The use of the microalgae Tisochrysis lutea in a green water system increases the weight of seahorse larvae Hippocampus reidi, Ginsburg, 1933

[Utilização da microalga Tisochrysis lutea em sistema de água verde aumenta o peso de larvas de cavalos-marinhos Hippocampus reidi, Ginsburg, 1933]

ABSTRACT

The study aimed to investigate the influence of different microalgae used in green water culture on the survival rate, performance and fatty acids profile of H. reidi larvae. The study consisted of four treatments composed of two microalgae (Tisochrysis lutea - ISO and Chaetoceros muelleri-CHO), used alone (TISO and TCHO) or combined (TIC, 1:1) in the culture water, and a treatment without microalgae (TWM) during the first 15 days of culture. The larvae were fed from the first to the seventh day with copepods (Parvocalanus crassirrostris) and rotifers (Brachionus rotundiformis), and from the eighth day onwards, the gradual inclusion of Artemia sp. nauplii. No differences in the survival rates were observed. The addition of specifically T. lutea in the TISO and TIC treatments affected weight, weight gain, specific growth rate and the condition factor, of the larvae compared to TWM. A higher concentration of (PUFA), DHA, ∑ n-3, n-3/n-6 ratio were found in TISO larvae after 15 days of rearing. In conclusion, the addition of microalgae T. lutea proved to be an advantageous alternative in a large-scale production system, since its inclusion affected the performance and the lipid composition of the larvae compared to the TWM.

Keywords:
larviculture; microalgae; Hippocampus reidi; lipid composition

RESUMO

O presente estudo teve como objetivo investigar a influência de microalgas utilizadas no cultivo em água verde na taxa de sobrevivência, no desempenho e no perfil de ácidos graxos de larvas de H. reidi. O estudo consistiu de quatro tratamentos compostos por duas microalgas (Tisochrysis lutea - ISO e Chaetoceros muelleri-CHO), utilizadas isoladamente (TISO e TCHO) ou combinadas (TIC 1:1) na água de cultivo, e um tratamento sem microalgas (TWM) durante os primeiros 15 dias de cultura. As larvas foram alimentadas do primeiro ao sétimo dia com copépodes (Parvocalanus crassirrostris) e rotíferos (Brachionus rotundiformis), e, a partir do oitavo dia, ocorreu a inclusão gradual de Artemia sp. náuplios. Não foram observadas diferenças nas taxas de sobrevivência. A adição específica de T. lutea nos tratamentos TISO e TIC afetou o peso, o ganho de peso, a taxa de crescimento específico e o fator de condição das larvas em comparação ao TWM. Uma maior concentração de (PUFA), DHA, proporção ∑ n-3, n-3/n-6, foi encontrada em larvas de TISO após 15 dias de criação. Conclui-se que a adição da microalga T. lutea mostrou-se uma alternativa vantajosa, uma vez que sua inclusão afetou o desempenho e a composição lipídica das larvas em comparação ao TWM.

Palavras-chave:
larvicultura; microalga; Hippocampus reidi; composição lipídica

INTRODUCTION

Seahorses are teleost fish of the genus Hippocampus, family Syngnathidae, generally found in shallow waters of tropical and temperate regions (Lourie et al., 2004). Due to habitat loss, and the high collection pressure in the natural environment, all seahorse species are on the International Union for Conservation of Nature - IUCN Red List of Threatened Species (THE IUCN, 2019). On the Brazilian coast, three species of seahorses can be found: Hippocampus erectus, H. patagonicus and H. reidi (Silveira et al., 2014). All of them are considered vulnerable by the list of endangered species of Brazilian fauna (MMA Nº. 148/2022). Although there is a market preference for captivity breed live seahorses, due to better adaptation to the consumption of inert foods and to captive conditions, the nutritional requirements and adequate feeding patterns, which directly influence the survival and performance of the larvae, are still important gaps for large-scale production. (Olivotto et al., 2008). H. reidi, as other species of seahorses, lack a functional stomach, being therefore considered agastric (Novelli et al., 2015), with low digestive enzymes activity (Souza et al., 2020), which makes food digestion difficult, especially in newborns. In this way, one of the key factors influencing larval survival is adequate nutrition, which is related to the ingestion, digestion and assimilation of the essential nutrients needed from the food offered (Lazo et al., 2007, Olivotto et al., 2011).

