Pruvotina impexa ( Pruvot, 1890 )

Cobo, M. Carmen, Farris, William J. & Kocot, Kevin M., 2024, New data on the biodiversity of Solenogastres (Mollusca, Aplacophora) in the Mediterranean Sea: findings from the program “ Our Planet Reviewed ” Corsica 2019 - 2022, Zoosystema 46 (21), pp. 543-576 : 558-566

Pruvotina impexa ( Pruvot, 1890)

Paramenia impexa Pruvot, 1890 : XXIII.

Pruvotina impexa – Cockerell 1903: 118.

MATERIAL EXAMINED. — Corsica (France) [7 specimens] • 4 specimens (used for sclerite preparations, DNA extractions and histology); CORSICABENTHOS 1, 2 and 3 ( Table 2 ); 50-122 m depth; MNHN-IM-2019-18281; GenBank: OR458919 (1 microscope slide with sclerites, 1 SEM stub; 11 slides with 5 µm serial sections); MNHN-IM-2019-1748; GenBank: OR458910 (1 microscope slide with sclerites, 1 SEM stub; 24 slides with 5 µm serial sections); MNHN-IM-2019-1746; GenBank: OR458910 (1 microscope slide with sclerites, 1 SEM stub; 26 slides with 5 µm serial sections); MNHN-IM-2019-16179; GenBank: OR458907 (1 microscope slide with sclerites, 1 SEM stub; 15 slides with 5 µm serial sections) • 1 specimen (used for sclerite preparations and DNA extraction); CORSICABENTHOS 2 ( Table 2 ); 50-122 m depth; MNHN-IM-2019-1745; GenBank: OR458909 (1 microscope slide with sclerites) • 2 specimens (preserved in 95% ethanol); CORSICABENTHOS 3 ( Table 2 ); 50-122 m depth; MNHN-IM-2019-18285; MNHN-IM-2019-18273 .

DESCRIPTION

Elongate body when alive, rounded in cross-section (4-12 × 0.5-0.8 mm), rounded ends. Length and shape, especially anterior body, notably changes animal movement and preservation ( Fig. 5 ). Four specimens reddish to orange color in the midbody region ( Fig. 5B, C, F ), when collected. Living animal observations showing coloration due to midgut content; some specimens expelled material from anterior end ( Fig. 5F ). While cutting the animals, colored material visible inside body, not the cuticle or epidermis. Color fading with time when preserved in ethanol ( Fig. 5D ). Two specimens when alive (MNHN-IM-2019-18281, MNHN-IM-2019-18285) ( Fig. 5A, E ). Sclerites protrude slightly from the cuticle, especially in the posterior region ( Fig. 5 A’), giving a slightly hirsute appearance. With hollow sclerites of three main types ( Fig. 8A, B ): hollow acicular sclerites with hook-shaped distal end, hollow acicular sclerites with pointed ends, and hollow acicular sclerites that are serrated distally. Hook-shaped sclerites of two types. Both with a spine located at the apex of the sclerite where it reflexes to form a hook with the same size (90-150 µm × 4-8 µm), differences between them in the length and shape of the distal region of the hook ( Fig. 8D ): in one type, longer (55-60 µm length), narrower and curved distally ( Fig. 8D, E, G ) whereas in the second type shorter and uniform in width (45 × 8 µm) ( Fig. 8D, E, F ). Most of the hollow acicular sclerites with pointed end ( Fig. 8B ) slightly curved (80 -210 × 4-8 µm) but some also straight and narrower (50-150 × 2-3 µm). Acicular sclerites creating a dense layer in the ventral region of the body, around the pedal groove, the ( Fig. 8B ). Hollow acicular sclerites that are slightly curved and with a serrated distal end (100 × 3-5 µm), less abundant type ( Fig. 8E ). With knife-shape scales of the pedal groove (45 × 8 µm), located between the acicular sclerites ( Fig. 8B ). With a pre-atrial dorsal sensory organ ( Fig. 11B ) and a bilobed atrium ( Fig. 11 A-C). Mouth ( Fig. 12 A-C) partially separated from the atrium by a wall with musculature but without a cuticular layer separating the two openings ( Fig. 11H, I ). The atrium with single and bilobed papillae ( Fig. 11 A-C). With a dorso-pharyngeal papilla gland ( Fig. 12 A-C) and ventrolateral foregut glands of type A / Pararrhopalia type ( Handl & Todt 2005) ( Fig. 12E ). Radula distichous with three to four median denticles ( Fig. 12D , D’). With a pedal fold that enters the mantle cavity.With seminal vesicles and a tripartite spawning duct in its middle region ( Fig. 12F ). With between 12 and 18 respiratory folds ( Fig. 12H ). With a pair of bundles of 12 to 16 abdominal spicules ( Fig. 12G , G’). Dorso-terminal sensory organ located in a terminal position ( Fig. 12I ). The posterior region (especially the respiratory folds and dorso-terminal sensory organ) difficult to study in the obtained serial sections of one of the specimens (MNHN-IM-2019-1748), but these structures perfectly observed in the other sectioned specimens (MNHN-IM-2019-1746 and MNHN-IM-2019-18281). Serial sections of MNHN-IM-2019-16179 not of adequate quality to characterize all the main diagnostic characters, but the existence of a dorso-pharyngeal papilla gland and remains of a radula determinate. Besides this, no noteworthy differences in the internal organs between the three examined specimens.

