Larval development of laboratory-reared Green Mandarinfish ...

AbstractLarvae of Synchiropus splendidus (Herre, 1927) were

obtained from natural spawnings of two wild-collectedpairs of adults. Egg and larval development of S. splendidusis similar to that described for other callionymids. Eggsmeasure 0.8 mm in diameter and hatch after 13-16 hoursat 26°C. Newly hatched larvae measure 1.56 ± .04 mm NLand begin feeding four days post-hatching (DPH) at 1.74± .06mm NL. The notochord accounts for 20% of thebody length at 12 DPH. Larvae lack heavy ventral pig-mentation, but have three distinctive lines of pigment onthe posterior portion of the body – one along the base ofthe dorsal fin, one along the base of the anal fin, and onealong mid-body. Larvae exhibit an ontogenetic colorchange from yellow to orange prior to settlement. Inci-dence of settlement was highest between 16-20 DPH.Ontogeny, including osteological development, is des -cribed from hatching to the juvenile stage.

ZusammenfassungDie untersuchten Larven von Synchiropus splendidus (Herre,

1927) entstammten dem natürlichen Laich von zwei in derNatur gefangenen erwachsenen Paaren. Die Ei- und Larven -entwicklung ähnelt bei S. splendidus der von anderen Callionymiden beschriebenen. Die Eier haben einenDurchmesser von 0,8 mm; bei 26° C schlüpfen die Larvennach 13 bis 16 Stunden. Die frisch geschlüpften Larvenhaben eine Länge von 1,56 ± 0,04 mm NL und fangen 4Tage nach dem Schlupf (DPH = days post-hatching) an zufressen, bei einer Länge von 1,74 ± 0,06 mm NL. Nach 12DPH macht die Rückensaite (Notochord) 20 % der Körper-länge aus. Bei den Larven fehlt eine starke ventrale Pigmen-tation, doch weisen sie drei deutliche Pigmentlinien im hin-teren Teil des Körpers auf – eine an der Basis der Rücken-flosse, eine an der Basis der Afterflosse und eine in Längsrich-tung in der Körpermitte. Im Laufe der Ontogenese zeigen dieLarven einen Farbwechsel von Gelb zu Orange kurz vor derAnsiedlung. Diese findet am wahrscheinlichsten nach 16 –

20 Tagen DPH statt. Die Ontogenese, einschließlich derKnochenentwicklung, wird vom Schlupf bis zum Jugendsta-dium beschrieben.

RésuméDes larves de Synchiropus splendidus (Herre, 1927) ont

été obtenues de pontes naturelles de deux couples d’adultescapturés dans leur milieu. Le développement de l’oeuf etde la larve de S. splendidus est semblable à celui décrit pourd’autres Callionymidés. Les oeufe font 0,8 mm de dia mè -tre et éclosent au bout de 13-16 heures, à 26°C. Les larvesqui viennent de naître font 1,56 ± ,04 mm NL et com-mencent à se nourrir 4 jours après l’éclosion à la taille de1,74 ± ,06 mm NL. La notocorde représente 20% de lalongueur du corps 12 H aprés l’éclosion. Les larves n’ontpas de forte pigmentation ventrale, mais ont trois lignesnettement pigmentées sur la portion postérieure ducorps – une le long de la base de la dorsale, une le long dela base de l’anale et une le long du milieu du corps. Leslarves montrent un changement de couleur ontogéniquedu jaune à l’orange avant fixation. La survenue de cette fi -xation a lieu majoritairement entre 16 à 20 heures après l’éclosion. L’ontogénie, y compris le développement ostéo -lo gique, est décrite depuis l’éclosion jusqu’au stade juvénile.

SommarioLarve di Synchiropus splendidus (Herre, 1927) erano ot -

tenute in condizioni naturali da due coppie di adulti raccoltiin natura. Lo sviluppo embrionale e larvale di S. splendidus èsimile a quello descritto per gli altri callionimidi. Le uova mi -su rano 0.8 mm di diametro e schiudono dopo 13-16 ore a26°C. Alla schiusa le larve misurano 1.56 ± 0.04 mm NL ecominciano a nutrirsi 4 giorni dopo la schiusa (DPH, dayspost-hatching) quando raggiungono 1.74 ± 0.06 mm NL.Dopo 12 DPH la notocorda comprende il 20% della lun -ghezza del corpo. Le larve mancano di pigmentazione ven-trale, ma posseggono tre marcate linee pigmentate sulla partepo steriore del corpo – una lungo la base della pinna dorsale,

aqua vol. 16 no. 1 - 20 January 20107

aqua, International Journal of Ichthyology

Larval development of laboratory-reared Green Mandarinfish, Synchiropus splendidus (Teleostei: Callionymidae)

