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

Abstract

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.

Keywords: Annelida; Naididae; Piwi; Pl10; Pristina longiseta; Vasa; asexual reproduction; fission; gametes; germline; gonad development; nervous system; posterior growth zone; sexual maturation; stem cells.

Figure 1

Schema of rapid paratomy and different stages of asexual reproduction in Pristina longiseta. (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. (D–F) Fission zone at different stages of asexual reproduction in Pristina longiseta, DIC. Early middle stage (D) and middle stage (E) of fission-zone development. Both parts of fission zone, cephalogenic part and somatogenic, become evident. (F) Late stage of fission zone development. p—prostomium, bc—blastemal cell mass, g—gut, nc—ventral nerve cord. Arrowheads indicate some of dividing cells. Black and white arrows show modified and intact integumentary epithelium, respectively. Red arrows point to body wall muscles. The red line marks the new developing tail region, the green line marks the new developing head region within the fission zone, and the asterisk marks the posterior growth zone of anterior zooid. Scale bar in (B,C) 25 µm. Scale bar in (D–F) 45 µm.

Figure 2

Gonad anlage and sexualized P. longiseta worms. In (B–F), the anterior is up. (A) Cross semi-thin section through the segment VII of the posterior zooid of an asexually reproducing worm, before physical separation of new individuals from each other. Black and red arrows show intact integumentary epithelium and body wall muscles, respectively. Staining with methylene blue. (B,E) A sexualized individual of P. longiseta, anterior (B) and trunk (E) regions, DIC. (C) Developing fission zone of the sexual mature P. longiseta worm showed in (B,E), early stage. (D) Another specimen of sexualized worm with a large egg in ovisac, DIC. (D) Germ cells in a mature Pristina longiseta specimen, DIC. (G,H) The cocoons of P. longiseta, DIC. cl—clitellum, eg—egg floating in coelomic cavity, o—growing oocyte, oc—cluster of oocyte, os—ovisac, ov—ovaries, t—testis, sp—sperm, sv—seminal vesicle. 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: 25 µm (A), 45 mkm (C,D,F,G), 70 µm (B,E), and 120 µm (H).

Figure 3

Plo-vasa expression during asexual reproduction in P. longiseta. Except for (A,F,G), all animals are oriented anterior to the left, while in (A,F,G), anterior is up. Lateral view for all panels. Plo-vasa is 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 (A,E,C,J). Plo-vasa appears to be de novo expressed in developing fission zone and is shown at various stages of asexual reproduction, from the earliest steps until physical separation of new individuals each from other (A,B,F–I). Arrows indicate additional domains of internal patches of Plo-vasa-positive cells in middle and posterior trunk segments (E,I,G). Vasa transcripts are found in anlage of gonads, testes and ovaries (C,D). st—stomach, t—testis. 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 except (A,B). Scale bar in (A,B), 70 µm.

Figure 4

Plo-pl10 expression during asexual reproduction in P. longiseta. All animals are oriented anterior to the left, lateral view. (A) Early-fission stage. (B–D) Mid-fission stage. (G) Late-fission stage. Plo-pl10 is strongly expressed in somatic tissue of the young segments and posterior growth zone; no expression is detected in the pygidium (A,B,E) and appears to be de novo expressed in developing fission zone (A–D,G). Plo-pl10 shows its activity in the nervous system, including ventral nerve cord and brain, as well as in circumpharyngeal domain. (F) Expression in the head region of an intact worm. br—brain, nc—ventral nerve cord, ph—pharynx, st—stomach. 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 except (A,B). Scale bar in (A,B), 70 µm.

Figure 5

Plo-piwi1 expression during asexual reproduction in P. longiseta. All animals are oriented anterior to the left. Lateral view for all panels. Plo-piwi1 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 (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.