REMARKS

Externally, and when analyzing the preserved material, the seven specimens were at first glance assumed to be four different morphospecies: 1) MNHN-IM-2019-1748, MNHN-IM-2019-1746, MNHN-IM-2019-18273, with orange coloration; 2) MNHN-IM-2019-1745, with shorter body, more rounded anterior end and pinkish to orange coloration; 3) MNHN-IM-2019-18285, MNHN-IM-2019-18281, white and elongated body; and 4) MNHN-IM-2019-16179, white-yellowish and shorter body. The results of the phylogenetic analysis and the species delimitation methods ( Fig. 2 ), along with the study of morphology ( Figs 5 ; 8 ; 11 ; 12 ) and our observations in the field, led to the identification of all of them as the same species: Pruvotina impexa ( Pruvot, 1890) .

Pruvotinidae is a diverse family and the only one in Solenogastres divided into subfamilies. The main differences between the subfamilies are the presence/absence of hook-shaped sclerites and the glands associated with the foregut ( García-Álvarez & Salvini-Plawen 2007). Species of the subfamily Pruvotininae Heath, 1911 have dorso-pharyngeal papilla gland and ventrolateral foregut glands of type A/ Pararrhopalia type, a distichous radula and hook-shaped sclerites ( García-Álvarez & Salvini-Plawen 2007; Pedrouzo et al. 2022). These characteristics place these specimens in the subfamily Pruvotininae Heath, 1911 , which includes three genera ( Table 4 ) and two described Mediterranean species Pruvotina impexa ( Pruvot, 1890) and Pararrhopalia pruvoti ( Pruvot, 1891) .

