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Spawning, early development, and first feeding in the lemonpeel angelfish Centropyge flavissimus Ike Olivottoa, Scott A. Holtb, Oliana Carnevalia, G. Joan Holtb,T a

Dipartimento di Scienze del Mare, Universita` Politecnica delle Marche, via Brecce Bianche, Ancona 60131, Italy b University of Texas at Austin Marine Science Institute, 750 Channel View Drive, Port Aransas, TX 78373, USA Received 9 August 2004; received in revised form 3 December 2004; accepted 7 December 2004

Abstract The present study investigates, for the first time, critical processes in early life history in captive rearing of the lemonpeel angelfish Centropyge flavissimus. Video recordings of courtship behaviors in the 300 L spawning tank showed several courtship behaviors to be similar to those described for wild fish, but courtship activities associated with harem maintenance were not observed in the captive fish. Typical courtship activity was seen on every day of observation, but gamete release occurred on only 7 of 10 observation days. The reason for the lack of gamete release was not obvious. Tank spawning began when temperature and photoperiod conditions were changed to spring and reached a maximum peak (in terms of egg number) during the summer, when the temperature was 28 8C. Egg production and fertilization rate remained relatively constant day to day and spawning continued for months at a time as long as temperature and photoperiod were maintained at optimum levels. Once the embryo hatched (15 h post fertilization) and the yolk sac stage was reabsorbed (48 h post hatching), larvae were ready to feed. Cultured Parvocalanus sp. nauplii or size-sieved wild zooplankton were suitable first food. Proper environmental conditions included rearing the larvae at 28 8C. This study provides the first data on the influence of temperature and first food offered on larval survival in the lemonpeel angelfish. D 2004 Published by Elsevier B.V. Keywords: Reef fish; Spawning behaviour; Embryos; Larvae; Parvocalanus; Copepods

1. Introduction Many fish collectors in tropical and subtropical countries employ cyanide to stun tropical fish, T Corresponding author. Tel.: +1 361 749 6716; fax: +1 361 749 6719. E-mail address: [email protected] (G.J. Holt).

making it easier to collect them, but widespread cyanide application harms coral reefs and marine ecosystems and threatens the food source of the local population. Therefore, in the last few years, a number of scientists have studied the reproduction of some of the species most commonly used in the aquarium trade for the purpose of rearing them in captivity.

0044-8486/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2004.12.009

AQUA-626279; No of Pages 9

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Centropyge angelfish are among the most highly prized of the coral reef fish. The family Pomacanthidae has circumtropical distribution with N 70 species in seven genera: Centropyge, Chaetodontoplus, Genicanthus, Holocanthus, Pomacanthus (Euxiphipops), Apolemichthys and Pygoplites (Fraser-Brunner, 1933; Chung and Woo, 1998). There has been recent interest in spawning and cultivating coral reef fish, especially angelfish, for which knowledge of reproductive biology is essential. According to Thresher (1984), before 1970 almost nothing was known of Pomacanthidae reproductive behavior bdespite their conspicuousness and prominence on the reef Q. Papers on Pomacanthids reported by Breder and Rosen (1966) describe spawning in captivity of only one species Pomacanthus arcuatus (Straughan, 1959a,b). But current interest in angelfish reproduction has resulted in numerous papers published in the last 25 years (Bauer and Bauer, 1981; Neudecker and Lobel, 1982; Thresher, 1982; Holt and Riley, 2001; Sakai et al., 2003). The small size and quiet behaviour of Centropyge suggests that they may be easier than other Pomacanthids to spawn in captivity (Ballard, 1970; Lobel, 1975; Bauer and Bauer, 1980). There are numerous critical processes in early life history where deficiencies could represent a limiting factor in captive rearing. These include spawning in captivity, embryo development, and the transition from endogenous to exogenous feeding. Recent research activities have resolved some of the problems related to egg quality, proper embryo development, and hatching (Olivotto et al., in press) by developing suitable diets and technologies (Carnevali et al., 1999). A major problem remaining is the optimisation of feeding schedules and environmental conditions for successful larval rearing (Holt, 2003; Olivotto et al., 2003). The aim of the present study was to describe reproductive behaviour of captive adults, egg development, and larval development, and determine appropriate larval rearing techniques for the lemonpeel angelfish.