Matthew L. Wittenrich1, Carole C. Baldwin2, Ralph G. Turingan1

1) Florida Institute of Technology, Department of Biological Science, 150 W. University Blvd., Melbourne, FL, 32901, USA

2) Division of Fishes, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, Washington, DC 20013-7012, USA

* Corresponding author: Matthew L. Wittenrich, Phone: 321-674-7222, Fax: 321-674-7222.Email: [email protected]

Received: 24 September 2009 – Accepted: 3 January 2010

un’altra lungo la base della pinna anale e la terza sulla parteme diana del corpo. Le larve mostrano una variazione onto -ge nica della colorazione dal giallo all’arancio prima dell’in se -dia mento. Il grado di insediamento era più elevato a 16-20DPH. L’ontogenesi, incluso lo sviluppo osteologico, è de -scritta a partire dalla schiusa fino agli stadi gio vanili.

INTRODUCTIONDragonets of the genus Synchiropus Gill, 1859, are

small, bottom-dwelling fishes from the tropicalIndian and Pacific Oceans and comprise approxi-mately 27 species (Fricke 1983). The Green Man-darin, Synchiropus splendidus (Herre, 1927), is a small(6 cm) species that occurs in shallow protectedlagoons and reefs in the western Pacific; it is distrib-uted from the Ryukyu Islands of southern Japan toNew Caledonia and Australia (Myers 1999).Together with S. picturatus (Peters, 1877), S. stellatusSmith, 1963, and S. ocellatus (Pallas, 1770), S. splen-didus is hugely popular in the world aquarium trade.To date, all specimens offered in the trade are cap-tured from the wild, and concern over the sustain-ability of this selective fishery has been raised (Sadovyet al. 2001, Vincent & Sadovy 1998). Despite initial successes in the propagation and

rearing of Synchiropus spp. (Wittenrich 2009; Mai2000, 2006; Sadovy et al. 2001; Wilkerson 1996),limited descriptive information is available pertain-ing to the development of these popular fishes.Improved larval rearing techniques and knowledgeof the life-history requirements of Synchiropus haverecently enabled continuous production of larvaland juvenile S. splendidus for observation. Sadovy etal. (2001) described the fishery, reproduction, andearly life history of S. splendidus. Herein, we expandon their description of larval development anddescribe for the first time the development of cer-tain osteological features. We describe morphology,color pattern, and growth of larvae and juveniles to50 days post-hatching (DPH). Additionally, wecompare larval S. splendidus to known larvae ofother Callionymidae. Developmental charactersmay be of value in future phylogenetic investiga-tions of callionymid fishes. We hope that this infor-mation will prove useful in providing a rearing pro-tocol for future scientific investigations and hatch-ery applications. Aquaculture potential and proto-cols will be addressed in future work. Cultured fishdo indeed exhibit long-term success in aquariumsand commercial production is underway at one US-based commercial hatchery, based on Wittenrich(2009).

MATERIALS AND METHODSLarvae described herein were obtained from natural

spawnings of two wild-collected pairs of Synchiropussplendidus. Eggs were collected shortly after spawningand transferred to cylindrical incubation tanks (15 l)provided with gentle aeration from an open-endedair diffuser. Two days after fertilization embryos weretransferred to 150 l black conical rearing tanks andreared at densities of no more than 1.5 larvae l-1.Water quality in each rearing tank was maintained bya biological filter tower, ultraviolet sterilizer, and pro-tein skimmer with an exchange rate of 0.5 l-1 min.Natural seawater filtered through a 10 µm closed-pore filter was maintained at 26°C, salinity 35 ppt,pH 8.2, NH3 and NO2 < 0.02 ppm, and NO3 < 10ppm (Red Sea marine lab test kit). Water changeswere performed every other day by replacing 10% ofthe total system water from the sump. Photoperiodwas maintained at 14 hrs light and 10 hrs dark withtwo 40W fluorescent bulbs (6500 K) mounted 20cm above the water surface. Greenwater was main-tained in the rearing tanks during all trials by adding0.5 ml of diluted Nannochloropsis oculata algae paste(Reed Mariculture) to the tanks each morning. Larvae were provided with rotifers (Brachionus pli-

