THE REEF & MARINE AQUARIUM MAGAZINE. Hippo Tangs. Butterflyfish Rearing Artemia Guide DRAGON WATCH: Oceanographers fear a waking methane monster

Transcription

1 THE REEF & MARINE AQUARIUM MAGAZINE Hippo Tangs Butterflyfish Rearing Artemia Guide DRAGON WATCH: Oceanographers fear a waking methane monster MAY/JUNE 2016

2 THE LONG ROAD TO BREEDING It was a sunny, flat calm day in March 2012, and I had just completed my fifth successful neuston tow along a reef drop off Oahu s west coast. The tow s target was pelagic fish eggs floating on the surface, part of the thin layer of organisms and gametes that rise out of the plankton for a portion of their life cycle. A short while later, back in the lab, I was staring through the glass of a dissecting scope, triumphantly admiring my diverse catch of fertile fish embryos, the identity of which was still a mystery. 46 CORAL

3 BUTTERFLYFISHES article & images by Frank Baensch A school of Milletseed Butterflyfish (Chaetodon miliaris) on a deep reef in Hawaii. JENNY HUANG CORAL 47

4 The Hawaii Larval Fish Project was just beginning. My mission was to find new aquarium reef fish species with a life history that might be adapted to aquaculture. Living close to the sea, I had come to realize that the most effective way to do this in my space-limited hatchery was to initially bypass the broodstock spawning bottleneck and obtain the fertilized eggs from the ocean. This allowed me to focus on what, for many species, is considered the most critical (and for me the most exciting) aspect of culturing a marine fish rearing the larvae. The eggs I collected hatched, and the larvae gradually showed the various morphologies, pigmentation patterns, and behaviors that make culturing them so thrilling. I recognized a couple of larvae just after hatching: the boxfish by its deep, rotund, dark-yellow body, and the moray eel by its distinctive head, fangs, and long, snake-like body. But most of the larvae were not readily identifiable with my novice eye. Inside the soup of mystery baby fishes, one form looked very similar to a Centropyge pygmy angelfish, a genus I had worked on for over a decade. But while preflexion Centropyge appear reddish, these larvae were distinctly yellow. One of the yellow Centropyge survived until about day 25, making it through flexion before dying. Examining the dead specimen under the microscope, I 48 CORAL

5 Right: Preflexion Centropyge and Chaetodon species. Here the author examines eggs under a stereoscope in his lab in Honolulu. noticed that the head was encased in bony plates. This unusual larval feature is characteristic of only two families. One is the scats (Scatophagidae), which do not occur in Hawaiian waters. The other family, which is important both on the reef and in our aquariums, is widespread here. Euphoria hit me as I realized this was a Chaetodon species, a member of the butterflyfishes the ideal candidate for the larval fish project! Butterflyfishes, with their amazing multicolored body patterns, are among the most conspicuous reef fishes worldwide. They play important ecological roles on coral reefs as corallivores, herbivores, and planktivores. The family comprises 130 species in 11 genera, with genus Chaetodon alone representing almost 90 species. Half of all butterflyfish species rely on coral polyps as part or all of their diet. Corallivorous butterflyfishes, nearly all of which are Chaetodon species, are often seen as indicators of reef health and have been heavily researched in the wild for that reason (Cole and Pratchett 2014). It is no surprise that butterflyfishes are adored favorites in our saltwater aquariums. These brilliant beauties consistently rank among the top six families in terms of their annual economic contribution to the aquarium trade (along with the angelfishes, damselfishes, surgeonfishes, wrasses, and gobies). Roughly 100 species are imported annually into the United States, many of which depend on coral polyps for food. Fortunately, the bulk of traded butterflyfishes do not require corals, making them more suitable for aquariums (Lawton et al. 2014). The harvesting of butterflyfishes poses a serious threat to natural populations, especially for corallivores that are already threatened by extensive habitat degradation. While the collection and grow-out of post-larval butterflyfishes using crest nets and light traps aims to provide some relief from wild harvesting, the technique does not deliver a reliable supply of specific species. In addition, the large-scale commercialization of post-larval collection would likely be destructive, since butterflyfish populations are maintained by very low rates of larval recruitment (Delbeek 2014). Captive-breeding butterflyfishes could be the most ideal, environmentally-sound way to meet the growing demand in the long term. There is little information on breeding butterflyfishes. They have a complicated reproductive biology and rarely reproduce in captivity. For that reason, few larval fish culturists have had the opportunity to even attempt to identify the rearing requirements of the larvae. We do know that the larvae are primitive at hatching and spend a long period up to two months in the open ocean before settling on the reef as juveniles. Undoubtedly, this makes them a challenge to rear. Of the handful of breeding accounts, only one reported success raising the CORAL 49

