Tolerance and growth of the longsnout seahorse Hippocampus reidi at different salinities

    Tolerance and growth of the longsnout seahorse Hippocampus reidi at different salinities Maik dos Santos Cividanes da Hora, Jean-Christophe Joyeux, Ricardo Vieira Rodrigues, L´ılia Pereira de Sousa Santos, Levy Carvalho Gomes, Mˆonica YumiTsuzuki PII: DOI: Reference:

S0044-8486(16)30238-1 doi: 10.1016/j.aquaculture.2016.05.003 AQUA 632131

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Aquaculture

Received date: Revised date: Accepted date:

1 October 2015 27 April 2016 3 May 2016

Please cite this article as: da Hora, Maik dos Santos Cividanes, Joyeux, Jean-Christophe, Rodrigues, Ricardo Vieira, de Sousa Santos, L´ılia Pereira, Gomes, Levy Carvalho, YumiTsuzuki, Mˆonica, Tolerance and growth of the longsnout seahorse Hippocampus reidi at different salinities, Aquaculture (2016), doi: 10.1016/j.aquaculture.2016.05.003

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TOLERANCE AND GROWTH OF THELONGSNOUT SEAHORSE Hippocampus reidi AT DIFFERENT SALINITIES

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Maik dos Santos Cividanes da Horaa,b,c*, Jean-Christophe Joyeuxb,c , Ricardo Vieira

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Rodriguesd , Lília Pereira de Sousa Santose, Levy Carvalho Gomesf, and Mônica

a

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YumiTsuzukia*

Laboratório de Peixes e Ornamentais Marinhos (LAPOM), Departamento de

Aqüicultura, Universidade Federal de Santa Catarina, 88040-970, Barra da Lagoa, b

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Florianópolis, SC, Brazil.

Laboratório de Ictiologia e Maricultura Ornamental (LabIMO), Base Oceanográfica,

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Universidade Federal do Espírito Santo, Coqueiral 29199-970, Caixa Postal 2030, Aracruz, ES, Brazil. c

Laboratório de Ictiologia, Departamento de Oceanografia, Universidade Federal do

Laboratório de Piscicultura Estuarina e Marinha, Instituto de Oceanografia,

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d

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Espírito Santo, Av. Fernando Ferrari, 514, Goiabeiras 29075-910, Vitória, ES, Brazil.

Universidade Federal do Rio Grande, CP 474, 96201-900 Rio Grande, RS, Brazil. Laboratório de Cultivo e Ecotoxicologia, Departamento de Oceanografia, Universidade

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e

Federal de Pernambuco, Av. Prof. Morais Rego, s/n, Recife, PE, Brazil. f

Laboratório de Ictiologia Aplicada, Universidade Vila Velha, Rua Comissário José

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Dantas de Melo, 21, Boa Vista, 29102770 - Vila Velha, ES, Brazil.

*E-mail: [email protected] (M.S.C. Hora), [email protected] (M.Y. Tsuzuki).

Abstract Hippocampus reidi is one of the most popular seahorse species in the aquarium trade. The commercial breeding of this species is an alternative to reducing the fishing pressure on natural populations. Two experiments with newly born juveniles were conducted in this study to assess salinity tolerance (Lethal Time for 50% of the population-LT50), survival and growth during the first 10 days of life. A third experiment determined the isosmotic point of adults. The highest LT50 was observed at 10 psu, followed by 15, 20, 25, 30, and 35 psu, and the lowest LT50 was recorded at 5 psu. A negative relationship between salinity and final weight and between salinity and

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final height was observed through the regressions performed, such that a decrease in salinity (until the lowest level analysed; 10 psu) implied a higher growth in height and weight. The highest survival rates were observed between the salinities 10 and 25 psu.

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The isosmotic point of the species was determined at 11.68 psu (303.38 mOsm/kg),

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which is in agreement with the salinity where the best salinity results were observed in the first two experiments. Using an intermediate salinity to produce H. reidi on a

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commercial scale would be advantageous because of survival and growth improvement in addition to requiring a lower seawater uptake and less cost for purchase artificial salt.

