Plankton Benthos Res 15(4): 337–341, 2020

Plankton & Benthos
Research© The Japanese Association of Benthology

Note

A morphological note on the pelagic polystiliferous
hoplonemertean Protopelagonemertes beebei(Nemertea: Pelagica)
Hiroshi Kajihara1,* & Atsushi Yamaguchi2,31Faculty of Science, Hokkaido University, Sapporo, N10 W8, Hokkaido 060–0810, Japan
Faculty of Fisheries Sciences, Hokkaido University, 3–1–1, Minato-cho Hakodate, Hokkaido 041–8611, Japan
3
Arctic Research Centre, Hokkaido University, Kita-21 Nishi-11 Kita-ku, Sapporo, Hokkaido 001–0021, Japan
2

Received 29 June 2020; Accepted 22 July 2020 Responsible Editor: Kensuke Yanagi
doi: 10.3800/pbr.15.337
Abstract: The three currently recognized species comprising the pelagic polystiliferous hoplonemertean genus Protopelagonemertes
Brinkmann, 1917 are supposedly distinguishable chieﬂy with respect to the number of proboscis nerves: 29 (varying from 22 to 30) in
P. hubrechti (Brinkmann, 1917); 19–21 (varying from 19 to 22) in P. beebei Coe, 1936; and 36 in P. joculatori Van der Spoel, 1988. A
single specimen collected oﬀ the Paciﬁc coast of Hokkaido, Japan, herein identiﬁed as P. beebei, was found to possess 19–23 primaryproboscis nerves. In addition to the primary proboscis nerves, the material also possessed 12–16 secondary proboscis nerves, which
are discernible from the primary nerves in that they coexist with putative glial cells. Although secondary proboscis nerves have been
identiﬁed in some other species in the Pelagica, they have not previously been described for any members of the Protopelagonemertes. Our ﬁndings thus prompted us to question the validity of morphological species delimitation within this genus, given that thedistinction between primary and secondary proboscis nerves has previously not been taken into consideration. Close examination of
the specimen in the living state revealed that it is characterized by a pair of cephalic furrows, a structure commonly found in benthic
representatives of the phylum, but herein conﬁrmed for the ﬁrst time among the Pelagica. A 658-bp partial sequence of the mitochondrial cytochrome c oxidase subunit I gene from the newly examined specimen was found to be identical to sequences derived fromtwo other specimens previously collected in Sagami Bay, thereby indicating that P. beebei may occur commonly in Japanese meso- tobathypelagic waters.
Key words: COI, Hoplonemertea, marine invertebrate, Polystilifera, taxonomy

Specimens of pelagic nemerteans are rarely encounteredin nature, and thus, their biology and systematics tend to bepoorly characterized. Among the 1300 species in Nemertea(commonly known as ribbon or proboscis worms), approximately 100 have been reported from the epi- to bathypelagiczones within the water column, whereas the remainder are
primarily benthic in distribution (Gibson 1995, Kajihara et al.
2008). With the exception of a few species belonging to the
Monostilifera (Wheeler 1934, Korotkevitsch 1961, Crandall &Gibson 1998, Chernyshev 2005, Crandall 2006, Chernyshev& Polyakova 2018, 2019), the majority of the pelagic nemerteans belong to the Pelagica within the suborder Polystilifera.
Since the description of Pelagonemertes rollestoni Moseley, 1875 (the ﬁrst representative of the Pelagica discovered
during the Challenger expedition), three major taxonomic
monographs have been published on this group of organisms* Corresponding author: Hiroshi Kajihara; E-mail, kajihara@eis.hokudai.
ac.jp(Brinkmann 1917a, Coe 1926, Korotkevitsch 1955). However,progress in the systematics of pelagic polystiliferans has been
hindered by their rarity, with 52 of the 96 described speciesof Pelagica being known from a single specimen (Maslakova& Norenburg 2001, Kajihara & Lindsay 2010). 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 (reviewed by Maslakova & Norenburg
2001). As of writing, there are only four barcode-sequence
entries for the Pelagica in the DDBJ/EMBL/GenBank databases to which species names have been tagged (Thollesson& Norenburg 2003, Bucklin et al. 2010, Kajihara et al. 2011,
Chernyshev & Polyakova 2019). Accordingly, to ensure thecorrect application of names to pelagic nemerteans, larger
amounts of data should be accumulated with respect to theirprecise morphology and DNA barcoding sequences, and for
as many species/specimens as possible.

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H. Kajihara & A. Yamaguchi

Fig. 1. Photographs of Protopelagonemertes beebei Coe, 1936 (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 thebody, dorsal view. Scale bars: 5 mm for (A); 1 mm for (B, C).

