Phylogenetic analysis using the Neighbor-Joining method was conducted to determine the evolutionary history of A. lata, resulting in a consensus tree (Fig. 1), in which Acanthobdella peledia was used as outgroup. This tree overall corroborated the relationships traditionally suggested for leeches by morphology . And, both A. lata CO1 sequences from Taiwan and South Korea were clustered together alongside the type species of Glossiphonia, Glossiphonia complanata (Linnaeus, 1758) within the Glossiphoniidae family.
The nomenclature used in the present study to describe the developmental stages of A. lata embryos follows the standard embryonic staging system devised for glossiphoniid leeches [3, 14–16]. A table listing the brooding period for several leeches species [3, 12, 17–23] including A. lata is provided (Table 1). Like all leeches, A. lata presents direct development. Embryos are ~0.5 mm in diameter and protected by a transparent cocoon on the ventral side of the parent leech. Cocoons contained from 15 to 116 embryos, with a mean of 47 ± 23 (n = 100). Direct observation showed that the number of embryos inside a cocoon increases with the size of the adult. A. lata embryos also present a light yellowish, at times greenish, coloration. Hypodermic insemination has been observed for many glossiphoniid species. During copulation, the spermatophores are usually released through an ejaculatory duct in the clitellar region of the concopulant or implanted anywhere in the posterior part of the leech body . The spermatozoa are then released from the spermatophore and reach the ovaries through the vector tissue . No spermatophores were observed to be attached to adult leeches in our laboratory population during this study. Prior to egg fertilization, adult specimens group together, with the dorsal side of one leech being covered by the ventral surface of the other.
Cleavage (stages 1 to 6)
After fertilization, meiosis is arrested at metaphase I until zygotes are deposited sequentially, leading to a slight asynchrony among the embryos of a single clutch. Before egg laying occurs, the parent leech squeezes a membranous sac, or cocoon, which encloses the incoming eggs. Consecutive extrusion of the two polar bodies, immediately followed by initiation of teloplasm formation  marks the completion of Stage 1 (Fig. 2a-d). Cleavage occurs until the formation of teloblasts, the ten embryonic stem cells that give rise to the segmental mesoderm and ectoderm. In A. lata embryos, two unequal cleavages segregate the teloplasm to the macromere D, constituting Stages 2 and 3 (Fig. 2e-g). An animal pole quartet of micromeres (a’ - d’) is then generated by the first, highly unequal, dextro-rotatory spiral cleavage; this constitutes Stage 4a (Fig. 2h). The formation of cells DNOPQ and DM is attributed to the obliquely horizontal cleavage of macromere D’ (Fig. 2i). The end of Stage 4b is marked by each of the three A, B, and C quadrants forming three micromeres (a’ - a’“, b’ - b’“, c’ - c’“) following which macromeres A’“, B’“, and C′“stop dividing, and then contribute to midgut endoderm formation during the later stages. Injection of dextran,tetramethylrhodamine (RDA) in DM" cells at Stage 4b confirmed the division of cell DM" into left and right M teloblasts, constituting the beginning of Stage 4c (Fig. 2j-k). The right M teloblast is located near the center of the vegetal pole, whereas the left M teloblast is visible from the animal pole. Stage 5 is characterized by the formation of more micromeres and the division of the cell DNOPQ”‘into left and right NOPQ cells (Fig. 2l). Subsequent division of the ectodermal precursors generates OP proteloblasts and Q teloblasts, marking Stage 6a (Fig. 2m). The differences in the lineage of the N teloblast and OPQ proteloblast were confirmed by double lineage tracer injection at this stage (Fig. 3a). By the end of Stage 6 (Fig. 2n), A. lata embryo comprises more than 38 cells as a result of further division of macromeres, which later contribute to the non-segmental, dorsal anterior ganglion of the nervous system. Through RDA injections on N and OPQ cells, it was possible to verify that during later stages of development, N cells differentiate into neuronal tissue and presumptive neuronal ganglions. On the other hand, OPQ cells differentiate into neuro-ectodermal-derived cells, which constitutes the exterior region of N lineage (Fig. 3a).
