Aquaculture 484 (2018) 351–360

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Aquaculture

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Sand star, Astropecten indicus Döderlein, 1888, as an alternative live diet for T captive harlequin , Hymenocera picta Dana, 1852

⁎ Jarunan Pratoomyota, , Siriwan Choosria, Vorathep Muthuwana, Nattawut Luangoona, Wiracha Charoendeea, Wilaiwan Phuangsanthiaa, Andrew P. Shinnb a Institute of Marine Science, Burapha University, Bangsaen, Chonburi, 20131, Thailand b Fish Vet Group Asia Limited, 21/359 Premjairard Road, Saensook, Muang Chonburi, Chonburi, 20130, Thailand

ARTICLE INFO ABSTRACT

Keywords: Harlequin shrimp, Hymenocera picta, are commonly maintained on a diet of comet seastars, Linckia multifora, Feeding behaviour however, difficulties in sourcing a ready supply throughout the year, represent limitations to their sole use Comet seastar within commercial scale production. In a two-step approach, to address their limited availability, firstly the Aquaculture acceptability of several alternative prey items (i.e. Astropecten indicus, Luidia maculata, Culcita novaeguineae and Luidia maculata Holothuria leucospilota) were assessed and then secondly, in-depth studies were conducted with the most ac- Culcita novaeguineae ceptable species identified from the first step. In the first trial, the feeding responses, i.e. time taken to attack Holothuria leucospilota Reproduction prey, by 120-day old, pre-starved shrimp were assessed. Shrimp quickly overturned specimens of A. indicus and began feeding within 5 min, while specimens of L. maculata and L. multifora were carefully assessed before a limb was selected and excised. As the seasonal availability of Linckia is limited, the performance of shrimp fed less frequently was explored. The second trial, therefore, investigated the growth, survival and reproductive capacity of shrimp fed different regimes of live L. multifora over a 5-month period compared to shrimp fed a favourable alternative prey identified from the first experiment, i.e. A. indicus. Shrimp were fed either: 1) live L. multifora daily (LMD); 2) live L. multifora every alternate week (LMW); 3) live A. indicus daily (AID); or, 4) live A. indicus every alternate week (AIW). There was 100% survival of those fed the LMD and LMW diets but 67% in those groups fed AID and AIW respectively; females displayed better growth than males but there were no significant differences (p < 0.05) in the growth performance of shrimp fed the different diets. Shrimp fed LMD, LMW and AID all spawned; AIW failed to spawn. There was a statistical difference in the time to first spawning between harlequin shrimp fed LMD (shrimp first spawned at 206 ± 12.78 days) and those fed LMW (shrimp first spawned at 236 ± 5.20 days); there were no other statistical differences. The study concludes that A. indicus appears to represent a favourable alternative live feed to the use of L. multifora that can be readily maintained within aquaria. During periods when the availability of live L. multifora is limited, a regime of either LMW or AID are appropriate for the maintenance of shrimp, however, minimising the time interval between feeding L. multifora would result in better performance. Statement of relevance: “Evaluating alternative live diets for harlequin shrimp”. The study assesses locally available alternative live feeds for the potential commercial-scale aquaculture of harlequin shrimp in Thailand, focusing on the use of Astropecten indicus as an appropriate live feed that can be supplemented when Linckia multifora is not available.

1. Introduction in the absence of compensatory restocking, such practices have placed this species in danger of overexploitation (Rhyne et al., 2007). There The harlequin or painted harlequin shrimp Hymenocera picta is an has, therefore, been a concerted effort to reduce the numbers currently attractive marine ornamental species that is popular with aquarists. taken from the wild and to establish practices for their aquaculture The rising demand for specimens within the trade of ornamental production as has been achieved with various species of Lysmata decapods has meant that wild stocks have decreased dramatically, and (Hippolytidae) and Stenopus (Stenopodidae) (see Lin et al., 2002;

⁎ Corresponding author at: Institute of Marine Science, Burapha University, Chonburi, 20131, Thailand. E-mail addresses: [email protected] (J. Pratoomyot), [email protected] (S. Choosri), [email protected] (V. Muthuwan), [email protected] (N. Luangoon), [email protected] (W. Charoendee), [email protected] (W. Phuangsanthia), andy.shinn@fishvetgroup.com (A.P. Shinn). http://dx.doi.org/10.1016/j.aquaculture.2017.07.023 Received 18 July 2016; Received in revised form 25 April 2017; Accepted 13 July 2017 Available online 26 July 2017 0044-8486/ © 2017 Elsevier B.V. All rights reserved. J. Pratoomyot et al. Aquaculture 484 (2018) 351–360

