals were cultured in Petri dishes with artificial spring water and Chlorophyta algae at 18 • C. Mashed spinach or dried spirulina powder was used as feed. As described previously [38], artificial illumination (16 h day, 8 h night) was used to optimize the intensity of asexual reproduction in cultures. We found that animals became occasionally sexualized under standard conditions for our laboratory culture. Thus, both fissioning and sexualized worms were collected from actively growing cultures.
Attempting to promote sexualization in P. longiseta, we have carried out a series of experiments with environmental shift. First, we have changed the photoperiod parameters by reducing daylight hours to 10 h (which corresponds to the length of the day in autumn or spring). In a series of parallel experiments, we varied the temperature by increasing it to 25 • C or decreasing it to 14 • C. Animals were observed every two days for 1 month. Worms continued to reproduce asexually, but showed no signs of mature gonads or gametes. The increased temperature led to the acceleration of asexual reproduction. Shorter daylight hours, as well as lower temperatures, slightly reduced the rate of population growth through asexual reproduction. Next, to assay the effect of starvation/refeeding, worms from the main laboratory culture were placed in separate Petri dishes with clean water. Three weeks later, these animals were fed. Starving animals stopped fissioning. After refeeding, they again began to reproduce asexually. Thus, the different conditions of these experiments affected the rate of asexual reproduction; the cues for initiation of sexual maturation are still unknown for P. longiseta.
To obtain materials for in situ hybridization and DIC analysis, worms were relaxed for 10 min in relaxant solution (10 mM MgCl 2 /5 mM NaCl/1 mM KCl/8% ethanol; see [44]) prior to fixation. For the DIC analysis of unlabeled intact objects, specimens were fixed overnight in 4% formaldehyde in 0.75× PBS/0.1% Tween-20 at 4 • C., and embedded in glycerol/PBS solution (9:1). For in situ hybridization experiments, the specimens were fixed in 4% PFA in PBS with 0.1% Tween 20 at +4 • C overnight and stored in MeOH at −20 • C.

Semi-Thin Sections Preparation
To prepare serial semi-thin sections, anesthetized fissioning worms were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2.5 h, washed in the same buffer, and postfixed in 1% OsO4 in 0.1 M cacodylate buffer. The fixed material was dehydrated in ethanol and embedded in Epon (Fluka). Semi-thin sections were prepared on a Leica EM UC7 ultramicrotome, poststained with methylene blue, and embedded in Histokitt medium (Roth Chemie, Karlsruhe, Germany).

Gene Cloning and Phylogenetic Analysis
To identify P. longiseta homologs of Vasa, Piwi, and Pl10, PCR reactions with degenerate primers and asexually reproducing worms' cDNA were performed. Extended fragments for Plo-vasa, Plo-piwi1, Plo-piwiA, Plo-piwi2, and Plo-pl10 were amplified by 5 -RACE and/or 3 -RACE PCR with gene-specific primers and cDNA prepared with the SMARTer RACE cDNA Amplification Kit (Clontech, Mountainview, CA, USA). All primers are given in the Supplementary Materials (Table S1). These amplified gene fragments were cloned into pCRII vectors (TOPO-TA cloning, Invitrogen, Waltham, MA USA) that were used in the transformation of chemically competent E. coli (One Shot ® TOP10). Plasmids with correct inserts were sequenced and used for RNA probe synthesis. The identity of cloned sequences was confirmed through phylogenetic analysis. Amino acid translations of the five P. longiseta genes were aligned to homologs and outgroups from other species (Supplementary Materials, Tables S2 and S3) using MUSCLE 3.8.31 [45] at the Phylogeny.fr web server (accessed on 26 June 2023) [46]. Bayesian phylogenetic analysis was conducted using the Markov-chain Monte Carlo method implemented in MrBayes 3.2.6 (http:// www.phylogeny.fr/ accessed on 26 June 2023) [46][47][48] as previously described [30]. The phylogenetic trees were handled using the FigTree program, v1.4.4 (http://tree.bio.ed.ac. uk/software/, accessed on 26 June 2023) (Supplementary Materials, Figures S1 and S2). The obtained sequences of Plo-vasa, Plo-pl10, Plo-piwiA, Plo-piwi2, and Plo-piwi1 were deposited in GenBank with the accession numbers JX264563-JX264567, and OR203685.

