BULLETIN OF MARINE SCIENCE. 89(2):635–642. 2013 http://dx.doi.org/10.5343/bms.2012.1050

NOTE

Bidirectional SEX CHANGE IN SEVEN OF (: )

Hisaya Manabe, Katsuya Toyoda, Kaori Nagamoto, Shin-ichi Dewa, Makoto Sakurai, Kiyoshi Hagiwara, Akihiko Shinomiya, and Tomoki Sunobe

ABSTRACT Bidirectional sex change in the gobies, Priolepis akihitoi Hoese and Larson, 2010, (Regan, 1908), Priolepis latifascima Winterbottom and Burridge, 1993, and (Valenciennes, 1837), was confirmed by rearing experiments. In female-female pairs, the larger individual changed sex to male, and in male-male pairs, the smaller changed to female. The gonads of these species simultaneously formed ovarian and testicular portions with an accessory gonadal structure. As the gonad structure in Priolepis borea (Snyder, 1909), Priolepis fallacincta Winterbottom and Burridge, 1992, and Priolepis inhaca (Smith, 1949) has the same form as in the above species, the latter are also likely to undergo bidirectional sex change. The natural occurrence of P. akihitoi, P. cincta, and P. semidoliata as solitary or paired (male and female) individuals inhabiting cave or rocky crevices suggests that the mating system of these species is monogamous.

Sex change or sequential hermaphroditism is well known among various teleost taxa, taking the form of protogyny (sex change from female to male), protandry (from male to female), or bidirectional sex change (Sadovy de Mitcheson and Liu 2008, Munday et al. 2010). The size-advantage hypothesis predicts that the evo- lution of protogyny or protandry is favored by the type of mating system, po- lygynous and random mating, respectively (Munday et al. 2006). For example, in Labridae the larger male maintains a territory so as to guard females or resources which attract females. In the absence of a male, the largest female changes sex and spawns with the remaining females (Kuwamura and Nakashima 1998). Although the mating systems of protandrous are not well studied, apart from the monogamous anemonefishes, the lizard flathead Inegocia japonica (see Appendix 1 for authorities) mates randomly (Shinomiya et al. 2003). Information on bidirectional sex change has been steadily accumulating in Gobiidae, Serranidae, Pseudochromidae, Pomacanthidae, Cirrhitidae, and Labridae (Munday et al. 2010), occurring in both polygynous and monogamous species. The polygynous gobiid Trimma okinawae changes sex from female to male by becoming the largest in the group, and from male to female by being subordinate (Sunobe and Nakazono 1993, Manabe et al. 2007). The coral-dwelling gobies, Paragobiodon echinocephalus and Gobiodon histrio, are monogamous, the larger individual changing to male in female-female pairs, and the smaller to fe- male in male-male pairs (Kuwamura et al. 1994, Munday 2002).

Bulletin of Marine Science 635 © 2013 Rosenstiel School of Marine & Atmospheric Science of the University of Miami 636 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013

The gobiid Priolepis, closely related to Trimma (Winterbottom and Burridge 1992, Thacker 2009), includes 34 species (Hoese and Larson 2010) dis- tributed on rocky and coral reefs of tropical and temperate waters in the Indo- Pacific and Atlantic oceans (Nogawa and Endo 2007). The preferred habitats of Prioleps spp. are mainly caves and rocky crevices (Sonoda and Imai 1971, Shiogaki and Dotsu 1974, Sunobe and Nakazono 1989, Wittenrich et al. 2007). Although the mating system of Priolepis is not well studied, Priolepis cincta is known to be monogamous (Sunobe and Nakazono 1999). The gonads of P. cincta, Priolepis hipoliti, and Priolepis eugenius include simulta- neously testicular and ovarian portions, suggesting bidirectional sex change (Cole 1990, 2010, Sunobe and Nakazono 1999, Munday et al. 2010). In the present study, we used rearing experiments and histological observations of gonads to examine the type of sex change of Priolepis akihitoi, P. cincta, Priolepis latifascima, and Priolepis semidoliata. In addition, we examined the gonad histology of Priolepis borea, Priolepis fallacincta, and Priolepis inhaca.