Calado, 2008). In nature, harlequin shrimp feed on the ambulacral 2. Materials and methods tissues and the tube feet of a range of asteroid echinoderms including the crown of thorns Acanthaster planci (Acanthasteridae), the sand 2.1. Source of Hymenocera picta sifting star Archaster typicus (Archasteridae), various species of As- terina (Asterinidae), the Indian seastar Fromia indica (Goniasteridae), The F1 juvenile progeny used for the first trials were derived from a the horned or chocolate chip starfish Protoreaster nodosus (Oreaster- single pair of adults that were obtained from the Andaman Sea in the idae), various species belonging to the genus Nardoa and Linckia coastal waters of Phuket, Thailand via a commercial vendor. The paired (Ophidiasteridae), most notably the blue Linckia starfish Linckia lae- adults (female = 1.0 g and 3.2 cm total length; male = 0.7 g and vigata and the comet seastar Linckia multifora, and a range of brit- 2.8 cm total length) were subsequently maintained under a natural tlestar species (see Fosså and Nilsen, 2000; Sprung, 2001; Calfo and photoperiod regime in a 30 L glass tank, linked to a main a recirculation Fenner, 2003; Maltry, 2003; Sewell, 2007). In Thai hatchery aqua- system, (total volume of the system exceeded 70 m3) within the re- culture though, live L. multifora is the preferred diet presented to search hatchery at Institute of Marine Science, Burapha University. The − harlequin shrimp based on the common observation of wild shrimp water passing through the tank at a flow rate of 6.0 L min 1 in this consuming Linckia spp.; a mature pair of shrimp may consume two to system was maintained within the following operational ranges: tem- three specimens of L. multifora (6–10 cm in diameter) each week. perature 28–32 °C, salinity 32–34 ppt, pH 8.0–8.4, dissolved oxygen −1 − − 1 Difficulties in sourcing a ready supply of L. multifora throughout the 6.7–7.5 mg L , total ammonia (NH3 N) 0.02–0.13 mg L , nitrite − −1 − −1 year coupled with their comparatively high price to other available (NO2 N) 0.00–0.02 mg L , nitrate (NO3 N) 0.01–2.6 mg L , alka- − species of seastar, however, represent limitations to the sole use of this linity 86–140 mg L 1; additional aeration was provided through a seastar within commercial scale production. Linckia multifora,forex- single 1″ air stone to each tank. The shrimp were fed an ad libitum diet ample, are difficult to obtain during the Thai monsoon season (Sep- of fresh, live L. multifora (i.e. a4–5 cm fresh piece of limb was replaced tember to December) and caught in the Andaman Sea or once the previous ration had been eaten or required refreshing) for a further afield need to be stockpiled, where resources allow. Specimens period of approximately 3 months before the progeny from one held in captivity for long periods of time (>2 months), however, spawning event were used for the current trials. become weaker to the point that they are less able to survive the After spawning, the berried female was isolated from the male until amputation of a limb. For culture facilities operating in the Gulf of the shrimp hatched, i.e. within approximately 2 weeks. Once hatched, Thailand, L. multifora are typically bought in Bangkok, where the price the H. picta zoea 1 were transferred to a 300 L static, circular fibreglass − varies from vendor to vendor (US$ 0.58–1.74 per 6–10 cm spe- tank at stocking density of 15 larvae L 1, with aeration and 20% daily − cimen 1). When live Linckia spp. are unavailable, then frozen mate- water exchange. The shrimp zoea were maintained on a mixed diet of − rial is used. The settling stage of captive bred harlequin shrimp (i.e. the rotifer, Brachionus rotundiformis, i.e. 10–15 rotifers mL 1, and, − shrimp that are ca. 40–44 days old post-zoeal development) can feed Artemia salina nauplii, i.e. 5 nauplii mL 1, for the first two weeks. on frozen Linckia spp. and Asterina spp. seastars, however, the shrimp Thereafter, the juvenile H. picta were transferred onto an Artemia have lower rates of survival and growth when compared to those fed nauplii only diet until the shrimp reached the settling stage, i.e. live seastars (Sean, 2010). During the Thai monsoon period, however, ~39 days' post-hatch. Mixed microalgae, Tetraselmis gracilis, other echinoderms such as the sand star Astropecten indicus can be Chaetoceros calcitrans and Isochrysis galbana were also added to the H. found more readily, either by directly harvesting them from the in- picta rearing tank and maintained at a density of ~20,000–40,000 cells − tertidal zone or can be purchased from fishermen (ca. US$ 0.35 spe- mL 1. Once the juvenile H. picta had reached the settling stage, they − cimen 1). Other species such as the eight armed seastar Luidia ma- were transferred and held individually in 1.2 L acrylic, adjacent tanks culata, which are dumped on the shore by fishermen as a by-catch, can linked to the main recirculation system. The H. picta were subsequently be collected free. maintained on a L. multifora diet, i.e. small excised pieces from live If the aquaculture production of harlequin shrimp is to be a viable specimens obtained locally, for a period of two months at which point commercial activity, then it is imperative that alternative feed items to the shrimp had attained an average weight of 0.23 ± 0.04 g (mean replace the use of live Linckia spp. during periods of their limited ± 1 S.D.) and an average total length of 2.07 ± 0.15 cm (n = 15). To availability can be found. Such alternatives must meet the nutritional avoid stressing the animals through direct handling, shrimp length was requirements of the species for growth and reproduction, must be determined from captured digital images of the 15 randomly selected available all year-round, should not be prohibitively priced and criti- specimens used for the prey preference trial by using the open access cally should be from sustainable populations or preferably cultured software package UTHSCSA Image Tool (IT) version 3 (University of sources. Texas Health Science Center, San Antonio, Texas 1996–2002; http:// The objectives of the current study were therefore to assess the compdent.uthscsa.edu/dig/itdesc. html). utility of locally available prey items that might serve as suitable re- placements to the use of Linckia. Here we investigate the acceptability 2.2. Sources of prey items presented to the Hymenocera picta and feeding behaviour of captive bred harlequin shrimp as they are presented to three alternative echinoderms namely, the sand star A. Live specimens of the ophidiasterid seastar L. multifora (ca. 6cm indicus,theeightarmedseastarL. maculata,thepincushionseastar, diameter) were obtained from a trader operating in Jatujak market, Culcita novaeguineae, and also to the black sea cucumber, Holothuria Bangkok (acquired from an undisclosed site in the Andaman Sea), the leucospilota, alongside a diet of comet seastar L. multifora,whichisa eight armed seastar L. maculata (ca. 20 cm diameter), the oreasterid common prey item in nature. It was hypothesised that A. indicus and L. cushion star C. novaeguineae (ca. 17 cm diameter) and the black sea maculata, as active moving species, might stimulate the shrimp to cucumber H. leucospilota (ca. 15 cm long) were obtained from fishermen predate upon them. To temper this, the comparatively slower moving working in the local coastal waters and landing their catch at Samaesan, C. novaeguineae and H. leucospilota were included as alternative prey Sattahip, Chonburi, Thailand (approximately 12°35′44.05″ N; items for assessment. In addition to assessing which prey items were 100°57′10.63″ E). Live specimens of the astropectinid sand star A. in- acceptable to the harlequin shrimp, the subsequent growth, survival dicus (ca. 5–6 cm diameter), however, were obtained at low tide at and maturation of the captive bred harlequin shrimp reared on the Bangsaen beach, Chonburi, Thailand (approximately 13°16′05.86″ N; most acceptable alternative diet will be compared to those fed L. 100°55′25.17″ E). The specimens were separated by species and held in multifora. separate tanks as part of the main recirculation system until required. The identity of all specimens was confirmed by Thai echinoderm expert Dr. S. Putchakarn, Institute of Marine Science, Burapha University.