catilis, average lorica length ranged from 180-277µm) and copepod-dominated, size-sorted wild plank-ton (55-90 µm) as initial feed 4 DPH and subse-quently provided with larger copepod-dominated,size-sorted wild plankton (110-500 µm) 8 DPHthrough settlement. Prey density of rotifers was main-tained from 3-5 DPH at 10 ml-1 and subsequentlylowered to 5 ml-1 until 12 DPH when rotifers wereremoved from the diet. Wild plankton was main-tained at 2 ml-1 throughout the larval period. Beginning at hatching (0 DPH), 20 larvae were

sampled from the rearing tank, initially every otherday and subsequently every 2-6 days, anesthetizedwith tricaine methanosulphonate (MS-222) andfixed in 5% seawater-buffered formalin for 24 hoursbefore transfer to 70% ethanol for later analysis.Notochord length (NL) or standard length (SL) offreshly collected larvae was measured using an ocularmicrometer fitted to a stereomicroscope prior topreservation. Twelve larvae from samples taken at 5,7, 9, 12, 15, 17 and 23 DPH were cleared andstained following the methods described by Potthoff(1984). Elements of the viscerocranium weredescribed following Hunt Von Herbing et al. (1996),Liu (2001) and Moteki (2002). References to osteo-logical development were standardized to bodylength. Preserved specimens of Paradiplogrammus

aqua vol. 16 no. 1 - 20 January 2010 8

Larval development of laboratory-reared green mandarin, Synchiropus splendidus (Teleostei: Callionymidae)

bairdi from Belize referenced in the “Discussion” arefrom ongoing larval-fish studies at the Smithsonian’smarine station at Carrie Bow Cay.

RESULTSEggs and hatching (Fig. 1)Eggs were released singly or clumped together in

chains, breaking apart as they swelled with water anddrifted in currents. Those released singly were spher-ical, whereas those that remained clumped wereoblong with triangular adhesion points to adjoiningeggs. In situ the eggs were clear and positively buoy-ant. They measured 0.8 mm in diameter and lackedoil globules. The chorion had a distinctive polygonalmesh-like appearance. Hatching began 13-16 hourspost fertilization.Morphological development (Table I; Figs 2-7) Newly hatched larvae measure 1.56 ± .04 mm

and possess a large oval yolk without oil globules.

The yolk is 52% body length. Eye placodes are pre-sent. At 2 DPH the larvae measure 1.67 ± .05mm,and the yolk has been reduced to 34% bodylength, giving the larvae a streamlined appearance.The anal pore and gut are visible, but are not con-nected to the finfold. At 3 DPH, pectoral-fin budsare visible, the anal pore has opened to the finfoldand the mouth is beginning to form. As the larvagrows, the body thickens, and the head becomesbroad. At 12 DPH the head width is 30% bodylength and expands to 36% body length by 23DPH. Age and growth of larvae and early juvenilesto 50 DPH is illustrated in Fig. 2. BehaviorNewly hatched larvae mostly drifted motionless but

swam in rapid short bursts a few times per minute.First feeding occurred 4 DPH at 26°C when the lar-vae measured 1.74 ± .06 mm and the yolk wasdepleted. At approximately 12 DPH, larvae exhib-


Matthew L. Wittenrich, Carole C. Baldwin, Ralph G. Turingan

Fig. 1A-B. (A) Synchiropus splendidus embryo 1 hr post fertilization. Note distinctive, polygonal mesh-like appearance ofchorion. (B) embryos at 10 hr post fertilization still attached by adhesion points (AP).

Table I. Mean measurements of laboratory-reared Synchiropus splendidus larvae. Measurements are expressed as a fraction ofbody length (notochord length, NL). DPH = days post-hatch. A dash indicates the measurement could not be made.