Materials and Methods

Collection of Specimens, Rearing Methods, and Preliminary Experiments.— Specimens were captured by hand net, using scuba or snorkeling. Eight individuals of P. akihitoi were collected at Sakurajima, Kagoshima Prefecture, Japan, on 12, 22, and 25 July, 2007. Four individuals each of P. cincta, P. latifascima, and P. semidoliata were collected at Tsuchihama and Tomori, Amami City, Kagoshima Prefecture, Japan, on 9–10 May, 2009. At the time of collection, the habitat and individual circumstance (solitary, pair, or group with three or more individuals) of each specimen was recorded. All specimens were brought to the laboratory of the Faculty of Fisheries, Kagoshima University, anesthetized with 100 ppm of MS222, measured for standard length (SL) to the nearest 0.1 mm, and identified on the basis of natural color patterns on the head and nape. Sex was determined from the shape of the urogenital papilla: either bulbous with several processes at the opening or tapered posteriorly, in females and males, respectively, as found in other gobiid species (Sunobe and Nakazono 1993, 1999, Kuwamura et al. 1994, Shiobara 2000, Manabe et al. 2008, Sakurai et al. 2009). An aquarium of 60 × 35 × 30 cm with two half-cut opaque vinyl chloride (OVC) pipes (10 cm inner diameter and 10 cm length) provided as a spawning nest was used for both P. akihitoi and P. cincta. Smaller aquaria (30 × 20 × 23 cm) with two similarly-sized pipes and two OVC pipes of 2 cm inner diameter and 5 cm length, respectively, were used for P. latifascima and P. semidoliata. Nests were placed directly on the substratum. A sheet of water proof paper, attached to the inner surface of the nest by a rubber band, was removed following spawning and examined microscopically so as to confirm whether or not fertil- ization of eggs had occurred. Water was circulated continuously by gravel filters. The water temperature of the aquar- ia containing P. latifascima and P. semidoliata was maintained at room temperature, be- tween 20 and 25 °C. Priolepis akihitoi and P. cincta water temperatures were cooled to nor- mal habitat levels: 22 °C in July, increasing gradually to 28 °C by late August and thereafter maintained at that level for P. akihitoi; from 25.5 °C in May to 26.5 °C by early August and thereafter maintained at that level for P. cincta. Fish were fed minced clams, minced fresh fish meat, formula food and/or Copepoda, and squid liver oil twice a day. To confirm the functional sex role of individuals sexed on the basis of urogenital papilla structure, preliminary rearing experiments were conducted for P. akihitoi (involving four heterosexual pairs), started on 26 July, 2007, and for P. cincta, P. latifascima, and P. semidoliata (two heterosexual pairs each), starting on 10 June, 2009. As the sizes of each NOTE 637 sex in naturally occurring pairs of P. cincta are similar (Sunobe and Nakazono 1999), experimental pairs consisting of fish caught individually also included similarly-sized individuals. However, individuals caught as a pair were retained as experimental pairs, regardless of any size differences. After deposition of an egg mass in the nest, the functional sexes of the pair were confirmed from the sex of the individual exhibiting parental care, because males of P. borea and P. cincta guard egg masses until hatching (Shiogaki and Dotsu 1974, Sunobe and Nakazono 1999). Fish-Exchange Experiments.—After the above treatment, fish were exchanged among the pairs to establish male-male and female-female pairs. Subsequent observations revealed whether or not male-to-female or female-to-male sex change had occurred (until 23 October, 2007, for P. akihitoi; until 9 October, 2009, for P. cincta, P. latifascima, and P. semidoliata). Completed sex change was indicated by subsequent spawning of the pair. If spawning was not observed before the end of the rearing experiments, sex was determined from the structure of the urogenital papilla. Gonad Histology.—At the completion of the experiments, all of the specimens were fixed in Dietrich’s solution for 24 hrs and then preserved in 70% ethanol. The abdomens of each were embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin. In addition to the above species, we examined the gonads of P. borea and P. fallacincta, from Yokosuka City Museum (YCM), and P. inhaca from Kanagawa Prefectural Museum of Natural History (KPM-NI), all preserved in 70% ethanol after fixation with 10% formalin. Specimens used included: P. borea (YCM-P39613), one male (27.2 mm SL) and one female (23.5 mm SL), collected at Hasama, Tateyama City, Chiba Prefecture, Japan, 4 August, 2001; P. fallacincta (YCM-P41440), one male (26.8 mm SL) and one female (25.5 mm SL), collected at Setouchi Town, Kagoshima Prefecture, Japan, 2 September, 2000; P. inhaca (KPM-NI 13472), one female (22.3 mm SL), collected at Zamami Isl., Okinawa Prefecture, Japan, 24 June, 2008. We dissected the specimens, extracted gonads, and prepared tissue followed the same method as outlined above.