352 J. Pratoomyot et al. Aquaculture 484 (2018) 351–360

2.3. Trial 1. Prey preference and feeding behaviour pieces of equivalent weight per pair. For pairs that were fed daily, a new ration was given once the previous day's diet had been consumed; Five × 22.5 L flow through, glass tanks, each measuring the diet, however, was never left for more than two days. For the 25 × 30 × 30 cm filled to a depth of 25 cm, were used for the trials. shrimp on the LMW feeding regime, they were fed in the same manner − For the trial, a flow rate of 1 L min 1 with additional aeration was as those on the LMD regime but after 7 days consecutive feeding, any used; the water flowing through each tank, however, was rendered to remaining diet was then removed so that the shrimp were starved for waste and not recycled. For each trial run, an individual two-month-old the next seven days before the next ration was given. The shrimp in post-settlement, captive bred harlequin shrimp, i.e. 0.23 ± 0.04 g and each tank were assessed twice per day (i.e. at 08:30 am and at 2.07 ± 0.15 cm, was used. Shrimp settle typically 39–54 days' post- 16:00 pm); any dead shrimp were removed and then weighed and their hatch; the age of the shrimp used here were ca. 120 days old at which length recorded. The feeding trial was conducted over the following point they were large enough to feed naturally on Linckia and the sex of five months during which spawning events were recorded, as were the each shrimp could be readily discerned from moults. Each shrimp was number of eggs, their development and hatch rate. starved for a period of one week prior to being transferred to the ex- perimental tank; starving was used to ascertain whether the alternative 2.4.3. Larval production prey items offered to shrimp were acceptable when shrimp were After moulting, the pair mate and the female spawns shortly hungry. For each trial run, a single juvenile shrimp was placed within a thereafter. If fertilisation occurs, then the female carries a clutch of eggs cylindrical acrylic enclosure in one corner of the tank to standardise its for 14–16 days. Once the eggs begin to turn transparent, the female was starting position. The shrimp was then left to acclimatise for a period of transferred to a static 30 L glass aquarium with aeration. In the trials 30 min before a single, live prey item was added to the centre of each conducted within the facility, the female typically stops eating the day tank, after which the acrylic enclosure was removed allowing the before the larvae hatch. When the eggs are ready to hatch, the female shrimp to interact with the prey item. The initial 20 min of interaction begins actively swimming mid column; egg hatching is synchronised was recorded using a Canon HD Legria HF20 video camera. The sub- and rapid (<5 mins; after sunset ca. 19.00 pm from multiple observa- sequent behaviour exhibited by each shrimp in response to their prey tions). The resultant number of zoea are calculated from five × 100 mL over the following 150 min, i.e. a total observation time of 180 min, samples taken at random points within each tank. Any unfertilised or was observed by eye and key behaviours (i.e. time taken to approach, unhatched eggs are recovered from the bottom of the tank and used to assess and initiate feeding on prey) were recorded using a digital stills calculate hatch rates. The subsequent development of the shrimp is camera mounted in front of the tank. The shrimp and the prey were followed by sub-sampling and by preparing specimens for examination then left together for a further 21 h, i.e. a total of 24 h of cohabitation, with a compound microscope. after which the damage to each prey item was assessed. Each prey item was assessed in triplicate using a new shrimp and a new prey specimen 2.5. Water quality for each interaction; shrimp and prey were randomly assigned to each tank. After each trial run, the tank water was disposed of, the tanks Throughout Trials 1 and 2, water temperature (Hach-sen Ion2), pH scrubbed and cleaned with the commercial detergent Sunlight® and (Hach-sen Ion2), and salinity (Salino-refractometer ATAGO-S/mill-E) were then allowed to dry completely before they were refilled and another checked daily, while the dissolved oxygen concentration monitored using trial run conducted. a Hach-sen Ion6, alkalinity was measured following the methods detailed by APHA (1992), ammonia nitrogen as determined by the phenol hypo- 2.4. Trial 2. Alternative live diets and their effect on feeding, survival, chlorite method (Strickland and Parsons, 1992), nitrite‑nitrogen de- growth and larval production termined by the azo dye method, and the nitrate‑nitrogen concentration as determined by the cadmium-reduction method (Strickland and Parsons, 2.4.1. Sex determination of the settle down stage of Hymenocera picta 1972) were analysed every two weeks. The water quality of each tank was Prior to the start of the experiment, the sex of each settle down maintained within the following operational ranges: temperature − shrimp was determined by examination of their cast moult under an 24.3–28.4 °C, salinity 32–37 ppt., pH 8.0–8.4, alkalinity 86–140 mg L 1, − − Olympus SZ30 dissecting microscope at ×1–4 magnification. From the dissolved oxygen 6.7–7.5 mg L 1, ammonia <0.1 mg L 1,nitrite − − moults of the ca. 70 to 90-day old captive bred harlequin shrimp, the <0.03mgL 1,and,nitrate<2.5mgL 1. morphology of the second pair of pleopods, which are used to separate the sexes, were examined to sex individuals; males develop an appendix 2.6. Statistical analysis masculina and an appendix interna while females develop only an ap- pendix interna. The shrimp were then held individually in 1 L plastic, All data are presented as the mean ± S.D. Differences between floating arenas constructed of 2 mm mesh that were placed within a samples were determined using either t-tests or a one-way analysis of larger 1000 L aquarium linked to the main recirculation system; all variance (ANOVA) and Duncan's post-hoc tests. The correlation coeffi- shrimp were maintained on L. multifora within this system for a further cients and slopes determined from the growth of each group of shrimp one month until the start of the trial. were calculated using Excel 2016 for Windows. Statistical significance was set at p < 0.05. 2.4.2. Feed and growth trial Twelve pairs of the 120-day old harlequin shrimp (0.24 ± 0.04 g 2.7. Ethics statement and 2.07 ± 0.15 cm), originating from the same parents, were main- tained as separate pairs in 24 × 15 × 15 cm glass aquaria, each of These experimental procedures were reviewed by and conducted which contained 5.6 L of 32–34 ppt seawater. The aerated aquaria, under the approval of Burapha University's internal ethical review which were linked into an 80,000 L recirculation system, had a flow board (ethics project certificate ID#065). − through rate of 1 L min 1. A randomised 4 × 3 design was used for the trial in which the harlequin shrimp were fed either live L. multifora or A. 3. Results indicus to satisfy four different feeding strategies: 1) feeding live L. multifora daily (LMD); 2) feeding live L. multifora every alternate week 3.1. Acceptability of the alternative live prey items (LMW); 3) feeding live A. indicus daily (AID); and, 4) feeding with live A. indicus every alternate week (AIW). On each feeding occasion, the The response exhibited by the harlequin shrimp to each prey item live diets were chopped, weighed and given at a rate of two 3 cm-long was scored on their searching, assessment, attacking and feeding

