Author: Carrera, Lili
Date published: March 1, 2013
(ProQuest: ... denotes formula omitted.)
The fine flounder Paralichthys adspersus (Steindachner, 1867) is a flatfish with high commercial value in Peru and Chile, and considered a new species for aquaculture taking into account the excellent quality of its filet, the decline of natural populations and the increasing local demand.
Paralichthys adspersus (Steindachner, 1867) is distributed from Paita (Peru) to Lota and Juan Fernández Islands (Chile) (Acuña & Cid, 1995). In the last years the interest in its culture has increased due to the high marketable price and the important national and international demand of the species. However, little knowledge of its biology and aquaculture exists (Silva & Flores, 1989; Chinchayán et al., 1997; Silva, 2001; Angeles & Mendo, 2005; Piaget et al., 2007) to allow the development of a profitable commercial culture.
Abundant literature exists on broodstock management and culture technology of other flatfishes of the genus Paralichthys such as P. olivaceus in Japan (Alam et al., 2002; Furuita et al., 2002; Hernández et al., 2007; Yamaguchi et al., 2007), P. microps in Chile (Silva, 2001), P. californicus (Conklin et al., 2003; Gisbert et al., 2004; Merino et al., 2007; Palumbo et al., 2007) and P. lethostigma (Smith et al., 1999; Watanabe & Carroll, 1999) in the United States, P. orbignyanus in Argentina and Brazil (Bambill et al., 2006; Müller et al., 2006; Lanes et al., 2009, 2010); although little is known about the use of Recirculating Aquaculture System (RAS) technology for broodstock culture of P. adspersus
RAS technology is very useful for fish production on a commercial scale due to the lower land and water demanded, the increased control over water quality, the maintenance of constant enviromental factors such as temperature, pH, salinity and photoperiod, the reduction of environmental impact and the increase in biosecurity. Thus, the present study was designed to establish the management of Paralichthys adspersus broodstock in captivity and the use of RAS for this species.
MATERIALS AND METHODS
Capture and transport of fish
Wild flounder fish were captured by means of cast nets (atarraya) in the north-central area of Lima, between November and December 2009 (Fig. 1). The fish were placed in 0.3 m3 fiber-glass tanks with constant aeration and temperature (15°C) using icepacks. In addition, 0.125 mL L-1 of sea water conditioner AquaSafe® was added to keep water in good conditions and to reduce the stress of the fish during transportation (6 h) to the Laboratory of Fish Culture of the Instituto del Mar de Perú (IMARPE) at Callao (Peru).
Facilities, RAS and water quality
Two 7 HP water pumps located in the IMARPE pier were used for water supply to RAS (Recirculating Aquaculture System). Water passed through four filters (sand, gravel, quartz and stone, placed in series) and stored in a 50 m3 reservoir tank (Fig. 2a). Wild flounders were placed in six 2.5 m3 tanks, connected to 2 RAS with 73 m2 surface area. Each RAS was composed by three blue colored, self-cleaning, cylindrical tanks, a 1/3 HP water pump, a 4.5 ft3 floating bed filter (FBF) working as a mechanical and biological filter, a 9000 BTU heat pump for temperature control, a 40 WUV light sterilizer (Fig. 2b), two 20 ìm cartridge filters (cuno) placed in parallel and a ½ HP blower to keep oxygen levels. Each tank was covered with a black geomembrane and a 150 W lamp was placed above the water surface and connected to a programmable lighting system to adjust the photoperiod.
Oxygen measured with a 3 Star Oxymeter (Thermo Scientific®), pH measured with a HI-98160 pH meter (Hanna®) and carbon dioxide (CO2), total ammonia nitrogen (TAN), nitrite (NO2) and nitrate (NO3) measured with LaMotte® kits, were recorded daily (early morning) and un-ionized ammonia nitrogen (NNH3) calculated using the method by Khoo et al. (1977). Temperature was recorded continuously using Tidbit temperature sensors, data loggers HOBO®, registering temperature values every 30 min, the data was downloaded using software HOBO ware Pro 2.7.2.
Additional groups of fish were kept in a 0.3 m3 circular fiberglass tank for periodic parasitological analysis (PAT).
The fish were starved for 15 days. After this period, the fish were fed once or twice per day (11:00 h and 13:00 h) with juvenile live fish (Odonthestes regia regia, Mugil cephalus), crustacean (Emerita analoga), fresh food (Engraulis ringens and Dosidicus gigas) and artificial feed. The artificial semi-moist feed was made by mixing fish and giant squid meal, fish oil, fresh anchovy, giant squid and vitamin premix. The fish were fed 0.5% of their body weight (BW) on Monday formulated feed, and on Wednesday and Friday fresh or live fish. Uneaten food was weighed in order to calculate daily food intake and the feeding rate was periodically adjusted according to fish growth.
