Aquaculture Research, 2011, 42, 892^897

doi:10.1111/j.1365-2109.2011.02824.x

SHORT COMMMUNICATION Endogenous feeding and morphological changes in hatchery-reared larval palm ruff Seriolella violacea (Pisces: Centrolophidae) under starvation
Claudia A Bustos1 & Alfonso Silva2
1 2

Programa de Doctorado en Acuicultura, Universidad Catolica del Norte campus Guayacan, Coquimbo, Chile Laboratorio de Cultivo de Peces, Facultad de Ciencias del Mar, Universidad Catolica del Norte campus Guayacan, Coquimbo, Chile

Correspondence: C A Bustos, Programa de Doctorado en Acuicultura, Universidad Catolica del Norte campus Guayacan, Larrondo1281, Coquimbo, Chile. E-mail: clausbustos@gmail.com

There is an increasing eort towards diversication of potential marine species for sh culture around the world (Aristizabal, Suarez, Vega & Bargas 2009; Cavalin & Weirich 2009; Leu, Liou, Wang, Yang & Meng 2009). However, to avoid negative impacts from the introduction of exotic species, the investigation of indigenous species becomes important and worthwhile (Ross, Mart| nez Palacios & Morales 2008). Species such as southern hake Merluccius australis (Bustos & Landaeta 2005; Bustos, Landaeta, BaySchmith, Lewis & Moraga 2007), amberjack Seriola lalandi (Moran, Smith, Gara & Poortenaar 2007) and palm ru Seriolella violacea Guichenot, 1848 have been considered to be excellent candidates for mariculture in Chile. Particularly, the palm ru, S. violacea, is a high-value marine nsh that inhabits subtropical waters, and it is distributed between 11S and 341S in the eastern Pacic Ocean (Iannacone 2003). Successful maintenance of wild-caught broodstock, natural spawning, larviculture and pilot production of juveniles have been achieved in recent years at Fish Culture Laboratory, Universidad Catolica del Norte, Chile. Nevertheless, it is necessary to improve the knowledge of the biology of its early life stages to optimize their larviculture. During larviculture, although plenty of live food is supplied to the culture tank, sh may still be suering from starvation due to poor vision and mouth gape limitation (Planas & Cunha 1999; Nakagawa & Miyashita 2008). After yolk resorption, energy reserves support the larvae for a limited period; the utilization

of some substances as energy sources by larvae faced with starvation promotes a series of changes in morphological characteristics, such as a decrease in their body height and degenerated gut height (Theilacker & Watanabe 1989; Yufera, Pascual, Polo & Sarasquete 1993), inducing poor performance in feeding and an adverse eect on growth (Blaxter & Ehrlich1974). Because of their ease of observation and measurement both in the laboratory and in the eld, the general collapse of gut and myotome development could be sensitive indicators for assessing larval starvation, especially at an early larval stage (e.g. irreversible starvation age) (Yufera et al.1993; Dou, Masuda,Tanaka & Tsukamoto 2002). Internally, starvation usually changes the shape of enterocyte cells in the intestine of several sh species, reducing the height of the midgut mucosal cells (Ehrlich, Blaxter & Pemberton 1976; Yufera et al. 1993; Chen, Qin, Carragher, Clarke, Kumar & Hutchinson 2007). Furthermore, the changes in the digestive tract, liver and pancreas may aect the synthesis of the digestive enzymes and functions of the digestive system (Gawlicka, Parent, Horn, Ross, Opstad & Torrissen 2000; Bolasina, Perez & Yamashita 2006). Information on starvation resistance and time to start external feeding is necessary to reduce the costs related to live feeds of larval palm ru and to reduce larval mortality in ongoing eorts to develop a commercial mass culture system for this species. The objectives of the work are to establish for the rst time, under laboratory-controlled conditions, larval

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Starvation eects on larval Seriolella violacea C A Bustos & A Silva

performance during endogenous feeding and the effect of early starvation on the morphological features and survival of larval S. violacea. Fertilized eggs were collected from captive adult S. violacea that spontaneously spawned during August and September 2008 in two12000 L broodstock tanks under ambient conditions (14 1 1C, 34 ppt) at Fish Culture Laboratory, from Universidad Catolica del Norte, Coquimbo, Chile. Eggs were then stocked at 500 eggs L 1 in one 500 L cylinder conical tank supplied with ltered seawater and gentle aeration. Temperature in the incubator tank ranged from 141 to 16 1C and a light regime of 12 L:12 D was provided throughout the embryo development and until hatching occurred. Hatching occurred 72^74 h after fertilization. Newly hatched larvae were then transferred into two cylindrical tanks (100 L), with sterilized and ltered seawater (1 mm), at an initial density of 50 larvae L 1. During the endogenous feeding, ten larval S. violacea were extracted daily from one of the tanks. Measurements of the larval length (LL), yolksac length and height were performed on freshly anaesthetized larvae with MS-222 under a stereomicroscope (Fig.1a).Yolk-sac volume was estimated considering the yolk-sac as an ellipsoid (V 5 4/3pab2), where a is half of the yolk-sac length and b is half of the yolk-sac height, Bustos et al. 2007). Then, an exponential negative curve was tted to calculate the yolk-sac absorption rate of larval S. violacea. Two days post hatching (2 DPH), larvae were stocked in two 60 L cylinder conical tanks with central drains of ltered seawater (1 mm) at 16 1C and a

