Pasipha atla, Negrete, Lisandro & Brusa, Francisco, 2016

Negrete, Lisandro & Brusa, Francisco, 2016, Land flatworms of the genus Pasipha (Platyhelminthes, Geoplanidae) in Argentina, with description of three new species, Zootaxa 4137 (2), pp. 187-210 : 188-195

Pasipha atla sp. nov.

( Figs. 1–4 )

Pasipha sp. 2: Negrete et al. 2014a

Type material. Holotype: MLP-He 6173. San Antonio Strict Nature Reserve, Misiones Province, Argentina, 29.X.2008, L. Negrete, coll.; cephalic region: transverse sections on 11 slides (7 µm); anterior region at level of ovaries: sagittal sections on 26 slides (7 µm); pre-pharyngeal region: transverse sections on 6 slides (6 µm); pharynx and copulatory apparatus: sagittal sections on 31 slides (7 µm).

Paratypes: MLP-He 6461–1. Urugua-í Wildlife Reserve, Misiones Province, Argentina, 22.VIII.2009, L. Negrete, coll.; cephalic region and anterior region at level of ovaries: transverse sections on 35 slides (8 µm); prepharyngeal region: transverse sections on 4 slides (8 µm); pharynx and copulatory apparatus: sagittal sections on 35 slides (8 µm). MLP-He 6461–2. Urugua-í Wildlife Reserve, Misiones Province, Argentina, 22.VIII.2009, L. Negrete, coll.; cephalic region: sagittal sections on 20 slides (8 µm); anterior region at level of ovaries: sagittal sections on 32 slides (8 µm); pre-pharyngeal region: transverse sections on 5 slides (8 µm); pharynx and copulatory apparatus: sagittal sections on 26 slides (8 µm).

Diagnosis. Dorsum dark brown with a whitish median stripe only present on the cephalic and posterior body regions. Eyes dorsal with clear halos. Pharynx cylindrical. Prostatic vesicle extrabulbar, tubular, with narrow lumen, and proximally forked. Male atrium twice the length of the female atrium, highly folded. Proximal part of male atrium with small folds. Ovaries anterior to the anteriormost testes. Distal sections of ovovitelline ducts ventral to the female atrium, joining behind it. Common ovovitelline duct vertical and posterior to the female atrium, and female genital canal antero-dorsally flexed. Female atrium with folded walls.

Type locality. San Antonio Strict Nature Reserve (26° 03’ S, 53° 46’ W), Misiones Province, Argentina.

Etymology. The scientific name is a combination of the initials of Urugua-í Wildlife Reserve park managers Ariel Tombo and Laura Aréjola, in gratefulness of their valuable assistance during fieldwork.

External morphology. The body is elongate with parallel margins. In living specimens, the anterior region gradually narrows, with blunt ending, and the posterior region is sharply pointed ( Fig. 1 A, B). The dorsal colour pattern is homogeneous dark brown ( Fig. 1 A). A thin whitish median stripe is distinguished in the cephalic region, extending to about 5 mm from the anterior tip ( Fig. 1 B), and close to the posterior tip (only visible under stereomicroscope). The ventral surface is greyish. After fixation, the dorsal colour pattern is light brown ( Fig. 1 B, C). The eyes, with clear halos, surround the anterior tip and extend uniserially on both body margins along 3–5 mm from the anterior tip, continuing bi- and tri-serially for 2–4 mm ( Fig. 1 A, C). Then the eyes extend pluriserially over the dorsal surface. At the pharyngeal level they become restrict to the body margins and less numerous, and extend to the posterior end. Length of the fixed specimens ranged from 35 to 48 mm, maximum width was 2.4–5.4 mm, and maximum height 1.5–2.2 mm. The mouth was located at a distance of 66–78% from the anterior tip, and the gonopore at 77–88% ( Table 1 ).

Internal morphology. Epidermis, secretions and musculature in the cephalic region. Dorsal epidermis (20–25 µm µmhigh) receives abundant rhabditogen and erythrophil fine granular secretions, and erythrophil and cyanophil amorphous secretions in less quantity. Ventral epidermis (30 µm high), ciliated in the creeping sole (~90% of body width), receives the same type of secretions. Sensory pits (20–25 µm deep), as simple invaginations, contour the anterior tip and spread on both body margins in a single row up to ~ 7 mm from the anterior tip. The cutaneous musculature has the same arrangement (see below) and similar thickness (10% to 15% of body height) as in the pre-pharyngeal region. The parenchymatic musculature is composed of the same layers as the pre-pharyngeal region, ranging from 5% to 10% of body height ( Fig. 2 A). There is no musculo-glandular specialization in the cephalic region.

