Species Diversity 21: 105–110 25 November 2016 DOI: 10.12782/sd.21.2.105

Occurrence and Molecular Barcode of the Freshwater Heteronemertean Apatronemertes albimaculosa (: Pilidiophora) from Japan

Hiroshi Kajihara1,4, Mariko Takibata2, and Mark J. Grygier3 1 Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan E-mail: [email protected] 2 Faculty of Psychology, Otemon Gakuin University, Ibaraki, Osaka 567-8502, Japan 3 Lake Biwa Museum, 1091 Oroshimo, Kusatsu, Shiga 525-0001, Japan 4 Corresponding author (Received 17 June 2016; Accepted 26 August 2016)

The freshwater heteronemertean Apatronemertes albimaculosa Wilfert and Gibson, 1974 has previously been reported from Germany, Austria, and the USA. All these records were from aquarium tanks with commercially distributed tropi- cal and/or subtropical water ; by hiding among their roots, the worms are thought to have been introduced from an unidentified place of origin. We report the occurrence of A. albimaculosa for the first time from Japan, based on specimens found in private home aquaria for guppy breeding, with a total of 18 of water plants grown for different periods and lengths of time but not all at the same time. Histological examination confirmed the presence of diagnostic features of this species, including a complex precerebral vascular system and hermaphroditism. The partial sequence of the mitochondrial cytochrome c oxidase subunit I gene (607 bp) of the Japanese material is identical with that of a heteronemertean speci- men from a freshwater tank in Madrid, Spain, which had been deposited in the public databases but left unidentified as to its species, , or family. Apatronemertes albimaculosa appears unlikely to be able to survive below 10°C for more than about a week, which suggests that its native locality is in the tropics or subtropics, not the temperate zone. The barcoding sequence herein determined will serve in future studies to help locate the natural place of origin. Key Words: Heteronemertea, COI, DNA barcoding, water plants, Potamogeton perfoliatus, Vallisneria, .

Beckers (2015) based on the type material, which had long Introduction been maintained by Mr Michael Wilfert, the first author of the original description, but which is now temporarily kept Apatronemertes albimaculosa Wilfert and Gibson, 1974 at the University of Bonn with the intent of depositing it in is one of the six species of fresh- and brackish-water Het- due course in a natural history museum (P. Beckers and J. eronemertea (Iwata 1970; Sundberg and Gibson 2008) that von Döhren pers. comm.). are currently known besides the approximately 500 ma- In this paper, we report the occurrence of A. albimacu- rine representatives of this group (Gibson 1995; Kajihara losa from Japan for the first time. Examination of the inter- et al. 2008). This species was originally described based on nal anatomy revealed diagnostic features that enabled us to specimens from freshwater tanks at the Aquarium der Stadt make a positive identification. To facilitate future studies to Düsseldorf and also at a “zoological shop (‘Zoo-Schäfer’)” locate the ‘fatherland’ of the species, we present a partial se- in Düsseldorf, Germany, which were almost certainly in- quence of the mitochondrial cytochrome c oxidase subunit troduced artificially from an unidentified tropical or sub- I (COI) gene, a marker that has been widely used for mo- tropical native locality [hence the generic name, derived lecular barcoding in nemerteans (e.g., Sundberg et al. 2010; according to its authors from the classic Greek α (a), nega- Fernández-Álvarez and Machordom 2013; Kvist et al. 2014). tive prefix, πατήρ (patēr), father, and Νημερτήϛ (Nēmertēs), a sea nymph, together meaning ‘ribbon worm without fa- therland’]. They were found together with commercially Materials and Methods distributed water plants including, in particular, Vallisneria spiralis L. (Wilfert and Gibson 1974). Senz (1993) subse- Six specimens were examined in this study, of which quently rediscovered the species in 1988 in pet-shop aquaria three remain as voucher specimens (serial histological sec- in Vienna, Austria. The name of the species also appears tions). DNA was extracted from five of them, and COI se- in Smith (2001), referring to specimens collected among quencing was attempted in three, but was successful in only roots of aquatic plants “Vallisnera and Echinodoris” [sic] one. All the specimens were collected alive by one of the (Vallisneria and Echinodorus, respectively) in 1994 in a pri- authors (MT) in private freshwater aquaria used for guppy vate home aquarium in Massachusetts, USA. The nervous- breeding, with the water temperature set at 25°C. Histologi- system morphology of the species was recently studied by cal preparation, DNA extraction, and PCR amplification

