SHORT COMMMUNICATION

Endogenous feeding and morphological changes in

hatchery-reared larval palm ruff Seriolella violacea

(Pisces: Centrolophidae) under starvation

Claudia A Bustos1 & Alfonso Silva2

1Programa de Doctorado en Acuicultura, Universidad Cato¤ lica del Norte campus GuayacaŁ n, Coquimbo, Chile2Laboratorio de Cultivo de Peces, Facultad de Ciencias delMar, UniversidadCato¤ lica del Norte campusGuayacaŁ n, Coquimbo, Chile

Correspondence: CA Bustos, Programa de Doctorado en Acuicultura, Universidad Cato¤ lica del Norte campus GuayacaŁ n, Larrondo1281,

Coquimbo, Chile. E-mail: clausbustos@gmail.com

There is an increasing e¡ort towards diversi¢cationof potential marine species for ¢sh culture aroundthe world (Aristizabal, SuaŁ rez, Vega & Bargas 2009;Cavalin & Weirich 2009; Leu, Liou, Wang, Yang &Meng 2009). However, to avoid negative impacts fromthe introduction of exotic species, the investigation ofindigenous species becomes important and worth-while (Ross, Mart|¤ nez Palacios & Morales 2008). Spe-cies such as southern hake Merluccius australis(Bustos & Landaeta 2005; Bustos, Landaeta, Bay-Schmith, Lewis & Moraga 2007), amberjack Seriolalalandi (Moran, Smith, Gara & Poortenaar 2007) andpalm ru¡ Seriolella violacea Guichenot, 1848 havebeen considered to be excellent candidates for mari-culture in Chile. Particularly, the palm ru¡, S. viola-cea, is a high-value marine ¢n¢sh that inhabitssubtropical waters, and it is distributed between 11Sand 341S in the eastern Paci¢c Ocean (Iannacone2003). Successful maintenance of wild-caughtbroodstock, natural spawning, larviculture and pilotproduction of juveniles have been achieved in recentyears at Fish Culture Laboratory, Universidad Cato¤ li-ca del Norte, Chile. Nevertheless, it is necessary to im-prove the knowledge of the biology of its early lifestages to optimize their larviculture.During larviculture, although plenty of live food is

supplied to the culture tank, ¢shmay still be su¡eringfrom starvation due to poor vision and mouth gapelimitation (Planas & Cunha1999; Nakagawa & Miya-shita 2008). After yolk resorption, energy reservessupport the larvae for a limited period; the utilization

of some substances as energy sources by larvae facedwith starvation promotes a series of changes in mor-phological characteristics, such as a decrease in theirbody height and degenerated gut height (Theilacker& Watanabe1989;Yufera, Pascual, Polo & Sarasquete1993), inducing poor performance in feeding and anadverse e¡ect on growth (Blaxter & Ehrlich1974). Be-cause of their ease of observation and measurementboth in the laboratoryand in the ¢eld, the general col-lapse of gut and myotome development could be sen-sitive indicators for assessing larval starvation,especially at an early larval stage (e.g. irreversiblestarvationage) (Yufera et al.1993; Dou, Masuda,Tana-ka & Tsukamoto 2002). Internally, starvation usuallychanges the shape of enterocyte cells in the intestineof several ¢sh species, reducing the height of themid-gut mucosal cells (Ehrlich, Blaxter & Pemberton1976;Yufera et al.1993; Chen, Qin, Carragher, Clarke,Kumar & Hutchinson 2007). Furthermore, thechanges in the digestive tract, liver and pancreasmay a¡ect the synthesis of the digestive enzymesand functions of the digestive system (Gawlicka, Par-ent, Horn, Ross, Opstad & Torrissen 2000; Bolasina,Pe¤ rez & Yamashita 2006).Information on starvation resistance and time to

start external feeding is necessary to reduce the costsrelated to live feeds of larval palm ru¡ and to reducelarval mortality in ongoing e¡orts to develop acommercial mass culture system for this species.The objectives of the work are to establish for the ¢rsttime, under laboratory-controlled conditions, larval