Figure 1 Larval Serioella violacea and morphological characteristics measured in larvae. (a) larva of 4.1mm NL and 3 days post hatching (DPH), (b) larva of 5.0 mm NL and 5 DPH, (c) larva of 7.3 mm NL and 14 DPH. NL, notochord length; HL, head length; ED, eye diameter; BD, body depth; MH, myotome height.

standpipe tted with a 150 mm mesh. Larvae in the latter tank were not fed and those in the other tank were fed enriched rotifers (Brachionus plicatilis) twice a day at a density of 10 rotifers mL 1. Rotifers were cultured using a batch culture system, fed a dry diet of bakers yeast combined with concentrated microalgae Nannochloropsis oculata and Isochrysis galbana. In addition, rotifers were enriched with AlgaMac 3050 (Aquafauna Bio-Marine, Hawthorne, CA, USA). The morphology of 10^20 larvae from both nonfed and fed groups was measured daily. Morphological measurements were performed on freshly anaesthetized larvae (MS-222) using a calibrated ocular micrometer tted to a stereomicroscope. Five parts of the body of the larvae were measured: notochord length (NL, from the tip of the mouth to the tip of the notochord), eye diameter (ED), body depth (BD, depth immediately posterior to the pectoral bud), head length (HL, from the tip of the snout to the opercular margin) and myotome height (MH, height immediately posterior to the anus) (Fig.1b). Comparisons were made for each measurement, at a daily scale, between fed and unfed larvae using Mann^Whitney U-tests. The daily mortality rate from yolk absorption time was dened as the percentage of the dead sh to the survivors at the beginning of each day. Instantaneous mortality rate (Z) was used to examine mortality among trials (fed vs. non-fed) and was dened as: Z 5 (ln N0^ln Nt)t 1, where N0 and Nt are the numbers of initial and surviving larvae, respectively, and t is the duration of the developmental stage in days. Newly hatched larvae [mean standard deviation (SD), 2.97 0.18 mm NL] had a yolk-sac 0.34 0.11mm3 in volume (range: 0.19^0.57 mm3) with a lipid droplet of 0.25 0.04 mm diameter (range: 0.15^ 0.31mm). The intercept of the growth linear model (i.e., the size at hatching) was xed at 2.97 mm. The estimated linear model was LL 5 0.316 A12.97, where LL is the larval length (mm) and A is the age (days). Therefore, larval growth in length during the endogenous period was fast, with an estimated linear rate of 0.316 mm day 1 (Fig. 2a). The yolk-sac was consumed quickly during the rst day and decreased exponentially after hatching, being almost completely absorbed on 4^5 days. The estimated exponential model was YSV 5 0.279e 0.573 A, where YSV is the yolk-sac volume (mm3) and A is the age (days) (Fig. 2b). The lipid droplet was completely absorbed at 10 DPH, and larvae survived until 14 DPH without food (Fig.1c). Starved larvae showed a reduction in growth in notochordal length between 6 and14 DPH compared

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Starvation eects on larval Seriolella violacea C A Bustos & A Silva

Aquaculture Research, 2011, 42, 892^897

Figure 2 (a) Larval growth of Seriolella violacea during endogenous feeding at 16 1C; (b) Larval rate of yolk-sac consumption during endogenous feeding in a warehouse reared at 16 1C. LL, larval length; A, age;YSV yolk-sac volume. ,

with normally fed larvae (Fig. 3a). Signicant dierences (Po0.01) were detected between fed and unfed larvae rst in the MH (9 DPH), BD (11DPH) and NL (12 DPH) (Fig. 3e, c and a) respectively. ED and HL were less sensitive to unfed conditions, because they showed signicant dierences only at the end of the experiment (14 DPH), when all the larvae died (Fig. 3a and d). Therefore, starved S. violacea larvae were smaller and more slender than normal fed 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; at 9 DPH unfed larvae showed a reduction in mortality (Fig. 3f), coinciding with the reduction in MH (Fig. 3e). Because mortality estimation data did not show normality (Shapiro^Wilks test, W 5 0.78, Po0.001), comparisons were made using the Mann^ Whitney U-test. A non-parametric test showed significant dierences in mortality estimations between fed and unfed larvae (U 5 2702, P 5 0.046). Several aspects of the early life history of S. violacea emerged from our study. First, during endogenous feeding, larvae grows fast in length (0.316 mm day 1), but when the yolk-sac is exhausted and exogenous feeding starts, larvae reduce their growth in length and increase the BD. Second, at 16 1C, starved larval S. violacea can survive at least 1 more week after yolk absorption. Third, MH is the rst morphological feature aected during starvation, and is probably the source of energy for the survival of larval S. violacea when food is not available.