6 A Pacific Double-Saddle Butterflyfish (Chaetodon ulietensis) in Papua New Guinea. larvae beyond the early first feeding stages (The Rising Tide Conservation Initiative raised Heniochus diphreutes to 37 days post hatching in 2011) (Delbeek 2014). Far more research needed to be done before butterflyfish farming could begin. My single Chaetodon larva had made it all the way through notochord flexion on the first try. Perhaps these fish were not that difficult to raise after all. I began towing in areas populated by butterflyfish aggregations, hoping to find more eggs. The twelfth neuston tow hit the jackpot: once they were in the larval tank, I identified almost two dozen first-feeding Chaetodon larvae close to the surface amongst a host of competitors. The larvae were carefully removed using a small beaker and transferred to their own personal 13-gallon (49-L) black round tank stocked with wild copepod nauplii and a fairly dense concentration of Isochrysis microalgae. So far, Chaetodon and Centropyge larvae seemed to have similar culture requirements, and I was hopeful. When I am working with limited egg quantities in small-volume larval tanks, my rearing technique focuses on maximizing the survival of individual larvae. It relies heavily on optimizing nutrition by feeding wild-collected rather than cultured copepods and maintaining optimal water quality by siphoning the bottom daily, doing water changes, and transferring the larvae to new tanks. This technique had proven effective for Centropyge and appeared to be working for the Chaetodon. Three weeks from hatching, eight larvae had survived beyond flexion. I had always produced juveniles with this number of larvae surviving postflexion, and I was ready to rejoice. Two weeks later, all of the larvae had died. Butterflyfish eggs lack distinct identifying features. A single oil globule, slight pigmentation, and a diameter of 50 CORAL

7 microns are common egg characteristics of many pelagic spawners. The larvae hatch at just over 2 mm TL and have dots of yellow pigment running along the notochord. They begin to feed by three days post hatching (dph) at just under 3 mm TL. The yellow bodies and surface orientation of first feeding larvae allows them to be easily separated. The distinctive bony plates become recognizable on the head as the larvae begin notochord flexion, between 18 and 22 dph. The body then becomes increasingly deep and ovoid in shape. Postflexion larvae are initially heavily pigmented and then gradually take on a silvery appearance, while the bony plates become larger, extending from the head onto the body. Over the next three years I raised Chaetodon larvae from 15 net tows, each with virtually the same frustrating survival pattern. Mortality was usually low during the early (preflexion) stage, followed by several spikes of expected mortality during the notochord flexion period, a critical period of development for many reef fishes. The postflexion larvae that survived fed and behaved normally for a while, but then hit a roadblock. Time and time again, they stopped feeding and perished as they fully developed their specializations to pelagic life (the bony head armor and spination and the elongate dorsal spines). The longest-living Chaetodon larva made it to 46 dph, just shy of 0.3 inch (8 mm) TL. The techniques that had worked to raise angelfishes, anthias, snappers, groupers, and triggerfishes were not working for the chaetodontids. Meanwhile, another research group, The Rising Tide Conservation Initiative, had developed protocols to spawn the Milletseed Butterflyfish (Chaetodon miliaris) at their research facility in Florida. Despite having access to fertile eggs on a biweekly basis, their results appeared to be similar, with larvae dying in the late postflexion stage. The Chaetodon culture bottleneck appeared to be the late larval stage. Fortunately, not all butterflyfish larvae are created equal. The project s 60th tow, made in August 2013, finally produced a breakthrough. The tow had collected butterflyfish eggs and, as usual, the larvae became identifiable shortly after first feeding. However, this time, most of the 20 or so surface-oriented, pencil-shaped bodies were darkly pigmented instead of yellow. As in previous Chaetodon kleinii, early larval stage. CORAL 51