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performance during endogenous feeding and the ef-fect of early starvation on the morphological featuresand survival of larval S. violacea.Fertilized eggs were collected from captive adult

S. violacea that spontaneously spawned during Au-gust and September 2008 in two12000 L broodstocktanks under ambient conditions (14 � 1 1C,34 ppt) atFish Culture Laboratory, from Universidad Cato¤ licadel Norte, Coquimbo, Chile. Eggs were then stockedat 500 eggs L�1 in one 500 L cylinder conical tanksupplied with ¢ltered seawater and gentle aeration.Temperature in the incubator tank ranged from 141to 16 1C and a light regime of 12 L:12 D was providedthroughout the embryo development and untilhatching occurred. Hatching occurred 72^74 h afterfertilization.Newly hatched larvae were then transferred into

two cylindrical tanks (100 L), with sterilized and ¢l-tered seawater (1 mm), at an initial density of50 larvae L�1. During the endogenous feeding, tenlarval S. violaceawere extracted daily from one of thetanks. Measurements of the larval length (LL), yolk-sac length and height were performed on freshlyanaesthetized larvae with MS-222 under a stereomi-croscope (Fig.1a).Yolk-sac volumewas estimated con-sidering the yolk-sac as an ellipsoid (V54/3pab2),where a is half of the yolk-sac length and b is half ofthe yolk-sac height, Bustos et al. 2007). Then, an ex-ponential negative curve was ¢tted to calculate theyolk-sac absorption rate of larval S. violacea.Two days post hatching (2 DPH), larvae were

stocked in two 60 L cylinder conical tanks with cen-tral drains of ¢ltered seawater (1 mm) at 16 1C and a

standpipe ¢tted with a 150 mm mesh. Larvae in thelatter tank were not fed and those in the other tankwere fed enriched rotifers (Brachionus plicatilis) twicea day at a density of 10 rotifers mL�1. Rotifers werecultured using a batch culture system, fed a dry dietof bakers yeast combinedwith concentrated microal-gaeNannochloropsis oculata and Isochrysis galbana. Inaddition, rotifers were enriched with AlgaMac 3050(Aquafauna Bio-Marine, Hawthorne, CA, USA).The morphology of 10^20 larvae from both non-

fed and fed groups was measured daily. Morphologi-cal measurements were performed on freshly anaes-thetized larvae (MS-222) using a calibrated ocularmicrometer ¢tted to a stereomicroscope. Five partsof the body of the larvae were measured: notochordlength (NL, from the tip of the mouth to the tip of thenotochord), eye diameter (ED), body depth (BD, depthimmediately posterior to the pectoral bud), headlength (HL, from the tip of the snout to the opercularmargin) and myotome height (MH, height immedi-ately posterior to the anus) (Fig.1b). Comparisonsweremade for each measurement, at a daily scale, betweenfed and unfed larvae using Mann^WhitneyU-tests.The daily mortality rate from yolk absorption time

was de¢ned as the percentage of the dead ¢sh to thesurvivors at the beginningof eachday. Instantaneousmortality rate (Z) was used to examine mortalityamong trials (fed vs. non-fed) and was de¢ned as:Z5 (ln N0^ln Nt)t

�1, where N0 and Nt are the num-bers of initial and surviving larvae, respectively, and tis the duration of the developmental stage in days.Newly hatched larvae [mean � standard deviation

(SD), 2.97 � 0.18mm NL] had a yolk-sac 0.34 �0.11mm3 in volume (range: 0.19^0.57mm3) with a li-pid droplet of 0.25 � 0.04mm diameter (range: 0.15^0.31mm). The intercept of the growth linear model(i.e., the size at hatching) was ¢xed at 2.97mm.The es-timated linear model was LL50.316 A12.97, whereLL is the larval length (mm) and A is the age (days).Therefore, larval growth in length during the endo-genous period was fast, with an estimated linear rateof 0.316mmday�1 (Fig. 2a). The yolk-sac was con-sumed quickly during the ¢rst day and decreased ex-ponentially after hatching, being almost completelyabsorbed on 4^5 days. The estimated exponentialmodel was YSV50.279e�0.573 A, where YSV isthe yolk-sac volume (mm3) and A is the age (days)(Fig. 2b). The lipid droplet was completely absorbed at10DPH, and larvae survived until 14DPH withoutfood (Fig.1c).Starved larvae showed a reduction in growth in