353 J. Pratoomyot et al. Aquaculture 484 (2018) 351–360 behaviours. The harlequin shrimp responded rapidly (i.e. average time over the surface of the seastar apparently appraising it, before moving to start feeding 3.3 ± 1.5 min; range 2–5 min) to fast moving speci- away. The shrimp repeated this process three or four times before mens of A. indicus, while their response and time to initiate feeding on starting to feed on either the end or the mid-point of a limb. The shrimp the comparatively slower moving L. multifora and L. maculata was much continued to feed on the limb once excised, allowing the specimen of L. slower, i.e. 64.7 ± 11.0 min (range 52–72 min) and 52.7 ± 10.2 min multifora to move away. (range 32–70 min) respectively (each based on three observations). The shrimp's behaviour and response time to specimens of L. ma- While the shrimp made initial assessments of the black sea cucumber H. culata were similar to those with specimens of L. multifora with the leucospilota, they neither attacked or attempted to feed and did not shrimp appraising the seastar several times before commencing feeding. return to reassess the sea cucumber within the 2-h observation period. The wounds following excision were observed to close immediately. For the harlequin shrimp that were individually exposed to the “near The harlequin shrimp's reaction to fast moving specimens of A. in- static” specimens of the oreasterid cushion star C. novaeguineae, in each dicus passing approximately 10–12 cm in front of them was immediate case the shrimp made no attempts to assess this seastar. with the shrimp pursuing the sand star. The subsequent sequence of events is detailed in Fig. 1 where the shrimp attempt to immobilise their 3.1.1. Feeding behaviour in response to the live prey items prey by turning them over. Once this had been successfully achieved, For the shrimp placed with live L. multifora, on removing the acrylic the shrimp began to feed, consuming the entire carcass with time enclosure thereby allowing the shrimp to interact with their prey, the (Fig. 2). The harlequin shrimp were disinterested by the near static shrimp quickly became aware of prey within their vicinity and began specimens of C. novaeguineae and H. leucospilota. exploring their aquarium. On encountering the L. multifora, they walked

Fig. 1. a–o. The hunting behaviour of harlequin shrimp, Hymenocera picta, in response to potential prey items – stills extracted from video footage. a-d, having spotted an actively moving sand star, Astropecten indicus, the shrimp moves on to the aboral surface of the sand star and makes an initial assess- ment; e-f, the harlequin attempts to flip over the sand star by positioning its body downwards, its claws (second pereiopods) on the substrate (e-f) and then by placing its legs, and if possible uropod, beneath the oral surface of the starfish (g), and then by extending, straightening its abdomen (h-i), flipping the sand star (j-k) in the process; l-o, as the sand star attempts to right itself to escape predation, the harlequin con- tinues to flip the sand star until the latter becomes exhausted. Thereafter the harlequin shrimp then be- gins to feed on the sand star.

354 J. Pratoomyot et al. Aquaculture 484 (2018) 351–360

Fig. 2. a–d. Harlequin shrimp, Hymenocera picta, feeding damage to a specimen of an eight armed seastar, Luidia maculata. a, harlequin shrimp moving over the seastar in a process of assessment; b, initial damage (arrowed) to the ambulacral plates inflicted by the claws on the harlequin shrimp's first pair of pereiopods; c, advanced limb damage with the softer tissues within the limb exposed. The seastar can inflict further damage to wounded limbs during the process of evading predation; d, once the limb of a seastar has been excised, the exposed face rapidly closes and seals the wound.

3.2. Survival and growth performance of Hymenocera picta weighed approximately ≥1 g at the time at which they first spawned but there were significant differences (p < 0.05) in the average age at The harlequin shrimp were fed to satiation at each feeding point and which they first spawned (LMD = 206 ± 12.77 cf. the amount they consumed was observed to increase steadily LMW = 236 ± 5.20 days' post-hatch), and, the number of zoea suc- throughout the 5-month trial. The average amount consumed of both cessfully hatching in the third spawning (LMD = 2600 ± 564 cf. seastar species was approximately 0.2–0.3 g/g body weight/day. All the LMW = 343 ± 592 and AID = 907 ± 810) (see Table 2 and Fig. 3). H. picta fed L. multifora, on either feeding regime (i.e. LMD, LMW), Shrimp fed the LMD had the highest number of spawnings and although survived. There were, however, some mortalities within the H. picta not significantly different from the other two regimes, those fed the A. reared on A. indicus daily - a single 280-day old male (160 days post- indicus had the lowest number. There were two mortalities among the 3 start of the trial; AID replicate 3 in Table 2) and a 255-day old female breeding pairs in those fed the AID diet, a female 135 days' post-start (135 days post-start of the trial; AID replicate 1 in Table 2). Although and a male 160 days' post start (see Table 2). there were no significant differences (p ≥ 0.05) in the initial weight and length of the 120-day old harlequin shrimp (Table 1), the sub- 4. Discussion sequent performance of the females was better than that of the males (see Table 1). 4.1. Feeding behaviour of harlequin shrimp on Astropecten indicus, Linckia multifora and Luidia maculata 3.2.1. Growth performance of the female Hymenocera picta There were no significant differences (p ≥ 0.05) in the growth Seastars belonging to the Class Asteroidae (e.g. Acanthaster, performance, i.e. in the weight, length, weight gain and length gain, of Archaster typicus, Asterina, Fromia indica, Linckia and Nardoa)arere- mature females (i.e. shrimp aged ca. 9-months post hatch) fed either L. ported to be the natural prey of harlequin shrimp (Calfo and Fenner, multifora or A. indicus daily. In a marked contrast to this, the juvenile 2003; Sean, 2010). From the current trials, specimens of A. indicus females fed A. indicus daily had a lower average weight and had a lower were rapidly predated upon and consumed; their impetus to attack weight gain than those fed on a regime based on L. multifora (Table 1). perhaps stimulated by the rapid movement and scent of A. indicus Among the shrimp fed L. multifora, the juvenile females fed every al- passing in front of the harlequin shrimp within the small aquaria. A ternate week showed a marked reduction in weight and weight gain study conducted by Rainbow (1974) with H. picta and L. multifora, when compared to those fed daily but the growth trends of the two however, indicated that only chemical cues played a role in the initial groups were not significantly different. Likewise, there were no sig- location of prey. The harlequin shrimp were also immediately stimu- nificant differences (p ≥ 0.05) between the female shrimp fed L. mul- lated by presence of a specimen of L. multifora in their aquaria once tifora every alternate week and those fed A. indicus on either a daily or the central tube was lifted, with the shrimp making an immediate alternate week basis (Table 1). move towards the seastar to assess it. The average time taken though for the shrimp to begin feeding on the L. multifora was not significantly 3.2.2. Growth performance of male Hymenocera picta ( p > 0.05) from the time taken to start feeding on the much larger There were no significant differences ( p ≥ 0.05) in the growth of specimens of L. maculata (64.7 ± 11.0 min for L. multifora versus the male H. picta between those reared on either regime of L. multifora 52.7 ± 10.2 min for L. maculata). The shrimp, however, were disin- or those fed A. indicus (Table 1). terested by the large specimens of pin cushion and sea cucumber placed in their aquaria, making almost no exploratory assessments of 3.2.3. Reproductive performance of the female harlequin shrimp each specimen. In addition to the chemical signals released from prey Only the females fed a diet of A. indicus given to them on alternate and their movement, the size of the prey items used may have also weeks failed to show signs of spawning throughout the trial (Table 2). played a role in the shrimp's assessment of prey and may explain the All the other females reared on their live diet regimes spawned; in each response time displayed by the harlequin shrimp. In this study, small, case, the eggs were transferred to their abdomens 2 h after mating and similarly sized L. multifora and A. indicus were used (ca. 5–6cmin these took approximately two weeks to develop. All the females diameter), while the specimens of L. maculata were much larger (ca.