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Keywords: Syngnathidae, ornamental fish, salinity tolerance, survival, isosmotic point

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Introduction

The longsnout seahorse Hippocampus reidi (Ginsburg, 1933) is found from

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North Carolina (USA), Bermuda and the Bahamas to Rio Grande do Sul (Brazil)

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(Froese and Pauly, 2014) and is one of the most popular species of the genus in the ornamental trade market. Since 2004, all seahorse species have been listed in Appendix

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II of CITES (2013) in view of the degradation of their natural habitats and, mainly, the high fishing pressure to supply the traditional Chinese medicine and the aquarium and craft trades. In Brazil, export quotas were established in 2004 for H. reidi and Hippocampus erectus (Silveira et al., 2014), aiming to control and reduce capture for

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international trade (Hippocampus patagonicus was recently recorded in Brazil, and collection is not permitted). Each company has the right to export 250 individuals of each species per year but there is no restriction on the domestic market. Another tool to reduce the fishing pressure on natural populations of H. reidi is commercial breeding, which is already performed in Brazil on a small scale (Hora and Joyeux, 2009; Koldewey and Martin-Smith, 2010). The life cycle of H. reidi in captivity is already known (Hora and Joyeux, 2009). However, there are few studies on the production of this species (Olivotto et al., 2008; Pham and Lin, 2013; Willadino et al., 2012; Melo-Valencia et al., 2013; Souza-Santos et al., 2013). This lack of studies causes many problems that generate high mortality rates, especially during the first days of life when the animals are more fragile and susceptible to disease (Koldewey and MartinSmith, 2010).

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H. reidi inhabits coastal and estuarine regions (Foster and Vincent, 2004) in salinity between 5 and 43 psu (e.g., Silveira, 2005; Mai & Velasco, 2012). Salinity is known to be one of the most important abiotic water parameters able to influence fish

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development at different life stages. Salinity may affect fertilization and egg incubation

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(Griffin et al., 1998; Zhang et al., 2010), larval growth (Ostrowski et al., 2011; Tsuzuki et al., 2008), and even the development of juveniles and adults (Lin et al., 2009; Resley

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et al., 2006). Marine and estuarine fish spend more energy for osmoregulation when in an environment with a salinity level different from their body concentration. This energy expenditure can vary from less than 10% up to 50% of their standard

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metabolism (Boeuf and Payan, 2001) and can negatively affect animal growth and survival. Thus, while Hippocampus kuda at the age of nine weeks show extreme salinity

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tolerance, growth and survival were better at intermediate salinities (Hilomen-Garcia et al., 2003). Nonetheless, growth and survival at different salinity levels were not tested in juveniles or other seahorse species (Lin et al., 2009), including H. reidi (Melo-

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Valença et al. , 2013).

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The objective of this study was to assess the tolerance to salinity and the growth of H. reidi juveniles during their first days of life and to determine the isosmotic point

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of the species. Such studies will contribute to improvements in seashore culture, there by meeting the needs of the H. reidi breeding industry and helping to reduce capture of

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these animals in the natural environment.

2. Methodology

Three experiments were conducted: 1) tolerance to salinity in H. reidi juveniles during the first days of life, 2) growth and survival at different salinity levels, and 3) determination of the isosmotic point of adult animals.

2.1 General maintenance conditions The experiments were performed at the Laboratory of Ichthyology and Ornamental Mariculture (Laboratório de Ictiologia e Maricultura Ornamental), located at the Oceanographic Base of the Federal University of Espírito Santo (Universidade Federal do Espírito Santo - UFES) (Aracruz, state of Espírito Santo- ES, Brazil) (authorization Ethic Committee for the Use of Animals UFES no.037-2011). Fifty-one

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H. reidi adults were captured in Espírito Santo Bay (Vitoria-ES, Brazil, 20°19′ S, 40°20′ W) (authorization ICMBio SISBIO no. 23924-1), including females and males in advanced pregnancy. The salinity was 28.0 ± 3.2 psu in the capture site.

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Animals allocated to experiments 1 and 2 were kept in 60 and 140 L tanks that

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were part of a saltwater recirculation system of approximately 4000 L equipped with a skimmer, a biological sand filter, and calcareous algae. Physical and chemical

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parameters were monitored daily. Temperature and salinity were measured with a multiparameter meter (model EC300, YSI, USA). The temperature ranged from 25.8 °C to 26.5 °C, and salinity was maintained at 30 psu. The pH was measured with a pH meter

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(model pH-700, Instrutherm, USA) and ranged from 8.3 to 8.4. Nitrite and total ammonia were measured by photocolorimetric method, and they remained below 0.05

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mg/L. The concentration of dissolved oxygen, measured by an ox meter (model MO910, Instrutherm, USA), was 6.6 ± 0.3 (mean ± standard deviation) mg/L. The water quality parameters were monitored with the same equipment in all experiments.