Data Visualization
Imaging of the mounted in-glycerol or in-Histokitt-medium specimens was conducted using DIC optics on an Axio Imager D1 microscope (Carl Zeiss, Oberkochen, Germany) equipped with digital camera AxioCam ICc5 (Carl Zeiss, Oberkochen, Germany). Pictures were acquired and edited with programs AxioVision 4.8 and Adobe Photoshop CS5.1.

Asexual Reproduction in Pristina longiseta
Ready to undergo paratomic fission, Pristina longiseta worms typically comprise 21-29 segments. A cephalic region consisting of the prostomium, peristomium, and specialized head (cephalic) segments is present at the anterior end of the worm. The posterior growth zone, that produces new segments during normal growth, is located just anterior to the pygidium at the posterior end. The number of head segments is constant and speciesspecific. In P. longiseta, it comprises six segments. The head segments are distinguished from all other segments by the absence of chloragogen cells and nephridia. Fission zones are typically formed between segments 14 and 18. Thus, there is no fixed segment for developing the paratomic zone (see also [38,49] for more details). Agametic reproduction in P. longiseta occurs by paratomy. It means that the new anterior and posterior ends of the two zooids develop prior to the physical separation of the fusioning individual ( Figure 1).
The paratomic fission zone forms in the one-third of a segment behind the anterior border of a trunk segment. Later, it is subdivided by a circular epidermal thickening into two parts, the cephalogenic and somatogenic parts ( Figure 1A,D,E). The cephalogenic part gives rise to the new cephalic region of the posterior zooid, and the somatogenic part gives rise to the new posterior end (with pygidium and growth zone) of the anterior zooid. The physical separation of the two daughter individuals occurs only after the complete formation of all these structures ( Figure 1F). Under optimal conditions, a worm can develop multiple fission zones, and every additional fission zone is usually initiated in progressively more anterior segments. Thus, P. longiseta reproduces by so-called rapid paratomy [5]. (A) Schema of rapid paratomy in P. longiseta. The first fission zone (fzI) usually appears within a segment within from 14 to 18, and gives rise to a new cephalic (head) region, and a new tail end with pygidium, growth zone (*), and some trunk segments. During progressive formation of fission zone I (fzI), an additional second fission zone (fzII) becomes evident in more anterior segment. After the new anterior and posterior ends of two-zooid development, the paratomy process is continued by the physical separation of the two daughter individuals. The blue color marks the head region and cephalogenic part of the fission zone; the pink color marks the somatogenic part of the fission zone and its derivatives. (B,C) Cross semi-thin sections through the cephalogenic part of the fission zone (B) and at the level of the boundary between the anterior and posterior zooids (C). Epidermal cells are modified and have morphology similar to the blastemal cells. Staining with methylene blue. The paratomic fission zone forms in the one-third of a segment behind the anterior border of a trunk segment. Later, it is subdivided by a circular epidermal thickening into two parts, the cephalogenic and somatogenic parts ( Figure 1A,D,E). The cephalogenic part gives rise to the new cephalic region of the posterior zooid, and the somatogenic part gives rise to the new posterior end (with pygidium and growth zone) of the anterior zooid. The physical separation of the two daughter individuals occurs only after the complete formation of all these structures ( Figure 1F). Under optimal conditions, a worm can develop multiple fission zones, and every additional fission zone is usually initiated in progressively more anterior segments. Thus, P. longiseta reproduces by so-called rapid paratomy [5]. At the early and middle stages of development, the paratomy zone differs significantly from other parts of the body in cell composition ( Figure 1B-E). It lacks such differentiated cell types such as chloragogen cells and cells of nephridia, while muscle fibers become modified and gradually break during fission (Figures 1B,C and 2A; see also [38,41] for more details). Only the intestinal epithelium remains seemingly unchanged. At the same time, a significant number of small, undifferentiated cells with a high nuclear-cytoplasmic ratio and intensively basophilic cytoplasm were observed within the fission zone on the semi-thin sections ( Figure 1B,C). Some of these cells proliferated ( Figure 1B,C). The integumentary epithelium of the paratomy zone undergoes significant modifications as well. After staining with methylene blue, the cytoplasm of epithelial cells in the paratomy zone was more basophilic than in other areas ( Figures 1B,C and 2A). These cells were columnar and dropshaped instead of the normal flattened morphology. Many cells displayed mitotic activity. The boundaries between the modified epithelium and the mass of deep undifferentiated cells were