Biometric sample and deworming
Fish were anesthetized with MS-222 solution (80 mg L-1) to record total length (cm), weight (g) and gonadal maturity every month. Fish were injected monthly a 10% enrofloxacin solution (0.05 mL kg-1) to prevent infections, whereas 3-5 min fresh water baths were used to eliminate external parasites. Recently catch (CAT), parasitological analysis (PAT) and RAS fish were sacrificed to check internal parasites. Different organs were dissected for analysis: brain, gills, heart, spleen, liver, kidney, muscle and skin, according to the methodology described by Millemann (1968).
Tagging and sexing
Fish were PIT (Passive Integrated Transporter) tagged on the dorsal muscle for individual identification. The codes of 121 fish were read with an Allflex® reader immediately after being tagged. Besides, two noninvasive techniques, visual inspection of genital opening and cannulation using a 0.8 mm diameter catheter (Watanabe & Carroll, 1999) were used for sex identification.
Total weight (WT) of fish was recorded every 45 days and used to calculate specific growth rate (SGR) and relative growth (RG) according to the following formula:
No mortality was recorded during transport, but 11.4% of the fish died during the acclimation period (cumulative mortality until September 2010). Surviving fish (121) were then distributed in two RAS units at a density of 2-3 kg.m2 and a sex ratio of 2:1 (female: male). Water flow rate was kept at 10-15 L min-1, with a total water change in the tank every 166- 250 min. Water parameters were stable and within the ranges recommended for other species of flatfishes (Poxton et al., 1982; Dinis et al., 1999; Smith et al., 1999; Katersky et al., 2006). Nitrogen levels showed the typical curve for the maturation of biofilters (Timmons et al., 2002) at the beginning but after that NH3 levels remained below 0.0125 mg L-1. The levels of N-NH3 and CO2 in the water only increased when feeding rate increased (Figs. 3a-3b).
PIT tagging and cannulation did not cause any mortality and no loss of tags was recorded. Although flounders do not show external dimorphism between males and females, females have genital opening with an "8" appearance with two pores located on the blind side (Fig. 4a), whereas in the case of males, it is smaller and narrower being located on the ocular side (Fig. 4b).
Fish feeding started with live fish given ad libitum, and then fresh fish and formulated feed were added. Feeding rate gradually increased according to consumption and increase in body biomass. Live fish and formulated feed given to the fish increased until a maximum of 2.8 and 2.4% of BW respectively, while fresh fish increased up to 4% of BW.
Formulated semi moist diet showed low stability dropping to the bottom of the tank where it disaggregated rapidly causing water quality problems. Although this problem was solved increasing the amount of the binder (carboxymethyl cellulose) from 0.5 to 2.5%, the diet had low palatability showing the fish a very low ingestion, thus the proportion of this diet was reduced with respect to live fish fed to flounders (Figs. 5a-5b).
The weight recorded for females and males was significantly different, with an initial average weight of 992 and 631 g, respectively. During the first 60 days of acclimation to captivity fish of both sexes lost weight showing a continuous increase afterwards (Fig. 6a). SGR values showed a clear positive increment in both sexes from the third month of captivity whereas in the case of RGR the values increased by August being the average weight 24.5% higher in males and 16.2% higher in females (Figs. 6b-6c).
Different internal and external parasites were observed in broodstock fish along the rearing (Fig. 7). Philometra sp. was the predominant internal parasite but its proliferation was prevented by installing 20 ìm filters in the water inlet to retain the larvae and intermediate hosts. Thus, Philometra sp. was eradicated from the system by July (Fig. 8a). Entobdella sp. was the most frequent external parasite (Fig. 8b) in this case to control and avoid spreading of this and other external parasites in the RAS, fish were treated weekly with freshwater baths.
The survival rate of P. adspersus during and after capture was 100%, due to the net used for fishing (atarraya) where the fish were individually caught. No physical damage was detected on the fish thanks to the short contact time (2 to 3 min) of the fish with the net that allowed no scale loss and stress. Silva & Oliva (2010) using another type of fishing gear (drive) got post-capture survival rates between 60 and 80%. In that case fish were captured in groups and physical damage due to overcrowding was observed in the fish.
