Biogeography 21. 37–42. Sep. 20, 2019

A record of utilization as a spawning bed for the invasive ascidian Ascidiella aspersa (Müller, 1776) newly introduced in the Pacific coast of northeastern Japan

Tomoaki Goto1, 2* and Yuki Oba2, 3

1 Sanriku Research Center, Iwate University, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan 2 Graduate School of Humanities and Social Sciences, Iwate University, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan 3 Present address: Fisheries Promotion Division of Iwate Prefectural Office, 75-1 Uchimaru, Morioka, Iwate 020-8570, Japan

Abstract: Recently, a newly introduced solitary ascidian, Ascidiella aspersa (Müller, 1776), has occupied rapidly in the fields for Patinopecten yessoensis (Jay, 1857) off the Pacific coast of northeastern Japan. In this study, utilization of the invasive ascidian by a native as a spawning bed was recorded on the basis of four specimens (41.2–74.3 mm in body length) collected from Ofunato Bay, Pacific coast of northern Hoshu, Japan, on January 22 and February 21, 2018. The egg derived from three of them had embryo with blackish eyes and opened mouth. Compared with the previously known utilizing solitary ascidians as spawning beds, the spawners of the eggs were thought as a native sculpin, Pseudoblennius cottoides (Richardson, 1848), based on the combination of clutch size, egg diameter, and melanophores in embryo. The host selectivity of the native sculpin to the newly introduced A. aspersa shown in the present study is likely due to an adaptation to the newly occupied species in its habitat.

Key words: Ascidiella aspersa, invasive, Pseudoblennius cottoides, solitary ascidian, spawning bed

Introduction is a hermaphroditic solitary species distributed originally in the northeastern Atlantic from the Mediterranean Sea to Nor- Introduction of nonnative species into land and aquatic way (Millar, 1952). This ascidian species is assessed harmful habitats has been recognized as a major problem (Bax et al., and moderate to serious threat as an invasive species in many 2001). If established populations of a nonnative species grow geographical locations such as the northwestern Atlantic and spread due to successful reproduction and recruitment of coast of North America, New Zealand, Southern Australia, subsequent generations into the breeding population, that spe- Tasmania, and India (Cohen et al., 2000; Lynch et al., 2016). cies may become prominent in the native biota, or ‘invasive’ In Japan, A. aspersa was found at first from artificial hanging (Parker et al., 1999; Richardson et al., 2000). Invasive species materials and bared shells for the aquaculture of scallop, Pat- may become numerically and ecologically dominant to native inopecten yessoensis (Jay, 1857), in Funka Bay, Pacific coast populations as a major threat to marine ecosystems as well, of Hokkaido, northern Japan, in 2008 (Kanamori et al., 2014; with dramatic effects on biological diversity and productivity, Nishikawa et al., 2014). Serious impacts on the aquaculture habitat structure, and fisheries (Carlton, 1999; Crooks, 2002; managements have been recorded in the Pacific waters off Davis & Thompson, 2000). The tunicate class Ascidiacea are northeastern Japan caused by its occupation in aquaculture diverse group, which inhabit worldwide marine environments fields with the rapid expansion of distribution since the first among the macro fouling communities in marine ecosystems, finding (Chida et al., 2011; Kanamori et al., 2014; Kanamori, attaching to natural and artificial substrates in the intertidal 2016). and subtidal zones of coastal habitats (Quesenberry et al., In 2018, four specimens of the recently introduced A. as- 2003; Shenkar & Swalla, 2011). Non-native invasive ascidians persa filled with fish eggs into the peribranchial cavity were are threat for coastal marine ecosystems due to the potential collected from Ofunato Bay, Pacific coast of northern Honshu, negative impacts on the biodiversity and ecological function Japan, during a field examination of A. aspersa in a scallop (Dijkstra et al., 2007; Lengyel et al., 2009; Morris et al., 2009; aquaculture field. We herein report the results on the examina- Carman et al., 2010; Wong et al., 2012; Colarusso et al., tions of the fish eggs and the inference of the host species. 2016). The European sea squirt Ascidiella aspersa (Müller, 1776) Materials and Methods −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− *Corresponding author: [email protected] This study was carried out based on the biological exami-