355 J. Pratoomyot et al. Aquaculture 484 (2018) 351–360

Table 1 The growth performance of the female and male harlequin shrimp, Hymenocera picta, fed either a daily live diet of comet seastars Linckia multifora or sand stars Astropecten indicus continuously or on alternate weeks for a total period of five months. The ages of the shrimp given are post-hatch. Statistical analyses found no differences between the shrimp fed each dietary regime.

Treatments L. multifora L. multifora fed A. indicus fed A. indicus fed fed daily daily but on daily daily but on alternate alternate weeks weeks

Female wt. (g) 120 days 0.21 ± 0.03 0.22 ± 0.07 0.20 ± 0.11 0.17 ± 0.05 150 days 0.44 ± 0.02 0.31 ± 0.07 0.37 ± 0.08 0.17 ± 0.03 180 days 1.44 ± 0.20 0.81 ± 0.11 0.97 ± 0.18 0.68 ± 0.17 210 days 1.51 ± 0.29 0.80 ± 0.01 1.00 ± 0.45 0.56 ± 0.22 240 days 2.21 ± 0.47 1.26 ± 0.18 1.49 ± 0.49 0.86 ± 0.36 270 days 2.64 ± 0.67 1.68 ± 0.41 1.73 ± 0.47 1.07 ± 0.57 Wt (g) gain 1.878 1.294 1.187 0.723 − cm 1

Female tot. len. (cm) 120 days 1.76 ± 0.5 1.79 ± 0.10 1.80 ± 0.11 1.69 ± 0.05 150 days 2.56 ± 0.17 2.37 ± 0.19 2.34 ± 0.20 2.07 ± 0.05 180 days 3.05 ± 0.03 2.57 ± 0.18 2.54 ± 0.37 2.23 ± 0.21 210 days 3.34 ± 0.22 3.13 ± 0.13 3.29 ± 0.51 2.81 ± 0.34 240 days 3.47 ± 0.32 3.26 ± 0.10 3.51 ± 0.33 3.10 ± 0.48 270 days 4.26 ± 0.34 3.63 ± 0.13 3.73 ± 0.27 3.30 ± 0.31 Len. (cm) 1.872 1.842 1.727 1.624 gain − g 1

Male wt. (g) 120 days 0.17 ± 0.04 0.17 ± 0.07 0.14 ± 0.10 0.13 ± 0.05 150 days 0.38 ± 0.13 0.25 ± 0.03 0.33 ± 0.13 0.14 ± 0.06 180 days 1.03 ± 0.17 0.75 ± 0.07 0.90 ± 0.25 0.61 ± 0.10 210 days 1.13 ± 0.35 0.62 ± 0.10 0.84 ± 0.30 0.57 ± 0.11 240 days 1.21 ± 0.44 0.76 ± 0.17 0.88 ± 0.35 0.70 ± 0.17 270 days 1.57 ± 0.54 1.25 ± 0.26 1.11 ± 0.16 1.00 ± 0.30 Wt (g) gain 1.043 0.936 0.539 0.53 − cm 1

Male tot. len. (cm) 120 days 1.70 ± 0.11 1.81 ± 0.19 1.79 ± 0.20 1.75 ± 0.10 150 days 2.41 ± 0.12 2.14 ± 0.02 2.29 ± 0.21 1.94 ± 0.10 180 days 2.70 ± 0.21 2.35 ± 0.17 2.66 ± 0.36 2.25 ± 0.41 210 days 3.19 ± 0.32 2.79 ± 0.07 3.03 ± 0.24 2.73 ± 0.12 240 days 3.40 ± 0.32 3.07 ± 0.20 3.31 ± 0.65 2.95 ± 0.39 270 days 3.76 ± 0.37 3.31 ± 0.40 3.50 ± 0.45 3.27 ± 0.35 Len. (cm) 1.629 1.751 1.612 1.659 gain − g 1

Data are mean ± standard deviation of triplicate samples.