One of the main bottlenecks of RAS is the elimination of toxic metabolites produced by feces, urine and microbial decomposition of uneaten food that need to be removed from the rearing water by the action of bio filters. All types of biofilters described in literature (Losordo et al., 1999) go through a process of maturation to form a colony of nitrifying bacteria that remove and transform ammonia in nitrites and then in nitrates. The biofilter used in the RAS described in the present study went through a maturation period of weeks, working efficiently afterwards and keeping the levels of ammonia in 0.004 mg L-1 on average, similarly to the levels obtained by Timmoms et al. (2002), Ingle de la Mora et al. (2003) and Summerfelt et al. (2004) and recommended for similar species such as Scophthalmus maximus L. (Poxton et al., 1982), Solea senegalensis (Dinis et al., 1999), Paralichthys lethostigma (Smith et al., 1999), P. dentatus (Katersky et al., 2006).
Sexual dimorphism between females and males is very clear for some fish species with differences in body shape, coloration, caudal fin shape, number of genital openings, etc. However, most flatfish do not show any morphological difference between sexes and either cannulation or ovarian biopsy is needed (Vecsei et al., 2003). In this study with P. adspersus the identification of females and males was carried out by cannulation.
Successful encoding of fish is considered by Thomassen et al. (2000) when low (1) mortality due to handling and (2) loss of tags is recorded. Thus, in the present study, PIT tagging of P. adspersus did not cause any fish mortality or loss of tags. This type of marking is used not only for aquaculture purposes but also in conservation programs, sport fishing and genetic improvement programs (Navarro et al., 2004, 2006; Astorga et al., 2005; Carrillo et al., 2009).
In teleost, fish maturation and successful reproduction are closely related to the nutritional status of the fish, thus poor nutrition prior or during the spawning period has clear effects either on the growth of the fish, their sexual maturation, and on the quantity and quality of spawning (Carrillo et al., 2000). In the present study P. adspersus feeding process went through several stages until the fish accepted fresh food and formulated diets, in an attempt to cover all their nutritional requirements, for a future evaluation of gonadal maturity and quality of eggs and larvae.
Furthermore, wild captured P. adspersus showed a high prevalence of endo and ectoparasites that affected food consumption, produced petechiae and consequently had a clear influence on fish condition. Other authors (Oliva et al., 1996) reported the parasites found in P. adspersus and cited mainly Entob- della sp. and Philometra sp. with higher prevalence in females than in males. Treatments with mechanical filters to avoid Philometra sp. and periodical freshwater baths for Entobdella sp. were enough to avoid parasite proliferation.
This research was supported by FINCyT (Programa de Ciencia y Tecnología) and IMARPE (Instituto del Mar del Perú) as part of project "Producción de semilla del lenguado Paralichthys adspersus en cautiverio: I Mejoramiento de la calidad y cantidad de desoves. Contrato No 051-FINCyT-PIBAP-2009".
Acuna, E. & L. Cid. 1995. On the ecology of two sympatric flounder of the Paralichthys in the bay of Coquimbo, Chile. Neth. J. Sea Res., 34(1-2): 1-11.
Alam, S., S. Teshima, S. Koshio & M. Ishikawa. 2002. Arginine requirement of juvenile Japanese flounder Paralichthys olivaceus estimated by growth and biochemical parameters. Aquaculture, 205: 127-140.
Angeles, B. & J. Mendo. 2005. Crecimiento, fecundidad y diferenciacion sexual del lenguado Paralichthys adspersus (Steindachner) en la costa central del Peru. Ecol. Aplic., 4(1-2): 105-112.
Astorga, N., J.M. Alfonso, M.J. Zamorano, M. Montero, V. Olivas, H. Fernandez & M.S. Izquierdo. 2005. Evaluation of visible implant elastomer tags for tagging juvenile gilthead seabream (Sparus auratus L.); effects on growth, mortality, handling time and tag loss. Aquacult. Res., 36: 733-738.
Bambill, G., M. Oka, M. Radoni., A. Lopez, M. Muller, J. Boccanfuso & F. Bianca. 2006. Broodstock management and induced spawning of flounder Paralichthys orbignyanus (Valenciennes, 1839) under a closed recirculated system. Rev. Biol. Mar. Oceanogr., 41(1): 45-55.
Chinchayan, M., G. Vera, R. Cisneros & L. Carrera 1997. Notas sobre el cultivo de los lenguados Paralichthys adspersus y Etropus ectenes en ambiente controlado. Inf. Prog. Inst. Mar. Peru, 64: 34-51.