Normally, in the early stages of seahorse culture, rotifers and Artemia are used as the main food items (Foster and Vincent, 2004; Olivotto et al., 2008, 2011; Koldewey and Martin-Smith 2010). However, these organisms have naturally low levels of highly unsaturated essential fatty acids (HUFAs), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), making it necessary to enrich them with live or dried microalgae, self-emulsified fish oil concentrate or commercial products to increase their nutritional value (Payne and Rippingale, 2000; Sorgeloos et al., 2001; Palma et al., 2011; Figueiredo et al., 2012). Some studies have already shown that the enrichment of live food provides good survival and growth of H. abdominalis (Woods, 2005; Martinez-Cardenas and Purser, 2007) and H. guttulatus (Palma et al., 2011). Despite the enrichment of live food, supplying the amount of highly unsaturated fatty acids necessary for the survival and larval growth, maintaining this food for a long period in clear water (without the addition of microalgae) can result in a gradual loss of the nutritional quality of these organisms (Wang et al., 2019). In addition to enrichment, the nutritional content of live food can be altered by adding microalgae to the culture water (Neori, 2011).

The presence of microalgae in the rearing water (green water) can improve the visual capacity of the larvae in the perception of prey in the culture water, prepare the larval digestive system for the first exogenous nutrition and also bring direct nutritional benefits, serving as a direct source of nutrients, in addition to providing essential nutrients such as vitamins and fatty acids for the offered live food, which will be consumed by fish larvae (Hjelmeland et al., 1998; Lee and Ostrowski, 2001), resulting in improved survival rates and larval growth (Naas et al., 1992; Palmer et al., 2007; Stuart and Drawbridge, 2011). Some species of microalgae naturally synthesize key omega-3 polyunsaturated fatty acids (e.g., EPA and DHA), which are crucial for the energy and growth needs of cultivated species (Shah et al., 2018). In this sense, microalgae present differences in their fatty acid profile: Tisochrysis lutea has higher levels of DHA, while Chaetoceros mulleri has higher levels of EPA (Martínez-Fernández et al., 2006) and, due to their profiles nutritional supplements, their combined use may result in better larvae production (Rico-Villa et al., 2006; Galley et al., 2010).

Therefore, it is possible to observe in marine fish larviculture, especially seahorses, that green water can be a viable and advantageous alternative in an intensive production system, since the presence of microalgae in the water can lead to an improvement in the enrichment of the food offered, in addition to attenuating the effect of light, which facilitates the capture of food, and the countless other benefits that this technique presents. Consequently, the present work aims to evaluate the effect of enrichment of the culture water with microalgae on the survival rate, performance and fatty acid profile of H. reidi larvae.

MATERIALS AND METHODS

This work was carried out at the Laboratory of Marine Fish and Ornamentals (LAPOM), located at the Elpídio Beltrame Mariculture Station, Aquaculture Department, Federal University of Santa Catarina, Florianopolis, SC, Brazil. The procedures performed during the experiment were in accordance with the protocol approved by the Ethics Committee on Animal Use (CEUA-UFSC / 5542220321).

H. reidi, Ginsburg 1933, breeders (n=24) were collected in the Espírito Santo Bay (Vitória-ES, Brazil, 20°19′ S, 40°20′ W), with ICMBio SISBIO authorization n° 76002-1. The animals were kept in 140-L aquariums connected to a saltwater recirculation system, equipped with a mechanical filter (bag), skimmer, biological and ultraviolet filters. The animals were fed twice a day with fresh water carid shrimp Palaemon sp. (Weber, 1795), they were enriched with a mixture of cod liver oil (total omega-3 fatty acids: 24%, DHA: 12%, EPA 8%; Mollers Tran, Norway) and dried spirulina (protein: 63.5%, carbohydrate: 16.1%, lipids: 0.8%; Alga Bloom microalgae, Brazil) in a 1:3 ratio, injected (65±0.02mg) into each shrimp with the aid of 5-mL syringes (adaptation of the methodology used in ICTOLAB).

The microalgae biomass C. muelleri (CCMP 1316) and T. lutea (CCMP 1324) used for technique “green water” were kept in and produced at the Algae Culture Laboratory (LCA)/UFSC in 2-L flasks containing the LCA-AM medium (Sales and Souza-Santos, 2020), the cultures were kept at a controlled temperature of 22 °C and constant aeration through the injection of compressed air enriched with 0.5% CO2 (v/v). C. muelleri and T. lutea were used in an exponential phase growth and for further fat acid analysis were centrifuged (10 min., 3500 rpm), frozen and kept at -80 °C (Table 1).

Four treatments were tested in triplicate: TWM - without addition of microalgae; TISO- addition of the microalgae T. lutea; TCHO- addition of the microalgae C. muelleri; TIC- addition of the microalgae T. lutea + C. muelleri in the proportion of 1:1. After hatching, 30 seahorse larvae were randomly distributed and kept in 12 circular PVC tanks (experimental units) with a total volume of 3 L, at a density of 10 larvae L^-1. The sampling units used in this study and the maintenance system for the larviculture were adapted from (Moorhead, 2015). The experiment lasted 15 days and was carried out in a recirculating system equipped with a skimmer, a ceramic media biological filter and a 100-μm filter bag. The water flow remained off during the day and on during the night to remove microalgae and residual live food.