– 37 – A record of utilization as a spawning bed for the invasive ascidian Ascidiella aspersa (Müller, 1776) newly introduced in the Pacific coast of northeastern Japan nation of A. aspersa in the mid Ofunato Bay (Fig. 1), Pacific coast of northern Honshu, Japan. The specimens of A. asper- sa were collected monthly from the ropes with bared scallop shells between 5–10 m in depth, hanged on May 23, 2017 at ca. 23 m depth in order to assess the biological characteristics. Each specimen of A. aspersa removed from the scallop shell was measured for body length (BL) followed by Kanamori (2016) and dissected after fixation with 10 % buffered forma- lin. Vertical profiles of water temperature and salinity were measured using STD (Rinko-Profiler, JFE, Advantec Co. Ltd.) Fig. 2. Vertical profiles of water temperature (A) and salinity (B) on January 22 (solid line) and February 21 (dashed line) at the in each sampling day. The present materials with concealed sampling position shown in Fig.1. fish-eggs were found from the ascidian specimens collected on January 22 and February 21, 2018. All of the eggs were removed from the peribranchial cavity and the total number was counted. Egg diameter was measured using micrometer for 20 eggs removed from each ascidian specimen. One–way analysis of variance (ANOVA) was employed to compare the egg diameter among host ascidian specimens examined. Sta- tistical analyses were performed by statistical software R3.5 (R Development Core Team, 2017).

Fig. 1. Map showing sampling location of Ascidiella aspersa (circle) in Ofunato Bay.

Fig. 3. Photographs showing dissected Ascidiella aspersa filled Results with fish eggs into the peribranchial cavity. A and B, right and left views of dissected mantle showing concealed eggs in a Samplings and morphological examinations specimen (74.3 mm BL), respectively. C and D, right view of Vertical profiles of water temperature and salinity are mantle surface and left view of partly dissected mantle showing concealed eggs in a specimen (64.2 mm BL), respectively. shown in Fig. 2. Water temperature at 10 m depth and bottom Abbreviations AT, BR, EG and IT indicate atrial siphon, (23 m depth) were 8.94 °C and 9.51 °C on January 22; and 7.73 branchial siphon, fish egg and intestine, respectively.

– 38 – Tomoaki Goto and Yuki Oba and 8.00 on February 21, respectively. Salinity at 10 m depth organs. and bottom (23 m depth) were 33.74 and 33.91 on January 22; Number of eggs removed from each ascidian specimen 33.80 and 33.92 on February 21, respectively. was 199 to 363 (mean: 265). The egg diameter (mean ± SD) Three (mean and range: 59.9 mm and 41.2–74.3 mm in BL) was 2.10 ± 0.08 mm, 2.09 ± 0.07 mm, 2.10 ± 0.08 mm and and one (55.2 mm BL) specimens of A. aspersa had fish eggs 2.09 ± 0.10 mm in diameter for the host ascidian specimen of into the peribranchial cavities among 37 (mean ± SD: 51.6 ± 74.3 mm BL, 64.2 mm BL, 41.2 mm BL and 55.2 mm BL, re- 14.5 mm BL) January and three (63.5 ± 9.1 mm BL) February spectively (Fig. 4). No significant difference was found in the specimens, respectively. Fig. 3 shows the lateral profiles of the egg diameter among the host specimens (One–way ANOVA: mantles containing fish eggs in two specimens of A. aspersa p=0.94). All the eggs removed from three host ascdians except collected on January 22 (A–B: 74.3 mm BL, and C–D: 64.2 74.3 mm BL, which was collected in January, have embryo mm BL). The fish eggs were completely or partly concealed with blackish eyes and opened mouth (Fig. 5). The egg from by the internal organs in right view (Fig. 3A, C), but com- 74.3 mm BL was uniformly pale yellow in color. The embryo pletely visible in left view for all specimens (Fig. 3B, D). The was same stage in each host ascidian specimen, characterizing egg mass was located in the posterior half of the perivitelline by having 3–6 melanophores on the top of the head; about 10 space posterior to the atrial aperture and left to the digestive melanophores on the dorsal surface of the yolk, and numerous

Fig. 4. Frequency distributions in diameter of egg removed from four specimens of Ascidiella aspersa. A, 74.3 mm BL. B, 64.2 mm BL. C, 41.2 mm BL. D, 55.2 mm BL.

Fig. 5. Egg with embryo from a specimen of Ascidiella aspersa (55.2 mm BL). A, 1.89 mm in egg diameter. B, 1.96 mm in egg diameter.