Carrillo, A., M. Herraez, S. Zanuy & B. Basurco. 2009. Advances in fish reproduction and their application to broodstock management: a practical manual for sea bass. In: A. Carrillo, M. Herraez, S. Zanuy & B. Basurco (eds.). Options mediterraneennes, series B: Studies and Research No 63, CIHEAM, Zaragoza, 95 pp.
Carrillo, M., S. Zanuy, F. Oyen, J. Cerda, J.M. Navas & J. Ramos. 2000. Some criteria of the quality of the progeny as indicators of physiological broodstock fitness. In: Recent advances in Mediterranean aquaculture finfish species diversification. (Cahiers Options Mediterraneennes, Seminar of the CIHEAM Network on Technology of Aquaculture in the Mediterranean, 1999/05/24-28). Zaragoza, 47: 61-73.
Conklin, D., R. Piedrahita, G. Merino, J. Muguet, D. Bush, E. Gisbert, J. Rounds & M. Cervantes. 2003. Development of California halibut, Paralichthys californicus, culture. J. Appl. Aquacult., 14: 143-154.
Dinis, M., L. Ribeiro, F. Soares & C. Sarasquete. 1999. A review on the cultivation potential of Solea senegalensis in Spain and in Portugal. Aquaculture, 176: 27-38.
Furuita, H., H. Tanaka, T. Yamamoto, N. Suzuki & T. Takeuchi. 2002. Effects of high levels of n_3 HUFA in broodstock diet on egg quality and egg fatty acid composition of Japanese flounder, Paralichthys olivaceus. Aquaculture, 210: 323-333.
Gisbert, E., R. Piedrahita & D. Conklin. 2004. Ontogenetic development of the digestive system in California halibut (Paralichthys californicus) with notes on feeding practices. Aquaculture, 232: 455- 470.
Hernandez, L.H., S. Teshima, S. Koshio, M. Ishikawa, Y. Tanaka & Md. Shah Alam. 2007. Effects of vitamin A on growth, serum anti-bacterial activity and transaminase activities in the juvenile Japanese flounder, Paralichthys olivaceus. Aquaculture, 262: 444-450.
Ingle de la Mora, G., E. Villarreal-Delgado, J. Arredondo-Figueroa, J. Ponce-Palafox & I. Barriga- Sosa. 2003. Evaluacion de algunos parametros de calidad del agua en un sistema cerrado de recirculacion, para acuicultura sometido a diversas cargas de biomasas de peces. Hidrobiologia, 13(4): 247-253.
Katersky, R., A. Myron & D. Bengtson. 2006. Oxygen consumption of newly settled summer flounder, Paralichthys dentatus (Linnaeus, 1766). Aquaculture, 257: 249-256.
Khoo, K., C. Culberson & R. Bates. 1977. Thermodynamics of the dissociation of ammonia ion in seawater from 5 to 40°C. J. Soln. Chem., 6: 281- 290.
Lanes, C., L. Sampaio & L. Marins. 2009. Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus. Theriogenology, 71: 525-533.
Lanes, C., M. Okamoto, A. Bianchini, L. Marins & L. Sampaio. 2010. Sperm quality of Brazilian flounder Paralichthys orbignyanus throughout the reproductive season. Aquacult. Res., 41: 1-9.
Losordo, T., M. Masser & J. Rakocy. 1999. Recirculating aquaculture tank production system: a review of component options. Southern Regional Aquaculture Center - SRAC. Publication No 453: 12 pp.
Millemann, R.E. 1968. Laboratory manual for FSH 490- parasites and diseases of Fish Departament of Fisheries and Wildlife. Oregon State University, Corvallis, Oregon, 153 pp.
Merino, G., R. Piedrahita & D. Conklin. 2007. The effect of fish stocking density on the growth of California halibut (Paralichthys californicus) juveniles. Aquaculture, 265: 176-186.
Muller, M., M. Radonic, A. Lopez & G. Bambill. 2006. Crecimiento y rendimiento en carne del lenguado Paralichthys orbignyanus (Valenciennes, 1839) cultivado en Argentina. En: IV Congreso Iberoamericano Virtual de Acuicultura, pp. 267-273.
Navarro, A., V. Oliva, M.J. Zamorano, R. Gines & J.M. Alfonso. 2004. Evaluacion del sistema de marcaje PIT (Passive Integrated Transporter) en alevines de dorada (Sparus auratus). I.T.E.A. 100A: 276-286.