Epidermis, secretions and musculature in the pre-pharyngeal region. Abundant rhabditogen cells with erythrophil secretion, numerous cells with fine granular and amorphous erythrophil secretions, and fine granular cyanophil secretion in less quantity discharge through dorsal epidermis (25–30 µm high) and body margins ( Fig. 2 C, E). Ventral epidermis (30 µm high), ciliated in the creeping sole (85–90% of body width), presents abundant small dermal rhabdites densely arranged at the apex of cells. Also, abundant amorphous cyanophil and fine granular erythrophil secretions, and less numerous amorphous erythrophil secretion are present in ventral epidermis ( Fig. 2 D). There is no glandular margin ( Fig. 2 B, E).

The cutaneous musculature consists of the three typical layers of Geoplaninae , a thin circular subepidermal layer, with the same thickness dorsally and ventrally, followed by an intermediate layer with diagonal fibres, and an internal thicker longitudinal layer arranged in bundles, which is thicker ventrally than dorsally ( Table 2 ). CMI ranges from 9% to 15%. Parenchymatic musculature is composed of three layers: a dorsal layer with decussate fibres (situated below the longitudinal cutaneous layer), which is the thickest, a supra-intestinal and a sub-intestinal transverse layer ( Fig. 2 B, C). Dorso-ventral fibres are arranged among intestinal branches. PMI ranges from 4% to 7% ( Table 2 ).

Digestive system. The pharynx (1.2–1.6 mm in length, about 3–4% of body length) is cylindrical, with the dorsal insertion slightly posteriorly displaced (100–350 µm) ( Fig. 2 F). The mouth is located in the posterior third of the pharyngeal pouch ( Fig. 2 F). The epithelium lining of the outer surface of the pharynx is cuboidal and densely ciliated, and the outer pharyngeal musculature is arranged in two layers: a thin longitudinal subepithelial layer (5– 7.5 µm thick) followed by an inner circular layer (10–25 µm thick). The epithelium lining the pharyngeal lumen is columnar and ciliated, and the inner pharyngeal musculature consists of a circular subepithelial layer (10–50 µm thick) followed by a subjacent longitudinal layer (15–50 µm thick). Secretory cells, the cell bodies of which are located anterior and lateral to the pharynx, discharge abundant erythrophil and cyanophil fine granular secretion in the pharyngeal epithelium ( Fig. 2 F).

The oesophagus (100–600 µm in length) ( Fig. 2 F) is lined by a columnar epithelium followed by a subjacent circular muscle layer (15–25 µm thick) and a longitudinal layer (10–15 µm thick). The oesophagus: pharynx ratio ranges from 19% to 32%.

Male reproductive system. The testes, ovoid in shape, are arranged in two to four irregular rows on each side of the body, being dorsal to the intestinal branches and located below the supra-intestinal parenchymatic muscle layer ( Fig. 2 B). Testes occupy 11–13% of the body height. They appear behind the ovaries and extend to the prepharyngeal region, being located at a distance between 21–27% and 61–75% of the body length from the anterior end ( Table 3 ). The sperm ducts are located among fibres of the sub-intestinal parenchymatic muscle layer or just below them, being slightly dorsal and medial to ovovitelline ducts in the pre-pharyngeal region ( Fig. 2 B). Behind the pharynx, sperm ducts are tortuous and distally dilated ( Fig. 3 ) with lumen full of spermatozoa. In the vicinity of the common muscle coat, sperm ducts bend toward the dorsum and forward, and open into the proximal region of the extrabulbar prostatic vesicle ( Figs. 3 , 4 B). The prostatic vesicle, located just behind the pharynx ( Figs. 2 F, 3), is tubular with a narrow irregular lumen and presents two anatomically and histologically distinguishable regions (see below) ( Figs. 3 , 4 A–C). The proximal region is proximally forked, communicating with sperm ducts ( Figs. 3 , 4 A– C). The distal region is ventro-posteriorly curved and opens into the ejaculatory duct exactly at the boundary of the common muscle coat ( Figs. 3 , 4 A, D). The ejaculatory duct is sinuous and opens into the bottom of the male atrium, sometimes slightly displaced ventrally ( Figs. 3 , 4 D). The walls of the most proximal part of the male atrium present numerous small folds ( Figs. 3 , 4 A, D, E) while the rest of the atrium is a cavity with large folds. Thus, its lumen is narrow ( Figs. 3 , 4 A). The male atrium is approximately twice the length of the female atrium ( Table 3 ).