8 The 130-gallon (492-L) broodstock tanks used to spawn butterflyfishes. Chaetodon fremblii on a Hawaiian reef. runs, the larvae were separated from potential competitors into 13 black round tubs and reared on wild, sizegraded copepods with Isochrysis as background algae. Forty-five days later the yellow Chaetodon larvae had died as expected, but the now even darker and more oblongshaped larvae were still voraciously feeding on smaller adult copepods. Fifty days later one of the larvae transitioned into a juvenile Pennant Bannerfish (Heniochus dephreutes)(baensch 2014)! Success with the H. disphreutes was an exciting surprise, but I made little headway in rearing the Chaetodon larvae. My neuston tows provided me with an incredible diversity of egg species; in fact, I have collected and grown several other butterflyfish species to the late stages. The problem was that egg collections from all but the most prolific spawning species (surgeonfishes and parrotfishes) were small and inconsistent. I needed more fertile eggs for experimentation. It was time to bring Chaetodon broodstock into the hatchery. The reproductive biology of Chaetodon species is diverse and complex. Most species spawn in pairs, some aggregate to spawn, and a few species even form harems, but the strength of the pair bond and the tendency to aggregate varies from species to species. Pairing butterflyfishes from individuals is complicated because they are not sexually dichromatic or dimorphic and likely do not change sex (gonochoristic). Species that form stronger monogamous bonds are less likely to accept a new mate. Pairing younger, immature fish is easier, but requires the fish to have the correct sex and reach maturity, which can take years. Conditioning is complicated for harder-to-keep butterflyfish species that depend on corals for food, those species that aggregate, and/or those that have distinct breeding seasons and require complex cues, such as lunar phases, to trigger reproduction. It is not surprising, then, that Chaetodon species have rarely reproduced in captivity. In fact, spawning reports are limited to just four species (C. miliaris, C. lunula, C. modestus, and C. nippon) (Delbeek 2014). All of these involved numerous fish in large-volume tanks. I knew that choosing the right Chaetodon broodstock would be critical. Ideally, the fish would have to be relatively small and easy to keep and would spawn in pairs/harems (not in aggregations), so that I could accommodate them comfortably in my modest-sized tanks. Since they could not be accurately sexed, they would have to be collected as pairs. Finally, since non-corallivore Chaetodon often feed individually or in large single- or mixed-species aggregations, the pairs would have to be collected just before spawning. I started my search in January because most of my wild Chaetodon eggs had been collected in the winter and spring, when most butterflyfishes likely spawn here. My first candidate was C. fremblii, a gorgeous Hawaiian endemic and one of the few chaetodontids that form harems. Chaetodon fremblii have been observed spawning in February and March (Lobel 1978). The two collected fish were found close together, 52 CORAL

9 Chaetodon kleinii broodstock (still frames from video). feeding on rubble bottom just off a reef drop, in about 80 feet (24 m) of water. Despite careful decompression, one of the fish had not properly equalized, and the decision was made to introduce the pair directly into the deep broodstock tank to reduce stress a fish can usually fix slight buoyancy issues itself in a deep tank without having to needle its swim bladder. I had bypassed quarantining fish I had personally collected several times in the past, and disease had never been an issue. A 130-gallon (492-L) tank (38 x 36 x 22 inches 3 /97 x 91 x 56 cm 3 ), which I had used to spawn angelfishes, triggerfishes, and wrasses in the past, proved adequate for the C. fremblii pair as well. Over a four-week period, the fish increasingly displayed spawning behavior during the late afternoon the male chased the female, nuzzling her abdomen, which seemed to swell more and more each day. One morning in late February, a single, large spawn of several thousand eggs in the egg collector pleasantly surprised me. But my joy was short-lived all of the eggs were infertile. Around that time, the fish became increasingly nervous, breathing heavily and rubbing against objects. A skin scrape revealed Cryptocaryon irritans. Reproductive behavior ceased shortly after the diagnosis; my careless fish introduction had come back to haunt me. A long in-tank treatment using salinity manipulation and chloroquine proved ineffective, and the parasites reappeared on the fish in full force. So I made the decision to release the pair on their home reef for their well-being. It was April, and C. fremblii were now found only singly on the reef. Wild spawning of this species appeared to have ceased. Two attempts to pair up single fish proved unsuccessful, resulting in no reproductive behavior. Meanwhile, my work on C. kleinii had begun. This planktivorous, relatively small species occurs throughout much of the tropical and sub-tropical Indo- Pacific, at depths from 10 to 400 feet (3 122 m). Chaetodon kleinii is hardy in captivity and reportedly has a beneficial attribute: it feeds on the marine aquarium pest Aiptasia. This species forms strong pair bonds while reproducing during the spring here in Hawaii. I clearly remember the day CORAL 53