Daily, before the first feeding, the microalgae were added to the experimental units at a concentration of 200.000 cell mL^-1 of each species (Kline and Laidley, 2015), estimated by counting under a microscope using a Neubauer chamber. H. reidi larvae were fed 3 times a day (09:00 AM, 01:00 PM and 05:00 PM), from the 1st to the 7th day with rotifers Brachionus rotundiformis (8 ind. mL ^-1), before being fed to the seahorse larvae, were enriched with the supplemental enrichment product Red Pepper (Bern Aqua NV, Belgium) for 6 hours according to the manufacturer's recommendation, being washed and filtered through a 60-μm mesh, and with copepods Parvocalanus crassirrostris (2 ind. mL ^-1) ,to harvest the copepods, a 45-μm mesh was used, and after filtering, a mixture of nauplii, copepodites and adults was washed and fed to the larvae and from the 8th day onwards Artemia nauplii, Artemia cysts (Porto Cyst, Natal-RN, Brazil)(2 ind. mL-1) were gradually offered, were placed for hatching from the 7th day of the experiment onwards, in an aerated and illuminated container, at a salinity of 35 and a temperature of 28°C, after 24 h, the nauplii were separated from the cysts, washed and offered to the seahorse larvae. Before feeding, the residual count of live food was performed, and live food was adjusted to maintain the ideal density. The photoperiod used was 14 hours of light (15 W fluorescent lamps), the physical and chemical parameters of the water were evaluated daily before the first feeding, the temperature and oxygen were measured with the oximeter (MO-920-Instrutherm) and remained around 27.80±0.05ºC, 6.30±0.07mg L-1, respectively, the salinity maintained at 30±0.09, verified with an optical refractometer (Soma SHR-10 ATC) and the pH remained at 8.03±0.01, measured using a bench pH meter (AZ-86505).

At the end of the experiment, the samples (seahorse larvae and microalgae) were frozen and kept at -80°C until analysis. Samples of seahorse larvae were composed of 6 to 8 individuals (~80mg) per replicate. For the evaluation of the microalgae, 60mg of wet weight biomass were used, the lipids were cold extracted according to (Bligh and Dyer, 1959), with modifications described by (Vendruscolo et al., 2022). The lipid fraction was subjected to alkaline transesterification, which was adapted from the ISO 5509 method described by the International Organization for Standardization (EN ISO…, 1978), the organic fraction containing fatty acid methyl esters (FAMEs) was reserved for chromatographic analysis. The technique of normalizing the areas of the chromatographic peaks was used to quantify the fatty acids, which had their areas corrected by the correction factors for the equivalent size of the fatty acid chain and the ester-to-acid conversion factor, according to (Visentainer, 2012).

Table 1
Fatty acid profile of the microalgae Tisochrysis lutea and Chaetoceros muelleri

Fatty acid (mg/g)	Tisochrysis lutea	Chaetoceros mulleri
10:0	0.53±1.57	0.19±0.29
11:0	0.18±0.07	0.11±0.06
12:0	0.06±0.02	0.14±0.27
13:0	0.09±0.27	0.12±0.37
14:0	14.25±1.51	10.88±2.24
14:1	0.20±0.34	0.11±0.00
15:0	0.84±0.79	0.96±0.08
16:0	19.94±2.50	17.25±5.88
16:1	32.02±14.84	4.97±5.09
17:0	1.24±0.05	1.69±0.65
18:0	9.97±3.27	3.71±9.52
18:1n-9	0.13±0.07	0.18±0.36
18:1n-9 cis	4.22±9.20	25.72±9.72
18:2n-6 cis	5.58±2.66	4.10±1.75
18:3n-3	0.93±1.56	3.64±1.56
18:3n-6	0.31±0.09	0.18±0.03
20:0	0.16±0.04	0.14±0.10
20:2n-6	0.13±0.08	9.31±3.24
20:3n-6	0.16±0.04	0.02±0.05
20:3n-3	0.08±0.04	0.06±0.12
20:4n-6 (AA)	3.57±2.36	0.20±0.57
20:5n-3 (EPA)	4.96±0.22	0.81±0.73
22:0	0.26±0.09	0.40±0.22
22:2	0.08±0.01	0.18±0.44
22:5n-3	0.05±0.10	0.02±0.00
22:6n-3 (DHA)	8.23±0.71	0.39±0.08
∑ SFA	47.59±5.45	39.96±4.06
∑ MUFAS	35.63±6.09	29.89±5.35
∑ PUFAS	23.25±0.09	19.98±1.05
∑ n-3	14.17±0.52	5.13±1.03
∑n-6	9.80±2.50	15.11±0.53
DHA/EPA	1.59±0.12	0.48±0.27
EPA/AA	1.39±0.21	3.05±1.43
n-3/n-6	1.84±0.66	0.32±0.06