2. Materials and methods 2.1. Animals All species of Centropyge studied to date form harem groups of one male and two to four females

(Bauer and Bauer, 1981). lemonpeel angelfish Centropyge flavissimus were maintained in pairs in 300 L tanks and fed twice daily (8 am and 4 pm) with commercial flake food and raw shrimp or fish; we observed that the inclusion of a second female resulted in the continued harassment of one of the females by the other two fish. Rocks covered with algae and invertebrates (live rocks) were stacked in the tanks to simulate the reef habit. Effluent water from spawning tanks was air-lifted into an external filter box with crushed oysters to buffer pH. Water returned from the filter box at a rate of 2.1 L/min, resulting in a total tank water exchange every 2.4 h. Photoperiod and temperature were manipulated using light timers and heat pumps to simulate seasonal changes. Fish were subjected to environmental manipulations that ranged from 10 h light and 20 8C for the simulated winter season to 13 h light and 28– 30 8C for summer. Spring and fall conditions were 11–12 h light and 24–26 8C. Spawning tanks were equipped with both overhead and underwater lights on separate timers to produce a 0.25 h dawn and dusk. Salinity was maintained at 32–36 ppt, pH 8.2, NO2, and NH3b0.03 ppm. 2.2. Behavioural observations Spawning activity was recorded on digital video tape on 10 evenings over a 16 day period during the bsummerQ season. The camcorder was mounted on a tripod in front of the tank. Recordings were made during the final 90 min of light each evening. A researcher started the recorder and immediately left the room so as not to disturb the spawning pair and did not reenter the room until the lights went out. The camera was removed from the tripod each evening but was replaced with a small silver-colored box so the object was present in front of the spawning tank throughout the day. Bauer and Bauer (1981) give a detailed account of the spawning behaviour of several species of Centropyge, including C. flavissimus, in both the wild and in captivity, and we followed their nomenclature in describing spawning behaviours. Each tape was reviewed in the laboratory, and frequency and duration of each behaviour were recorded.

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2.3. Embryo collection, development, and hatching rate Eggs were collected in a 150 Am mesh bag (1020 cm) attached to the intake pipe inside the filter box (Holt and Riley, 2001). A subsample of embryos was placed in a beaker (2 L) of 5 Am filtered sea water at 26 8C, pH 8.2, salinity 32–36 ppt (to match the spawning salinity). Only floating embryos were considered fertilized and percent hatch was calculated as the number of viable larvae on the morning after hatch divided by the number of fertilized eggs. The main developmental stages were photographed under the microscope and the timing was recorded. 2.4. Larval rearing The larval rearing tank was a 150-L fiberglass conical-shaped tank (Holt, 2003). The water was only gently aerated from day 6 post hatching, since the larvae seemed extremely sensitive to turbulence. The light reflection was reduced by the grey colour of the tank, while the phytoplankton Isochrysis galbana was used to bgreen upQ the larval tanks until the bottom of the tank could no longer be seen. Approximately 15% of the water was replaced each morning from day 6 post hatching, and a few drops of a 5% potassium iodine (KI) solution were added once a day. 2.5. Evaluation of live prey Different strains of wild and cultured zooplankton were evaluated to find a suitable food for rearing the larvae. Foods selected for evaluation were some of the plankton species that typically represent first food for fish larvae: copepod nauplii and dinoflagellates. The two copepod species selected here have a different naupliar size and, in particular, they may be considered possible natural preys for angelfish larvae since Acartia tonsa is a cosmopolitan species and Parvocalanus spp. are circumtropical. The heterotrophic dinoflagellate Oxyrrhis marina was chosen because of its small size relative to the copepod nauplii and the importance of dinoflagellates in the diets of fish larvae in the wild. Two temperatures (26 8C and 28 8C) were chosen for

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evaluation that represent bcoolQ and bwarmQ temperatures typical of the tropical environments where angelfish spawn. Wild plankton was collected twice a day during high tide at the University of Texas Marine Science Institute (UTMSI) pier laboratory in the Aransas Pass (Port Aransas, TX, USA) with a 25 Am plankton net. The plankton was screened through different size mesh and the 25–75 Am fraction was collected for feeding. At the end of each collection, the most abundant zooplankton species were recorded. Copepod stock cultures of A. tonsa and Parvocalanus spp. originally isolated from plankton collections near Port Aransas, TX, USA, were maintained in a temperature-controlled room (27F0.5 8C) in two different 200 L tanks with gentle aeration under the following conditions: salinity 32–36 ppt, pH 8.2, NO2, and NH3b0.03 ppm. A 12:12 light–dark cycle was maintained in the culture room and water quality was continuously monitored. Water was completely replaced every 2–3 weeks. Both cultures were fed daily to excess with the microalgae I. galbana. Culture densities were monitored daily and when nauplii reached a concentration of 1 individuals/ml, they were used to feed the fish larvae. Naupliar stages were separated from copepodites and adult stages using different sized mesh (150–100–75–25 Am). O. marina was cultured in 25 L tanks on I. galbana (50000 cells/ml) at 28 8C and 33 ppt salinity. Water was gently aerated and a 12:12 light–dark cycle was used to raise the dinoflagellate. 2.6. Experimental design Egg batches were divided into different groups to study the effects of different diets of cultured copepods and the dinoflagellate on the survival rate of the larvae. Six egg clutches (about 1200 embryos) were used to estimate the role of temperature and diet on survival rate of the lemonpeel angelfish larvae. All larvae were maintained at the same salinity (32–36 ppt) as the spawning tank and kept on a 24 h light regime. Larvae, 200F10 tank 1, were divided into groups and reared using three different diet combinations at two different temperatures. Live prey were added to the larval tank twice a day (8 am and 5 pm).

