Sunday 10 January 2021

Examining the anatomy of the pelagic Nemertean Protopelagonemertes beebei.

Specimens of pelagic Nemerteans are rarely encountered in nature, and thus, their biology and systematics tend to be poorly characterised. Among the 1300 species in Nemertea (commonly known as Ribbon or Proboscis Worms), approximately 100 have been reported from the epi- to bathypelagic zones within the water column, whereas the remainder are primarily benthic in distribution. With the exception of a few species belonging to the Monostilifera, the majority of the pelagic Nemerteans belong to the Pelagica within the suborder Polystilifera. Since the description of Pelagonemertes rollestoni by Henry Nottidge Moseley in 1875 (the first representative of the Pelagica discovered during the Challenger expedition), three major taxonomic monographs have been published on this group of organisms, that of August Brinkmann in 1917, that of Wesley Coe in 1926, and that of Vera Korotkevich in 1955. However, progress in the systematics of pelagic Polystiliferans has been hindered by their rarity, with 52 of the 96 described species of Pelagica being known from a single specimen. Consequently, for many species the degree of intraspecific variation in taxonomic characters has yet to be established. Moreover, taxonomic descriptions have often been based on material that had become markedly distorted or damaged during retrieval from deep waters. As June 2020, there are only four barcode-sequence entries for the Pelagica in the DDBJ/EMBL/GenBank databases to which species names have been tagged. Accordingly, to ensure the correct application of names to pelagic Nemerteans, larger amounts of data should be accumulated with respect to their precise morphology and DNA barcoding sequences, and for as many species/specimens as possible.

As with the case of other pelagic Nemertean taxa, the taxonomy of the Polystiliferan genus Protopelagonemertes is problematic. Traditionally, the number of proboscis nerves has been one of the major taxonomic characters used to distinguish between the three species that constitute this genus; 29 in  Protopelagonemertes hubrechti; 19–21 (varying within a single specimen) in Protopelagonemertes beebei; and 36 in Protopelagonemertes joculatori. However, variations in the numbers of proboscis nerves have been subsequently documented for different specimens identified as Protopelagonemertes hubrechti: 22, 24, 25, and 26 nerves in four specimens collected in the Southeast Atlantic, off South Africa; 26, 27–29, and 30 nerves in three specimens from the Northwest Atlantic, off Nonsuch Island, Bermuda; and 30 nerves in a specimen from Antarctica. In contrast, no further information has been forthcoming with regards to the variability of this character in two additional specimens identified as Protopelagonemertes beebei, which were collected in the Northeast Pacific, off Baja California, and the Northwest Pacific, off the Kuril Islands, respectively. However, the number of proboscis nerves in a specimen from Sagami Bay, Japan, identified as Protopelagonemertes beebei, was found to vary from 19 to 22 (or, seemingly up to 25), thereby indicating that the variation in this character is completely continuous between that which has previously been reported for Protopelagonemertes beebei and Protopelagonemertes hubrechti. Protopelagonemertes joculatori was described based on a single specimen, and accordingly, there is no indication as to the intraspecific variation of this character in this species. Moreover, it seems highly probable that a proportion, if not all, of the previous studies on these specimens have failed to make a distinction between primary and secondary proboscis nerves. Secondary proboscis nerves were first mentioned by Wesley Coe in 1926 for Planktonemertes agassizii; Pelagonemertes joubini; Plionemertes plana; and Proarmaueria pellucida, and have also been referred to as 'small' proboscis nerves for Neoarmaueria laticeps and Neoarmaueria tenuicauda; Balaenanemertes parvula; Chuniella compacta; Nectonemertes tenuis; Obnemertes solida; and Obnemertes maximovi. In 1992 Alexi Chernyshev described 7–8 'large' and 6–8 'small' proboscis nerves in Zinarmaueria platonovae, and subsequently presented a histological photomicrograph of secondary ('false') proboscis nerves in Planktonemertes sp. More recently, Alexi Chernyshev and Neonila Polyakova have presented a confocal laser scanning micrograph image (labeled with phalloidin, 5-HT, and α-tubulin antibodies) for Nectonemertes cf. mirabilis, depicting primary (larger) and secondary (smaller) proboscis nerves. Despite the findings of these previous studies, the distinction between the primary and secondary nerves is not necessarily obvious. In this reguard, Wesley Coe reasoned that the primary nerves represent the true proboscis nerves, given 'the fact that their branches divide the longitudinal muscular layer into the same number of separate bundles'; however, this can equally apply to secondary nerves. Later, Alexi Chernyshev remarked that the primary nerves can be distinguished from secondary nerves based on the fact that the former invariably come into contact with the inner circular muscle layer of the proboscis.

In a paper published in the journal Plankton & Benthos Research on 18 November 2020, Hiroshi Kajihara of the Faculty of Science at Hokkaido University, and Atsushi Yamaguchi of the Faculty of Fisheries Sciences and the Arctic Research Centre at Hokkaido University, describe  a single specimen of an orange pelagic Polystiliferan, approximately 2 cm long and 7 mm wide in the anesthetised state, identified as Protopelagonemertes beebei, was collected between 3.35 and 4.10 pm Japan Standard Time on June 9, 2020, during a research cruise of the training vessel Ushio-Maruusing a vertical tow of a 80-cm ring net with 63 μm mesh, from a depth of 830 m (20 m above the sea floor) to the surface off the Pacific coast of Hokkaido, northern Japan.