Pelagic stage Benthic juvenile

Age (DPH) 5 7 9 12 15 17 23

Mean body length 2.01 NL 2.19 NL 2.64 NL 2.99 NL 3.08 NL 3.33 NL 3.26 NLProportionsSnout to anus length 0.45 0.55 0.57 0.53 0.55 0.55 0.62Snout to dorsal length – – – 0.43 0.42 0.39 0.46Snout to pectoral length – – – 0.33 0.32 0.30 0.34Head length 0.27 0.28 0.31 0.34 0.35 0.34 0.37Head width 0.15 0.16 0.21 0.30 0.30 0.33 0.36Body depth 0.25 0.25 0.25 0.28 0.28 0.26 0.29Eye diameter 0.11 0.11 0.12 0.13 0.14 0.14 0.15

ited a conspicuous behavior that persisted untilsettle ment: when a larva approached prey, the poste-rior portion of the body curled to one side of thebody, which potentially aids maneuverability andsteering (Sadovy et al. 2001). Intermittent settlingbehavior was exhibited near 12 DPH when the lar-vae rested on the vertical walls and floor of the rear-ing tank for short periods before returning to theopen water. Most larvae settled to the floor of therearing tank by 16-20 DPH. In subsequent larvalrearing trials sufficient wild zooplankton densitieswere difficult to collect due to storm events. Duringthose times, when prey concentration in the openwater of the rearing tank was limited, larval settle-ment occurred earlier, around 14 DPH.

Body pigmentation (Figs 3-7)At hatching, the body and yolk are covered with

stellate and dendritic xanthophores, with a heavyconcentration on the dorsal and anal finfolds poste-rior to the yolk. Scattered melanophores are presentalong the dorsal surface of the yolk, on the dorsal fin-fold and on the head and trunk. The notochord tipand caudal finfold lack pigment. By 3 DPH the clearposterior finfold tip is conspicuous relative to the restof the body, which is largely yellow. The head andtrunk are primarily covered by stellate xanthophores,whereas the posterior portion of the body, beyondthe yolk, is dominated by dendritic xanthophoresinterlaced with larger melanophores. Dense xan-thophores are present on the ventral finfold edge



Fig. 2. Age and growth of S. splendidus to 50 DPH. Vertical dashed lines represent: FF, first feeding; MET, metamorphosis.

Table II. Mean counts of laboratory-reared Synchiropus splendidus larvae. DPH = days post-hatch.

Pelagic Benthic juvenile

Age (DPH) 5 7 9 12 15 17 23

Neural arches 4 15 18 18 18 18 18Haemal arches 0 7 13 12 13 13 13Dorsal pterygiophores 0 0 0 12 12 12 12Anal pterygiophores 0 0 0 6 7 6.5 7Dorsal-fin rays 0 0 0 0,4 0,6 IV,8 IV,8Anal-fin rays 0 0 0 4 7 7 7Pelvic-fin rays 0 0 0 6 6 6 6Pectoral-fin rays 0 0 0 0 0 0 0Caudal-fin rays 0 0 0 6 11 12 15



from the anal pore to the last myomere, forming adistinct yellow edge. This yellow edge is mirrored onthe dorsal finfold by a matching posterior yellow line.A similar, but smaller yellow edge is present on the

dorsal finfold above the posterior edge of the yolk.The body is almost solid yellow at 4 DPH, at whichtime eye pigmentation first appears and the iris iri-dophores reflect silver. The dorsal and anal finfolds

Fig. 3A-E. (A) side view and (B) ventral view of newly hatched S. splendidus larvae exhibiting stellate and dendritic xan-thophores. (C) 2 DPH larva with reduced yolk, anal pore developing and clear notochord tip. (D) side view and (E) dorsalview of 3 DPH larvae with pectoral-fin buds and proliferation of stellate and dendritic xanthophores. Scale bar = 1 mm.



Fig. 4A-J. Larvae of S. splendidus illustrating morphological development and live and alcohol-preserved pigmentation. (A)5 DPH live larva, (B) 5 DPH preserved, (C) 8 DPH live, (D) 8 DPH preserved, (E) 12 DPH live, (F) 12 DPH preserved,(G) 15 DPH live, (H) 15 DPH preserved, (I) 17 DPH live, (J) 17 DPH preserved. Scale bar = 1 mm

melano phores increase in density, and the bodybecomes almost solid orange. A conspicuous brownspot appears on the trunk over the posterior edge ofthe gut and anal pore. The head and trunk are adense, dark, uniform orange by 12 DPH, and thedark trunk spot fades. The median fins are clear. By8 DPH iris iridophores reflect gold, which then dark-ens through settlement.Settlement-stage larvae become mottled with dark

orange to brown irregular bands on the trunk. After



are densely covered with clusters of xanthophores,and the distinctive yellow edges are lost. The analpore is bright yellow. The posterior finfold, beyondthe last myomere, remains unpigmented. Melano -phores remain sparse. Between 6 and 8 DPH,

Fig. 7. Juvenile S. splendidus at 50 DPH showing adult col-oration and morphology, 10 mm SL.

