1 National Marine Fisheries Service Fishery Bulletin First U.S. Commissioner established in 1881 of Fisheries and founder NOAA of Fishery Bulletin Abstract—The aim of this study was to The power of ultrasound: observation of make ultrasound observations of 2 cap- tive reef manta rays (Mobula alfredi), nearly the entire gestation and embryonic thereby collecting the first known data regarding the embryonic devel- developmental process of captive reef manta opment of reef manta rays throughout rays (Mobula alfredi) almost the entire gestation period. The gestation period and water tempera- 1 ture at parturition were ~1 year and Kiyomi Murakumo (contact author) 1,2 ~27°C, both of which are consistent Rui Matsumoto with observations of wild individuals. Taketeru Tomita1,2 On the basis of embryonic features Yousuke Matsumoto1 observed through ultrasound, 3 devel- 2 opmental stages were recognized: early Keiichi Ueda (0–80 d after copulation), middle (80–150 d after copulation), and late Email address for contact author: k- [email protected] (150–360 d after copulation). The mid- dle stage was distinguished from the 1 Okinawa Churaumi Aquarium early stage by the presence of large, Ishikawa 424 wing- shaped pectoral fins, cephalic Motobu, Okinawa 905-0206, Japan lobes, and buccal pumping, whereas 2 the late stage was distinguished from Okinawa Churashima Research Center the middle stage by the overlapping Okinawa Churashima Foundation of the right and left pectoral fins and Ishikawa 888 cessation of motion of the entire body Motobu, Okinawa 905-0206, Japan in utero. These criteria will be useful for determining the gestation stages of wild reef manta rays. Studies on the life history of large of wild reef manta rays and estimated aquatic vertebrates, including the the gestation period for this species, reef manta ray (Mobula alfredi) and on the basis of the duration between 2 the giant manta (M. birostris), are consecutive parturition events for the extremely difficult because such spe- same individual. However, their data cies move fast and have extensive were based on observations of females ranges in vast marine environments in the field, and no internal anatomi- and because technology for studying cal or embryonic developments were free- roaming aquatic animals does not observed. The only documented obser- exist (Castro, 2016). In the aquarium, vation of an embryo of a live reef manta observations are also difficult, because ray can be found in Tomita et al. (2012), of the slow growth of animals, which who conducted ultrasound examina- can take decades to reach maturity, tions of a pregnant manta ray at the and because they are difficult to main- Okinawa Churaumi Aquarium, located tain for such long periods. Life history in Okinawa, Japan, and described traits concerned with female reproduc- its embryonic respiratory behavior. tion, gestation cycle, gestation period, However, this report provided only a Manuscript submitted 21 July 2019. and fecundity are essential for studies “snapshot” of this species’ embryonic Manuscript accepted 18 December 2019. on the conservation and management development during late- term gesta- Fish. Bull. 118: 1–7 (2020). Online publication date: 24 January 2020. of large aquatic vertebrates. tion, and this species’ entire gestation doi: 10.7755/FB.118.1.1 Knowledge of the female reproduc- process, from early to late term, has not tive biology of manta rays (Mobulidae) been reported. The views and opinions expressed or is still limited (Couturier et al., 2012). Long- term captivity and successful implied in this article are those of the Marshall and Bennett (2010) observed breeding of reef manta rays at the Oki- author (or authors) and do not necessarily reflect the position of the National the breeding behavior (e.g., courtship nawa Churaumi Aquarium have pro- Marine Fisheries Service, NOAA. display, copulation, and parturition) vided a unique opportunity to monitor 2 Fishery Bulletin 118(1) the entire gestation process of this species. In 2007, a reef manta ray gave birth in the Kuroshio Tank (which has a volume of 7500 m3). This event was the first captive birth A of this species (Matsumoto and Uchida, 2008), and since us p then, 2 females have given birth to a total of 8 more off- spring at the aquarium. To the best of our knowledge, this report is the first to define almost the entire gestation process and embryonic ex development of the reef manta ray. This study would not have been possible without the recent development of B underwater ultrasound (Tomita et al., 2019). Some pre- vious studies have demonstrated that ultrasound is a powerful technique for observing the embryonic condition of captive, viviparous elasmobranchs (e.g., Carrier et al., 2003; Daly et al., 2007). However, the device used in this study is unique in that it