• SFA- saturated fatty acids; MUFA- monounsaturated fatty acids; PUFA- polyunsaturated fatty acids; DHA- docosahexaenoic acid; EPA- eicosapentaenoic acid, AA- arachidonic acid.
• Data presented as mean ± standard deviation

The growth of seahorse larvae a total length (mm) and wet weight (mg) was determined using the mean of 30 individuals on the day of birth and the mean of 15 individuals per treatment at the end of the experiment (15th day). The larvae were euthanized with clove oil (1.5mL L^-1) before biometric measurements. Total length (mm) was defined as the sum of crown height, trunk height and tail length (Lourie et al., 2004), measurements were performed using the Dino Capture software, from a USB Dino-Eye camera coupled to a binocular stereoscopic loupe for measuring larvae. The weight of the individuals was measured using a precision analytical balance (0.001 g).

Using these biometric data, the following were calculated:

Specific growth rate (SGR):100x [ln (final weight) - ln (initial weight) /time]

Specific development rate (SDR):100x [ln (final height) - ln (initial height) /time]

Condition factor (CF): (Weight /Height³) x100

Weight gain (WG): (final weight - initial weight) / initial weight

Survival (S%): 100x (number of survivors on the day) / (initial number of fish - number of fish taken for sampling on the day)

The Shapiro-Wilk test was used to analyze data normality and distribution. As the data showed normal distribution, an analysis of variance (ANOVA - One Way) was performed to determine possible differences between variables. Percentage data were transformed using the arcsine function before being analyzed and Tukey's test at the 5.0% significance level was also applied using the Sigma Plot software14. Data are presented as mean ± standard deviation.

RESULTS

Survival rates did not differ significantly between the different treatments with and without the addition of microalgae (P>0.05), ranging from 77.3% (TCHO) to 88.0% (TIC) at 15 days of larviculture (Table 2).

Table 2
Survival (mean ± SD) of Hippocampus reidi larvae with 15 days submitted to different treatments: TWM - without addition of microalgae; TISO- addition of the microalgae Tisochrysis lutea; TCHO- addition of microalgae Chaetoceros muelleri; TIC- addition of the microalgae T. lutea + C. muelleri

Treatments	Survival (%)
TWM	84.00 ± 4.00
TISO	84.00 ± 2.30
TCHO	77.30 ± 3.50
TIC	88.00 ± 4.00

Seahorse larvae were born with a weight of 2.01±0.21 mg and a total length of 6.68 ± 0.55mm. After 15 days of larviculture, higher and similar weight values were obtained in treatments with the microalgae T. lutea (TISO) (25.91±4.07mg) and the combination of the two microalgae (T. lutea and C. muelleri) (TIC) (25.47±2.64 mg) if compared with values of the treatment without the addition of microalgae (TWM) (20.91±2.93 mg) and the treatment with the microalgae C. muelleri (TCHO) (21.68±2.75mg) (P<0.05), which did not differ from each other (Fig. 1).

Regarding the total length (mm) of the larvae, at the end of the experiment none of the treatments with the addition of microalgae induced significant changes in this parameter which varied between 18.72±0.74mm and 19.24±1.00mm, no significant differences were detected in the values of specific development rate (% day ^-1). The values of specific growth rate (% day ^-1) and weight gain (mg) of larvae at the end of the experiment in TISO and TIC treatments were higher and significantly different when compared to TWM (P<0.05), but similar in relation to TCHO. Regarding the Condition Factor, larvae showed significantly higher and similar values in all treatments with the addition of microalgae in relation to the treatment without addition of microalgae (TWM) (Table 3).

The profile of saturated fatty acids (SFA) was significantly higher in TCHO larvae than in other treatments at the end of the experiment, the (MUFAS) values were significantly higher in the larvae from the TIC treatment when compared to the other treatments and the (PUFA) values were significantly higher in the TISO larvae than in the other treatments (P<0.05). Values the AA and EPA, as well as the DHA/EPA and EPA/AA ratio did not differ between the larvae in different treatments. However, higher and significant DHA values and ∑ n-3, n-3/n-6 ratio were found in TISO larvae after 15 days of larviculture (P<0.05) (Table 4).