Fig. 5. Dorsal and lateral views of 23 DPH S. splendidus.Scale bar = 1 mm.

Fig. 6. Juvenile S. splendidus at 43 DPH, 7 mm SL.

Fig. 8A-D. Cleared and stained S. splendidus larvae (A) 5DPH, (B) 9 DPH, (C) 12 DPH, (D) 23 DPH.

settlement the body turns pale tan to brown withdarker vertical bands and blotches across the body.The pelvic and anal fins are the first to gain partialbrown pigment near 25 DPH. At 30 DPH the tanand brown mottled pattern has become more con-spicuous and faint green bands are present on thehead and eyes. Forty-day old juveniles exhibit well-delimited brown bars on the body, faint green band-ing on the head and brown anal and pelvic fins. Thecaudal, dorsal and pectoral fins remain clear. At 50DPH the body color has turned bright orange witha characteristic blue banding pattern resembling theadult. The dorsal, anal, pelvic, and caudal fins arenow bright orange with a blue outline. Body pigmentation of 5-17 DPH larvae fades

quickly after alcohol preservation. Small mela no -phores are scattered loosely on the frontal, gut, andanus of 5 DPH larvae. Concentrated melanophoresalso appear on the dorsal and ventral edges of thetrunk. From 8-17 DPH melanophore concentrationincreases on the frontal and nape regions, dorsal andventral melanophore stripes become more promi-nent and a midbody stripe appears. Largemelanophores appear scattered over the opercularregion in 12 DPH larvae, and a dark spot is visibleon the dorsal trunk below the dorsal anlagen. Settlement-stage larvae, from 23-50 DPH, have

brown body pigmentation, and scattered melano -phores on the frontal, nape and operculum. The spotbelow the dorsal fin remains until near 50 DPH.Dorsal, anal and midbody stripes have disappeared.At 50 DPH the body pigmentation resembles theadult, and while the color intensity fades in preserv-ative the banding pattern remains.

Skeletal Development (Table I-III; Fig. 8)Jaws. Meckel’s cartilage is present in 5 DPH larvae

(1.95 mm ± .21mm NL). The dentary and retroarti -c ular begin to differentiate at 9 DPH (2.5 ± .19 mmNL) and are beginning to ossify at 12 DPH (2.85 ± .39 mm NL). The premaxilla and maxilla arevisible at 9 DPH (2.5 ± .19 mm NL). Neither theupper nor lower jaw is completely ossified at 23DPH (3.51 ± .23 mm SL). The premaxilla and den-tary lack teeth throughout larval development. Suspensorium. The hyomandibulosymplectic carti-

lage, consisting of cartilaginous precursors to thehyomandibular and symplectic, is visible at 5 DPH.The palatoquadrate cartilage is present at 9 DPH.Dorsal and ventral ossification of the hyomandibularand initial ossification of the symplectic, palatine andquadrate are evident by 23 DPH (3.51 ± .23 mmSL). The cartilaginous metapterygoid begins to ossify

by 17 DPH (3.05 ± .23 mm SL). The ectopterygoidand endopterygoid first appear as ossifying bone at23 DPH (3.51 ± .23 mm SL).Hyoid arch. Rudimentary hyoid bars are present at

first feeding. Ceratohyal and epihyal cartilages con-tinually expand until 9 DPH when the hyoid reachesits maximum depth and has the full complement ofsix branchiostegal rays. The interhyal and epihyalbegin ossifying at 17 DPH followed by the cerato-hyal and basihyal at 23 DPH. Opercular series. All elements of the opercular series

form as dermal bone. The opercle is the first toappear, at 15 DPH, followed by the interopercle andsubopercle at 17 DPH. The preopercle appears at 23DPH. The preopercular spine is not present until thejuvenile stage, approximately 30 DPH. Branchial arch. Cartilaginous basibranchial and cer-

atobranchials 1 and 2 are present at 5 DPH, but donot begin ossifying until approximately 17 DPH.Epibranchials and hypobranchials differentiatearound 12 DPH. Ossification of the gill arches is notcomplete by 23 DPH. Appendicular skeleton. The cleithrum, present at first