20 cm). marine shrimp based studies (e.g. Rainbow, 1974; Lee and Meyers, While the use of both visual and chemical cues in searching for 1996; Archdale and Anraku, 2005). On encountering a specimen of relevant food items has been reported for many marine either L. multifora or L. maculata, the harlequin shrimp were observed (Hindley, 1975; Glynn, 1980; Jensen, 2011), not all crustaceans use moving over the body of the seastar as they assessed which limb to both in seeking out prey. The horned shrimp, P. echinata, for example, begin feeding on (Fig. 2a). This assessment would suggest that the requires only visible cues (Jensen, 2011), while juvenile banana harlequin shrimp evaluate the chemical cues released from their prey prawns, P. merguiensis need only chemical cues (Hindley, 1975). The before either initiating feeding or continuing their search. Harlequin level and types of chemicals that are released by potential prey, for shrimp have been reported to favour the soft tissues of their prey, i.e. example, nucleotides, amino acids etc., will influence the response of the tube feet and the ambulacral grooves (Sprung, 2001; Sewell, 2007); the hunters to then either accept and to pursue the prey or to reject and in the present study, they were observed to crack through the calcar- ignore it (Hindley, 1975; Lee and Meyers, 1996; Archdale and Anraku, eous ambulacral plates using their claws on the first pair of pereiopods 2005). While low levels of these organic chemicals can facilitate the (Fig. 2b) and then using their second pair of pereiopods to feed on the detection, the location of and the discrimination of different prey, softer tissues within (Fig. 2c). During the process of trying to evade higher levels of the same chemicals can motivate the shrimp to consume predation, the seastar may inflict further damage to its wounded limbs the prey (Hindley, 1975; Zimmer-Faust et al., 1996). causing them to break off (Fig. 2d). Once a limb had been excised, the In the current trial, once the acrylic arena surrounding the harlequin wound on the exposed limb still attached to the main body was ob- shrimp was removed, then the shrimp's antennules and antennae im- served to seal itself within 2 mins; the seastar then continued to move mediately began twitching in assessment of the chemicals cues from the away from the shrimp. The shrimp's response to specimens of A. indicus, prey items placed in the aquaria. This twitching was observed in the however, was markedly different with the shrimp immediately pursuing presence of all five potential prey items, as each shrimp explored its the comparatively fast moving sand star, flipping it over and then aquarium – this is a behaviour that has been noted in several other starting to feed without the same level of apparent assessment seen on

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Table 2 The reproductive performance of the female harlequin shrimp, Hymenocera picta, fed either a comet seastar, Linckia multifora, or a sand star, Astropecten indicus, based live diet for a period of five months. The top table summarises the first three consecutive spawning events for each group of experimental shrimp. The lower table provides the timing of spawning event (in days' post-start) for each replicate pair of shrimp on the three dietary regimes and in parentheses provides the number of zoea hatching from each spawning event. The spawning events (days post-hatch) are presented graphically. Abbreviations: AID = A. indicus given daily; ABW = A. indicus given daily but on alternate weeks; dph = days post-hatch; LMD = L. multifora given daily; LMW = L. multifora given daily but alternate weeks. Different superscripts in the same row signify statistical significant differences (p < 0.05). The shrimp were 120 days old when the trial started. LMD LMW AID ABW Dietary regime (n = 3) (n = 3) (n = 3) (n = 3) Av. age at first spawning (days) 206 ± 12.77a 236 ± 5.20a 221.7 ± 22.19 – Av. time to next moult after spawning (d) 1.8 ± 0.6 2.0 ± 0.6 2.0 ± 0.6 – Av. egg development time (d) 14 ± 0.6 14 ± 0.7 14 ± 0.4 –

encountering Linckia specimens. strategies of either feeding live prey daily without interruption or al- These prey assessment trials were performed in triplicate and while ternatively for 7-days followed by withholding food for 7-days was a larger number of replicates would have been desirable, this was in explored. The decision to withhold feeding for up to one week set to part curtailed by the initial costs of procuring broodstock (a single explore the extremes in feeding. An earlier study conducted by breeding pair of adult H. picta currently starts at around US$ 70), and Luangoon (unpublished data), with adult, wild H. picta found no dif- then by the number of F1 progeny that survived to two months' post- ference in the regularity of spawning when fed daily and when fed daily settlement (i.e. 120-days post hatch) and their allocation to the different for one week followed by a week of no feed. A longer starvation period, components of this study. however, was found to impact on spawning rate. In rationalising the use For the trial, live specimens of A. indicus were collected from of live feed, the study set out to reduce the number of live seastars Bangsaen beach, Chonburi and stocked into a flow through 500 L fi- needed by having periods of feeding alternating with periods of non- − breglass tank (flow rate 6 L min 1;28–32 °C; 32–34 ppt) to provide a feeding. While a similar reduction in the number of animals used in the ready supply of live animals. During their maintenance, the animals “one-week on” followed by “one-week off” could have been achieved by spawned multiple times and a captive-bred population was established; feeding them on every alternate day, the trial set out to explore the this culture was maintained for at least three generations (i.e. F3; extremes of a feeding regime (i.e. when seastars are in limited supply, >1 year) and stopped only on conclusion of the parent project. The are not readily available and have to be ordered in). A further, un- details of their aquaculture, however, will be presented in a future published study by the current authors explored the utility of feeding manuscript. frozen seastar material to shrimp but found that shrimp performed poorly; the use of frozen feed was not, therefore, included as an alter- native feeding regime within this study. 4.2. Growth and survival of Hymenocera picta The juvenile harlequin shrimp used for the trial were reared on L. multifora until they were ca. 120 days' post-hatch. While this feeding For the 5-month long growth trial, A. indicus was selected as the history may have affected the results seen in Trial 2, prior to this study alternative live diet for evaluation and the performance of this was no recognised, alternative live feeds for the culture of H. picta were measured against a live diet of L. multifora, as the control. The feeding

357 J. Pratoomyot et al. Aquaculture 484 (2018) 351–360

Fig. 3. The average number of zoea hatching per female (mean ± s.d.; n = 3) from their first three consecutive spawning events. Each group of females were raised on a different regime of live prey in terms of the species presented, i.e. Linckia multifora or Astropecten indicus, and in the frequency with which prey was given, i.e. daily on a continuous basis or daily for 7 days followed by 7 days over which feed was withheld. Statistically significant differences (p < 0.05) between the numbers of zoea hatching between each experimental group within a spawning event are denoted by different letters.