As with the case of other pelagic nemertean taxa, the taxonomy of the polystiliferan genus Protopelagonemertes
Brinkmann, 1917b 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 (Coe 1936, Van der Spoel 1988): 29 in P.
hubrechti (Brinkmann, 1917a); 19–21 (varying within a single
specimen) in P. beebei Coe, 1936; and 36 in P. joculatori
Van der Spoel, 1988. However, variations in the numbers ofproboscis nerves have been subsequently documented for different specimens identiﬁed as P. hubrechti: 22, 24, 25, and 26
nerves in four specimens collected in the Southeast Atlantic,
oﬀ South Africa (Wheeler 1934); 26, 27–29, and 30 nervesin three specimens from the Northwest Atlantic, oﬀ Nonsuch
Island, Bermuda (Coe 1936); and 30 nerves in a specimen
from Antarctica (Wheeler 1940). In contrast, no further information has been forthcoming with regards to the variabilityof this character in two additional specimens identiﬁed as P.
beebei, which were collected in the Northeast Paciﬁc, oﬀ Baja
California, and the Northwest Paciﬁc, oﬀ the Kuril Islands,
respectively (Coe 1954). However, the number of proboscis
nerves in a specimen from Sagami Bay, Japan, identiﬁed as
P. 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 P. beebei and P. hubrechti (Kajihara

et al. 2011). Protopelagonemertes joculatori was describedbased on a single specimen (Van der Spoel 1988), and accordingly, there is no indication as to the intraspeciﬁc variationof 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 Coe (1926) for Planktonemertes agassizii Woodworth, 1899; Pelagonemertes joubini
Coe, 1926; Plionemertes plana Coe, 1926; and Proarmaueria
pellucida Coe, 1926, and have also been referred to as “small”(маленький) proboscis nerves for Neoarmaueria laticeps(Korotkevitsch, 1955) and Neoarmaueria tenuicauda (Korotkevitsch, 1955) (Korotkevitsch 1955); Balaenanemertes parvula Korotkevich, 1964; Chuniella compacta Korotkevich,
1964; Nectonemertes tenuis Korotkevich, 1964; Obnemertes
solida Korotkevich, 1964; and Obnemertes maximovi Korotkevich, 1960 (Korotkevitsch 1964, 1966). Chernyshev (1992)
also described 7–8 “large” (крупный) and 6–8 “small”(маленький) proboscis nerves in Zinarmaueria platonovae
Chernyshev, 1992, and subsequently presented a histologicalphotomicrograph of secondary [“false” (ложный)] proboscis
nerves in Planktonemertes sp. (Chernyshev 2011, pl. XX, ﬁg.
5). More recently, Chernyshev & Polyakova (2018) have presented a confocal laser scanning micrograph image (labeledwith phalloidin, 5-HT, and α-tubulin antibodies) for Nectone-

Morphology of Protopelagonemertes beebei

mertes cf. mirabilis Verrill, 1892, 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 thisregard, Coe (1926, p. 40) reasoned that the primary nerves
represent the true proboscis nerves, given “the fact that theirbranches divide the longitudinal muscular layer into the same
number of separate bundles”; however, this can equally apply to secondary nerves. Later, Chernyshev (2011) 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.
Very recently, on June 9, 2020, during a research cruiseof the training vessel Ushio-Maru, a single specimen of anorange pelagic polystiliferan, approximately 2 cm long and
7 mm wide in the anesthetized state (Fig. 1A), herein identiﬁed as Protopelagonemertes beebei, was collected between
15 : 35 and 16 : 10 (JST), using a vertical tow of a 80-cm ring
net with 63 µm mesh, from a depth of 830 m (20 m above thesea ﬂoor) to the surface at 41°58.13′N, 141°39.79′E, oﬀ the Paciﬁc coast of Hokkaido, northern Japan. On board, the specimen was acquired by Atsushi Yamaguchi, maintained at 3°C
in a 500-mL bottle, and subsequently sent to Hiroshi Kajihara
on June 13, 2020. On arrival at the laboratory, the specimen
was anesthetized in MgCl2 solution isotonic to seawater, during which the proboscis was partially protruded, and subsequently photographed (Fig. 1A, B). The posterior end of thebody, which was slightly notched along its medial portion in
dorsal view (Fig. 1C), was cut for a few millimeters and ﬁxed
in 99% EtOH for DNA extraction. The remainder of the body
was ﬁxed in Bouinʼs ﬂuid for 24 h, dehydrated in 99% EtOH,
cleared in xylene, embedded in paraﬃn, serially sectioned ata thickness of 7 µm, stained using Malloryʼs trichrome method (Gibson 1994), and embedded in Entellan New (Merck).
This voucher specimen has been deposited in the Invertebrate
Collection of the Hokkaido University Museum (ICHUM),
with the catalogue number 6146.
Histological observation of the proboscis of ICHUM 6146
revealed that the secondary proboscis nerves in this taxon areaccompanied 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 (Fig. 2A, B). We counted 19–23 primary proboscis nerves and 12–16 secondary proboscis nervesin the anterior proboscis chamber in the present material (Fig.
2A). The number of the primary proboscis nerves in ICHUM
6146 overlaps with that reported for P. beebei, at 19–21 (Coe1936). However, when combined, the number of the primaryand secondary nerves in certain portions add up to 36, thereby encompassing the numbers reported for P. hubrechti (29,varying from 22 to 30) and P. joculatori (36). Accordingly, P.
beebei, and possibly P. joculatori, may be synonymous with
P. hubrechti. Our observations thus prompted us 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