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3. Results 3.1. Courtship and spawning During the first 6 months period in captivity, the lemonpeel angelfish couple was subjected to environmental changes that mimic typical seasonal changes. After fall and winter, spawning began when photoperiod and temperature were changed to spring conditions, reaching a maximum peak (in terms of egg number) during the summer, when the temperature was 28 8C. Spawning was confirmed to occur through the collection of newly fertilized eggs from the aquarium following the release of a gamete cloud by the spawning pair. Subsequent spawns were confirmed by the observation of a gamete release seen on the tape. The fish were observed to spawn in 7 of 10 observation periods.

The courtship ritual was defined herein to begin with the initiation of the circling behaviour. Courtship lasted 19–39 min and actual spawning occurred from 30 to 52 min before final lights-out. Courtship in the lemonpeel angelfish consisted of randomly alternating series of Quiver Displays, Circling, and Spawning Rise. The major differences observed between our observations and those of Bauer and Bauer (1981) were that neither fish of the pair ever underwent blanching. Most significantly, it was primarily the female rather than the male that did most of the Circling behaviour. For each Spawning Rise, both individuals assumed the spawning posture where the male tilted his body upward at approximately 458 angle with his snout pressed against the females vent while the female maintained a near-horizontal posture (Fig. 1). In this position they would rise up in the water column over a particular rock in the aquarium. In two spawning events, spawn rises

Fig. 1. Spawning behavior of lemonpeel angelfish. (A) Spawning Rise: male angles his body at 458 upwards and presses snout against vent of female. (B) Thrust and Turn: male twists his body to bring the genital orifices of both fish together and eggs and sperm are released simultaneously. (C) Post spawning, both individuals turn and dart to the bottom.

ARTICLE IN PRESS I. Olivotto et al. / Aquaculture xx (2005) xxx–xxx Table 1 Fertilization and hatch rate in the different experimental groups Experimental groups

Fertilization rate (%)

Hatch rate (%)

A B C D E F G

89 90 91 88 92 90 90

98 99 97 97 99 98 98

occurred constantly, approximately every 10–15 s on average, with a high count of 120 rises. In most of the spawning events, however, the number of spawn rises

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was much lower, with an average of about 40 rises per event and a low of only 11 intermittent rises in one episode. In these cases, there was more circling. The intensity, duration, and frequency of these behaviors increased over the course of courtship. Initial spawning rises lasted b1 s and only traversed a distance of a few centimeters, while latter rises lasted for N3 s and covered much of the 20 cm water column in the tank. The final spawn rise in which spawning occurred was usually the longest rise. On 7 of 10 observations, the final Spawning Rise culminated in the thrust and turn where the male rapidly twists his body over so the genital orifices of both individuals are essentially in contact, and eggs and sperm are released in an often

Fig. 2. Centropyge flavissimus embryo development at 26 8C: (A) 1 h post fertilization (pf); (B) 3 h pf—the gastrula stage; (C) 8 h pf— beginning of organogenesis; (D) 10.5 h pf; (E) 14.5 h pf embryo prior to hatching.

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visible gamete cloud. Following the spawning act, both fish dart to the bottom and no further courtship activity is observed that evening. 3.2. Fertilization and hatch rate The average number of eggs per spawn was 200. The small (0.7 mm) spherical eggs contained a single oil droplet and embryos hatched after approximately 15 h pf (post fertilization) at 26 8C. Fertilization rate, estimated by the number of sinking and floating embryos, was about 90%. Hatch rate, calculated as the number of viable larvae on the morning after hatching divided by the number of fertilized eggs, was estimated to be 98% (Table 1). 3.3. Embryo development All the eggs were encased in a flexible, transparent, spherical capsule. Ten F2 embryos obtained from a single egg clutch were used to describe embryo development. At about 1 h pf, the 16-cell segmentation stage was evident (Fig. 2A). At 3 h pf, the gastrula stage was reached (Fig. 2B), and after 8 h pf, the beginning of organogenesis was evident (Fig. 2C).