known and so it was essential to raise shrimp to the same standard 4.2.1. Reproduction and larval production of Hymenocera picta before starting this first assessment of shrimp performance on an al- There were several factors that may influence the reproductive ternative live diet. The results show that harlequin shrimp reared on A. performance of crustaceans that include, for example, water tempera- indicus can mature and spawn. This finding supports the rationale for ture and sea levels (Annala and Bycroft, 1988; Chen and Kennelly, conducting further trials where the growth and survival of H. picta 1999; Hartnoll, 2001 cited by Chockley and St. Mary, 2003). The H. raised soley on A. indicus are compared to counterparts reared only on picta used here were seen to spawn regularly (i.e. every 2 weeks) when L. multifora. the water temperature was 27-28 °C but this was interrupted when the The trial results, however, demonstrated that the growth and sur- water temperature dropped below 25 °C. A study on the reproduction of vival of H. picta are affected when shrimp were transferred onto a non- the caridean shrimp Gnathophylloides mineri found that the shrimp re- L. multifora diet. Specifically, all the H. picta that were fed L. multifora produce throughout the year and that the reproductive cycle, which (i.e. LMD and LMW) survived, while those fed the A. indicus on both the includes moulting, mating, egg transfer and incubation on the abdom- daily and discontinuous diets (i.e. AID and AIW) had lower rates of inal plates for a period of 14 days, was about 16 days long (Macia and survival. For the growth trial, pairs of H. picta were used; all the ex- Robinson, 2012). The results from the current trial were similar to that perimental females grew faster than the males. This observation is not of wild H. picta in that having spawned, the female then moulted within unusual as the females of many marine ornamental shrimp species are a day or two days, mating then occurred within 2 h following the moult. reported to be larger than their mates, for example Plesionika izumiae Within a further 2 h, the eggs were transferred to the abdominal plates (see Ahamed and Ohtomi, 2012), Haliporoides sibogae (see Baelde, where they were then incubated over the following for 14 days. These 1994), Pandalus borealis (see Bergström, 1992), and, Stenopus hispidus timings are also similar for the cleaning shrimp, Lysmata amboinensis (see Chockley and St. Mary, 2003). One reason for this rapid growth whose reproductive cycle is ca.14 days (Cunha et al., 2008) while the and larger size is that the female shrimp are responsible for egg pro- egg development time until hatch of the ornamental shrimp, Stenopus duction and carrying them until they hatch (Berglund, 1981). From the hipidus is ca.16 days (Chockley and St. Mary, 2003). From the trial current study, there were no significant differences (p > 0.05) in the findings, it has been possible to show that the reproductive cycle (over growth rates in the males nor the females maintained on the different the first three spawning events at least) that the periodicity of the re- live diets (Table 1). Available energy from the diet is first used for the productive cycle of H. picta was unaffected by Astropecten as an alter- maintenance of bodily functions and activity but once these energy native live feed when given continuously, however, drops in water requirements have been met, then additional energy can be utilised for temperature below 25 °C were sufficient to cause interruptions. Female growth (Jobling, 1993; Anger, 2001). It is interesting to note that when H. picta that were fed on Astropecten on a discontinuous regime (i.e. the average amount of prey (g) eaten per shrimp per day is calculated AIW), however, failed to spawn. If, however, the number of zoea and averaged across the 5-month feeding period (data not shown), then hatching from each spawning event are considered (see Fig. 3), then the the average daily intake for each individual species is similar regardless female harlequin shrimp that were fed L. multifora continuously had of the feeding regime used but there were differences between L. mul- significantly (p < 0.05) higher numbers of zoea, i.e. over double the − tifora and A. indicus, i.e. 0.1625 g shrimp d 1 for LMD, 0.1625 g shrimp number of zoea hatching from the second and third spawnings, than did − − − d 1 for LMW, 0.24 g shrimp d 1 for AID, and 0.215 g shrimp d 1 for the females fed L. multifora discontinuously or Astropecten continuously. AIW. This suggests that shrimp increase their feed intake to compensate While sequential spawning events could result in decreased spawning for the non-feeding period. Despite using similarly sized L. multifora and quality (Palacios et al., 1999, 2000), from the current trial the number A. indicus (ca. 5–6 cm diameter), the intake of A. indicus was almost of zoea that hatched gradually rose over the first three spawning events 50% higher than that of L. multifora perhaps suggesting that the intake but this rise may merely be a result of increasing female size with time of A. indicus is not sufficient to satisfy their dietary needs; this may also and the ability to produce more eggs. This correlation between female account for the decreased survival of the shrimp in these groups. This is size and egg clutch number has been observed in other studies, for demonstrated by the inferior growth and survival of the female H. picta example in those conducted with S. hipidus (see Chockley and St. Mary, fed Astropecten although there were no significant differences in their 2003) and Farfantepenaeus duorarum (see Emerencíano et al., 2012). growth rates when compared to those fed L. multifora. The females that were fed discontinuously (LMW) were the poorest performers of the three spawning groups and in addition to producing the lowest number of zoea at each spawning event, the age of the

358 J. Pratoomyot et al. Aquaculture 484 (2018) 351–360 females at their first spawning was av. 236 days compared to 206 and be used to supplement their diet when L. multifora is not available. This 222 days respectively for the females fed continuously on either L. research by no means represents a conclusion to the problem of the multifora (LMD) or A. indicus (AID). There were also two mortalities harlequin shrimp's dependency on L. multifora; the research continues among the three breeding pairs reared on the AID diet; as only a limited with investigations into the development and formulation of a pelleted number of replicates was possible within this trial, it is not known if diet that negates the need for a live diet altogether. these mortalities were due to poor nutrition or to other factors. Har- lequin shrimp, for example, have been maintained within our research 5. Conclusions aquaria for more than three years and so the mortalities seen here are considered premature. All the trial results must, therefore, be inter- Ensuring a year-round supply of live L. multifora for captive reared preted with caution and future trials using a larger number of replicates harlequin shrimp is a challenge. When presented with a range of al- must look to validate the observations made here. In fish, maturation ternative prey items, harlequin shrimp respond quickly to the presence can be delayed by dietary nutrient deficiencies in the early life stages of A. indicus and initiate feeding; L. maculata also appears to be initially (Luquet and Watanabe, 1986 cited by Harrison, 1990), and while this acceptable as a prey item. A subsequent 5-month growth trial com- may be the case in the current trial in which young H. picta (i.e. 2- paring the performance of harlequin shrimp reared on a daily or dis- month old post-settlement shrimp) were started on their relevant continuous regime of L. multifora or A. indicus found no difference in the dietary regimes, it may also be a consequence of the subsequent diet growth rates of shrimp on the different regimes. Harlequin shrimp ap- being insufficient to meet the daily nutritional demands of the growing pear to compensate for starvation periods by increasing their daily in- and maturing shrimp. Improvements to the reproductive performance take when food is available. The average daily intake of A. indicus (g − of H. picta may, therefore, be achieved by ensuring that the shrimp are shrimp d 1), however, was 50% higher than that of the shrimp fed L. fed to satiation when fed and, also by further trials to define the multifora. Female harlequin shrimp reared on a discontinuous regime of minimum starvation period without detrimental impact on re- A. indicus failed to spawn; females reared on a discontinuous diet of L. productive performance. While such studies are informative in situa- multifora were delayed in spawning which was not seen for those reared tions when the availability of appropriate prey items are limited, it is on a daily diet of either L. multifora or A. indicus. The females reared on important to stress that the welfare of any captive reared is a daily diet of L. multifora, however, produced significantly more zoea. paramount and that they should not be denied access to feed unless While the study indicates that A. indicus may serve as an alternative live under extenuating circumstances. In the current study, all the H. picta diet when L. multifora is unavailable (i.e. shrimp should be fed daily or females fed the LMD, LMW and AID diets were able to produce larvae every alternate day), a one week on one week off regime should not be once their body weight was approximately ≥1 g; this was also the case for sustained periods. Future trials will set to further explore feeding the females reared on the LMW diet although their time to first regimes to maximise feeding efficiency including investigations into the spawning was delayed (see Table 2; Fig. 3). utility of captive bred A. indicus and in the formulation of artificial diets to match the nutritional composition of L. multifora thereby negating 4.3. The aquaculture of Astropecten indicus the current reliance on live feeds.