Figure 6

Plo-piwiA and Plo-piwi2 expression during asexual reproduction in P. longiseta. Except for (E), all animals are oriented anterior to the left, while in (E), anterior is up. Lateral view 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 (A–D). Plo-piwiA is also strongly expressed at the posterior end of the worms, including somatic tissue of the young segments, posterior growth zone and in the posterior part of the ventral nerve cord (A); no expression is detected in the pygidium (A,E,F). Its transcripts are shown in the posterior part of the ventral nerve cord (A). Arrows indicate additional domains of internal Plo-piwiA-positive cells in the posterior trunk segments, including cells underlying the gut ((F), deep focal plane). (G) Plo-piwi2 is de novo expressed at a very low level during the early mid stage of fission zone formation. 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 except (A) and (F). Scale bar in (A), 70 µm. Scale bar in (F), 35 µm.

Figure 7

Plo-vasa expression in the cells along the ventral nerve cord in asexually reproducing worms. Except for (F), all animals are oriented anterior to the left, while in (F), anterior is up. Lateral view for all panels, deep focal plane. Plo-vasa-positive ventral cells, various in number, are shown on the ventral nerve cord within the trunk segments. (A–C) Middle trunk segments. (D) More posterior trunk segments. (E) Enlarged view of the boxed region shown in (D). (F) the most anterior trunk segments. st—developing stomach in new individual (posterior zooids). 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 except (E). Scale bar in (E), 15 µm.

Figure 8

Plo-piwi1 expression in the cells along the ventral nerve cord in asexually reproducing worms. All animals are oriented anterior to the left. Lateral view for all panels, deep focal plane. Plo-piwi positive ventral cells (arrows), various in number, are found on the ventral nerve cord within the trunk segments. (A,B) Anterior trunk segments. (C,D,F) Middle trunk segments. (E) More posterior trunk segments. (F) A magnified view of the mid-body region. St—stomach. 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 except (F). Scale bar in (F), 18 µm.

Figure 9

Plo-vasa expression in sexually mature P. longiseta. Except for (G), all animals are oriented anterior to the up direction, while in (G), anterior is on the left. Lateral view for all panels, deep focal planes. (B–D) Enlarged view of the gonadal segments of the animal shown in (A). (E–H) Another sample of sexually mature worms at the late stages of gametogenesis. (D) An example of the gene expression at early-to-mid stage of testis development. (I) An example of Plo-vasa expression at early stage of testis development. cl—clitellum, o—growing oocyte, oc—cluster of oocyte, ov—ovaries, t—testis, sp—sperm, st—stomach, sv—seminal vesicle. Scale bar, 45 µm for all panels except (A). Scale bar in (A), 70 µm.

Figure 10

Plo-pl10 expression in gonadal segments of the sexually mature P. longiseta worms. Except for (F), all animals are oriented anterior to the left, lateral view; in (F), anterior is up, ventral view. Deep focal planes for all panels. Animals are at a different stage of sexual maturation: early stage (A), mid stage (B,C), and late stage (C–F). cl—clitellum, o—growing oocyte, oc—cluster of oocyte, ov—ovaries, t—testis, st—stomach, sv—seminal vesicle. Scale bar, 45 µm for all panels.

Figure 11

In contrast to Plo-piwiA, Plo-piwi1 is expressed in the gonadal segments of the sexually mature P. longiseta worms. All animals are oriented anterior to the left. Lateral view for all panels, deep focal planes. Animals are at a different stage of sexual maturation: early stage (A), mid stage (B–E), and late stage (F–H). mRNA of Plo-piwi1 is detected in testes and clusters of male and female germ cells; Plo-piwi1 expression disappears at the late gametogenesis (F–G). No expression of Plo-piwiA is detected at any stage of sexual maturation of P. longiseta, including late stage (H). cl—clitellum, o—growing oocyte, oc—cluster of oocyte, ov—ovaries, t—testis, st—stomach, sv—seminal vesicle. Scale bar, 45 µm for all panels.

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.