not obvious, and cells of these two domains were not clearly distinguished from each other. entiated cell types such as chloragogen cells and cells of nephridia, while muscle fibers become modified and gradually break during fission (Figures 1B,C and 2A; see also [38,41] for more details). Only the intestinal epithelium remains seemingly unchanged. At the same time, a significant number of small, undifferentiated cells with a high nuclear-cytoplasmic ratio and intensively basophilic cytoplasm were observed within the fission zone on the semi-thin sections ( Figure 1B,C). Some of these cells proliferated ( Figure 1B,C). The integumentary epithelium of the paratomy zone undergoes significant modifications as well. After staining with methylene blue, the cytoplasm of epithelial cells in the paratomy zone was more basophilic than in other areas ( Figures 1B,C and 2A). These cells were columnar and drop-shaped instead of the normal flattened morphology. Many cells displayed mitotic activity. The boundaries between the modified epithelium and the mass of deep undifferentiated cells were not obvious, and cells of these two domains were not clearly distinguished from each other.  Later, when an external epithelial furrow grows, and a constriction between the anterior (somatogenic) and posterior (cephalogenic) cell masses of the fission zone become evident ( Figure 1C), structures of new cephalic region begin to form ( Figure 1G,E) including the presumptive prostomium, as a growing epidermal fold, and the primordia of the cerebral ganglion, as a derivate of deep undifferentiated cells. The cephalogenic mass of deep cells, having increased greatly in size, segments to form bilateral pairs of chaetal sacs. Simultaneously, the posterior growth zone of the anterior zooid begins to produce new trunk segments ( Figure 1F).
During the final growth of the paratomic zone, the old segments behind the new head display a segment identity shift. The gut in the segment immediately behind the fission zone thickens and forms a new stomach by morphallaxis [28,44,49,50]. Another important manifestation of this shift is the establishment of female gonad-like structures in previously non-gonadal segments ( Figure 2A).
By analyses of the semi-thin sections through the VII segment of the posterior zooid (an old segment that became the first trunk segment), the ovary-like structures were found on both sides of the ventral nerve cord close to the anterior septum. Each ovary was composed of germ and somatic cells in close association ( Figure 2A). The germ cells were interpreted as oogonia. At the same time, in the most posterior new-formed cephalized segment of the posterior zooid (segment VI), some male-specific structures became evident (see below).
We also found that many components of male and female reproductive systems are usually reduced or absent in the worms that actively propagated asexually.

Sexually Matured Form of Pristina longiseta
Naidid species in the wild reproduce both asexually and via sexualized individuals [5,15] but they are mostly not known to reproduce sexually under laboratory conditions [37,38,42,43]. For example, P. longiseta worms were successfully propagated asexually only over a period of time of observation in our laboratory. Attempting to promote sexualization in P. longiseta, we have carried out a series of experiments with environmental shift. In particular, we have changed the photoperiod parameters as well as temperature and food availability. The different conditions of these experiments affected the rate of asexual reproduction but we were not able to induce sexualization and obtain fully mature individuals. Nevertheless, we found over forty sexualized individuals in several Petri dishes of our laboratory cultures. These mature worms were used for both morphological and molecular research. These specimens became sexualized under standard conditions for our laboratory culture. Thus, the cues for initiation of sexual maturation are still unknown for P. longiseta. All these sexualized worms demonstrated various stages of maturation. Most of them developed sexual anatomy ( Figure 2B,D-F). Sexualized worms were hermaphroditic and developed variable-in-size gonads in two consecutive segments, a pair of testes in segment VI, and a pair of ovaries in segment VII. The fully matured animals developed a complete set of sexual apparatus including spermatheca, atrium, seminal vesicles, and ovisac. They also had a clitellum, the collar of thickened epithelium around the sexual segments. Germ cells (sperm and oocytes) were found inside the corresponding parts of the reproductive system in these specimens ( Figure 2F). Altogether, the data suggest that the laboratory strain of P. longiseta maintains the ability to become fully sexually mature and to establish germline products even after a long period of agametic reproduction. During the intensive search, we found several egg-and ellipsoid-shaped cocoons ( Figure 2G,H), but all of them were empty, without an egg or embryo. Thus, the question if these animals are really able to develop embryonically is still open.