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The experiments were conducted under a 12-hour photoperiod (12 hours light

30 cm above the tanks.

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and 12 hours dark). The light originated from 15-W fluorescent lamps that were located

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The animals were fed four times a day, twice in the morning and twice in the afternoon until satiation. The food consisted of live marine caridean shrimp collected in the natural environment and marine mysids (Mysidium gracile) collected and frozen. The tanks were siphoned three times daily in the afternoon before supplying food and

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one hour after the fourth feeding to remove food debris and faeces from the bottom.

2.2. Experiment 1- Salinity tolerance of juveniles In this species, newborn juveniles are large-sized (6 to 8.5 mm in height; Hora & Joyeux, 20009; Willadino et al., 2012; Souza-Santos et al., 2013). Immediately after birth, 720 juveniles were randomly captured among more than 1000 siblings from a single brood of a single male and were directly transferred to the experimental units to test their resistance to abrupt salinity change. The experimental units consisted of 24 3L tanks with moderate aeration, each tank holding 30 individuals (10 individuals/L). Eight salinity treatments were tested (0, 5, 10, 15, 20, 25, 30, and 35 psu) with three replicates per treatment. Salinity 0 was included in the experiment because it may be a common occurrence in some localities where this coastal and estuarine seahorse is

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found. It also is a salinity remarkably close to the lowest salinity reported in the wild for the species. Deionised water was added to reduce salinity. Conversely, to increase salinity, the water was heated to accelerate the evaporation process until reaching the

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desired salt level (Sampaio & Bianchini, 2002). The animals were not fed throughout

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the experimental period in order not to introduce any bias caused by zooplankton tolerance to salinity (newborns only accept live food). Thirty percent of the water was

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renewed daily, the temperature was maintained at 25.0 ± 0.4 °C, the pH was 8.1 ± 0.3 and nitrite and ammonia levels were below 0.05 mg/L. Dead individuals were counted and removed every six hours during the first 72 hours, and then every 12 hours. The

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experiment was ended when all individuals had died. Death was defined as the absence

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of opercular movement or the absence of a reaction to mechanical stimulation.

2.3. Experiment 2 - Survival and growth of juveniles at different salinities

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2.3.1 Experimental design

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The survival and growth of the H. reidi juveniles were evaluated during the first 10 days of life at seven salinities: 5, 10, 15, 20, 25, 30, and 35 psu. This period of initial

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life was chosen because the newly born juveniles are planktonic (Hora and Joyeux, 2009) and because it is when the highest mortality under culture conditions occurs, therefore considered the most critical period in the production of this species (Olivotto

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et al., 2011). Treatments were performed in triplicate using newborns from ten males (breeders). Each male provided two groups from a single brood (except for a brood from which three groups were separated). Each group comprised 60 randomly selected offspring. These two (or three) groups produced by a single male were placed in replicates of different salinities. The experiment was conducted in 40-L tanks with moderate aeration (1.5 seahorse/L). All juveniles were transferred in the same water where they were born (salinity of 30 psu) to a tank, and the salinity was gradually decreased by dripping freshwater at a rate of 2.5 psu per hour, for acclimation at each salinity. Once the water reached the desired salinity level, the juveniles were allocated to their corresponding treatment. The same method of experiment 1 was used to increase the salinity, also adopting the same rate. The zero salinity was not analysed because all animals subjected to this treatment rapidly died during the tolerance test (see results: Experiment 1).

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The temperature in the tanks during the experiment was 25.3 ± 0.5 °C. The pH was 7.6 ± 0.6, and the nitrite and ammonia levels remained below 0.05 mg/L. Fifty percent of the water was renewed daily.