Traditionally, the three genera within Pruvotininae ( Pruvotina Cockerell, 1903 ; Pararrhopalia Simroth, 1893 ; Labidoherpia Salvini-Plawen, 1978 ) are distinguished by a combination of characters related to the atrio-buccal cavity, respiratory folds, and copulatory stylets ( Table 4 ) ( García-Álvarez & Salvini-Plawen 2007; Zamarro et al. 2013: Table 1 ; Pedrouzo et al. 2022). A solenogaster is said to have a common atrio-buccal cavity when the mouth opens at the posterior region of the atrium cavity. On the contrary, a clear separation between the atrium and mouth occurs when there are two separated cavities with a ridge or septum (with musculature and cuticular covering) between them ( Zamarro et al. 2013). In certain species, a musculature-supported ridge lacking cuticle partially separates the mouth and atrium ( Zamarro et al. 2013). Labidoherpia (monospecific genus constituted by the species L. spinosa ( Thiele, 1913)) has a common atrio-buccal cavity, in Pararrhopalia the mouth and atrium are separated, and in Pruvotina the mouth and atrium are partially separated ( García-Álvarez & Salvini-Plawen 2007; Pedrouzo et al. 2022). Nevertheless, a review of the available descriptions casts doubts on the reliability of this character. Salvini-Plawen (1978) mentions a ridge between the mouth and atrium without sclerites in Labidoherpia spinosa , which corresponds with a partial separation as the one described for Pruvotina species (e.g., Salvini-Plawen 1978, Zamarro et al. 2013). In the description of the type species of Pararrhopali a (Pararrhopali a pruvoti ; misidentified as “ Proneomenia vagans” Kowalevsky & Marion, 1887 in Pruvot 1891), it is pointed out that atrium seemed to be separate from the mouth in the sections, something not observed in the living animals, and it is suggested that “this seems to be due only to the retraction of the cephalic extremity at the time of death” ( Pruvot 1891). In addition, Pedrouzo et al. (2022) did not observe a cuticular layer in the septum between the mouth and atrium in the specimens they identified as Pararrhopalia pruvoti . Similarly, in the description of Pararrhopalia oscari Pedrouzo & Urgorri, 2022 it is stated that “atrium and mouth are separated by a small muscular groove (ridge) without cuticle” ( Pedrouzo et al. 2022). In view of all this, we consider that the atrio-buccal cavity is not a valid character to differentiate between genera in the subfamily Pruvotininae , and that its taxonomic value should be revaluated. Respiratory folds are diagnostic of Pruvotina and Labidoherpia , while they are absent in Pararrhopalia ( García-Álvarez & Salvini-Plawen 2007; Pedrouzo et al. 2022). Pruvotina is the only genus of the subfamily without copulatory stylets. Taking all this together, the specimens from Corsica were classified within Pruvotina : mouth and atrium partially separated, respiratory folds present and without copulatory stylets.

The comparison of our specimens and the literature led to the identification of our specimens as Pruvotina impexa ( Pruvot 1890, 1891; Cockerell 1903; Simroth 1893; Todt 2006; García-Álvarez & Salvini-Plawen 2007; Salvini-Plawen 2008; Pedrouzo et al. 2022). Nevertheless, we also found some juxtaposition of characters with Pararrhopalia pruvoti and thus a discussion considering both Mediterranean species is necessary. Remarkably, the only available descriptions of Pruvotina impexa are based on the type material ( Pruvot 1890, 1891). On the contrary, for Pararrhopalia pruvoti there are updated redescriptions and new records ( Todt 2006; Salvini-Plawen 2008; Pedrouzo et al. 2022). In the original comparison of the two species, Pruvot (1891) pointed out that they are very similar with the only difference in the anterior region being the atrio-buccal cavity, with the aforementioned nuances ( Pruvot 1891: fig. 55). In the Corsica specimens, the mouth and atrium are partially separated ( Figs 11 ; 12 A-D) as the ridge or septum between both openings lacks a cuticular layer ( Fig. 11H vs Fig. 12D ). In the live animals, like Pruvot (1891), we did not observe two independent cavities, but there is a muscular septum arising from each wall of the opening in its central region ( Fig. 5 A’, A’’).

The main difference between the two species is the existence of respiratory folds in Pruvotina impexa , whose presence was used as justification for the definition of the genus ( Simroth 1893). Although the absence of respiratory folds in Pararrhopalia pruvoti was corroborated by a redescription of the species based on newer material ( Salvini-Plawen 2008), another recent work ( Pedrouzo et al. 2022) described folds in the mantle cavity of a specimen identified as Pararrhopalia pruvoti (around 10 folds; Pedrouzo pers.comm.). The interpretation of these folds as respiratory organs cannot be discarded. Moreover, Pedrouzo et al. (2022) described a folded wall in the pallial cavity of another Pararrhopalia species, P. oscari , “without forming real respiratory folds.” In addition, in the description of Pararrhopalia fasciata , the absence of respiratory folds is followed by a question mark ( Salvini-Plawen 1978). In two of the Corsica specimens (MNHN-IM-2019-1746 and MNHN-IM-2019-18281) the respiratory folds correspond with what was described for Pruvotina impexa ( Pruvot 1891: Plate XXV, fig. 5a). In a third specimen (MNHN-IM-2019-1748), however, the respiratory folds are not so evident. In view of this, we consider that the apparent absence of respiratory folds should be assessed with caution.