Interestingly, many sexualized individuals undergo paratomic fission at the same time ( Figure 2C,E). The animals showed various stages of fission-zone formation. Most of them were at the early or middle stages, but several were at the late stage and ready for the physical separation of the two daughter individuals. By this way, the processes of asexual reproduction and sexual maturation do not compete with each other. On the other hand, although the animals showed various stage of fission-zone formation, no one sexualized worm had multiple fission zones developing at once.

Vasa, Piwi, and Pl10 Homolog Expression in Growing Adults and Asexually Reproducing Pristina longiseta
One of the main goals of this study was to investigate how germline/multipotency markers are expressed during paratomic fission and sexual maturation in the annelid P. longiseta. In this work, we identified in P. longiseta three homologs of Piwi (Plo-piwi1, Plo-piwiA, and Plo-piwi2), one homolog of Vasa (Plo-vasa), and one homolog of PL10 (Plo-pl10), and examined their developmental patterns by WMISH.
Plo-piwi2 is the only gene whose mRNA is not detected by in situ hybridization in growing adult P. longiseta worms. All other genes show strong expression in the superficial and deep cells of young segments and the posterior growth zone in both growing adult worms and asexually reproducing animals. The expression domains of Plo-vasa, Plo-piwi1, Plo-piwiA, and Plo-pl10 are especially wide on the lateral and ventral sides; however, Plo-piwiA signal is more diffuse. None of these gene transcripts were detected in the pygidium ( Figures 3A,B,E,J, 4A,B,E, 5F and 6A,E). Plo-piwiA and Plo-pl10 transcripts were found in ventral-nerve-cord cells ( Figures 4A,B,F and 6A,E). In addition, Plo-piwiA-positive cells were also seen in the hindgut region ( Figure 6F).
question if these animals are really able to develop embryonically is still open.
Interestingly, many sexualized individuals undergo paratomic fission at the same time ( Figure 2C,E). The animals showed various stages of fission-zone formation. Most of them were at the early or middle stages, but several were at the late stage and ready for the physical separation of the two daughter individuals. By this way, the processes of asexual reproduction and sexual maturation do not compete with each other. On the other hand, although the animals showed various stage of fission-zone formation, no one sexualized worm had multiple fission zones developing at once.

Vasa, Piwi, and Pl10 Homolog Expression in Growing Adults and Asexually Reproducing Pristina longiseta
One of the main goals of this study was to investigate how germline/multipotency markers are expressed during paratomic fission and sexual maturation in the annelid P. longiseta. In this work, we identified in P. longiseta three homologs of Piwi (Plo-piwi1, Plo-piwiA, and Plo-piwi2), one homolog of Vasa (Plo-vasa), and one homolog of PL10 (Plo-pl10), and examined their developmental patterns by WMISH.
Plo-piwi2 is the only gene whose mRNA is not detected by in situ hybridization in growing adult P. longiseta worms. All other genes show strong expression in the superficial and deep cells of young segments and the posterior growth zone in both growing adult worms and asexually reproducing animals. The expression domains of Plo-vasa, Plo-piwi1, Plo-piwiA, and Plo-pl10 are especially wide on the lateral and ventral sides; however, Plo-piwiA signal is more diffuse. None of these gene transcripts were detected in the pygidium ( Figures 3A,B,E,J, 4A,B,E, 5F and 6A,E). Plo-piwiA and Plo-pl10 transcripts were found in ventral-nerve-cord cells ( Figures 4A,B,F and 6A,E). In addition, Plo-piwiA-positive cells were also seen in the hindgut region ( Figure 6F).      developing fission zone and transcripts of this gene are shown at various stages of asexual reproduction, from the earliest steps to the latest stage (A-E). Expression is observed in an additional fission zone (D). Plo-piwi1 is also strongly expressed at the posterior end of the worms, in somatic tissue of the young segments and posterior growth zone, but not in the pygidium, in which no expression is detected (F). Arrows indicate additional domains of internal and superficial patches of Plo-piwi1-positive cells in middle and posterior trunk segments (E,F). The asterisk marks the posterior growth zone, the red line marks the new developing tail region, and the green line marks the new developing head region within the fission zone. Scale bar, 45 µm for all panels. Plo-piwiA appears to be expressed de novo in the developing fission zone and transcripts of this gene are shown at various stages of asexual reproduction, from the earliest steps to the latest stage In segments of the animal's body without a fission zone, the expression of Plo-vasa and Plo-piwi1 shows a character that is more complex. In the posterior third of the body of P. longiseta, the domains of Plo-vasa and Plo-piwi1 expression look like small bilaterally (mostly ventrolaterally) located patches of cells of the integumentary epithelium or internal cells ( Figures 3E-G,I,J and 5C,E). The anterior border of such an expression pattern corresponds typically to the position of a fission zone or is located slightly anteriorly.