Results

Priolepis akihitoi, P. cincta, P. semidoliata, and P. latifascima were found only in caves or rocky crevices, all individuals of the first three species being captured either as a pair (male and female) or singly. Priolepis latifascima occurred only as solitary individuals (Table 1). None of the aforementioned species occurred in groups of more than two individuals. In the preliminary experiments, spawning occurred in all pairs, indicating that the individuals sexed on the basis of genital papilla shape functioned normally as respective males or females. In the exchange experiment, spawning was observed in pairs Pa1-Pa4, Pa5-Pa7, Pa2-Pa8, Pc1-Pc3, Pl1-Pl3, Pl2-Pl4, and Ps1-Ps3, dem- onstrating that bidirectional sex change occurred. Other pairs (Pa3-Pa6, Pc2-Pc4, and Ps2-Ps4), which did not spawn were also confirmed to have changed sex ac- cording to the shape of the urogenital papillae. Thus, bidirectional sex change was observed in all four species. In all cases, the larger and smaller individuals functioned as males and females, respectively (Table 1). Histological observations indicated that the gonad in P. akihitoi comprised si- multaneously ovarian and testicular portions plus an accessory gonadal structure (AGS; Cole 1990; Fig. 1). In females, the ovarian portion of the gonad contained oocytes in various stages of development, while the testicular portion and AGS 638 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013 ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♂ ♀ ♂ ♀ Sex role papillae Tapered Tapered Tapered Tapered Tapered Tapered Tapered Tapered Tapered Tapered Bulbous Bulbous Bulbous Bulbous Bulbous Bulbous Bulbous Bulbous Bulbous Bulbous Shape of urogenital – – – Exchanging experiment Oct 10, 2007 Oct 01, 2007 Sep 11, 2009 Sep 11, Sep 24, 2009 Sep 27, 2007 Aug 17, 2009 Aug 28, 2009 Date of spawing Sep 06, 2007 Sep 06, 2007 Sep 06, 2007 Sep 06, 2007 Aug 07, 2009 Aug 07, 2009 Aug 07, 2009 Aug 07, 2009 Aug 07, 2009 Aug 07, 2009 Start of pairing Larger Larger Larger Larger Larger Larger Larger Larger Larger Larger Smaller Smaller Smaller Smaller Smaller Smaller Smaller Smaller Smaller Smaller Relative size Pl1 Pl3 Pl2 Pl4 Ps1 Ps3 Ps2 Ps4 Pa1 Pa4 Pa3 Pa6 Pa5 Pa7 Pa2 Pa8 Pc1 Pc3 Pc2 Pc4 Code no. ♂ ♀ ♀ ♂ ♂ ♀ ♂ ♀ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ Sex role Jul 9, 2009 Jul 09, 2009 Jul 31, 2009 Jul 16, 2009 Jun 25, 2009 Aug 16, 2007 Aug 31, 2007 Aug 30, 2007 Aug 03, 2009 Aug 16, 2007 Date of spawing Preliminary experiment Jul 26, 2007 Jul 31, 2007 Jul 26, 2007 Jul 26, 2007 Jun 10, 2009 Jun 10, 2009 Jun 10, 2009 Jun 10, 2009 Jun 10, 2009 Jun 10, 2009 Start of pairing Pa2 (46.9) Pa3 (36.4) Pa4 (41.0) Pa5 (43.4) Pa6 (33.8) Pa7 (42.8)* Pa8 (44.4)* Pc1 (25.3) Pc2 (33.0) Pc3 (44.6)* Pc4 (46.8)* Pl1 (19.4) Pl2 (19.2) Pl3 (19.6) Pl4 (21.4) Ps1 (26.3) Ps2 (30.5) Ps3 (25.5)* Ps4 (25.6)* Pa1 (47.3) Table 1. Results of the rearing experiments. * indicates collected as a pair. Table Priolepis cincta Priolepis latifascima Priolepis semidoliata Code no. (mm SL) Priolepis akihitoi NOTE 639

Figure 1. Gonadal structures of (A) male and (B) female Priolepis akihitoi. O = ovarian portion; T = testicular portion; AGS = accessory gland structure. Scales are 0.5 mm. were undeveloped. In males, the testicular portion of the gonad was filled with well-developed spermatozoa; the ovarian portion with a few young oocytes, and a fully developed AGS. Gonads of P. cincta, P. borea, P. fallacincta, P. inhaca, P. lati- fascima, and P. semidoliata showed similar histology to those of P. akihitoi.