Another purported distinction between these two species involves the presumed presence of copulatory stylets in Pararrhopalia pruvoti , absent in Pruvotina impexa . Pruvot (1891) identified four specimens as “ Proneomenia vagans ” Kowalesky & Marion, 1887 (now accepted as Dorymenia vagans ) based on the presence of copulatory stylets that Simroth (1893) later named as a new species: Pararrhopalia pruvoti . The putative copulatory stylets of these specimens were described as bundles of 16 spicules ( Pruvot 1891; Plate XXX, fig. 60a), that differs from the rounded single and long copulatory stylets of Dorymenia vagans (Kowalesvky & Marion 1887: Plate V, fig. 27; fig. 14B in the present work). There is no reference to copulatory stylets or abdominal spicules in Pruvot’s work concerning Pruvotina impexa , and thus the lack of these structures has been inferred, although his description of the animal’s posterior region is incomplete ( Pruvot 1891). Notably, the specimens from Corsica identified as P. impexa exhibit paired bundles of hard copulatory structures numbering between 12 and 16 ( Fig. 12H , H’). These bundles correspond in size and position to what was called as abdominal spicules in other Pruvotina species, such as P. glandulosa ( Pedrouzo et al. 2022) and their appearance in the serial sections and number of spicules in the bundles is comparable with the structures referred to as copulatory stylets in P. pruvoti ( Pruvot, 1891: fig. 60a; Pedrouzo et al. 2022: fig. 5D) ( Fig. 12H , H’). Therefore, both species have hard copulatory structures. The nomenclature for these structures can be ambiguous. In general, abdominal spicules are short and numerous, located at both sides of the opening of the pallial cavity. Copulatory stylets, on the other hand, are longer structures, that emerge from the pallial cavity in a more central position. They are typically single or double structures but may have accessory spicules and have muscular reinforcement. For some families as for example Proneomeniidae , the distinction between these structures is clear (e.g., Scheltema et al. 2012). The nomenclature used in Pruvotinidae requires revision for consistency.

Besides, the external aspect of the examined specimens corresponds with the description of Pruvotina impexa by Pruvot (1891), particularly in the way the sclerites protrude from the cuticle in the posterior region ( Pruvot 1891). This was especially evident in observations of living specimens ( Fig. 5 A’). There is not a previous mention of the orange color that we found in some of the animals, but this is likely because it fades after preservation. Pruvotina impexa is described as a white animal ( Pruvot 1890), which correspond with some of our specimens (MNHN-IM-2019-18281, MNHN-IM-2019-18285). The mantle sclerites are also comparable with Pruvot’s descriptions who did not find differences between the sclerites of Pruvotina impexa and Pararrhopalia pruvoti ( Pruvot 1891: fig. 5a). Nevertheless, in one of the specimens from Corsica (MNHN-IM-2019-18281), we found serrated sclerites, as Pedrouzo et al. (2022) did for the specimen they identified as Pararrhopalia pruvoti . The hook-shaped sclerites with the longer, narrower, and distally curved distal region of the hook, which were very abundant in the Corsica specimens, were not described before. We attribute this to the fact that the thinner region is easily broken when scraping sclerites onto microscope slides (as we have seen in our own optical microscope slides produced for this species).

Taken together,and especially considering the respiratory folds and habitus, the identification of the specimens from Corsica as Pruvotina impexa is certain. However, our redescription adds important details for the species (hard copulatory structures and observations of the habitus), highlighting overlap in the diagnostic characters of Pruvotina impexa and Pararrhopalia pruvoti . Therefore, more studies comparing the two species are needed, including molecular analysis. Especially significant, because of their importance to pruvotinid taxonomy, is the need of a better understanding of the respiratory folds and copulatory stylets / abdominal spicules.