can be used by scuba divers at d water depths of 10 m or more, allowing the observation of pregnant females without handling the specimens and, therefore, minimizing potential stress. ex Materials and methods Experimental animals The 2 female reef manta rays (specimens 1 and 2) examined Figure 1 were originally captured by using a set net off Okina wa Photographs of the water-surface ultrasound device and Island, Japan. Specimen 1 was 205 cm in disc width (DW) technique used to make observations of the gestation and when captured in 1998 and had grown to 387 cm DW by embryonic developmental process of captive reef manta 2013, whereas specimen 2 was 227 cm DW when captured rays (Mobula alfredi) during 2007–2015 at the Okinawa in 2008 and had grown to 368 cm DW by 2015. Specimen 1 Churaumi Aquarium in Japan. (A) The probe (p) of the gave birth 7 times from 2007 through 2013, and specimen 2 ultrasound device (us) was extended with a PVC pipe (ex). gave birth once in 2015. (B) An aquarium staff member persuades the reef manta ray to swim to the surface by serving krill with a long- Ultrasound examinations handled dipper (d), while another staff member obtains ultrasound footage from above. Ultrasound examinations were conducted in the Kuro- shio Tank at the Okinawa Churaumi Aquarium, by using a FAZONE M1 ultrasound device (Fujifilm Global, Tokyo, observations was timed differently for each pregnancy, Japan) and a transducer in a 3.525-MHz linear array and starting from 12 h to 31 d after mating. at depths up to 24 cm. The examinations were conducted Analysis of ultrasound footage was conducted by using by using either 1) water- surface ultrasound (Fig. 1) or software designed to edit images in the DICOM file for- 2) underwater ultrasound (Fig. 2). For water- surface mat (OsiriX, vers. 3.9.2–3.9.4, Pixmeo SARL, Geneva, ultrasound, the ultrasound probe was attached to the Switzerland). Through analysis of footage, we confirmed tip of a PVC pipe that was ~2 m long, and the observer the developmental condition of the embryo, such as the approached the specimens from above during feeding. presence or absence of yolk sac and cephalic lobes and the When the animals swam near the surface, the trans- size of the pectoral fins. In addition, we observed the kine- ducer was placed on the skin of the dorsal surface above matics of the embryo, confirming the presence or absence the uterus. For underwater ultrasound, the device was of buccal movements. housed in a water- and pressure-resistant case (Tomita et al., 2019), and scuba divers collected ultrasound data by placing the transducer on the skin of the dorsal sur- Results face above the uterus. We conducted ultrasound experiments for 93 d, about We tracked a total of 7 pregnancies in 2 female reef manta once every month. The beginning of the ultrasound rays, and 6 of these pregnancies resulted in successful births. Mating events were observed for all pregnancies. 1 Mention of trade names or commercial companies is for identi- The gestation periods, calculated from the 6 pregnancy fication purposes only and does not imply endorsement by the events of the 2 captive females from 2007 through 2015 National Marine Fisheries Service, NOAA. with the assumption that fertilization occurred on the day Murakumo et al.: Ultrasound observation of gestation and embryonic development of captive Mobula alfredi 3 as indicated by the diameter of the internal uterine cav- ity (maximum of ~10 cm; Fig. 3A). The embryo was not A yet visible, and yolk could be observed. Later (~50 d after copulation), the motionless embryo could be observed in utero (Fig. 3B). The amount of uterine fluid was greater during the middle stage than during the early stage, as indicated by the diameter of the internal uterine cavity (maximum of 30 cm). In addition, the pectoral and dorsal fins and cephalic lobes were well developed (Fig. 4B), and the yolk sac was slender, cylindrical, and easily identifiable in ultrasound images (diameter of ~2 cm) (Fig. 4C). Further- more, the tips of the right and left pectoral fins were bent p dorsally or ventrally along the curve of the uterine wall but never overlapped, and the embryo rotated its body B inside the uterine cavity and exhibited buccal pumping (Fig. 4D). During the late stage, the relative amount of uter- ine fluid decreased, as the size of the embryonic body increased, and a conspicuous bulge was observed on the dorsal surface of the maternal body. The uterus was greatly expanded; therefore, the entire uterine image could not be acquired by ultrasound, and uterine diame- ter was no longer available. The yolk sac was still present p but was rarely observed in ultrasound images, possi- bly because of its small size, and the pectoral fins were folded dorsally, with the right and left pectoral fins largely overlapping (Fig.