Larval growth during early development, particularly before rst feeding, is slow in several taxa (Paralabrax maculatofasciatus, o0.05 mm day 1, Pena & Dumas 2005; Merluccius australis, 0.03^0.04 mm day 1, Bustos et al. 2007). Other species, particularly with small larvae at hatching (i.e., Sparus aurata, $ 0.1mm day 1, Yufera et al. 1993), show a rapid early development at similar water temperatures. Estimations of larval growth in length were large for S. violacea at 16 1C and may be a strategy to increase chances of rst feeding after yolk-sac absorption, which compensates the high mortality estimates (Fig. 3f) during early ontogeny. The lipid droplet was completely absorbed by fed and unfed larvae at 10 DPH. Probably a combination of fatty acids from the lipid droplet and proteins from musculature kept unfed larvae alive until14 DPH. On the contrary,Yufera et al. (1993) found that unfed larval S. aurata were unable to completely absorb the lipid droplet, and it remains in dying larvae. Although simple, morphological measurements provide a rapid, real and signicant representation of the changes caused by the lack of food in the early life stages of sh, there is also a disadvantage in the morphological method due to the lack of validation of the criteria derived from laboratory studies for use with natural populations. The starvation due to the non-availability of feed or delayed initial feeding can have an adverse eect on the growth of larval sh, and it is very important to understand the appropriate time to introduce the rst feeding (Gawlicka et al. 2000; Dou, Masuda, Tanaka & Tsukamoto 2005;

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Aquaculture Research, 2011, 42, 892^897

Starvation eects on larval Seriolella violacea C A Bustos & A Silva

Figure 3 Temporal variability in the morphometric measurements of fed and unfed larval Seriorella violacea reared at 16 1C. Asterisks indicate from which day signicant dierences (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).

Kailasam, Thirunavukkarasu, Selvaraj & Stalin 2007). In the case of palm ru larvae, we observed that 4 DPH is the appropriate time to introduce the rst feeding and that starved individuals showed reduced growth in length and their trunk musculature was aected. Similarly, Dou et al. (2002) reported for Japanese ounder larvae Paralichtys olivaceus that MH and BD decreased signicantly between unfed and fed larvae. The utilization of body tissues as an energy source produces considerable alterations in the digestive tract and adjacent glands in starved larvae of marine species such as larval sardine Sardinops 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 enterocytes and a reduction in the height of enterocyte cells both in the midgut and in the hindgut (Theilacker & Watanabe 1989; Bisbal & Bengtson 1995), lack of supranuclear vacuoles in the hindgut (Oozeki, Ishii & Hirano 1989; Crespo, Marin de Mateo, Santamaria, Sala, Grau & Pastor 2001), degeneration of the cellular structure both in the liver and in the pancreas (OConnell 1976; Kjorsvik, van der Meeren, Kryvi, Arnnnson & Kvenseth 1991) and disorder of trunk musculature (Bisbal & Bengtson 1995; Gisbert,

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Aquaculture Research, 2011, 42, 892^897

Conklin & Piedrahita 2004). Coincidently, the rst effect observed in larval S. violacea occurred in the musculature or MH, considering all the observations made in the study, because probably in other tissues an early damage occurred. In fed larvae, there was a continuous increase in MH, but in unfed larvae the data were signicantly lower (Fig. 3e). A similar pattern 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 severe muscle bre separation and lack of intermuscular tissue, which directly aect MH and BD. Additionally, the reduction in the musculature due to starvation after rst feeding may aect the swimming capabilities of sh larvae, both for escape predators and for pursuit preys (Yin & Blaxter 1987; Skiftesvik 1990), reason for which in a culture it is predictable that these larvae are destined to die. Knowledge of the early life history of S. violacea is scarce and this rst eort will help to understand the larval dynamics under hatchery conditions. Biochemical and histological examination of the larvae during starvation was needed to validate the practicability and applicability of the morphological indicators suggested by this study. Future investigations are necessary to answer questions about the variability among batches during the reproductive season, reduce mortality and optimize its larviculture.

Acknowledgments The authors thanks two anonymous referees for suggestions on an early version of this manuscript, and Mauricio F. Landaeta and Marcia Oliva for their help in the laboratory and data analysis. This investigation was funded by project FONDEF DO2I1161 Research and development of a base technology for culture and production of the palm ru (Seriolella violacea) adjudicated to Dr. A. Silva. During the writing of the manuscript, CAB was partially funded by a CONICYT doctoral scholarship.

References
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Keywords: yolk, lipid droplet, morphometrics, Chile

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