© 2016 The Japanese Society of Systematic Zoology 106 Hiroshi Kajihara et al. and COI sequencing were done by HK, following the meth- University, MCZ 58906; from a private home aquarium ods of Kajihara et al. (2011). in the Brighton section of Boston, Massachusetts, USA, The first specimen was found on 31 January 2010. On 4 D. G. Smith leg. in July 1994]; Beckers 2015: fig. 4a [type February 2010, MT brought it alive to MJG, who identified specimen]. it as a nemertean. After digital photography using a Fuji- “Undescribed freshwater heteronemertean”: Andrade et film Finefix S1Pro camera attached to an Olympus SZH10 al. 2012: 159 [MCZ DNA106130; from a freshwater fish research stereomicroscope, the living worm was relaxed in tank in Madrid, Y. Lucas Rodriguez leg. on 30 November

MgCl2. Then its anterior part was fixed in Bouin’s fluid for 2009]. histological observation and the posterior region was fixed in 99.5% ethanol for DNA extraction. This material was Material examined. ICHUM 5112, serial transverse then sent to HK for serial paraffin sectioning and DNA se- sections of anterior half of body cut at 8 µm, mounted in quencing. The histological voucher specimen has been de- Entellan® New (Merck, Germany), and stained by Mal- posited in the Invertebrate Collection, Hokkaido University lory trichrome method, 8 slides, 31 January 2010, MT leg., Museum, Sapporo, Japan (ICHUM). among roots of freshwater plants in a private home aquari- The second specimen was collected on 15 February 2010; um in Takatsuki, Osaka, Japan, 29 mm long in life; ICHUM after a detailed inspection for the diagnostic white spots 5113, serial transverse sections of entire cut at using the above-mentioned microscope at highest (70×) 8–10 µm and stained as above, MT leg. on 20 July 2015, si- magnification and incident illumination, it was fixed as phoned up from amidst tank-bottom gravel, 10 mm long in above by MJG on 16 February, and sent to HK for detailed life; LBM1340000137, serial transverse sections of anterior study. The serial sections of the anterior half of the body half of body cut at 8–10 µm and stained as above, 5 slides, have been deposited in the Lake Biwa Museum, Kusatsu, MT leg. on 15 February 2010 from the same environment, Japan (LBM). 10–12 mm long in life. For comparison, serial digital images The third and fourth specimens (both ∼4 cm in body of the transversely sectioned type slides of A. albimaculosa length), collected on 22 March 2010, and the fifth, collected provided by P. Beckers were also examined. on 8 September 2011, were sent to HK and used entirely for Brief description. Body reddish pink, posteriorly DNA extraction; a partial COI sequence (607 bp) from the paler (Fig. 1A, C), 1–4 cm long, up to 0.5 mm wide; epider- third specimen has been deposited in the DNA Databank of mis with numerous rice-grain-shaped white dots (Fig. 1B), Japan with the accession number LC095809. about 30 µm long and 10 µm wide, varying in shape and size The sixth specimen, collected on 20 July 2015 by MT, was according to body contraction and extension, these being sent to HK and observed under a Nikon SMZ 1500 stereo- particularly abundant in anterior half of body, but not in far microscope. Photographs were taken with a Nikon D5200 anterior end; cephalic slits rudimentary; mouth situated at digital camera attached to the SMZ 1500 by NYPIXS2-3166, a distance (about 2.0–2.5 times width) from tip of head; no HY1S-FA, and NY1S-1501750 adapters (Micronet, Japan), caudal cirrus. Precerebral vascular system with outer ves- with external lighting by a pair of Hikaru Komachi Di sels (Fig. 2A). Proboscis with muscle crosses (Fig. 2B), pre- strobes (Morris, Japan). It has been serially sectioned and sumably two in number, but this not obvious due to small deposited in ICHUM. size (one cross was easily observed as shown in Fig. 2B, but Additional specimens were collected from the same the presence of another was difficult to confirm). Excretory aquaria by MT, mostly by using a siphon vacuum aquarium system composed of amorphous tissue distributed between gravel cleaner or by disentangling the worms from the filtra- alimentary canal, blood vessels, rhynchocoel, and gonads tion apparatus of one of the tanks, but also once in sediment throughout postcerebral region (Fig. 2C). Hermaphroditic; scooped up from among the roots of water plants (Potamo- testes 50–80 µm in diameter (Fig. 2C), only found in fore- geton perfoliatus L.). These were sporadically sent off alive gut region and near posterior tip of body, largely absent in several times to HK between 2010 and 2015, but in all of intestinal region; single oocyte, up to 250 µm in diameter, those trials the worms died in transit, almost certainly due contained in each ovary (Fig. 2D), latter arranged in row to low water temperature (5–9°C on arrival). on each side throughout intestinal region; about 50 ovaries present in each row in ICHUM 5113 (complete specimen of 10 mm body length). In life, posteriorly-moving peristaltic Results and Discussion waves of body thickening and thinning observed in anterior region of ICHUM 5112. Apatronemertes albimaculosa Wilfert and Gibson, 1974 Remarks. Wilfert and Gibson (1974) reported that the (Figs 1–2) largest specimen from Düsseldorf reached 12 cm in body length, which is three times greater than the longest speci- Apatronemertes albimaculosa Wilfert and Gibson, 1974: men we observed. They reported the red pigmentation to 89–105, figs 1–6 [from freshwater aquaria in Düsseldorf]; be darker posterior whereas in our material the posterior Senz 1993: 74; 1998: 11–12, 30; 2003: 35, 38 [Naturhisto- region was palest. The minute white spots in the epidermis risches Museum Wien, NHMW-EV 3488–3491; pet-shop (Fig. 1B), to which the specific name refers, are noticeable aquaria in Vienna, W. Senz leg. in 1988]; Smith 2001: 111, only when observed with higher magnification in the liv- figs 5.3, 5.4 [Museum of Comparative Zoology at Harvard ing state, and probably represent glandular cells. As Wilfert Freshwater nemertean from guppy tanks 107