Along the ventral nerve cord, dorsally to it, there are single Plo-vasaand Plo-piwi1positive cells. These cells are spindle-shaped and have a high nuclear-cytoplasmic ratio. The most anterior localization of such cells is at the level of the first postlarval (trunk, not head) segment. The number of such cells in the segments, as well as in different samples, varies greatly and may correlate with the feeding conditions of the animals (Figures 7 and 8).
nal cells (Figures 3E-G,I,J and 5C,E). The anterior border of such an expression pattern corresponds typically to the position of a fission zone or is located slightly anteriorly.
Along the ventral nerve cord, dorsally to it, there are single Plo-vasa-and Plo-piwi1positive cells. These cells are spindle-shaped and have a high nuclear-cytoplasmic ratio. The most anterior localization of such cells is at the level of the first postlarval (trunk, not head) segment. The number of such cells in the segments, as well as in different samples, varies greatly and may correlate with the feeding conditions of the animals (Figures 7 and  8).  Plo-vasa, Plo-pl10, Plo-piwi1, Plo-piwiA, and Plo-piwi2 are expressed de novo in the area of paratomy (Figures 3,4,5 and 6). Plo-vasa appears to be expressed earlier than other genes in this area, being present, already, at the very early stages of development of the fission zone ( Figure 3F). Plo-pl10 and Plo-piwi1 transcripts appear within the paratomy zone a little later and Plo-piwiA occurs even later and less intensively. A very low level of diffuse Plo-piwi2 expression can only be detected at the early mid-fission stage ( Figure 6G). The first signs of expression of Plo-vasa, Plo-pl10, Plo-piwi1, and Plo-piwiA appear in the cells of Plo-vasa, Plo-pl10, Plo-piwi1, Plo-piwiA, and Plo-piwi2 are expressed de novo in the area of paratomy (Figures 3-6). Plo-vasa appears to be expressed earlier than other genes in this area, being present, already, at the very early stages of development of the fission zone ( Figure 3F). Plo-pl10 and Plo-piwi1 transcripts appear within the paratomy zone a little later and Plo-piwiA occurs even later and less intensively. A very low level of diffuse Plo-piwi2 expression can only be detected at the early mid-fission stage ( Figure 6G). The first signs of expression of Plo-vasa, Plo-pl10, Plo-piwi1, and Plo-piwiA appear in the cells of the modified epidermis; then, as blastemal masses develop, large expression domains are formed corresponding to the internal masses of undifferentiated cells. The expression level remains very high throughout the middle stage and the beginning of the late paratomy in both the developing new caudal end and the developing head region. High levels of Plo-vasa, Plo-pl10, Plo-piwi1, and Plo-piwiA transcripts are seen in the posterior growth zone of the anterior zooid ( Figures 3G-I, 4G, 5D and 6C,D), during the late stage of asexual reproduction, a distinct domain of Plo-pl10 expression appears in the intestine at the level of the future seventh segment of the posterior zooid. The expression of all these genes gradually disappears in the cells of the integumentary epithelium, and then in the internal cells of the cephalogenic part of the fission zone. Nevertheless, low levels of Plo-vasa and Plo-pl10 transcripts are found in the head region of the posterior zooid immediately after physical separation from the anterior (parent) individual ( Figure 7F). At the end of morphallactic remodeling of the gut into the stomach (segment VII), Plo-vasa expression is no longer detected in the head region, but Plo-pl10 continues to be expressed in the anterior nerve ganglia, as well as in a group of cells dorsally and laterally adjacent to the cerebral ganglia and buccal pharynx ( Figure 4F,J).