Discussion

A pair is regarded as monogamous if (1) the male and female confine most of their spawning acts (copulations) to the same partner, and/or (2) they remain partners after fertilization, until the young no longer require their care (Barlow 1988). Sunobe and Nakazono (1999) reported that the mating system of P. cincta was monogamous because four pairs repeatedly spawned for 49 days. Male-female pairs of both P. akihitoi and P. semidoliata were found in caves and rocky crevices as observed in P. cincta (Sunobe and Nakazono 1999, present study), suggesting that despite the lack of continuous confirmatory observations, the mating systems of P. akihitoi and P. semidoliata are also monogamous. Bidirectional sex change in P. akihitoi, P. cincta, P. latifascima, and P. semidoliata was confirmed in our fish-exchange experiments. Bidirectional sex change has been observed previously in a related genus: T. okinawae, Trimma grammistes, Trimma kudoi, and Trimma yanagitai (Sunobe and Nakazono 1993, Shiobara 2000, Manabe et al. 2008, Sakurai et al. 2009). Histological observations on these species indicated that the gonads included both ovarian and testicular portions, concurrently. Gonads of P. borea, P. fallacincta, and P. inhaca (this study), and P. hipoliti and P. eugenius (Cole 1990, 2010), having similar histology to the above species, suggest that these species also have the ability for bidirectional sex change. In both the monogamous coral-dwelling gobies, P. echinocephalus and G. histrio, a pair bond is established with the closest occurring individual after the loss of a mate, since long-distance movement between host corals is risky due to increased predation (Kuwamura et al. 1994, Munday 2002). Bidirectional sex change may be 640 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013 adaptive if the closest individual is of the same sex (Kuwamura et al. 1994, 2011, Munday 2002). Our observations Priolepis spp. indicated a similar situation. The exchange experiments herein demonstrated that the larger and smaller in- dividuals of same-sex pairs changed to male and female, respectively. The male and female in each pair were usually similar sized in natural populations of P. cincta (Sunobe and Nakazono 1999), which is also suggested in P. akihitoi and P. semidoliata (Table 1). In female-female pairs of P. echinocephalus, the larger in- dividual changes to male and in male-male pairs, the smaller individual changes female. The faster growth of females compared with males during the non-spawn- ing period results in the mostly equal sizes between the sexes in each breeding pair of P. echinocephalus (Kuwamura et al. 1994). Our results suggest that there may be a similar difference in growth rate between males and females in pairs of Priolepis spp.

Acknowledgments

We are grateful to H Senou (KPM-NI) for permission to dissect specimens of P. inhaca. Thanks are also due to M Matsuoka, M Deki, and S Shibata for their field assistance. This study was supported by Grant-in-Aid to TS (no. 16570025 and 19570016) from the Japan Society for Promotion of Science. This paper is dedicated to our colleague, the late K Toyoda.

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Date Submitted: 19 July, 2012. Date Accepted: 15 February, 2013. Available Online: 6 March, 2013.

Addresses: (HM) General Education, Kagoshima University, 1-21-30 Korimoto, Kagoshima 890-0065, Japan. (KT, KN, AS) Faculty of Fisheries, Kagoshima University, Kagoshima 890- 0056, Japan. (S-ID) Diving Service Umi-Annai, 7-7 Masagohonmachi Kagoshima 890-0067, Japan. (MS) Kagoshima Immaculate Heart College, 4-22-1 Toso, Kagoshima 890-8525, Japan. (KH) Yokosuka City Museum, 95 Fukada-dai, Yokosuka 238-0016, Japan. (TS) Laboratory of Fish Behavioural Ecology, Tateyama Station, Field Science Center, Tokyo University of Marine Science and Technology, 670 Banda, Tateyama 294-0308, Japan. Corresponding Author: (TS) Email: . 642 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013

Appendix 1. Species names and authorities mentioned in the present study.

Gobiodon histrio (Valenciennes, 1837) Inegocia japonica (Cuvier, 1829) Paragobiodon echinocephalus (Rüppell, 1830) Priolepis akihitoi Hoese and Larson, 2010 Priolepis borea (Snyder, 1909) Priolepis cincta (Regan, 1908) Priolepis eugenius (Jordan and Evermann, 1903) Priolepis fallacincta Winterbottom and Burridge, 1992 Priolepis hipoliti (Metzelaar, 1922) Priolepis inhaca (Smith, 1949) Priolepis latifascima Winterbottom and Burridge, 1993 Priolepis semidoliata (Valenciennes, 1837) Trimma grammistes (Tomiyama, 1936) Trimma kudoi Suzuki and Senou, 2008 Trimma okinawae (Aoyagi, 1949) Trimma yanagitai Suzuki and Senou, 2007