Navarro, A., V. Oliva, M.J. Zamorano, R. Gines, M.S. Izquierdo, N. Astorga & J.M. Afonso. 2006. Evaluation of PIT system as a method to tag fingerlings of gilthead seabream (Sparus auratus L.): effects on growth, mortality and tag loss. Aquaculture, 257(1-4): 309-315.
Oliva, M., R. Castro & R. Burgos. 1996. Parasites of the flatfish Paralichthys adspersus (Steindachner, 1867) (Pleuronectiformes) from northern Chile. Mem. Ins. Oswaldo Cruz, Rio de Janeiro, 91(3): 301-306.
Palumbo, A., J. Linares, W. Jewell, S. Doroshov & R. Tjeerdema. 2007. Induction and partial characterization of California halibut (Paralichthys californicus) vitellogenin. Comp. Biochem. Physiol., Part A, 146: 200-207.
Piaget, N., A. Vega, A. Silva & P. Toledo. 2007. Effect of the application of β-glucans and mananooligosaccharides (βG MOS) in an intensive larval rearing system of Paralichthys adspersus (Paralichthydae). Invest. Mar., Valparaiso, 35(2): 35-43.
Poxton, M., R. Murray & T. Linfoot. 1982. The growth of turbot (Scophthalmus maximus L.) in recirculating systems. Aquacult. Eng., 1: 23-34.
Silva, A. 2001. Advances in the culture research of small-eye flounder, Paralichthys microps, and Chilean flounder, P. adspersus, in Chile. J. App. Aquacult., 11: 147-164.
Silva, A. & M. Oliva. 2010. Revision sobre aspectos biologicos y de cultivo del lenguado chileno (Paralichthys adspersus). Lat. Am. J. Aquat. Res., 38(3): 377-386.
Silva, A. & H. Flores. 1989. Consideraciones sobre el desarrollo y crecimiento larval del lenguado (Paralichthys adspersus, Steindachner, 1987) cultivado en laboratorio. Rev. Pacifico Sur (Numero Especial): 629-634.
Smith, T., D. McVey, W. Jenkins, M. Denson, L. Heyward, C. Sullivan & D. Berlinsky. 1999. Broodstock management and spawning of southern flounder, Paralichthys lethostigma. Aquaculture, 176: 87-99.
Summerfelt, S., G. Wilton, D. Roberts, T. Rimmer & K. Fonkalsrud. 2004. Developments in recirculating systems for Arctic char culture in North America. Aquacult. Eng., 30: 31-71.
Timmons, M., J. Ebeling, F. Wheaton, S. Summerfelt & B. Vinci. 2002. Sistemas de recirculacion para la acuicultura. Fundacion Chile, Santiago de Chile, 748 pp.
Thomassen, S., M. Ingemann-Pedersen & G. Holdensgaard. 2000. Tagging the European eel Anguilla anguilla L. with coded wire tags. Aquaculture, 185: 57-61.
Yamaguchi, T., S. Yamaguchi, T. Hirai & T. Kitano. 2007. Follicle-stimulating hormone signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during gonadal sex differentiation in Japanese flounder, Paralichthys olivaceus. Biochem. Biophys. Res. Commun., 359: 935-940.
Vecsei, P., M. Litvak, D. Noakesa, T. Rienc & M. Hochleithnerd. 2003. A noninvasive technique for determining sex of live adult North American sturgeons. Environ. Biol. Fish., 68: 333-338.
Watanabe, W. & P. Carroll. 1999. Recent progress in controlled reproduction of southern flounder Paralichthys lethostigma. UJNT Tech. Rep., 28: 141- 14.
Received: 10 January 2012; Accepted: 10 January 2013
Lili Carrera1, Noemí Cota2, Melissa Montes2, Enrique Mateo3, Verónica Sierralta3 Teresa Castro3, Angel Perea4, Cristian Santos1, Christian Catcoparco2& Carlos Espinoza2
1Laboratorio de Cultivos Marinos, Instituto del Mar del Perú
Esquina Gamarra y General Valle s/n, Chucuito, Callao, Perú 2Proyecto FINCyT, Instituto del Mar del Perú, Esquina Gamarra y General Valle s/n
Chucuito, Callao, Perú
3Laboratorio de Patología Acuática, Instituto del Mar del Perú
Esquina Gamarra y General Valle s/n, Chucuito, Callao, Perú
4Laboratorio de Biología Reproductiva, Instituto del Mar del Perú
Esquina Gamarra y General Valle s/n, Chucuito, Callao, Perú
Corresponding author: Lili Carrera (firstname.lastname@example.org)