Germinal band formation (stages 7 to 8)
Stage 7 begins with the equal division of OP proteloblasts, forming pairs of O/P teloblasts (Fig. 4Aa). Multiple unequal divisions of each teloblast give rise to blast cells, which will form bandlets that later constitute the germinal bands (Fig. 4Ab-c). These then come in contact with each other through their distal ends at the region where the future head of embryo will develop . The germinal bands start forming during the later part of Stage 7. The diameter of the formed O/P cells decreases as they start producing blast cells, observed by light microscopy as more transparent bands growing from each cell. RDA injection to M cells confirms that the pair of mesoteloblasts (M) gives rise to the mesodermal-derived tissue, namely, muscle fiber and prickle cell (Fig. 3a). By the time Stage 8 begins, the ectodermal and mesodermal bandlets reach the surface of the embryo; the ectodermal bandlets on each side seem to crawl distally along the ipsilateral m bandlet to start forming the germinal bands, culminating in the formation of the germinal plate (Fig. 4Ae-i). Epiboly constitutes the main event in Stage 8. Additionally, during the later part of Stage 8, the embryonic attachment organ is formed at the anterior end of the germinal plate and is the first part of the embryo to emerge from the vitelline membrane (Fig. 4Aj). This organ can be pushed into the ventral surface of the parent leech, allowing the developing embryo to be carried by the parent adult until hatching, which happens by the time the suckers are strong enough to clamp onto the ventral surface of the parent .
Organogenesis and hatching (stages 9 to 11)
Stage 9 starts with the completion of the germinal plate formation. It is distinguished by the appearance of bilateral pairs of coelomic cavities within the mesoderm, progressing from the anterior to the posterior end, and it culminates after the ventral cord is visible and connects anteriorly to the dorsal ganglion by the circumesophageal connective nerves (Fig. 4Ba-b). The beginning of Stage 10 is characterized by the formation of the posterior-most coelomic cell layer, whose proliferation lead to the lateral and dorsal expansion of the edges of the germinal plate; this, gradually displaces the provisional epithelium formed during Stage 8, toward the dorsal midline. The proboscis differentiates into an everted position (Fig. 4Bc-d). Immunostaining using anti-acetylated tubulin allowed the visualization of the formation of the ventral ganglion and peripheral nerve fibers during this stage (Fig. 3b). Stage 11 starts when the lateral edges of the germinal plate have met all along the dorsal midline. During Stage 11, the proboscis invaginates. Development of the ventral ganglion and peripheral nerves is completed, and the crop ceca, intestine, posterior sucker, and pigmented eye spots are formed (Fig. 4Be). Only after A. lata develops eye spots and retracts its tubular proboscis inside the mouth orifice, it hatches from the vitelline membrane (Fig. 4Bf). A. lata develops three pairs of eye spots, with the anterior-most pair being close together. The newly formed eye spots are colored red and turn dark brown soon after the embryo hatches (Fig. 5d-e).
Post-embryonic stage and parental care
The beginning of juvenile stage is marked by the exhaustion of the yolk from within the crop . Starting from mid Stage 11, A. lata grows distinctively flat (Fig. 5a-c). Body coloration of the growing adults is fawn to pale, slightly translucent with a body length of 10–22 mm . Parental care in A. lata ends after the juveniles that have exhausted their yolk start leaving the adult. After brooding is finished, the parent adults feed on snails and reproduce at least two times more before dying.
Calsensin expression patterns
Chemical in situ studies for Ala-calsensin expression were conducted during the later stages of A. lata development. No expression was detected during Stage 9 (Fig. 6a). During Stage 10, expression of mRNA transcripts was detected in the developing segmental ganglia (Fig. 6b). Then, during Stage 11, Ala-calsensin was expressed in the segmental ganglia and the peripheral neurons in the body wall (Fig. 6c-d). In addition, a phylogenetic tree was constructed that clusters Ala-calsensin and a Helobdella robusta calsensin ortholog together to form a monophyletic group alongside Hma-calsensin within Hirudinea (BP > 50%) (Additional file 1).