Figure 1
Weight (mean ± SD) of 15-day-old Hippocampus reidi larvae submitted to different treatments: TWM - without addition of microalgae; TISO- addition of the microalgae Tisochrysis lutea; TCHO- addition of microalgae Chaetoceros muelleri; TIC- addition of the microalgae T. lutea + C. muelleri. Different letters indicate significant differences between treatments (P<0.05).

Table 3
Zootechnical parameters of 15-day-old Hippocampus reidi larvae submitted to different treatments: TWM - without addition of microalgae; TISO- addition of the microalgae Tisochrysis lutea; TCHO- addition of microalgae Chaetoceros muelleri; TIC- addition of the microalgae T. lutea + C. muelleri

	TWM	TISO	TCHO	TIC
TL1	18.72±0.67a	19.24±1.00a	18.72±0.74a	19.16±0.78a
SGR2	15.45±0.60b	16.72±0.14a	15.95±0.23ab	16.84±0.46a
SDR3	6.98±0.27a	7.13±0.44a	6.88±0.17a	7.09±0.54a
WG4	9.14±0.91b	11.29±0.26a	9.95±0.39ab	11.53±0.86a
CF5	0.31±0.01b	0.36±0.01a	0.33±0.02a	0.36±0.03a

• 1= Total Length; 2= Specific Growth Rate; 3= Specific Development Rate; 4 = Weight Gain; 5= Condition Factor. Different letters on the same line indicate significant differences between treatments (P<0.05). Data presented as mean ± standard deviation.

Table 4
Fatty acid profile of Hippocampus reidi larvae after 15 days of cultivation in treatments: TWM - without addition of microalgae; TISO - addition of the microalgae Tisochrysis lutea; TCHO - addition of microalgae Chaetoceros muelleri; TIC - addition of microalgae T. lutea + C. muelleri

Fatty acid (mg/g)	TSM	TISO	TCHO	TIC
8:0	1.25±0.11b	2.80±0.45a	3.02±0.59a	0.74±0.05b
10:0	0.07±0.00b	0.18±0.03a	0.12±0.04ab	0.05±0.02b
13:0	0.21±0.02c	0.35±0.01a	0.28±0.06ac	0.10±0.01b
14:0	1.40±0.15 a	1.70±0.06 a	1.18±0.16 a	1.55±0.41 a
15:0	1.27±0.08c	1.88±0.28a	1.62±0.19 ac	0.73±0.08b
16:0	21.82±0.62a	21.85±0.79a	23.39±0.51a	19.32±0.99b
16:1	4.50±0.08 a	4.91±0.09 a	4.78±0.20 a	5.62±0.93 a
17:0	1.94±0.05 a	1.88±0.12 a	2.03±0.30 a	1.98±0.34 a
17:1	1.24±0.13 a	1.14±0.01 a	1.08±0.07 a	1.15±0.23 a
18:0	21.33±0.73b	20.51±0.37 b	22.68±0.00 a	17.81±0.21c
18:1 trans isomer	0.51±0.07a	0.44±0.01 a	0.48±0.01 a	0.22±0.02 b
18:1n-9	14.48±0.08bc	15.13±0.12 c	13.68±0.58 b	20.64±0.26 a
18:2n-6	5.17±0.15 a	5.05±0.10 a	4.53±0.29 a	5.65±0.77 a
18:3n-3	0.53±0.24 a	0.64±0.13 a	0.55±0.12 a	0.81±0.19 a
18:3n-6	0.18±0.01 a	0.24±0.03 a	0.20±0.05 a	0.25±0.05 a
20:0	0.22±0.00b	0.22±0.02b	0.25±0.01b	0.45±0.04a
20:1n-9	0.30±0.00 a	0.28±0.02 a	0.28±0.01 a	0.28±0.02 a
20:2n-6	0.13±0.00b	0.20±0.00a	0.17±0.01c	0.20±0.00a
20:3n-6	0.14±0.00 a	0.12±0.02 a	0.10±0.03 a	0.12±0.03 a
20:3n-3	0.06±0.00b	0.09±0.01a	0.05±0.02b	0.06±0.00b
20:4n-6 (AA)	9.29±0.42 a	9.19±0.15 a	8.76±0.07 a	9.03±0.99 a
20:5n-3 (EPA)	3.33±0.11 a	3.24±0.15 a	3.19±0.31 a	3.33±0.11 a
22:0	0.41±0.03a	0.24±0.00b	0.33±0.01a	0.40±0.06a
22:5n-3 (DPA)	0.56±0.01a	0.53±0.01ab	0.46±0.03b	0.56±0.06a
22:6n-3 (DHA)	4.98±0.27b	7.04±1.63a	4.85±0.79b	4.59±0.37b
∑ SFA	49.96±0.95b	51.62±0.51b	54.92±0.95a	45.55±1.27c
∑ MUFAS	21.04±0.22b	21.90±0.24b	20.30±0.86b	27.43±0.07a
∑ PUFAS	24.39±0.35c	26.36±0.01a	22.84±0.09b	24.86±0.79c
∑ n-3	9.465±0.06b	11.01±0.83a	9.105±0.30b	9.505±0.44b
∑ n-6	14.93±0.29a	14.80±0.30ab	13.73±0.39b	14.68±0.67ab
DHA/EPA	1.50±0.13 a	2.15±0.40 a	1.54±0.40 a	1.36±0.26 a
EPA/AA	0.35±0.00 a	0.35±0.01 a	0.36±0.03 a	0.38±0.03 a
n-3/n-6	0.62±0.07b	0.73±0.07a	0.66±0.01c	0.61±0.04b