Vasa, Piwi, and Pl10 Homolog Expression in Sexually Matured Pristina longiseta
In actively asexually reproducing P. longiseta, small ventrolateral clusters of Plo-vasa-, Plo-pl10-, and Plo-piwi1-positive cells are found close to the anterior septum of segment VI, and sometimes segment VII. According to the available data on the structure of the reproductive system and gametogenesis in oligochaetes, naidids in particular [40,41], the anlages of the testes and ovaries appear in segments VI and VII, respectively, on the border of the larval (head region) and postlarval body. During P. longiseta sexual maturation, the testes increase in size; the clitellum, seminal and ovarian vesicles, and other structures appear. The germ cells of oligochaetes leave the gonads very early. Clusters of 8-16 spermatogonia, which are products of incomplete cytokinesis, separate from the testes. They enter the body cavity and then into the seminal vesicles. Spermatogonia in each cluster actively proliferate, and as a result, large masses of cells are formed. In the ovaries, the development of female germ cells occurs up to the stage of early oocytes, which also form groups of 16-32 cells. Further development of female germ cells occurs in the coelom of the ovarian segment, and later in the ovisac. In each of the groups of oocytes, only one cell becomes an egg. It accumulates yolk and undergoes meiosis. Thus, the gonads themselves do not exist for long and quickly disappear, especially in naidids. Male gonads are always formed earlier than female gonads (protandric hermaphroditism) [40,41].
The results of in situ hybridization show the differential character of the active expression of the Plo-vasa, Plo-pl10, and Plo-piwi1 genes at the stages of gonadal development and functioning (Figures 9-11). mRNAs of these genes are detected already at the earliest stages of male germ-cell formation. A very high level of transcripts is characteristic of the Plo-pl10 gene, which is observed, probably, until the stage of spermiogenesis, i.e., the completion of spermatozoa formation in the seminal vesicles ( Figure 10A-C). mRNA of other genes disappears in spermatocytes much earlier. Expression of Plo-piwi1 fades in cell clusters that have left the testes, and Plo-vasa expression is limited by the time of formation of such clusters in the testes. No gene expression was detected in spermatozoa ( Figures 9A,D,F, 10F and 11F).      During P. longiseta sexual maturation, Plo-piwiA expression is not associated with either gonadal development or germ cell establishment and maintenance ( Figure 11H).

Reproductive Strategies in Pristina longiseta
Although virtually all multicellular animals are capable of embryonic development, many of them, from sponges to placental mammals, are also capable of asexual reproduction [2][3][4][5][6]11,14,18,27,29,35,39]. The very phenomenon of disintegration of the whole organism, in which an individual physically divides its body, and the acquisition by its parts of the status of individuals remains largely incomprehensible at the contemporary level of biology. The variety of forms of asexual reproduction and their distribution across taxa is evidence of independent repeated acquisition of this ability in different taxa [3,5,51]. Oligochaetes of the family Naididae are convenient models to study morphogenetic and evolutionary aspects of this problem. It is shown that in these worms, the paratomy type of transverse division occurred in several evolutionary lines on the basis of regenerative abilities [39,52]. In oligochaetes, paratomic fission is represented by two forms: slow paratomy and rapid paratomy. Slow paratomy is accompanied by the formation of chains from no more than two zooids (for example, Nais communis), while rapid paratomy leads to the formation of chains from several zooids (P. longiseta) [38,49]. In the case of P. longiseta, we observe one of the extreme forms of transition from sexual to predominantly asexual reproduction. In most other relevant examples, these forms of reproduction are in competitive relationships, i.e., individuals typically exhibit only one reproductive mode (agametic propagation or sexual reproduction) at a time [5,41,53]. Our work has illustrated this phenomenon in a new way. In P. longiseta, we found the initiation of gonad development in the segment of the body adjacent to the fission zone. The appearance of cell clusters, interpreted as prospective gonads, has also been described in a closely related species, P. leidyi [42,43]. It is noteworthy that the formation of clusters of gametogenic cells occurs in the old segment of the body undergoing morphallaxis. Such a restructuring of old tissues is much more complex and different from embryogenesis [1]. On the other hand, although tissue remodeling by morphallaxis occurs during both fission and sexual maturation, the mechanisms by which this is achieved should differ in the two contexts in naidid species. First, gonads are formed de novo by morphallaxis of previously non-gonadal segments. Second, development of reproductive system structures can be regulated under environmental and endogenous control, probably via endocrine regulation, and finally, the germ cells usually have a specific origin [27,28,54].