notochordal length between 6 and14DPH compared

Figure 1 Larval Serioella violacea and morphologicalcharacteristics measured in larvae. (a) larva of 4.1mm NLand 3 days post hatching (DPH), (b) larva of 5.0mm NLand 5 DPH, (c) larva of 7.3mm NL and14 DPH. NL, noto-chord length; HL, head length; ED, eye diameter; BD, bodydepth; MH, myotome height.

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with normally fed larvae (Fig. 3a). Signi¢cant di¡er-ences (Po0.01) were detected between fed andunfed larvae ¢rst in the MH (9 DPH), BD (11DPH)and NL (12 DPH) (Fig. 3e, c and a) respectively. EDand HL were less sensitive to unfed conditions, be-cause they showed signi¢cant di¡erences only at theend of the experiment (14 DPH), when all the larvaedied (Fig. 3a and d). Therefore, starved S. violacealarvae were smaller and more slender than normalfed larvae.Unfed larvae also showed higher mortality rates

(0.419 � 0.367 day�1) than larvae fed with rotifers(0.320 � 0.324 day�1), particularly up to 8 DPH; at9 DPH unfed larvae showed a reduction in mortality(Fig. 3f), coinciding with the reduction in MH(Fig. 3e). Because mortality estimation data did notshow normality (Shapiro^Wilks test, W50.78,Po0.001), comparisons were made using the Mann^WhitneyU-test. A non-parametric test showed signif-icant di¡erences inmortality estimations between fedand unfed larvae (U52702, P50.046).Several aspects of the early life history of S. violacea

emerged from our study. First, during endogenousfeeding, larvae grows fast in length (0.316mmday�1),but when the yolk-sac is exhausted and exogenousfeeding starts, larvae reduce their growth in lengthand increase the BD. Second, at 16 1C, starved larvalS. violacea can survive at least 1more week after yolkabsorption. Third, MH is the ¢rst morphological fea-ture a¡ected during starvation, and is probably thesource of energy for the survival of larval S. violaceawhen food is not available.

Larval growth during early development, particu-larly before ¢rst feeding, is slow in several taxa(Paralabrax maculatofasciatus, o0.05mmday�1,Pen� a & Dumas 2005; Merluccius australis,0.03^0.04mmday�1, Bustos et al. 2007). Other spe-cies, particularly with small larvae at hatching (i.e.,Sparus aurata, � 0.1mmday�1, Yufera et al. 1993),showa rapid early development at similar water tem-peratures. Estimations of larval growth in lengthwere large for S. violacea at 16 1C and may be a strat-egy to increase chances of ¢rst feeding after yolk-sacabsorption, which compensates the high mortalityestimates (Fig.3f) during early ontogeny.The lipid droplet was completely absorbed by fed

and unfed larvae at 10 DPH. Probably a combinationof fatty acids from the lipid droplet and proteins frommusculature kept unfed larvae alive until14 DPH. Onthe contrary,Yufera et al. (1993) found that unfed lar-val S. auratawere unable to completely absorb the li-pid droplet, and it remains in dying larvae.Although simple, morphological measurements

provide a rapid, real and signi¢cant representationof the changes caused by the lack of food in the earlylife stages of ¢sh, there is also a disadvantage in themorphological method due to the lack of validationof the criteria derived from laboratory studies for usewith natural populations. The starvation due to thenon-availability of feed or delayed initial feeding canhave an adverse e¡ect on the growth of larval ¢sh,and it is very important to understand the appropri-ate time to introduce the ¢rst feeding (Gawlicka et al.2000; Dou, Masuda, Tanaka & Tsukamoto 2005;

Figure 2 (a) Larval growth of Seriolella violacea during endogenous feeding at16 1C; (b) Larval rate of yolk-sac consump-tion during endogenous feeding in awarehouse reared at16 1C. LL, larval length; A, age;YSV, yolk-sac volume.