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2.3.2. Feeding

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Juveniles were fed exclusively on live wild zooplankton from the first to fifth

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day of age. The zooplankton was sampled at the Piraquê-Açu River estuary (Aracruz ES, Brazil 40°09'14.41'' W, 19°57'01.13'' S) with a conical plankton net with a 100-µm mesh size. After sampling, the zooplankton was sorted (300 µm) to remove larger organisms. The estuarine zooplankton community at Piraquê-Açu is widely dominated

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by copepods (Acartia lilljeborgi, Temora turbinata, Parvocalanus crassirostris, Oithona oswaldocruzi, Oithona oculata, Euterpina acutifrons and Paracalanus parvus)

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(Pereira J.B. & Nunes R.A. personal communication). Barroso (2004) reports that the salinity at the estuary mouth (near the zooplankton sampling site) ranges from 11.60 to 34.05 psu, with an average of 30.45 (± 4.41 psu). The collected zooplankton was

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acclimated to each tested salinity and directly offered to fish (non- enriched). The feed

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was offered two to four times daily until satiation.

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Metanauplii of Artemia sp. Enriched with SuperSelco® (INVE, USA) were offered from day 5 until the end of the experiment, following the manufacturer's methodology. The feed replacement was gradual according to the modified protocol of Hoar and Joyeux (2009) as follows. On the first transition day (fifth day of life of the

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juveniles), the feed consisted of 75% wild zooplankton and 25% Artemia sp. On the second transition day, the feed consisted of 50% each. On the third transition day, the feed consisted of 25% wild zooplankton and 75% Artemia sp., and 100% Artemia sp. was provided on the fourth transition day (eighth day of life).

2.3.3. Data sampling Mortality was assessed daily while the tanks were siphoned. The animals remaining at the end of each experiment were counted. When a difference between the numbers of dead animals in the two estimates was observed, missing juveniles were considered to have died on the first day because at this age dead seahorses degrade easily, often hindering visualisation. Survival was calculated as:

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Survival = 100(number of survival at the day/(number of juveniles at the beginning – number of juveniles collected)) Every other day (second, fourth, sixth, eighth, and tenth day), six to eight

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individuals of each replicate were randomly euthanized using the anaesthetic

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benzocaine (30 mg/L) and preserved in 70% alcohol (authorised by the Ethics and

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Animal Use Committee [Comitê de Ética e Uso de Animais – CEUA] – UFES no. 037/2011). On day 0 (day of birth), eight animals not being used in the experiment were collected to determine the initial height and weight. Due to full mortality in one of the three replicates at salinity 10 (for no identified reason), the replicate was removed from

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the analysis.

Height (in mm) was defined as the sum of the crown height, trunk length, and

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tail length (Lourie et al., 1999) and was estimated using graduated photos through the SigmaScan Pro 5 software. Seahorses were centred in the photos to prevent the effect of the distortion at the edges(parallax) from influencing the measurements. The growth

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rate(in mm/day) in each replicate was calculated as the slope (b) of the linear regression

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(height = a + b. age) between zero and 10 days using the individual data (n = 48). The specimens were weighed (wet weight) with an analytical precision scale (0.001 g). The

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specific growth rate in weight (SGR; %day-1) was calculated as:

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SGR = 100((ln(final weight) – ln(initial weight))/time)

2.4. Experiment 3 – Determination of the isosmotic point Forty male and female adult seahorses (18.3 ± 2.4 cm and 23.6 ± 7.8 g), were exposed to five salinities (6, 12, 18, 24, and 30 psu), in duplicate, for 12 days in 25-L tanks with moderate aeration and with four individuals per replicate. The animals were acclimated for each salinity at an approximate rate of 0.33 psu per hour. The specimens were fed frozen mysid M. gracile throughout the experiment. The tanks were siphoned to remove food debris and faeces twice a day, and 60% of water was renewed daily. Water quality parameters analysed were as follows: pH of 7.6 ± 0.3, ammonia and nitrite levels remained below 0.05 mg/L, and oxygen ranged from 6.3 to 7.3 mg/L. Twelve days later, 100 mL of water from each tank was collected; the animals were anaesthetised with eugenol (50 mg/L), and the blood was removed with a 3-mL syringe with heparin sodium (Hepamax). Plasma was obtained by centrifuging the blood at

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6000 ×g (Micro 22R Hettich Zentrifugen, Global Medical Instrumentation, Ramsey, MN, USA) for 10 minutes. The osmotic plasma and water concentrations were

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measured in a vapour pressure osmometer (Vapro 5600 Wescor, Utah, USA).

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2.5. Statistical analysis

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For the tolerance test, the lethal time of 50% of the population (LT50) and its confidence interval were determined through a probit analysis. The smallest period during which 50% of the individuals had died in each treatment was used for each

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salinity because the experiment was conducted until all the animals of each salinity had died.