Embryonic precursors of the head and the body were well developed after 10 1/2 h pf (Fig. 2D). At 14 1/2 h pf, the embryos were ready to hatch; numerous myomers were formed along the mid-body region, and the head, eyes, tail, and spinal chord were evident. The yolk mass was still very large and the heart, located in front of the yolk sac, in a postero-ventral position to the head, could be seen beating (Fig. 2E). The retina of the eye was still not pigmented, and the embryo was active and moving within the capsule. At 15 h pf, all the embryos had hatched. Newly hatched prolarvae were long and slender, lacked functional jaws and pigment in the eyes, and had scattered melanophores and a moderate-sized yolk sac containing a single oil droplet. Average size of the prolarvae was 2.3 mm. Feeding begins approximately 48 h post hatch (ph). By this time, the larvae have well developed eyes, jaws, month, and gut (Fig. 3A). 3.4. Larval development Four days post hatch (ph), larvae were well developed and very active swimming near the surface of the water. The yolk sac was almost completely absorbed and pigmentation was very light. Pectoral

Fig. 3. Centropyge flavissimus larval development: (A) 48 h post hatch with eyes, mouth, jaws, and gut developed; (B) 4 days post hatch with well developed pectoral fins; (C) 10 days post hatch with swim bladder and gills.

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fins were well developed. Scattered stellate melanophores along the ventral and lateral midline and on the fin-folder were evident (Fig. 3B). At 6 days ph, the body had become deeper and the gut was well distended, indicating that larvae were feeding. Ten days ph, larvae had well developed swim bladders and the gills are evident (Fig. 3C). From days 11 to 14, the body became deeper and rounder, especially in the area of the gut, and the dorsal part of the body darkened. Due to low survival rate, no photo documentation is available from this time period. 3.5. Effect of temperature and diet on larvae survival Larvae did not survive past day 6 in the experimental groups reared at 26 8C temperature (Groups, A, C, and E, respectively) (Table 2). Also larvae reared at 28 8C fed on A. tonsa nauplii and O. marina survived only until day 6. Ten percent of the larvae survived for 14 days only when fed wild plankton plus O. marina or Parvocalanus sp. nauplii at a rearing temperature of 28 8C (groups D and F; Table 2). The main species present in the 25–75 Am wild Table 2 Experimental groups with rearing temperature and food regime for each group Experimental groups

Temperature (8C)

Diet

Survival

A

26

Did not survive past day 6

B

28

C

26

D

28

E

26

F

28

O. marina 10 individuals/ml; A. tonsa nauplii 5 individuals/ml O. marina 10 individuals/ml; A. tonsa nauplii 5 individuals/ml O. marina 10 individuals/ml; wild plankton 5 individuals/ml O. marina 10 individuals/ml; wild plankton 5 individuals/ml Parvocalanus sp. nauplii 5 individuals/ml Parvocalanus sp. nauplii 5 individuals/ml

Did not survive past day 6 Did not survive past day 6 10% Survival rate until day 14 Did not survive past day 6 10% Survival rate until day 14

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plankton fractions collected during the experiment period were A. tonsa nauplii, Parvocalanus sp. nauplii, and the two tintinnids Amphorides quadrilineata and Codonella sp.

4. Discussion Tropical latitudes generally have little temperature fluctuation relative to temperate environments, due to the large ocean surfaces and absence of a cold season (McGregor and Nieuwolt, 1998). For example in the Indo-Pacific Ocean, the sea surface temperature fluctuates 4–6 8C seasonally (McGregor and Nieuwolt, 1998). Accordingly, temperature variation of only a few degrees represents a proportionally large change for organisms that are adapted to this relatively stable thermal environment. As a consequence, small changes in temperature could have a disproportionately greater impact on development of tropical fish larvae than larvae in temperate systems with naturally large temperature variations. Although limited data are available about spawning seasons of Centropyge, it is evident that there is a seasonal change in reproductive activity. Field studies report that spawning occurs in the early spring through summer (Moyer and Nakazono, 1978, Lobel, 1978; Munro et al., 1973; Collier et al., 2004). In our study, spawning activity began in late spring and reached a maximum peak in the summer when water temperatures exceed 27 8C. A 300 L tank is more than adequate as spawning habitat for a pair of lemonpeel angelfish despite the fact that several courtship behaviors seen in wild fish (Bauer and Bauer, 1981) were not part of the spawning ritual of the captive fish. Bauer and Bauer (1981) describe 13 distinct behaviours in the spawning ritual of all Centropyge studied, including C. flavissimus. Five of those involve the male patrolling his territory and visiting each of the several females in his harem, but none of those activities was observed (or at least was not distinguishable from normal swimming activities) in the small confines of the 300 L aquarium and in the presence of only one female. Typical courtship activity was seen on every day of observation but gamete release occurred on only 7 of 10 observation days. The reason for the lack of gamete release was not obvious as there was no