• SFA- saturated fatty acids; MUFA- monounsaturated fatty acids; PUFA-polyunsaturated fatty acids; DHA-docosahexaenoic acid; EPA- eicosapentaenoic acid, AA- arachidonic acid. Different letters on the same line indicate significant differences between treatments (P < 0.05)
• Data presented as mean ± standard deviation.

DISCUSSION

In the present study, the addition of microalgae, specifically T. lutea (TISO and TIC) in the rearing water positively affected the weight, the weight gain, the specific growth rate and the condition factor of the larvae, especially in relation to the treatment without the addition of microalgae (TSM). These results reflect the importance of the microalgae T. lutea, possibly due to its nutritional profile, especially the high levels of DHA present in it (Martínez-Fernández et al., 2006). DHA and EPA play important roles in maintaining the membrane fluidity during fish larval development, incorporated into the nervous tissue, and when supplied in adequate amounts, it promotes the development and maturation of the digestive system, growth, survival and normal morphogenesis (Zambonino and Cahu, 1999; Sargent et al., 2003, Cahu et al., 2003; Villeneuve et al., 2005; Tocher, 2010), while its deficiency can induce physiological stress (Lund et al., 2012).

After 15 days of rearing, and larvae from the TISO treatment showed the highest values of DHA, ∑ n-3 and n-3/n-6 ratio. Previous studies with marine fish larvae have shown greater nutritional requirements for n-3 than n-6, as well as DHA playing a bigger role than EPA as an essential fatty acid (Izquierdo et al., 1989; Watanabe et al., 1989; Mourente et al., 1993; Faleiro and Narciso, 2010; Izquierdo and Koven, 2011). The nutritional requirements of seahorses are still unknown, but a well-balanced fatty acid fraction of AA, EPA and DHA plays a key role in seahorse nutrition, reflecting on survival and growth (Faleiro and Narciso, 2010; Planas et al., 2020). In the larviculture of the Atlantic cod (Gadus morhua), the use of the microalgae I. galbana in the culture water improved survival, in addition to showing a higher percentage of DHA in the larval lipid composition, indicating the importance of the indirect nutritional benefits that the algae can provide to the larvae (Van der Meeren et al., 2007).

In general, fish larvae have an absolute need or requirement for dietary phospholipids during early developmental stages, mainly due to the lack of a digestive tract (Bell et al., 2003) and the seahorses belong to the agastric fish group, not having a proper stomach (Wilson and Castro, 2010). The importance of DHA for increasing the growth and survival of various marine fish larvae has been extensively studied (Izquierdo, 2005; Izquierdo and Koven, 2011), as well as in seahorse larviculture (Filleul, 1996; Chang and Southgate, 2001; Planas et al., 2020). An insufficient supply of DHA during larviculture has been shown to cause malformations, slower growth, and increased mortality in fish (Tocher, 2010; Lund et al., 2014). In addition to the greater amount of DHA that T. lutea presents and the benefits of its supplementation in the larviculture of H. reidi observed in this study, the use of a variety of food items, as well as the inclusion of the copepod P. crassirostris as part of the diet in the early stage of H. reidi larviculture possibly contributed to the positive growth results obtained. Other studies have also shown that supplementation of copepods as live food for seahorses significantly maximizes survival and growth rates than when fed only rotifers or Artemia, or a combination of both (Payne and Rippingale, 2000; Randazzo et al., 2018; Hora and Joyeux 2009; Olivotto et al., 2010; Willadino et al., 2012; Palma et al., 2014; Zhang et al., 2015; Blanco and Planas, 2015).