In our case, it was possible to find not only the initiation, but also a quite complete development of the sexual organ system. The appearance not only of mature gametes, but also clitellum and cocoon shells confirm this acquisition of sexual maturity. It is noteworthy that this spontaneous event occurred simultaneously in a large number of individuals that resembles the seasonal peak of sexual reproduction in other naidids [15]. Although a common pattern is that agametic propagation occurs in animals that are not yet sexually mature, and that such animals completely stop asexual reproduction before reproducing sexually, P. longiseta demonstrated the ability for both reproductive modes simultaneously. Thus, the processes of asexual reproduction and sexual maturation are not mutually exclusive in these animals. On the other hand, we did not find any embryos, and these data could be evidence of possible suppression of gamete formation (at final stages) or embryogenesis in the animals under laboratory conditions. In future studies, it will be worthwhile to determine the cues for the initiation of sexual maturation in P. longiseta to answer the question whether these animal are capable of embryonic development.
Our data on P. longiseta corroborate older descriptions and showed that the ovaries in naidids are inconspicuous and germ cells detach from the ovaries and form groups of cells that float within the coelomic cavity and ovisac [40,41,55,56]. The fact that only the very beginning of oogenesis occurs in the ovaries suggest the organization of the female gonads in naidids differs essentially from that found in other oligochaete annelids [57].
Our results are consistent with data on the involvement of the Vasa, Pl10, and Piwi homologs in the formation of new proliferative tissues, such as the posterior growth zone and the fission zone [6,24,31,43,60,62]. Moreover, we have shown that P. longiseta has three homologs of Piwi, and that all three are expressed in the fission zone, although Plo-piwi2 expression is very weak and transient. In contrast, in P. leidyi, only one of the two Piwi homologs is expressed in the fission zone [43]. In this work, we showed, for the first time, the involvement of the Pl10 homolog in the formation (and thus, presumably, functioning) of the posterior growth zone and fission zone. In addition, this gene seems likely to be important for morphallactic remodeling of the gut during stomach formation in the posterior zooid, and can also have some function in the nervous system ( Figure 12). that the expression of GMP genes in the posterior growth zone, fission zone, and regeneration blastema is an ancestral feature for annelids. Moreover, recent work has shown that piwi-positive cells can give rise to variable differentiation patterns in adult P. leidyi worms [61], raising even more intriguing questions.
Our results are consistent with data on the involvement of the Vasa, Pl10, and Piwi homologs in the formation of new proliferative tissues, such as the posterior growth zone and the fission zone [6,24,31,43,60,62]. Moreover, we have shown that P. longiseta has three homologs of Piwi, and that all three are expressed in the fission zone, although Plo-piwi2 expression is very weak and transient. In contrast, in P. leidyi, only one of the two Piwi homologs is expressed in the fission zone [43]. In this work, we showed, for the first time, the involvement of the Pl10 homolog in the formation (and thus, presumably, functioning) of the posterior growth zone and fission zone. In addition, this gene seems likely to be important for morphallactic remodeling of the gut during stomach formation in the posterior zooid, and can also have some function in the nervous system ( Figure 12). Figure 12. Summary of gene expression during P. longiseta asexual reproduction (fission zone formation) and sexual maturation (germ cells/gonadal development). Schematic representation of Plo- Figure 12. Summary of gene expression during P. longiseta asexual reproduction (fission zone formation) and sexual maturation (germ cells/gonadal development). Schematic representation of Plo-vasa, Plo-pl10, Plo-piwi1, Plo-piwiA, and Plo-piwi2 expression patterns is shown in different colors for each gene. Lateral view, anterior to the left for all panels. During asexual reproduction, a region of new tissue referred to as a fission zone forms within a mid-body segment, developing a new tail and a new head. All five are expressed de novo in the area of paratomic fission zone. Plo-vasa and Plo-piwi1 are also expressed in ventrolateral patches of cells (internal and epidermal), as well as in cells that are distributed along the ventral nerve cord and are likely migrating and associated with gonads. Plo-vasa, Plo-pl10, and Plo-piwi1 genes are differentially expressed at the stages of gonadal development and gametogenesis, while transcripts of Plo-piwiA and Plo-piwi2 are not detected in the gonadal segments (VI and VII) during the sexual maturation of animals. See text for more details.
In P. longiseta, we found that two of the five GMP genes we investigated, Plo-vasa and Plo-piwi1, are expressed in isolated spindle-shaped cells distributed along the ventral nerve cord. The morphology of these cells suggests they are migrating cells. Similar Piwi-positive cells were also shown in P. leidyi; however, such Vasa-positive cells were undetectable by WMISH in this species. These cells appear to be associated specifically with the fission process and gonads [42,43].