Sperm ducts are lined by a ciliated cuboidal epithelium surrounded by a circular muscle layer (5 µm thick). The prostatic vesicle is lined with ciliated columnar epithelium, traversed in its proximal region by abundant fine granular pale cyanophil secretion, and abundant coarse granular erythrophil secretion in its distal region ( Fig. 4 A– D). The glandular cell bodies are located in the surrounding parenchyma laterally and anteriorly to the prostatic vesicle. The prostatic musculature of both regions comprises intermingled longitudinal and circular fibres (25–55 µm thick). The ejaculatory duct is lined by a ciliated columnar epithelium, which receives scarce fine granular cyanophil secretion, followed by a subjacent muscular layer with circular fibres intermingled with some longitudinal fibres (10–20 µm thick).

The lining epithelium of the small folds of the most proximal part of the male atrium is columnar and ciliated, with the apex of the cells filled with strongly stained fine granular erythrophil secretion ( Fig. 4 E). The epithelium of the rest of the male atrium is cuboidal and non-ciliated except in the distal portion, which is columnar. The male atrium receives abundant fine granular cyanophil secretion along its entire length, and in its middle third also receives abundant fine granular erythrophil secretion ( Fig. 4 F, G). Cell bodies of both types of glands are located below the epithelial lining of the atrium. The muscularis consists of a subepithelial circular layer (10–25 µm thick) followed by a longitudinal layer (5–10 µm thick). Longitudinal fibres, forming a not well organized coat (40–100 µm thick) (named eigenmusculatur by Graff (1899); here named intermediate muscle fibres), are located between the muscularis and the common muscle coat ( Figs. 3 , 4 A). The common muscle coat consists of longitudinal and less abundant circular and oblique fibres, thicker dorsally (20–50 µm thick) than ventrally (10–20 µm thick).

Female reproductive system. The ovaries (300–500 µm in length, 200–400 µm high), ovoid in shape, are situated at a distance of 13–22% of the body length from the anterior end ( Fig. 2 G, Table 3 ). They are located between the sub-intestinal parenchymatic muscle layer and the nervous plate ( Fig. 2 G). The ovovitelline ducts emerge from the medial dorsal side of the ovaries, and run posteriorly, being located just above the nervous plate in the pre-pharyngeal region ( Fig. 2 B, G). Their distal sections are located ventrally to the female atrium. Just behind this atrium, they contour the common muscle coat and run to the sagittal plane in a slight ascending course. The ovovitelline ducts join behind the atrium to form a common glandular ovovitelline duct ( Figs. 3 , 4 H). It is almost vertically oriented and distally curves forward, passing through the common muscle coat, and opens into the female genital canal ( Figs. 3 , 4 H). The latter continues ascending and opens into the bottom of the female atrium, dorsally displaced ( Fig. 3 , 4 H). The female atrium presents folded walls, although it is not as richly folded as the male atrium ( Figs. 3 , 4 A). At the level of the gonopore there is a dorsal fold that separates the atria ( Fig. 3 ).

The ovovitelline ducts are lined with a ciliated cuboidal epithelium, followed by a circular muscle layer (5 µm thick). Both the distal portions of the ovovitelline ducts and the common glandular ovovitelline duct receive abundant secretion from the shell glands ( Figs. 3 , 4 A, H). The lining epithelium of the latter, which also receives scarce fine granular cyanophil secretion, is columnar and ciliated followed by a circular muscle layer (10 µm thick). The female canal is lined by a non-ciliated columnar epithelium, which receives abundant fine granular cyanophil secretion ( Fig. 5 I). The muscularis of the female canal consists of circular and longitudinal intermingled fibres (10–20 µm thick). Like the female canal, the lining epithelium of the female atrium is columnar and nonciliated, but in the latter, the apical part of the epithelium is filled with abundant fine granular erythrophil and cyanophil secretions ( Fig. 4 J). In the distal portion of the female atrium erythrophil secretion is more abundant. Cell bodies of these glands are subepithelial, located in the surrounding parenchyma within the common muscle coat. The muscularis of the female atrium consists of a thin layer of intermingled circular and longitudinal fibres (5–10 µm thick). There is a thick muscle layer with intermingled circular and some longitudinal and oblique fibres (35–100 µm thick) (here named intermediate muscle fibres) located between the atrial muscularis and the common muscle coat ( Figs. 3 , 4 A). The common muscle coat consists of longitudinal fibres, thicker dorsally (35–50 µm thick) than ventrally (15–25 µm thick). Vitellaria are well developed in all specimens. At the pre-pharyngeal level, vitelline follicles are arranged dorsally and ventrally to the intestinal branches and among them ( Fig. 2 B).

Parasitism. Nematode larvae were found in all study specimens, located in the parenchyma among intestinal branches and vitellaria of the anterior body region.

Figures

FIGURE 1. Pasipha atla sp. nov. (A) Photograph of a live specimen (MLP-He 6173), with detail of the cephalic region. Dorsal (MLP-He 6173) (B) and lateral (MLP-He 6461 – 1) (C) view of anterior body region of preserved specimens.