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Kailasam, Thirunavukkarasu, Selvaraj & Stalin2007). In the case of palm ru¡ larvae, we observedthat 4 DPH is the appropriate time to introduce the¢rst feeding and that starved individuals showed re-duced growth in length and their trunkmusculaturewas a¡ected. Similarly, Dou et al. (2002) reported forJapanese £ounder larvae Paralichtys olivaceus thatMH and BD decreased signi¢cantly between unfedand fed larvae. The utilization of body tissues as anenergy source produces considerable alterations inthe digestive tract and adjacent glands in starved lar-vae of marine species such as larval sardine Sardi-nops sagax (Uriarte & Balbont|¤ n 1987), S. aurata

(Yufera et al. 1993) and Seriola lalandi (Chen et al.2007), among others.Starvation usually results in shrinkage of entero-

cytes and a reduction in the height of enterocyte cellsboth in the midgut and in the hindgut (Theilacker &Watanabe 1989; Bisbal & Bengtson 1995), lack of su-pranuclear vacuoles in the hindgut (Oozeki, Ishii &Hirano 1989; Crespo, Marin de Mateo, Santamaria,Sala, Grau & Pastor 2001), degeneration of the cellu-lar structure both in the liver and in the pancreas(O’Connell 1976; Kjorsvik, van der Meeren, Kryvi,Arn¢nnson & Kvenseth 1991) and disorder oftrunk musculature (Bisbal & Bengtson1995; Gisbert,

Figure 3 Temporal variability in the morphometric measurements of fed and unfed larval Seriorella violacea reared at16 1C. Asterisks indicate from which day signi¢cant di¡erences (Po0.01) between fed and unfed groups were detected.(a) Notochord length (NL), (b) eye diameter (ED), (c) body depth (BD), (d) head length (HL), (e) myotome height (MH) and(f) daily mortality. Bars indicate one standard deviation (SD).

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Conklin & Piedrahita 2004). Coincidently, the ¢rst ef-fect observed in larval S. violacea occurred in themusculature or MH, considering all the observationsmade in the study, because probably in other tissuesan early damage occurred. In fed larvae, there was acontinuous increase in MH, but in unfed larvae thedata were signi¢cantly lower (Fig. 3e). A similar pat-tern has been observed in unfed larval Sparus aurata(Yufera et al.1993) and Paralicthys olivaceus (Dou et al.2002). Typical damages in starved larvae are severemuscle ¢bre separationand lackof intermuscular tis-sue, which directly a¡ect MH and BD. Additionally,the reduction in the musculature due to starvationafter ¢rst feeding may a¡ect the swimming capabil-ities of ¢sh larvae, both for escape predators and forpursuit preys (Yin & Blaxter 1987; Skiftesvik 1990),reason for which in a culture it is predictable thatthese larvae are destined to die.Knowledge of the early life history of S. violacea is

scarce and this ¢rst e¡ort will help to understand thelarval dynamics under hatchery conditions. Bio-chemical and histological examination of the larvaeduring starvationwas needed to validate the practic-ability and applicability of the morphological indica-tors suggested by this study. Future investigations arenecessary to answer questions about the variabilityamong batches during the reproductive season,reduce mortality and optimize its larviculture.

Acknowledgments

The authors thanks two anonymous referees for sug-gestions on an early version of this manuscript, andMauricio F. Landaeta and Marcia Oliva for their helpin the laboratoryand data analysis.This investigationwas funded by project FONDEF DO2I1161 ‘Researchand development of a base technology for cultureand production of the palm ru¡ (Seriolella violacea)’adjudicated to Dr. A. Silva. During the writing of themanuscript, CABwas partially funded bya CONICYTdoctoral scholarship.

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Keywords: yolk, lipid droplet, morphometrics,Chile

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