The data from experiment 2 were analysed using quadratic and linear regressions

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at a 5% significance level (Zar, 1996). The coefficient of determination (Pearson r and r-squared, r2) was used to indicate the goodness-of-fit of the regression. Differences in

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initial weight and height of juveniles of the different breeders were tested using a one-

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way ANOVA, followed by Turkey’s test. All statistical tests were analysed using the SPSS version 7.5 software and are expressed in the text as the mean ± standard error.

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The isosmotic point was determined by linear regression analysis through calculating the regression equation between plasma and water osmolality. The intersection between the straight line and the isosmotic line is considered the isosmotic

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point (Sampaio and Bianchini, 2002).

3. Results

3.1. Experiment 1- Salinity tolerance of juveniles All individuals subjected to salinity 0 psu died before six hours of exposure. In all other treatments, juvenile seahorses exhibited resistance to abrupt changes in salinity. The probit analysis divided the treatments into four homogeneous groups (Figure 1) according to the LT50 value. Group d comprised fish kept at 5 psu, which died earlier than fish at other salinities. Group a comprised individuals kept at a salinity of 10 psu and that showed the longest survival time (LT50 of 166.2 hours) and where the last individual died on the tenth day due to fasting. Group b comprised individuals kept at

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salinities 15 and 20 psu with an intermediate LT50, and group c comprised individuals kept between 25, 30, and 35 psu and with an even lower LT50.

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3.2. Experiment 2 - Survival and growth of juveniles at different salinities

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Height (8.4 ± 0.2 mm) and weight (12.70 ± 1.50 mg) of the H. reidi juveniles at birth did not differ among the different breeders used (ANOVAs, both P> 0.05).

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The survival was dependent on salinity during the fifth and tenth days of life of the juveniles, according to the quadratic regressions(both P < 0.05) (Figure 2). At salinity 5 psu, 100% mortality of the juveniles was observed on the second day of

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exposure, and thus, this salinity level was disregarded. Even after acclimation, the wild zooplankton was not able to survive at 5 psu. The best final survival results were

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observed between 10 and 25 psu (Figure 2).

Height and weight at the end of the experiment were negatively correlated with salinity (P< 0.05; rHeight= 0.970; rWeight= 0.921) (Figure 3). The final height at 10 days

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decreased at an approximate rate of 0.1 mm psu-1 (slope value b; Figure 3). The

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reduction in growth was only detectable as of the eighth day (P< 0.05; rHeight= 0.73; b=0.091 and was undetected on days 2, 4, or 6 (data not shown). The highest average final

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heights were observed at 10 psu (18.13 ± 0.27 mm), 15 psu (18.18 ± 0.62 mm) and 20 psu (17.39 ± 0.28 mm), and the highest final weights were observed at 10 psu (20.50 ± 1.83 mg) and 15 psu (20.75 ± 2.68 mg). A negative correlation was also observed for the growth rate in height and SGR in

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weight (P< 0.05; rmm/day= 0.95; r%/day= 0.93) (Figure 4). 3.3. Experiment 3 – Determination of the isosmotic point of Hippocampus reidi Water osmolality showed a significant linear relationship (P< 0.05; r= 0.98) with water salinity, while the relationship with plasma osmolality was lower but also significant (P<0.05; r = 0.29), and a positive linear relationship with water salinity was observed (Figure 5). The estimated isosmotic point was 303.38 mOsm kg-1H2O, corresponding to a salinity of 11.68 psu (Figure 5).

4. Discussion It has been suggested that the earliest cause of heavy mortality of juveniles of H. subelongatus, H. trimaculatus, H. kuda and H. reidi (Payne and Rippingale, 2000;

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Sheng et al., 2007; Lin et al., 2008; Hora and Joyeux, 2009) may have been fasting since birth. The newly born H. reidi proved to be resistant to an extended period of inanition, as well as to an abrupt change in salinity. Fish kept at 10 psu showed a LT50

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of 166.2 hours (approximately seven days), whereas those kept at 30-35 psu, a salinity

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level commonly used for culturing the species during experiments (Olivotto et al., 2008; Willadino et al., 2012; Souza-Santos et al., 2013), had a LT50 value of near 100 hours

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(four days). At a salinity level of 34.0 (± 1.2 psu), Willadino et al. (2012) observed that 50% of unfed newly born H. reidi died within 120 hours (five days). Few individuals died with in the first 24 hours when H. reidi was transferred