and Gibson (1974) suggested, they do not represent flecks of white pigment, but are bodies only slightly less translucent than the surrounding tissue, and their appearance depends on the illumination. They are most clearly seen on the body margins in tangential view when the worm is viewed by in- cident light (not visible at all by transmitted light), appear- ing as somewhat translucent-pale granules against the pink background. They are, however, visible over the whole body surface when illuminated by horizontally directed strobe lights for photography (e.g., Fig. 1B). In our specimens they seem distinctly smaller than the “numerous white spots of about 50–200 µm in diameter” noted by Wilfert and Gibson (1974: 90), and also uniform in size. Similar white spots are, in any case, often found in other, marine species (e.g., Oto- typhlonemertes spp.), imparting a velvety appearance to the epidermis, and thus are not specific for A. albimaculosa. In general, the precerebral vascular system in heterone- merteans is comprised of a single lacunar tube either hang- ing over or lying just beneath the rhynchodaeum, or a pair of vessels on both sides flanking the rhynchodaeum, or a mixture of these conditions. In A. albimaculosa, however, on each side an additional, narrow vessel [=‘outer ves- sel’ in Wilfert and Gibson’s (1974) terminology] lies lateral to the normal, main vessel [‘inner vessel’] (Fig. 2A); there- fore, four vessels can be found in a cross section, while the outer vessels merge with the inner vessels at their anterior end and also in the brain region. Such a complex precerebral vascular system has never been reported in any other het- eronemertean, and thus can be regarded as a species-specific character for A. albimaculosa. The presence of the outer ves- sels in our material thus strengthens our conclusion about the species identification despite the coloration differences noted above. In marine heteronemerteans, the excretory system typi- cally consists of collecting tubules that are often ramified and closely associated with blood vessels in the foregut re- gion, leading to paired efferent ducts that open to the exteri- or through the epidermis (Oudemans 1885). In contrast, the excretory system of A. albimaculosa comprises amorphous tissue spread among various internal organs and discharges into the intestine via numerous specialized cells with granu- lar contents (Fig. 2D). Similar tissue has been reported in the freshwater heteronemertean Amniclineus zhujiangensis Gibson and Qi, 1991 and the brackish-water heteronemer- tean Yininemertes pratensis (Sun and Lu 1998), which sug- gests that this morphology is the result of adaptation to a low-salinity environment. In freshwater forms, hermaphroditism is common among hoplonemerteans (Gibson and Moore 1976) but has rarely been confirmed among heteronemerteans. Of the six spe- cies of fresh- and brackish-water heteronemerteans, only A. albimaculosa is known to be hermaphroditic: A. zhujiangen- sis and Y. pratensis are dioecious (Gibson and Qi 1991; Sun

Fig. 1. Apatronemertes albimaculosa Wilfert and Gibson, 1974, ICHUM 5113 (A, B), 5112 (C), photographs taken in life. A, Entire body; B, magnification of body surface in intestinal region, show- ing minute oval inclusions in epidermis, these appearing white with lateral strobe illumination; C, anterior region. 108 Hiroshi Kajihara et al.