During the feed and growth trial, the specimens of A. indicus held Acknowledgments under captive conditions began spawning and were taken through three successive generations. The culture was stopped when the parent pro- The authors gratefully acknowledge the National Research Council ject concluded and the aquaria were required for other research in- of Thailand who provided financial support through research grants itiatives. The ability to culture A. indicus under aquaculture conditions managed by Burapha University (no. 2554A10803041 (20.8/2554) and is therefore an asset in promoting this as an alternative live feed. There 2555A10803034 (70/2555)). The authors would also like to thank the is interest, however, to re-establish cultures and to repeat the trials anonymous referees for their invaluable comments in the revision of using aquaculture reared A. indicus. While many conscientious harle- this manuscript. quin shrimp breeders have discontinued the culture of this ornamental shrimp species until the current dependency on live wild L. multifora References can be overcome, the demand for harlequin shrimp remains and shrimp are supplied from a variety sources. The unregulated and unsustainable Ahamed, F., Ohtomi, J., 2012. Growth patterns and longevity of the pandalid shrimp practice of removing harlequin shrimp and L. multifora, from the wild Plesionika izumiae (: ). J. Crustac. Biol. 32, 733–740. http://dx.doi. org/10.1163/193724012X645853. continues and is evidenced by the commonplace sale of wild shrimp in Anger, K., 2001. Biology of Decapods Larvae, Crustacean Issues. Balkema markets throughout Asia. There is therefore an ethical dilemma of Publishers, Rotterdam, Netherlands90 265 1828 5. continuing the aquaculture of harlequin shrimp to supply enough ani- Annala, J.H., Bycroft, B.L., 1988. Growth of rock lobsters (Jasus edwardsii) in Fordland, New Zealand. N. Z. J. Mar. Freshw. Res. 22, 29–41. http://dx.doi.org/10.1080/ mals to curtail the number that would otherwise be removed from the 00288330.1988.9516275. wild but given the shrimp's stenophagous feeding habits this is being APHA, 1992. Standard Methods for the Examination of Water and Wastewater, 18th ed. done at the cost of the unsustainable practice of harvesting live L. American Public Health Association, 1015 15th Street, NW, Washington, DC 20005. multifora from the wild. The culture of A. indicus, therefore, offers a Archdale, M.V., Anraku, K., 2005. Feeding behavior in Scyphozoa, Crustacea and Cephalopoda. Chem. Senses 30 (Suppl. 1), 1303–1304. http://dx.doi.org/10.1093/ stepping stone towards finding a sustainable diet for the culture and chemse/bjh235. maintenance of H. picta. Astropecten indicus represents a readily ac- Baelde, P., 1994. Growth, mortality and yield-per-recruit of deep-water royal red prawns ff ceptable live prey; the shrimp were quick to respond to their presence (Haliporoides sibogae)o eastern Australia, using the length-based MULTIFAN method. Mar. Biol. 118, 617–625. http://dx.doi.org/10.1163/193724012X645853. within their aquaria and to begin feeding. Harlequin shrimp maintained Berglund, A., 1981. Sex dimorphism and skewed sex ratios in the prawn species Palaemon on a wholly A. indicus diet matured and produced zoea, i.e. there were adspersus and P. squilla. Oikos 36, 158–162. http://dx.doi.org/10.2307/3544440. fi ff Bergström, B., 1992. Growth, growth modeling and age determination of Pandalus bor- no statistically signi cant di erences in the average age of shrimp to – fi ealis. Mar. Ecol. Prog. Ser. 83, 167 183. http://dx.doi.org/10.3354/meps083167. rst spawning (i.e. between those fed LMD and AID), in their sub- Calado, R., 2008. Marine ornamental shrimp. In: Biology, Aquaculture and Conservation. sequent egg development time or in the time to moult post-spawning Wiley-Blackwell, UK978-1-4051-7086-4, . (see Table 2). It is worth noting that the shrimp receiving a daily ration Calfo, A., Fenner, A., 2003. Reef Invertebrates: An Essential Guide to Selection, Care and Compatibility. Reading Trees, PA, USA (400 pp. ISBN 10: 0967263034, ISBN 13: of A. indicus outperformed the shrimp given a weekly ration of L. 9780967263038). multifora but further trials are required to validate this observation. Chen, Y., Kennelly, S.J., 1999. Growth of spanner crabs, Ranina ranina,off the coast of While it is true that A. indicus does not match the performance of Australia. Mar. Freshw. Res. 50, 319–325. http://dx.doi.org/10.1071/MF98023. Chockley, B.R., St. Mary, C.M., 2003. Effects of body size on growth, survivorship, and shrimp reared on L. multifora, these findings suggest that A. indicus can

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