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disturbance of the fish (during courtship) on those days. Egg production and fertilization rate remained relatively constant day to day and spawning can continue for months at a time as long as temperature and photoperiod are maintained at optimum levels. The importance of temperature on larval survival of lemonpeel angelfish was also shown. Small differences in temperature (F2 8C) resulted in a large variation in larval activity and survival. Low temperature may affect predatory skills and swimming activity of the larvae such that this small decrease in temperature reduces survival of these fish larvae. In fact, it is during development in the pelagic environment that fish are the most susceptible to changes in temperature (Rombought, 1997). All experimental feeding groups were subjected to an extended photoperiod (24L/0D) since many studies demonstrated that in these conditions, fish feed for a longer time, yielding higher rates of growth and development (Tandler and Helps, 1985; Duray and Kohno, 1988; Olivotto et al., 2003). The nutritional requirements of marine fish are more rigorous than those of fresh water fish. In fact numerous studies show that a diet rich in HUFAs during the early larval stages is extremely important for optimal nervous system function, in addition to being essential to the development of the brain and the maintenance of its functional efficiency in adults (Harrocks and Yeo, 1999; Sargent et al., 1999, Vagelli, 2004). Tropical marine zooplankton (mostly composed of copepods, dinoflagellates, and tintinnids), normally the first food for marine fish larvae, has a very high content of HUFA. The present study provides information on the potential of cultured zooplankton strains as first food for lemonpeel angelfish larvae. The ubiquity of paracalanoid copepods in coastal tropical habits and their occurrence in wild fish diets (Mitchell, 1991) make them attractive as prey for larviculture. Moreover, species such as Parvocalanus sp. can occur in very high densities in tropical waters (N150/L; McKinnon and Klumpp, 1998) and Acartia sp. is a very common and cosmopolitan species, making these two copepods suitable prey for fish larvae. Our data indicate that initial suitable food and a temperature of 28 8C are better for larval survival. The most interesting data demonstrate that survival rate changes dramatically when larvae fed with certain

plankton strains are subjected to different temperatures. Larvae survived for 14 days only when offered wild plankton or Parvocalanus sp. nauplii, and no long-term survival was observed when larvae were fed solely on A. tonsa nauplii. An important attribute in choosing an appropriate larval diet is the body size of the prey in relation to the mouth size of the fish species to be reared. While our cultured Parvocalanus sp. nauplii have an average naupliar stage dimension of 7045 Am, A. tonsa nauplii have an average size of 9070 Am and are probably too large for the lemonpeel angelfish larvae. Due to its small size, Parvocalanus sp. may be a suitable and innovative first food for small coral reef fish larvae. The same encouraging results that were achieved using Parvocalanus sp. nauplii were also attained with 25–75 Am size fraction of wild plankton. Thus, size and species composition are likely important for improving the larval survival of high value tropical fish like the lemonpeel angelfish.

5. Conclusions This study represents an important first step for successful spawning and rearing of the lemonpeel angelfish. Spawning was fairly straight forward but temperature and size of copepods nauplii were found to be crucial in the early rearing of lemonpeel angelfish larvae. Significant improvements in both rearing conditions and first feeding were achieved in this study and may represent a starting point for possible commercial production of this species.

Acknowledgements Funding for the study was provided by the World Wildlife Fund, and Hugh and Angela McAllister Foundation to G.J. Holt; bFondi Di Ateneo 2003Q to Oliana Carnevali; and bBorsa di Studio Dottorato di Ricerca in Biologia ed Ecologia MarinaQ to Olivotto Ike. Special thanks to Kurt Chambers for the video recordings, to Cammie Hyatt for zooplankton expertise, and to Cynthia Faulk, Matt Palmtag, and all the staff of UTMSI Fisheries and Mariculture Laboratory for help with various stages of this project. This is

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contribution no. 1330 from The University of Texas at Austin Marine Science Institute.

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