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from 30 psu to 5 - 35 psu. This ability to regulate plasma ions upon a rapid change in salinity is an adaptation of estuarine fish to survive the constant changes that occur in

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the estuary (McCormick, 1995). Thus, H. reidi and other estuarine seahorse species (e.g., Hippocampus abdominalis, H. capensis and H. kuda) are highly adapted to salinity variations (Foster & Vincent, 2004), with salinity change having no detectable

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influence on survival and growth (Hilomen-Garcia et al., 2003). Other juveniles and

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larvae of marine and euryhaline teleost fish, such as Siganus guttatus (Young and Dueñas, 1993), Lutjanus argentimaculatus (Estudillo et al., 2000), and Rachycentron

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canadum (Faulk and Holt, 2006), have also been shown to be resistant to an abrupt salinity change.

In the natural environment, a large river discharge in the estuary may cause a

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major decrease in environmental salinity, as could occur where the culture of H. reidi in net cages in saltwater shrimp tanks is being proposed (Fonseca et al., 2015). A large freshwater inflow may cause high mortality rates, especially if this condition lasts for long period (Russell, 1994; Bell et al., 2003). Similar to our study, Hilomen-Garcia et al. (2003) found that between four and 24 hours, all individuals died abruptly when nine-week-old H. kuda juveniles were transferred from salinity 33 - 35 to 0 psu. For animals kept in floating net cages, ensuring survival would require transfer to safe salinity areas or to recirculation systems. Juveniles in tolerance tests also did not survive much more than 48 hours at salinity 5 psu, while all specimens died before the end of the study in the growth experiment. The wild zooplankton offered during the growth experiment at salinity 5 psu also did not withstand this saline condition and died. Seahorses have an efficient renal reabsorption system, which prevents the loss of excess salts at the same time that

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the gills work to uptake water ions (Karnaky, 1998; Hwangand Lee, 2007). However, because teleosts tend to lose ions in a hypotonic medium, wild zooplankton would be important in the replacement of ions and homeostatic maintenance.

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The effect of salinity on the growth of marine and estuarine teleosts may vary

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considerably among species and with ontogeny (Blanco Garcia et al., 2014). However,

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many of these organisms exhibit better growth (Woo and Kelly, 1995; Dutil et al., 1997; Tsuzuki et al., 2007; Imsland et al., 2008), feed conversion (Imsland et al., 2008), larval development (Griffin et al., 1998; Zhang et al., 2010), and enzyme activity (Le François et al., 2004; Tsuzuki et al., 2007) at intermediate salinities, close to the salinity of the

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internal osmotic medium. This study achieved better LT50, survival, and growth results at salinities near the isosmotic point of the species (11.68 psu), thus corroborating

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previous studies.

Several other marine and estuarine species such as Dicentrarchus labrax (Pickett et al., 2004), Paralichthys orbignyanus (Sampaio and Bianchini, 2002), Siganus

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rivulatus (Saoud et al., 2007), Dicologoglossa cuneata (Herrera et al., 2009), Sparus

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aurata (Bodinier et al., 2010), Pagrus pagrus (Ostrowski et al., 2011), and Seriola lalandi (Blanco Garcia et al., 2014) have an osmotic point at approximately one-third of

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the seawater salinity (11-14 psu; near 350 mOsm/kg) (Boeuf and Payan, 2001; Nordlie, 2009). Fish tend to spend less energy for osmoregulation when exposed to an environment isosmotic with the internal medium, allocating the extra energy for growth

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(Boeuf and Payan, 2001, O'Neill et al., 2011). However, physiological changes in the growth of marine teleosts at intermediate salinities appear to be more complex than a simple reduction in the metabolic cost for osmoregulation. Swanson (1998) suggests that the fish use flexible energy distribution strategies to maintain activity and growth in response to environmental variations. Growth rates at salinities 25, 30, and 35 psu (0.80 ± 0.03; 0.78 ± 0.11 and 0.69 ± 0.12 mm/day) in this study were equivalent to those observed by Hora and Joyeux (2009) (0.74 ± 0.02 mm/day) for H. reidi cultured at 26.5 to 29.0 psu until the beginning of sexual maturation (60 days). The highest average growth rate, final height, and final weight were observed for salinities 10 to 20 psu. Hilomen-Garcia et al. (2003) also reported a better development at intermediate salinities (15 and 20 psu) for H. kuda subjected to salinities ranging from 0 to 85 psu. Other studies (Lin et al., 2009; Melo-

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Valença et al., 2013) tested very short range of salinities that are not comparable with ours. The present study revealed that the best salinity for culturing H. reidi juveniles,

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considering tolerance to salinity, survival, and growth, is between 10 and 20 psu; the

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osmotic point of the species was estimated at 11.68 psu. Thus, using an intermediate

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salinity for the production of this species on a commercial scale would be advantageous because it maximises species survival and growth, in addition to requiring a lower

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uptake of seawater and less purchase of salt.