FIGURE 2. Pasipha atla sp. nov. (MLP-He 6173). (A) Half of a transverse section of the cephalic region. (B) Schematic drawing of half of a transverse section of the pre-pharyngeal region. (C – D) Detail of the dorsal (C) and ventral (D) portion of a transverse section in pre-pharyngeal region. (E) Detail of the marginal portion of a transverse section in pre-pharyngeal region. (F) Sagittal section of the pharynx. (G) Sagittal section of the anterior region. (di) dorsal insertion; (dp) dorsal parenchymatic muscle layer; (i) intestine; (lc) longitudinal cutaneous muscle layer; (mo) mouth; (n) nervous plate; (od) ovovitelline duct; (oe) oesophagus; (ov) ovary; (ph) pharynx; (phl) pharyngeal lumen; (php) pharyngeal pouch; (pv 1) proximal prostatic vesicle; (rh) rhabditogen glands; (sbp) sub-intestinal parenchymatic muscle layer; (sd) sperm duct; (spp) supra-intestinal parenchymatic muscle layer; (t) testes; (vi) ventral insertion; (vit) vitellaria.

FIGURE 3. Pasipha atla sp. nov. (MLP-He 6173). Sagittal reconstruction of the copulatory apparatus. (cgd) common glandular ovovitelline duct; (cm) common muscle coat; (ej) ejaculatory duct; (fa) female atrium; (fc) female genital canal; (im) intermediate muscle fibres; (g) gonopore; (ma) male atrium; (od) ovovitelline duct; (php) pharyngeal pouch; (pv 1) proximal prostatic vesicle; (pv 2) distal prostatic vesicle; (sd) sperm duct; (sg) shell glands.

FIGURE 4. Pasipha atla sp. nov. (MLP-He 6173). (A) Sagittal section of the copulatory apparatus. (B – G) Details of the male reproductive system, in sagittal view. (H – J) Details of the female reproductive system, in sagittal view. (cgd) common glandular ovovitelline duct; (cm) common muscle coat; (ej) ejaculatory duct; (fa) female atrium; (g) gonopore; (im) intermediate muscle fibres; (ma) male atrium; (od) ovovitelline duct; (pv 1) proximal prostatic vesicle; (pv 2) distal prostatic vesicle; (sd) sperm duct; (sg) shell glands.

FIGURE 5. Pasipha johnsoni sp. nov. Photographs of live specimens, in dorsal view. (A) Holotype (MLP-He 6489). (B) Immature specimen (MLP-He 6488).

Tables

TABLE 1. Measurements (mm) of fixed specimens of Pasipha atla sp. nov. (DG) distance of gonopore from anterior end, (DM) distance of mouth from anterior end, (DMG) distance between mouth and gonopore. The numbers in parentheses represent the position relative to body length (%).

	MLP-He 6173	MLP-He 6461–1	MLP-He 6461–2
Length	35.3	42	35
Maximum width	2.6	2.4	5.4
Maximum height	1.5	1.6	2.2
DM	27.5 (78%)	32 (76%)	23 (66%)
DG	30 (85%)	37 (88%)	27 (77%)
DMG	2.5	5	4
Creeping sole (%)	90%	90%	90%

TABLE 2. Thickness of cutaneous (CM) and parenchymatic (PM) musculatures (µm), and CMI and PMI indices at prepharyngeal region of Pasipha atla sp. nov.

	MLP-He 6173	MLP-He 6461–1	MLP-He 6461–2
CM dorsal
circular	2.5	5	5
diagonal	10	12.5	17.5
longitudinal	60	75	62.5
CM ventral
circular	2.5	5	5
diagonal	15	25	32.5
longitudinal	85	125	80
CMI (%)	12%	15%	9%
PM dorsal	30	40	50
PM supra-intestinal	25	35	25
PM sub-intestinal	25	30	25
PMI (%)	5%	7%	4%

TABLE 3. Measurements (mm) of reproductive organs of Pasipha atla sp. nov. LCGD, length of common glandular ovovitelline duct; LFA, length of female atrium; LFC, length of female canal; LMA, length of male atrium; LPV, length of prostatic vesicle. The numbers in parentheses represent the position relative to body length (%).

	MLP-He 6173	MLP-He 6461–1	MLP-He 6461–2
Anteriormost testes	7.5 (21%)	12 (29%)	9.4 (27%)
Posteriormost testes	24.7 (70%)	31.5 (75%)	21.4 (61%)
LPV (proximal)	0.6	0.85	0.95
LPV (distal)	0.6	0.9	1.2
LMA	3	3.25	2.5
Location of ovaries	4.5 (13%)	9.2 (22%)	7.6 (22%)
LCGD	0.35	0.6	0.6
LFC	0.35	0.37	0.4
LFA	1.2	2	1.1