339

Fig. 2. Transverse sections of the proboscis anterior chamber ofProtopelagonemertes beebei Coe, 1936 (ICHUM 6146) showing
primary (indicated by white arrowheads) and secondary (indicatedby 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 theouter 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).

descriptions of the three congeners P. hubrechti, P. beebei,
and P. joculatori (Brinkmann 1917a, b, Coe 1936, Van der
Spoel 1988). In addition, a connection between what is probably a secondary proboscis nerve and the outer circular muscle
layer is observed in ICHUM 6146 (Fig. 2C), which is inconsistent with the criterion proposed by Chernyshev (2011).
Hence, with a view toward a more systematic classiﬁcationof this genus, morphological and molecular data from reliably identiﬁed material, ideally from type localities, should beaccumulated for each of the nominal species Bathynemertes
hubrechti Brinkmann, 1917b (type locality: North Atlantic),

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H. Kajihara & A. Yamaguchi

Protopelagonemertes beebei Coe, 1936 (type locality: oﬀ
Nonsuch Island, Bermuda), and Protopelagonemertes joculatori Van der Spoel, 1988 (type locality: Banda Sea).
We conﬁrmed the presence of cephalic furrows in ICHUM
6146, which is probably the ﬁrst time this feature has been
described in the Pelagica (Fig. 1B). Although the function ofthese 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 arecommonly found in benthic nemerteans and may also be present in pelagic types. However, in pelagic nemerteans, the epidermis is often dislodged during collection (e.g., Coe 1926),
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, we
propose a new Japanese name, dai-dai-oyogi-himomushi. A
megablast search (Zhang et al. 2000) at the DDBJ website(http://blast.ddbj.nig.ac.jp) based on the LC565011 sequence(658 bp, cytochrome c oxidase subunit I [COI] gene) derived
from ICHUM 6146 resulted in a 100% match with AB587263(Kajihara et al. 2011) and HQ848618 (Andrade et al. 2012)
which are sequences derived from two specimens collected at
a depth of ~1300 m in Sagami Bay (Kajihara et al. 2011). To
determine the sequence of LC565011, total DNA was extracted from the EtOH-preserved tissue using a DNeasy Blood &Tissue Kit (Qiagen). PCR ampliﬁcation was performed using
the primer pair LCO1490/HCO2198 (Folmer et al. 1994) under the following thermal cycling conditions: an initial denaturation at 98°C for 1 min; followed by 35 cycles of 98°C for
10 s, 45°C for 30 s, and 72°C for 1 min; with a ﬁnal extension
at 72°C for 4 min. 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 (Applied
Biosystems).
Acknowledgements
We thank the crew of the T/V Ushio-Maru for their assistance in obtaining the specimen. We also thank Haruki
Ishiyama (Hokkaido University) for assistance in molecular
work, and two anonymous reviewers for their constructive
comments on the manuscript. HK is particularly grateful toProfessor Alexei V. Chernyshev (A.V. Zhirmunsky Instituteof Marine Biology, National Scientiﬁc Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences; concurrently Far Eastern Federal University) for important literature information. This study was conducted with the supportof the Arctic Challenge for Sustainability (ArCS) Project(Program Grant Number JPMXD1300000000) and ArCS
II. Part of this study was also supported by Grants-in-Aid
for Scientiﬁc Research 20K06780 (C) to HK, 20H03054 (B)to AY, 19H03037 (B) to AY, 17H01483 (A), and 20K20573(Pioneering) to AY from the Japan Society for the Promotionof Science (JSPS).

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