Pruvotina impexa was described from Banyuls-sur-Mer (South of France, Mediterranean Sea) and collected at 80 m depth ( Pruvot 1890, 1891). With the specimens included in this work we add a new location and depths (60-70 m) for the species in the Mediterranean Sea.

Figures

FIG. 5. — Habitus of Pruvotina impexa (Pruvot, 1890): A, MNHN-IM-2019-18281 (photo of the preserved specimen); A’, MNHN-IM-2019-18281 (photo of the living animal); A’’, detail of the atrio-buccal region of MNHN-IM-2019-18281, arrow indicates the muscular ridge separating the atrium and pedal pit; B, MNHN- IM-2019-1746 (photo of the living animal); C, MNHN-IM-2019-1748 (photo of the living animal); D, MNHN-IM-2019-1745 (photo of the preserved specimen); E, MNHN-IM-2019-18285 (photo of the preserved specimen); F, MNHN-IM-2019-18273 (photo of the living animal and expelled gut contents); G, MNHN- IM-2019-16179 (photo of the preserved specimen). Scale bar: 1 mm. Symbols: stars, the anterior end of the animals.

FIG. 8. — SEM images of the sclerites of A-G, Pruvotina impexa (Pruvot, 1890): A, general view of the sclerites in the dorsal body region (MNHN-IM-2019-18281); B, acicular sclerites and scales of the pedal groove (arrow) in the ventral region of the body (MNHN-IM-2019-18281); C, general view of the sclerites in the dorsal body region (MNHN-IM-2019-1746); D, two types of hook-shaped sclerites (star, long distal region of the hook; dot, short distal region of the hook) (MNHN- IM-2019-1746); E, hook-shaped sclerites with long distal region of the hook (star) and the serrated sclerites (encircled) (MNHN-IM-2019-18281); F, hook-shaped sclerites with short distal region of the hook (MNHN-IM-2019-18281); G, hook-shaped sclerites with long distal region of the hook (MNHN-IM-2019-18281); H-J, Pruvotina impexa (MNHN-IM-2019-16179): H, general view of the sclerites in the dorsal region of the body; I, hook-shaped sclerite; J, scales of the pedal groove; K, L, acicular sclerites of Pruvotia cf. sopita (MNHN-IM-2019-1744). Scale bars: 20 µm.

FIG. 11. — Histological sections of the anterior region of Pruvotina impexa (Pruvot, 1890) (MNHN-IM-2019-1746): A-D, atrial cavity; E-H’, region between the atrial opening and the mouth; H´, detail of the ventral region between the opening of the atrium and the mouth, without cuticular layer. Abbreviations: see Material and methods. Scale bars: 100 µm.

FIG. 12. — Histological sections of Pruvotina impexa (Pruvot, 1890) (MNHN-IM-2019-1746) A-E, anterior region (continuation of Fig. 11): A-C, mouth and dorsal gland; C’, detail of the ventral cuticle between the opening of the mouth and the pedal pit; D, radular region of the foregut; D’, detail of the radula; E, section of the glandular esophagus; F, G, posterior region: F, mid region of the spawning duct; G, section of the abdominal spicules; G’, detail of the abdominal spicules; H, respiratory folds; I, posterior region of the body with the dorsoterminal sensory organ. Abbreviations: see Material and methods. Scale bars: 100 µm.

FIG. 2. — Maximum likelihood phylogenetic reconstruction based on COI. MNHN CORSICABENTHOS specimens are shaded with colors corresponding to those used in Fig 1. BOLD (ALPNB #s) and NCBI accession numbers precede species names for publicly available sequences and both NCBI accession numbers and museum catalog numbers precede the species names for the Corsica specimens. Symbols: *, the taxa considered as reference sequences based on Kocot et al. (2019); gray blocks, species delimitation results according to Assemble Species by Automatic Partitioning (ASAP, Puillandre et al. 2021).