– 39 – A record of utilization as a spawning bed for the invasive ascidian Ascidiella aspersa (Müller, 1776) newly introduced in the Pacific coast of northeastern Japan melanophores along the ventral contour of the tail (Fig. 5). traits (Uchida, 1932; Kimura et al., 1987, 1988; Nishida et al., 2008). Awata et al. (2019) described the clutch size of each Discussion egg mass and the egg diameter for seven species identified us- ing a genetic identification based on the whole genomic DNA Some of the parasitic animals that inhabit aquatic environ- for 120 egg masses derived from 1212 host solitary ascidians. ments preferentially deposit their eggs in live invertebrates According to Nishida et al. (2008) and Awata et al. (2019), the (known as ‘ostracophils’: Balon, 1975; Leung, 2014). Some clutch size deposited into a single host ascidian has relatively ascidian tunicates have been known to provide spawning beds large intraspecific variation. Nishida et al. (2008) pointed out for some ostracophilous sculpin and tubesnout species into that a positive correlation between clutch size and size of host their peribranchial cavities (Uchida, 1932; Yabe, 1997; Mune- ascidian is present as the specific reproductive behaviour in P. hara, 1991; Akagawa et al., 2004, 2008; Nishida et al., 2008; percoides despite it is still unclear in other species. While it is Awata et al., 2019). To date, four species of Pseudoblennius, difficult to identify the species based on the clutch size caused i.e., P. cottoides (Richardson, 1848), P. percoides Günther, by such intraspecific variations, the clutch from the present 1861, P. zonostigma Jordan and Starks, 1904 and P. sp. called ascidian specimens contained apparently smaller number and “Kirin-anahaze”, Furcina osimae Jordan and Starks, 1904, larger number of eggs than that in P. sp. called as “Kirin-ana- and Aulichthys japonicus Brevoort, 1862 distributed around haze” and P. percoides, respectively (Table 1). Although early Japanese archipelago have been recorded as the egg bearing development in the egg has been poorly known for P. zon- species to solitary ascidians (Nishida et al., 2008; Awata et al., ostigma, the present results represent somewhat greater clutch 2019). In Pacific coast of northern Honshu, Japan, the - size and larger egg diameter (Table 1). Embryo in the present ing season of A. japonicus is apparently later than the present egg specimens is different from P. percoides by having greater study (Table 1), and its clutch size and egg diameter are also number of melanophores on the dorsal surface of head (3–6 in different from those in the present materials (Table 1). Thus, present materials vs. 0–2 in P. percoides: Kimura et al., 1988; the fish eggs from A. aspersa were possibly spawn by the egg Nishida et al., 2008; Kojima, 2014), and from P. zonostigma bearing sculpins. Whereas the spawning season of the scul- by lack of melanophores anterior to anus (2 in P. zonostigma: pins are almost equal to the present study period, the present Kojima, 2014). Therefore, the fish eggs derived fromA. asper- results on clutch size and egg diameter are much greater than sa are thought to be spawn by P. cottoides based on the com- F. osimae (Table 1). In the genus Pseudoblennius spawning parison of morphological features in egg among the previously into solitary ascidians, four species consisting of P. cottoides, known fishes utilizing solitary ascidians as spawning beds. P. percoides and P. sp. called “Kirin ana-haze” have been re- The present results suggested that the invasive A. aspersa corded from the Pacific coast of northeastern Japan, including recently introduced in the study area, was selected by a native the present study area (Nakabo & Kai, 2013). Since morpho- cottid species, P. cottoides, as spawning beds. In the Pacific logical identification for the eggs concealed into ascidians is coast of northeastern Japan including study area, A. apsersa difficult, the parent fish species have been identified based on has rapidly dispersed after its introduction, and occupied the the limited observations of spawning behavior or development sessile organism habitat in the aquaculture fields for scallop

Table 1. Comparison of spawning characteristics between the present materials and the previously known fish species baering eggs into solitary ascidians. P. sp. Aulichthys Species Present study P. cottoides P. percoides P. zonostigma Furcina osimae (Kirin-anahaze) japonicus 2) 1) 1) 1) 1) Spawning January–February December–January December–January December–February December–February February–March May–June in Otsuchi Bay season December1) December–March2) February in Abratsubo Bay1) Clutch size 265 (199-363) ca. 1201) ca. 70-5001) 7–338(mean 137)1) 395–1200(mean 744)1) 73–83 (mean 77)1) 15–441) 66–440(mean 247)3) 37–161(mean 111)2) 55–110(mean 68)2) Egg diameter 2.09 (1.88–2.28) 1.85–1911) 1.4–1.81) 1.7–2.01) 1.9–2.01) 1.6–1.71) 2.3–2.82) 1.60-1.982) 1.6–2.03) 1.9-2.02) 1.8-2.04) Host species Ascidiella aspersa Halocynthia ritter4) Halocynthia hispida2) Unknown Halocynthia ritter1) Pyura sacciformis1) Halocynthia roretzi1) Pyura sacciformis4) Halocynthia ritter2) Halocynthia roretzi1) Ciona intestinalis1) Pyura sacciformis2) Styela picata1) Styela clava1) References 1) Shiogaki & Dotsu (1974) 1) Nishida et al. (2008) 1) Awata et al. (2019) 1) Awata et al. (2019) 1) Awata et al. (2019) 1) Akagawa et al. (2004) 2) Kimura et al. (1987) 2) Awata et al. (2019) 2) Awata et al. (2019) 2) Awata et al. (2019) 3) Nishida et al. (2008) 4) Awata et al. (2019)

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