The survival rates of H. reidi larvae after 15 days in the treatments with microalgae (TISO - T. lutea; TCHO - C. muelleri; TIC - T. lutea + C. muelleri) and without microalgae (TWM) in the rearing water, numerically, the survival was quite high for the species at this study, ranging from 77.3% to 88.0 %. This result is possibly related to the diversity of food items offered (copepods, rotifers and Artemia). Randazzo et al. (2018), when evaluating the influence of diet (copepods and brine shrimp) on the initial development of H. reidi, they found that the group fed a mixed diet of copepods (Acartia tonsa) and brine shrimp nauplii achieved a survival of 59%, while only with brine shrimp it was 20.8%, and with copepods alone it was 66.7%. Olivotto et al. (2008) also observed greater survival in H. reidi when they included cultivated copepods (Tisbe spp.) as a food supplement, and groups fed with a combined diet of rotifers, copepods and brine shrimp nauplii showed greater survival than those fed the standard diet (14% with rotifer and 10% with brine shrimp nauplii).

In addition to the inclusion of copepods as a food supplement, the species used in this study (Parvocalanus crassirostris) may also have influenced the high survival rates. Schubert et al. (2016) when evaluating the effect of live food species and HUFA composition on the survival and growth of H. reidi found that the species of live food can be a key factor for the survival and growth of juvenile seahorses. Previous works suggest that calanoid copepods, such as the P. crassirostris species used in this study, are an ideal food for marine fish and seahorse larvae (Payne and Rippingale, 2000; Randazzo et al., 2018) mainly due to their pelagic habit, consequently more available as a prey (Payne and Rippingale, 2000; Olivotto et al., 2008). Celino et al. (2012) suggests that in addition to offering a mixed diet (copepods and rotifers) during H. kuda larviculture, supplementation with green water enables the rearing of juveniles in captivity, providing an adequate diet and strategies that can potentially be applied in large-scale seahorse breeding.

CONCLUSION

In conclusion, the addition of microalgae, specifically T. lutea in the treatments (TISO-T. lutea ; TIC - T. lutea + C. muelleri), in the rearing water during the first 15 days of life of the seahorse H. reidi, proved to be an advantageous alternative in a large scale production system, since its inclusion positively affected the performance (weight, specific growth rate and weight gain), and the lipid composition of the larvae compared to the treatment without the addition of microalgae (TWM).