The specimen was acquired by Atsushi Yamaguchi, maintained at 3°C in a 500-mL bottle, and subsequently sent to Hiroshi Kajihara on 13 2020. On arrival at the laboratory, the specimen was anesthetised in magnesium chloride solution isotonic to seawater, during which the proboscis was partially protruded, and subsequently photographed. The posterior end of the body, which was slightly notched along its medial portion in dorsal view, was cut for a few millimeters and fixed in 99% ethanol for DNA extraction. The remainder of the body was fixed in Bouin's fluid for 24 h, dehydrated in 99% alcohol, cleared in xylene, embedded in paraffin, serially sectioned at a thickness of 7 μm, stained using Mallory's trichrome method, and embedded in Entellan New. This voucher specimen has been deposited in the Invertebrate Collection of the Hokkaido University Museum, with the catalogue number 6146.

 
Photographs of Protopelagonemertes beebei (ICHUM 6146), taken of the specimen in an anesthetized state. (A) Entire body, dorsal view. (B) Anterior end of the body, ventral view, showing cephalic furrows (indicated by arrowheads). (C) Posterior end of the body, dorsal view. Scale bars: 5 mm for (A); 1 mm for (B), (C). Kajihara & Yamaguchi (2020).

Histological observation of the proboscis of ICHUM 6146 revealed that the secondary proboscis nerves in this taxon are accompanied by cell bodies, putatively glial cells (or otherwise neuronal perikarya). In contrast, the primary proboscis nerves are devoid of cell bodies and appear to consist exclusively of axonal fibers. Kajihara and Yamaguchi counted 19–23 primary proboscis nerves and 12–16 secondary proboscis nerves in the anterior proboscis chamber in the present material. The number of the primary proboscis nerves in ICHUM 6146 overlaps with that reported for Protopelagonemertes beebei, at 19–21. However, when combined, the number of the primary and secondary nerves in certain portions add up to 36, thereby encompassing the numbers reported for Protopelagonemertes hubrechti and  Protopelagonemertes joculatori. Accordingly, Protopelagonemertes beebei, and possibly Protopelagonemertes joculatori, may be synonymous with Protopelagonemertes hubrechti. Kajihara and Yamaguchi's observations thus prompted them to question the validity of morphological species delimitation in Protopelagonemertes, given that no distinction has been made between primary and secondary proboscis nerves in the original descriptions of the three congeners Protopelagonemertes hubrechti, Protopelagonemertes beebei, and Protopelagonemertes joculatori. In addition, a connection between what is probably a secondary proboscis nerve and the outer circular muscle layer is observed in ICHUM 6146, which is inconsistent with the criterion proposed by Alexi Chernyshev. Hence, with a view toward a more systematic classification of this genus, morphological and molecular data from reliably identified material, ideally from type localities, should be accumulated for each of the nominal species Bathynemertes hubrechti (type locality: North Atlantic), Protopelagonemertes beebei (type locality: off Nonsuch Island, Bermuda), and Protopelagonemertes joculatori (type locality: Banda Sea).

 
Transverse sections of the proboscis anterior chamber of Protopelagonemertes beebei (ICHUM 6146) showing primary (indicated by white arrowheads) and secondary (indicated by black arrowheads) proboscis nerves. (A) Twenty-three primary nerves and 13 secondary nerves. (B) Magnification of #5 and #6 primary and #2 secondary nerves in (A), the latter of which contains putative glial cells. (C) A secondary nerve extending to the outer circular muscle layer. Abbreviation: OCM, outer circular muscle layer of the proboscis. Scale bars: 200 μm for (A); 50 μm for (B); 20 μm for (C). Kajihara & Yamaguchi (2020).

Kajihara and Yamaguchi have confirmed the presence of cephalic furrows in ICHUM 6146, which is probably the first time this feature has been described in the Pelagica. Although the function of these furrows is uncertain, it can be speculated that they may act as chemical or physical receptors, conceivably by perceiving changes in water pressure attributable to vibrations/waves generated by prey and predator species. Cephalic furrows are commonly found in benthic nemerteans and may also be present in pelagic types. However, in pelagic Nemerteans, the epidermis is often dislodged during collection, thereby rendering the detection of cephalic furrows difficult or impossible.

Protopelagonemertes beebei appears to be common in Japanese meso- to bathypelagic waters; for this species, Kajihara and Yamaguchi propose a new Japanese name, dai-dai-oyogi-himomushi. A megablast search at the DDBJ website based on the LC565011 sequence (658 base pairs, cytochrome c oxidase subunit I gene) derived from ICHUM 6146 resulted in a 100% match with AB587263 and HQ848618 which are sequences derived from two specimens collected at a depth of about 1300 m in Sagami Bay. To determine the sequence of LC565011, total DNA was extracted from the ethanol-preserved tissue using a DNeasy Blood & Tissue Kit. Polymerase chain reaction amplification was performed using the primer pair LCO1490/HCO2198 under the following thermal cycling conditions: an initial denaturation at 98°C for 1 minute; followed by 35 cycles of 98°C for 10 seconds, 45°C for 30 seconds, and 72°C for 1 minute; with a final extension at 72°C for 4 minutes. Nucleotide sequencing was carried out using the same primer pair with an ABI BigDye Terminator ver. 3.1 Cycle Sequencing Kit and a 3130 DNA Analyzer.

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