Plo-vasa, Plo-pl10, and Plo-piwi1 genes are differentially expressed at the stages of gonadal development and gametogenesis ( Figure 12). As in P. leidyi, the expression of these genes suggests the development of prospective gonads, both testes and ovaries, in asexually reproducing P. longiseta. The expression of these genes is very characteristic for male germ-cell formation. The Plo-pl10 gene is expressed in a prolonged period form the earliest steps of testis formation until the stage of spermiogenesis, i.e., completion of spermatozoa formation in seminal vesicles. Plo-vasa and Plo-pl10 transcripts were found in oocytes floating in the coelomic cavity and even in yolk-rich eggs. On the other hand, Plo-piwi1 expression does not last long in oocytes or spermatocytes. In contrast to these three genes, the expression of Plo-piwiA and Plo-piwi2 is not associated with either gonadal development or germ-cell establishment and maintenance.
Similar results were shown for E. japonensis. In this oligochaete, transcripts of both Ej-vlg1 and Ej-vlg2 (homologs of Vasa) were found in the testes, seminal vesicles, and ovaries of mature worms. Ej-vlg1 was expressed in spermatogonia and spermatocytes, but not in spermatids or in sperms. In contrast, Ej-vlg2 transcripts were detected in more restricted cells in the seminal vesicle. mRNA of both genes, Ej-vlg1 and Ej-vlg2 were observed in oogonia and oocytes in the ovary. Ej-piwi expression was also found in the testis, seminal vesicle, and ovary. As in the case of Ej-vlg1, Ej-piwi mRNAs were detected in spermatogonia and spermatocytes, but not in secondary oocytes [22][23][24]. In E. japonensis, it was also found that germ-cell precursors are present in the prospective gonadal region, even in asexually growing animals. After architomy, a kind of fission where an animal splits into fragments before the new head and the new tail develop, gonads can regenerate in each fragment [22].
The emergence of gonad material after such a long period of agamic reproduction raises intriguing questions. Two explanations are theoretically possible. First, we can hypothesize the presence of a specific stem-cell population such as vasaand piwi-positive cells in E. japonensis [22][23][24]. Indeed, both P. leidyi and P. longiseta demonstrate the presence of piwi-positive cells migrating along the ventral nerve cord ( [43,63], and this work). Moreover, in contrast to P. leidyi, we found vasa-positive cells of similar morphology and localization in P. longiseta. This scenario seems to be the most probable, although it does not explain the variability in the presence of such cells. Secondly, it is possible that primordial germ cells form directly from somatic ones by dedifferentiation. Our previous published data suggest a possible migration of dedifferentiated epidermal cells into the segment followed by active cell proliferation which then results in blastemal mass formation in P. longiseta [6]. Epidermal cells at the fission zone and in the posterior growth zone showed not only ultrastructural characteristics but also expression patterns of pluripotency markers vasa, pl10, and piwi, very similar to those of blastema cells. Therefore, at least two different scenarios for the origin of the gonad material after a long period of agamic reproduction are possible. Future studies shall involve the identification of evidence for these alternative mechanisms.
Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/jdb11030034/s1. Table S1. Primer sequences used to clone fragments of the Plo-vasa, Plo-pl10, Plo-piwi1, Plo-piwiA, and Plo-piwi2 genes presented in the paper; Table S2. GenBank access numbers for sequences used for PL10 and VASA amino acid alignments; Table S3. GenBank access numbers for sequences used for PIWI amino-acid alignments; Figure S1. Phylogenetic analysis of Pristina longiseta Vasa and Pl10 homologs. Bayesian consensus tree of the Helicase domains of metazoan Vasa and Pl10 genes; Figure S2. Phylogenetic analysis of Pristina longiseta Piwi homologs. Bayesian consensus tree of the Piwi domain of metazoan Piwi genes.
Author Contributions: Conceptualization, R.P.K.; methodology, R.P.K.; formal analysis, investigation, visualization R.P.K. and N.P.S.; writing-original draft preparation, writing-review and editing, R.P.K.; preparation of illustrations, R.P.K.; supervision, R.P.K.; funding acquisition, R.P.K. All authors have read and agreed to the published version of the manuscript.