Acknowledgments

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MSCH and JCJ dedicate this work to the memory of Thiony E. Simon (1985 – 2016), a great friend and a great scientist. We thank E.A. Rossi for assistance in capturing the seahorses and collecting wild zooplankton; Pablo and Alejandro, business

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owners Demarcompany Ltda ME, for donations and equipment loan to the LabIMO;

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J.H.C. Guabiroba, F.S. Magalhães and C.S. Rotta for their support in implementing the experiments; F.A. Delunardo and L.N. Simões for sampling blood from the seahorses.

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The study was partially funded by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico; CNPq) and the Ministry of Fisheries and Aquaculture (Ministério da Pesca e

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Aquicultura; MPA) as part of the project "Sistemas de Produção do Cavalo-Marinho Hippocampus reidi (Syngnathidae) [Production Systems of the Longsnout Seahorse Hippocampus reidi (Syngnathidae)]" coordinated by LPSS. The first author received scholarships from the Brazilian Federal Agency for Support and Evaluation of Graduate Education (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; CAPES) awarded by the postgraduate programmes in Environmental Oceanography/UFES (Masters) and in Aquaculture/UFSC (Doctoral). References Bell, E. M., Lockyear, J. F., McPherson, J. M., Marsden, A. D. & Vincent, A. C. J., 2003. The first field studies of an endangered South African seahorse, Hippocampus capensis. Envir. Biol. Fishes 67, 35–46. Blanco Garcia, A., Partridge, G.J., Flik, G., Roques, J. a C., Abbink, W., 2014. Ambient salinity and osmoregulation, energy metabolism and growth in juvenile yellowtail

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kingfish (Seriola lalandiValenciennes 1833) in a recirculating aquaculture system. Aquac.Res. 45 1-9.

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Willadino, L., Souza-Santos, L.P., Mélo, R.C.S., Brito, A.P., Barros, N.C.S., AraújoCastro, C.M.V., Galvão, D.B., Gouveia, A., Regis, C.G., Cavalli, R.O., 2012. Ingestion rate, survival and growth of newly released seahorse Hippocampus reidi fed exclusively on cultured live food items. Aquaculture 360-361, 10-16.

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Captions of figures Figure 1 – Lethal time of 50% of the population (LT50) determined by probit analysis (mean ± error). Letters a-d indicate homogeneous groups of treatments.

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Figure 2 – Average survival (mean ± standard error) of Hippocampus reidi juveniles under different salinities at five and 10 days of age. The evolution in average survival as a function of salinity may be described by the quadratic equations shown. Figure 3 – Final height and weight (mean ± standard error) of Hippocampus reidi subjected to different salinity treatments for 10 days. The equations concern the quadratic (final weight) and linear (final height) regressions on salinity.

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Figure 4 – Growth rate in height and specific growth rate (SGR) in weight (mean ± standard error) of Hippocampus reidi subjected to different salinity treatments for 10 days. The equations of the linear regressions between growth and salinity are given.

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Figure 5 – Osmolality of water and of the plasma of Hippocampus reidi subjected to different salinities for 12 days. The isosmotic point was estimated as 11.68 psu (303.38 mOsm/kg).

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Highlights 

We studied the tolerance and the growth of newborn seahorse Hippocampus reidi to salinities in 5 psu increments between within 0-35, and determinated the

The longest LT50 of newborns was observed for salinity 10 psu, decreasing at

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higher salinities. LT50 was shortest at 0 and 5 psu.

Weight and height at 10 days of age decreased with salinity within the interval 10-35 psu.

The isosmotic point of the species was determined as 303.38 mOsm / kg, equivalent to 11.68 psu.

Intermediate salinities therefore offer the best conditions for cultivating this

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species during the first days of life.

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isosmotic point from wild-caught adults adapted to various salinities.