feeding as a thin band, thickens with growth. At 9DPH, the post- and supracleithrum are visible. Allcleithral elements begin to ossify around 12 DPH.The scapula and coracoid are visible at 5 DPH. Nei-ther element begins to ossify until after settlement,near 4 mm SL. Pectoral radials are not formed at 23DPH. Pectoral-fin rays have not begun differentiat-ing at 23 DPH. The pelvic basipterygium is visible at9 DPH and begins ossifying at the distal end atapproximately 17 DPH. Pelvic-fin rays appear at 12DPH. Axial skeleton. Two to five neural arches are visible

in 5 DPH specimens, 15 are present at 7 DPH and18 by 9 DPH. At 7 DPH five to seven haemal archesare present and number 13 by 9 DPH. Initial ossifi-cation of the vertebrae and neural and haemal spinesis observed at 9 DPH and progresses in an anterior-to-posterior direction. All but the two posteriormostvertebral centra, neural and haemal arches are ossi-fied in 12-15 DPH specimens. Caudal complex. Development of the caudal com-

plex begins at 9 DPH when the cartilaginous parhy-pural appears anterior to a small triangular ventralhypural plate. The cartilaginous neural arch andspine of PU2 (NPU2) is present opposite the parhy-pural. At 12 DPH the ventral hypural plate hasexpanded and supports three to five caudal rays. Thenotochord is straight and elongate, the tip account-ing for roughly 20% body length. The dorsal





Table III. Osteological development of laboratory-reared Synchiropus splendidus larvae. Open bars indicate first appearanceof a feature. Gray bars indicate onset of ossification. Skeletal structures borne of cartilage replacement bone (C) or dermalbone (D).

hypural plate is now visible as a thin cartilage pro-jection and supports a single caudal ray. NPU2 hasexpanded dorsally from its attachment point on thevertebra, and the autogenous, cartilaginous epural ispresent. At 15 DPH the ventral hypural plate sup-ports five caudal rays, and the parhypural supportsa single ray. The dorsal hypural plate is now a tri-angle of cartilage that supports three caudal rays.Notchord flexion is evident at 17 DPH as a weakupward bend from the horizontal. At 23 DPH,flexion is complete, and the posterior edges of thehypural plates form a vertical margin. The urostyleis now less than 5 % body length. Ossification ofthe caudal complex is not apparent at 23 DPH.The full complement of caudal-fin rays (15) is pre-sent at 23 DPH.Dorsal and anal fins. Dorsal and anal anlagen appear

at 9 DPH, and the full complement of cartilaginousptergyiophores is present at 12-15 DPH. At 23DPH, the full complement of dorsal and anal-fin ele-ments is present.

DISCUSSIONComparisons with early life-history stages of other

callionymidsEgg and larval development of S. splendidus is sim-

ilar to that described for other callionymids (Leis &Rennis 1983, Houde 1984, Powell & Greene 2000,Sadovy et al. 2001). Callionymid eggs are typicallyspherical, colorless, pelagic, range in size from 0.55-0.97 mm and are released at the height of a spawn-ing ascent from single-pair spawning events (Leis &Rennis 1983, Thresher 1984, Houde 1984, Sadovyet al. 2001). Sadovy et al. (2001) reported that eggsof S. splendidus are lightly adhesive, buoyant andremain together as they drift away with passing cur-rents. Takita (1983) reported that eggs of Calliony-mus calliste Jordan & Fowler, 1903, are shed in adhe-sive masses that break apart into individual pelagiceggs as they near hatching. In our experience, S.splendidus, as well as S. picturatus, S. ocellatus, and S.stellatus (M. L. Wittenrich, unpublished data) spawnin a similar fashion with eggs being shed singly orclumped together in long chains that break apartshortly after spawning. Eggs lack oil globules, have apartially separated yolk, and most species exhibit apolygonal mesh-like sculpturing on the chorion.Sadovy et al. (2001) suggested that the mesh-likeappearance of the S. splendidus chorion is more dis-junct than that of other callionymids. Hatching of S.splendidus embryos is influenced by temperature andranges from 12.5 h at 28.5° C to 16 h at 24° C