10 Chaetodon kleinii eggs (neurula stage). the pair was collected. It was March and I was diving on a small patch reef off Oahu s south shore in about 40 feet (12 m) of water. A school of C. kleinii was loosely hanging together under a small reef ledge. This species often aggregates while feeding. The tide was slack and the group was probably taking a break from picking plankton out of the water column, which is more abundant during tide changes. I noticed two fish hanging together closely, apart from the others. They were equal in size, but in one fish the abdomen was slightly swollen hopefully with eggs. Collection would have to be executed properly to keep these fish from rejoining their group. A minute later, after a quick maneuver with my two hand nets, the two fish were peering through the vents of my collecting bucket. I was thankful to be diving shallow, which made decompression of the gravid female shorter and less stressful. A couple of hours later at my hatchery, I carefully examined the pair through the glass pane of a 30-gallon (114-L) quarantine tank, where they would spend the next six days; I had learned my lesson with C. fremblii. Maintaining the fish in spawning state was critical, which meant providing them with ample feedings, minimizing stress, and replicating the photoperiod and water temperature on the reef. Prophylaxis would have to be mild. I opted for the no-chemical transfer method, moving the fish to a new tank every morning to break the parasite life cycle. The abdomen of the gravid female was more swollen the morning I introduced the pair to the broodstock tank. They quickly adjusted to their more spacious new home. The fish had been fed twice a day in quarantine and were soon gorging themselves on a mix of adult Mysis and chopped fresh squid, shrimp, and scallops. By afternoon, the more slender fish was observed closely following the gravid fish, nuzzling his snout against her lower abdomen. Luck was on my side this time. The mated pair spawned that very same evening, producing close to 900 fertile eggs. I quickly got to work. Chaetodon larvae had consistently survived better when subjected to lower light levels and higher tank turbidities and when smaller, rotifer-sized live foods were fed longer into the late larval phase. Larval rearing bottlenecks are most often related to nutrition, so I wanted to test various combinations of small live-food organisms. I set up three 13-gallon (49-L) black round tubs, each holding about 300 eggs. Larvae in tank 1 predominantly received SS-rotifers (5 10/ml) and a mix of wild and cultured nauplii and copepodites (less than 1/ml). Larvae in tank 2 predominantly received cultured copepod nauplii and copepodites (5 10/ml) and a low density of SS-rotifers (less than 1/ml). Larvae in tank 3 were predominantly fed fresh, wild, size-graded plankton (mostly nauplii and copepodites, 5 10/ml) every other day and small numbers of SS-rotifers (less than 1/ml). In all tanks, Isochrysis were maintained at high densities throughout the larval phase. Light levels were gradually reduced as the larvae grew, but most significantly near the later stages. The tanks were siphoned every morning to clean the bottom, detect mortality, and change the water. The density of the live foods was controlled daily by filtering the water through a 50-micron screen. The results in the first two tanks were not surprising. The larvae in the rotifer tank survived well initially, but experienced high mortality during notochord flexion, 54 CORAL

11 A female Chaetodon kleinii, ripe with eggs. The fruitful reproduction of my C. kleinii broodstock was short-lived. Embryo production ceased after that initial fertile spawn. The desire to reproduce was there, as evidenced by lateafternoon chasing behavior and the sporadic release of infertile eggs. The fish were healthy and feeding well and the water quality parameters were good. The photoperiod and water temperature matched the natural conditions at the time. Something else was amiss! Like me, another biologist had witnessed gravid C. kleinii with no larvae surviving past this critical period. The larvae in the rotifer plus cultured copepod tank survived to the mid-postflexion stage, with the last larva dying with an empty gut near 40 dph, just under 0.3 inch (7 mm). The larvae in tank 3 had also suffered significant losses by this time; fewer than 10 larvae remained. The groundbreaking difference was that six larvae were much larger inch (10 11 mm) TL and still feeding. The barrier had been broken. I was in new territory. My survivors became increasingly nervous, skipping on the surface and swimming erratically around the tank. By day 60, their bodies were more coin-shaped and appeared silver, and the tholichthys plates had grown from the head onto the midsection. At this time, the larvae finally began actively feeding on adult copepods and newly hatched Artemia nauplii. The black bar through the eye and the white posterior margin on the dorsal first appeared near 65 dph (0.47 inch/12 mm TL). Two accidents caused further losses during the 20-day transitional stage that followed. Three larvae jumped out one evening when the water cleared from an algae crash, and one died from flash shock during a documentation session in the photo tank. The two survivors were moved to the juvenile system by 84 dph (0.9 inch/24 mm TL) and immediately began accepting finely grated frozen seafood. Ten days later the fish were fully colored juveniles. The Chaetodon larval phase had finally been completed! Chaetodon kleinii, late larval stage. 56 CORAL