Fig. 2. Apatronemertes albimaculosa Wilfert and Gibson, 1974, ICHUM 5112, photomicrographs of transverse sections. A, Precerebral re- gion, showing outer vessel (inner vessels ventrally anastomosed here); B, proboscis musculature, showing muscle-cross fibre (indicated by ar- rowheads) connecting outer circular muscle layer and inner myoepithelium; C, testis containing sperm; D, intestinal region, showing oocytes and amorphous excretory tissue (indicated by asterisks) discharging into intestine via specialized cells (indicated by arrowheads). and Lu 1998), sexually mature Planolineus exsul Beauchamp, of the partial COI sequence from the Japanese specimen 1928 and Siolineus turbidus Du Bois-Reymond Marcus, (LC095809) at the NCBI website (http://blast.ncbi.nlm.nih. 1948 have been found only as females (Beauchamp 1928, gov) yielded a complete match (100% in query cover and 1929; Du Bois-Reymond Marcus 1948), and Hinumane- identity) with HQ848584 from an “undescribed freshwater mertes kikuchii Iwata, 1970 is known only by an immature heteronemertean” (Andrade et al. 2012) that was collected specimen (Iwata 1970; Chernyshev 2014). That our material in a freshwater fish tank in Madrid, Spain, with voucher was hermaphroditic (Fig. 2C, D) lends support to our inter- DNA deposited in MCZ (MCZ DNA106130). From this pretation of its specific identity. specimen, Andrade et al. (2012) also provided sequences of Unexpectedly, a BLAST search (Altschul et al. 1990) other gene markers: JF293030 (18S rRNA), HQ856860 (28S Freshwater nemertean from guppy tanks 109 rRNA), JF277733 (histone H3), JF277666 (histone H4), and known place of origin, the rest of the freshwater/brackish- JF277587 (16S rRNA). We think it reasonable to identify water heteronemerteans known to date are all distributed in this Spanish specimen as A. albimaculosa based on the com- Asia: P. exsul in Bogol, Indonesia (Beauchamp 1928, 1929); plete match of its COI sequence with that of our material. So an unidentified form from Taiwan, reported by Takakura far as we are aware, this represents the first example of ret- (1932) (for English translation see Kajihara 2004); H. ki- rospective identification by molecular sequence of nemer- kuchii from Lake Hinuma, Japan (Iwata 1970) and Primor- teans in public databases that have been left unidentified to sky Krai, Russia (Chernyshev 2014); A. zhujiangensis in the the species level. The presence of a myriad of such unidenti- lower part of the Zhujiang (Pearl) River, China (Gibson and fied barcoding sequences of various groups of organisms in Qi 1991); and Y. pratensis from the Chiangjiang (Yangtze) GenBank can be important in biodiversity studies, if correct River Estuary, China (Sun and Lu 1998). The ‘fatherland’ of is provided post hoc. The assertion that “DNA Apatronemertes may also be somewhere in tropical Asia. barcoding should accompany taxonomy” (Sundberg et al. 2010: 274) is thus true here. It is likely that A. albimaculosa has been living in fresh- Acknowledgments water aquaria all around the world where commercially distributed tropical water plants are grown. While Wilfert We are grateful to Patrick Beckers and Jörn von Döhren and Gibson (1974) observed a particular association of A. (University of Bonn) for their help and effort in providing albimaculosa with the roots of Vallisneria spiralis, this the information about the type specimen of A. albimacu- species was not cultured in MT’s aquaria. Instead, on one losa, and also to Ms Tomoko Ikari (now T. Miyamoto: for- occasion, two worms were simultaneously found among merly Lake Biwa Museum) for taking digital photographs of roots of Potamogeton perfoliatus. The other water-plant spe- ICHUM 5112 in life. cies hitherto cultured in MT’s water tanks, which must be regarded as additional potential carrier(s) of A. albimacu- losa, include Anubias barteri Schott var. nana (Engl.) Cru- References sio, Bacopa caroliniana B. 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