(Sadovy et al. 2001). Incubation time in the presentstudy was 13-16 h at 26 °C. Larvae of callionymids are among the smallest

recorded marine fish larvae at hatching, rangingfrom 1.0-2.1 mm in length (Leis & Rennis 1983,Houde 1984). Leis & Rennis (1983) noted that thesmall size, long notochord tip and heavy ventralpigmentation of larval callionymids distinguishthem from larvae of other families. Larval S. splen-didus are typical callionymid larvae in that newlyhatched larvae are 1.5-1.6 mm TL, and the noto-chord accounts for 20% of the body length at 12DPH. However, they are not so heavily pigmentedventrally as described and illustrated by Leis & Ren-nis (1983: 224, Fig. 66) for other callionymid lar-vae. Heavy ventral pigmentation may not be char-acteristic of most callionymids: for example,although larvae of the western Atlantic Diplogram-mus pauciradiatus (Gill, 1865) have heavy ventralpigment on the lateral surface of the gut, larvalParadiplogrammus bairdi (Jordan, 1888) lacks heavypigmentation ventrally (Powell & Green 2000).The 2.9- and 5.1-mm unidentified callionymid lar-vae illustrated by Leis & Rennis (1983: Fig. 66B-C)have three distinctive lines of pigment on the pos-terior portion of the body-one along the base of thedorsal fin, one along the base of the anal fin, andone along mid body. That same pattern of pigmen-tation is apparent in S. splendidus larvae (see Fig.3B, D, F), but it is not present in preserved P. bairdilarvae from Belize, which may have melanophoresalong the lateral midline and a few blotches associ-ated with the dorsal-fin base but lack melanophoresalong the anal-fin base.Our description of osteology should serve as a base-

line for future comparative work within the Cal-lionymidae. Certain developmental features such asthe elongate notochord tip and sequence of ossifica-tion should prove valuable in future studies of cal-lionymid relationships. We found the incidence of settlement was highest

between 16-20 DPH, whereas Sadovy et al. (2001)reported settlement at 14 DPH at 24-26° C. One ofthe authors (MLW) noticed in subsequent rearingtrials that food availability influenced time to settle-ment (although not quantified). When prey concen-tration in the open water of the rearing tank was low,larval settlement occurred near 14 DPH. Whetherthis is an artifact of captivity or suggestive of the abil-ity of late-stage larvae to alternate between pelagicand benthic lifestyles to exploit food resourcesremains unknown. Settlement in callionymids in



general ranges from 14-20 DPH at body lengths of3.5-10 mm TL (Sadovy et al. 2001, Thresher 1984). A notable observation of S. splendidus larvae is the

ontogenetic color transformation (from yellow toorange) that occurs at 8 DPH. Sadovy et al. (2005)showed that the epidermis of adults is rich in mucouscells and has a distinct sacciform cell type that theyproposed may secrete a noxious mucous functioningas a predator deterrent. The vibrant color pattern ofadult S. splendidus may be a warning to potentialpredators. Little is known about ontogenetic colorchanges in live callionymid larvae because those colors fade in alcohol preservation. Settlement-stage(or nearly so) larvae of Paradiplogrammus bairdi fromBelize are bright orange, but coloration of earlier lar-val stages is unknown, and adults are drab relative toadult S. splendidus. There are conspicuous xan-thophores on the spinous dorsal fin in some P. bairdilarvae, and possibly smaller larvae are more uni-formly yellow as are larvae of S. splendidus. Thepelagic-stage coloration of bright orange that occursin the Indo-Pacific S. splendidus and the Atlantic P.bairdi may represent a novel predator deterrent func-tion and a conserved phylogenetic peculiarity.The ontogenetic color change in S. splendidus is

accompanied by the development of mucous cells.Although histological and chemical analyses werebeyond the scope of this study, mucous productionwas evident during sampling and microscopy work.Orange has been suggested to be an aposematicwarning color in the larvae of some marine organ-isms (e.g. Lindquist 2002, Young & Bingham 1987),and many marine organisms incorporate poisonousor noxious substances into gametes that serve as bit-ter or distasteful deterrents (e.g. McClintock & Ver-non 1990, Gladstone 1987, Pillsbury 1957). Furtherinvestigation of the ontogenetic shift to orange inSynchiropus, including histological and chemicalanalyses, is needed.

ACKNOWLEDGEMENTSWe thank A. Didoha for assistance with raising lar-

vae. This is contribution number 865 of theCaribbean Coral Reef Ecosystems Program (CCRE),Smithsonian Institution, supported in part by theHunterdon Oceanographic Research Endowment.

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Larval development of laboratory-reared Green Mandarinfish ...

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