12 DON T MISS THE SATURDAY July Cranbrook Institute of Science Woodward Ave, Bloomfield Hills, Mi Each year we strive to bring in speakers to cover a wide variety of topics related to captive breeding, 2016 will be no different! Current speakers include: Chad Callan Martin Moe Tickets available now. females in the wild (Colin 1989). Both likely spawning periods coincided with a new moon phase. Could the reproduction of this species be tied to a lunar cycle? Using a dimmable low-wattage blue incandescent light (used during the Centropyge days) I began to manually simulate the moon phase as best I could. About a month later the first fertile spawn was collected. Spawning occurred for several days while the moonlight was off. Spawns of up to 2,500 eggs with 30 percent fertility have been recorded. In summary, it appears that three main factors contributed to raising Chaetodon larvae. The first, finally starting a run with more than just a handful of eggs, is obvious. The second, feeding a mix of smaller foods later into the larval phase, should have been obvious as well. Unlike the vast majority of difficult-to-rear reef fish larvae that can be raised on nutritious adult copepods, late-stage Chaetodon species have a smaller mouth size. The third, and likely most important feeding a variety of fresh plankton every other day illustrates the high nutritional requirements of the larvae. My rearing technique leaves lots of room for improvement, as shown by the low survival rate of the first successful run. Despite a lot of effort, know-how, and the best nutrition that I could provide, the larvae often fed poorly, sometimes behaved abnormally, and took a very long time to become juveniles 30 days longer than C. kleinii s natural post-larval phase (Stier et al. 2014). The fresh, wild copepod nauplii and copepodites, lower light levels, and high tank turbidities no doubt helped the larvae cross the critical culture barrier in my small tanks, but this is probably not what they prefer. Reportedly, wild late-stage Chaetodon are very active swimmers that feed on chaetognaths, though this finding is based on the stomach contents of just two larvae (Sampey et al. 2007). Raising Chaetodon larvae in larger tanks would allow more eggs to be utilized and provide a calmer, more stable rearing environment for the older, more active larvae. This, combined with feedings of wild plankton or a variety of equally nutritious, smaller live foods, and perhaps even a quality dry diet, would likely improve survival and reduce larval duration. Every group of captive-bred fish was difficult to breed at one time, even the first clownfish. This research sheds 58 CORAL

13 Manage your own subscription It s quick and user friendly A Chaetodon kleinii adult. Go to Click on the SUBSCRIBE tab. some light on the various culture bottlenecks of another valuable group of fishes and brings us one step closer to making their aquaculture a reality. The broodstock qualities of C. kleinii make it a model species for the experimental rearing of Chaetodon larvae, to further grind through the larval rearing bottlenecks of this group. More Chaetodon larval rearing is planned, but I became sidetracked working on another important aquarium-fish family that had been a challenge to rear from my wild egg collections the wrasses. More on this in a future report! Here you can: Change your address Renew your subscription Give a gift Subscribe Buy a back issue Report a damaged or missing issue ON THE INTERNET The Hawaii Larval Fish (Culture + Imaging) Project REFERENCES Baensch, F The Hawaii Larval Fish Project. CORAL 11 (2): Cole, A.J. and M.S. Pratchett Diversity in Diet and Feeding Behaviour of Butterflyfishes: Reliance on Reef Corals versus Reef Habitats. In M.S. Pratchett, M.L. Berumen, and B. Kapoor, eds, Biology of Butterflyfishes, pp CRC Press, Boca Raton, FL. Colin, P.l Aspects of the spawning of western Atlantic butterflyfishes (Pisces: Chaetodontidae). Environ Biol Fishes 25 (1 3): Delbeek, J.C Captive Care and Breeding of Coral Reef Butterflyfishes. In M.S. Pratchett, M.L. Berumen, and B. Kapoor, eds, Biology of Butterflyfishes, pp CRC Press, Boca Raton, FL. Lawton, J.L., M.S. Pratchett, and J.C. Delbeek Harvesting of Butterflyfishes for Aquarium and Artisanal Fisheries. In M.S. Pratchett, M.L. Berumen, and B. Kapoor, eds, Biology of Butterflyfishes, pp CRC Press, Boca Raton, FL. Lobel, P.S Diel, lunar, and seasonal periodicity in the reproductive behavior of the pomacanthid fish, Centropyge potteri, and some other reef fishes in Hawaii. Pacif Sci 32: Sampey, A., A.D. McKinnon, M.G. Meekan, and M.I. McCormick Glimpse into guts: overview of the feeding of larvae of tropical shorefishes. Mar Ecol Prog Ser 339: Other options: us at: customerservice@ coralmagazineservice.com CALL: Or WRITE: Coral Magazine 2406 Reach Road Williamsport, PA CORAL 59