REFERENCES

• BELL, J.G.; MCEVOY, L.A.; ESTEVEZ, A.; SHIELDS, R.J.; SARGENT, J.R. Optimising lipid nutrition in first-feeding flatfish larvae. Aquaculture, v.227, p.211-220, 2003.
• BLANCO, A.; PLANAS, M. Mouth growth and prey selection in juveniles of the European Long-snouted Seahorse, Hippocampus guttulatus. J. World Aquac. Soc., v.46, p.596-607, 2015.
• BLIGH, E.G.; DYER, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., v.37, p.911-917, 1959.
• CHANG, M.P.; SOUTHGATE, C. Effects of varying dietary fatty acid composition on growth and survival of seahorse, Hippocampus sp., juveniles. Aquarium Sci. Cons., v.3, p.205-214, 2001.
• CAHU, C.; INFANTE, J.Z.; TAKEUCHI, T. Nutritional components affecting skeletal development in fish larvae. Aquaculture, v.227, p.245-258, 2003.
• CELINO, F.T.; HILOMEN-GARCIA, G.V.; NORTE-CAMPOS, A.G.C. Feeding selectivity of the seahorse, Hippocampus kuda (Bleeker), juvenile under laboratory conditions. Aquac. Res., v.43, p.1804-1815, 2012.
• EN ISO 5509: animal and vegetable fats and oils: preparation of methyl esters of fatty acids. London: International Organization for Standardization, 1978.
• FALEIRO, F.; NARCISO, L. Lipid dynamics during early development of Hippocampus guttulatus seahorses: searching for clues on fatty acid requirements. Aquaculture, v.307, p.56-64, 2010.
• FIGUEIREDO, J.; LIN, J.; ANTO, J.; NARCISO, L. The consumption of DHA during embryogenesis as an indicative of the need to supply DHA during early larval development: a review. J. Aquac. Res. Dev., v.3, p.2-7, 2012.
• FILLEUL, M.A. Optimising growth of juvenile big bellied seahorse Hippocampus abdominalis leeson. Unpublished B. App. Sci., University of Tasmania, Australia, 1996. 100p.
• FOSTER, S.J.; VINCENT, A.C.J. Life history and ecology of seahorses: implications for conservation and management. J. Fish Biol., v.65, p.1-61, 2004.
• GALLEY, T.H.; BATISTA, F.M.; KING, R.J.; BEAUMONT, A.R. Optimisation of larval culture of the mussel Mytilus edulis (L.) Aquac. Int., v.18, p.315-325, 2010.
• HJELMELAND, K.; PEDERSEN, B.H.; NILSSEN, E.M. Trypsin content in intestines of herring larvae, Clupea harengus, ingesting inert polystyrene spheres or live crustacean prey. Marine Biol., v.98, p.331-335, 1998.
• HORA M DOS, S.C.; JOYEUX, J.C. Closing the reproductive cycle: growth of the seahorse Hippocampus reidi (Teleostei, Syngnathidae) from birth to adulthood under experimental conditions. Aquaculture, v.292, p.37-41, 2009.
• IZQUIERDO, M.S. Essential fatty acid requirements in Mediterranean fish species. Cahiers. Options Mediterranéennes, v.63, p.91-102, 2005.
• IZQUIERDO, M.S.; KOVEN, W.M. Lipids In: HOLT, J. (Ed.). Larval fish nutrition. Oxford: Wiley-Blackwell, John Wiley and Sons, 2011. p.47-82.
• IZQUIERDO, M.S.; WATANABE, T.; TAKEUCHI, T.; ARAKAWA, T.; KITAJIMA, C. Requirement of larval red seabream Pagrus major for essential fatty acids. Nippon Suisan Gakkaishi, v.55, p.859-867, 1989.
• KOLDEWEY, H.J.; MARTIN-SMITH, K.M. A global review of seahorse aquaculture. Aquaculture, v.302, p.131-152, 2010.
• KLINE, M.D.; LAIDLEY, C.W. Development of intensive copepod culture technology for Parvocalanus crassirostris: Optimizing adult density. Aquaculture, v.435, p.128-136, 2015.
• LAZO, J.P.; MENDOZA, R.G.; HOLT, J.; AGUILERA, C.; ARNOLD, C.R. Characterization of digestive enzymes during larval development of red drum (Sciaenops ocellatus). Aquaculture, v.265, p.194-205, 2007.
• LEE, C.S.; OSTROWSKI, A.C. Current status of marine finfish larviculture in the United States. Aquaculture, v.200, p.89-100, 2001.
• LOURIE, S.A.; FOSTER, S.J.; COOPER, E.W.T.; VINCENT, A.C.J. A guide to the identification of seahorses. Project seahorse Traffic North America. Washington D.C.: University of British Columbia and Word Wildlife Fund., 2004. 114p.
• LUND, I.; SKOV, P.V.; HANSEN, B.W. Dietary supplementation of essential fatty acids in larval pikeperch (Sander lucioperca); short and long term effects on stress tolerance and metabolic physiology. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol., v.162, p.340-348, 2012.
• LUND, I.; HÖGLUND, E.; EBBESSON, L.O.E.; SKOV, P.V. Dietary LC-PUFA deficiency early in ontogeny induces behavioural changes in pike perch (Sander lucioperca) larvae and fry. Aquaculture, v.432, p.453-461, 2014.
• MARTÍNEZ-FERNÁNDEZ, E.; ACOSTA-SALMÓN, H.; SOUTHGATE, P.C. The nutritional value of seven species of tropical microalgae for black-lip pearl oyster (Pinctada margaritifera, L.) larvae. Aquaculture, v.257, p.491-503, 2006.
• MARTINEZ-CARDENAS, L.; PURSER, G.J. Effect of tank colour on Artemia ingestion, growth, and survival in cultured early juvenile potbellied seahorses (Hippocampus abdominalis). Aquaculture, v.264, p.92-100, 2007.
• MOORHEAD, J.A. Research-scale tank designs for the larval culture of marine ornamental species, with emphasis on fish. Aquacult. Eng., v.64, p.32-41, 2015.
• MOURENTE, G.; RODRIGUEZ, A.; TOCHER, D.R.; SARGENT, J.R. Effects of dietary docosahexaenoic acid (DHA; 22:6n−3) on lipid and fatty acid compositions and growth in gilthead sea bream (Sparus aurata L.) larvae during first feeding. Aquaculture, v.122, p.79-98, 1993.
• NAAS, K. E.; NRESS, T.; HARBOE, T. Enhanced first feeding of halibut larvae (Hippoglossus hippoglossus L.) in green water. Aquaculture, v.105, p.143-156, 1992.
• NEORI, A. Green water microalgae: the leading sector in world aquaculture. J. Appl. Phycol., v.23, p.143-149, 2011.
• NOVELLI, B.; SOCORRO, J.A.; CABALLERO, M.J. et al. Development of seahorse (Hippocampus reidi, Ginsburg 1933): histological and histochemical study. Fish Physiol. Biochem., v.41, p.1233-1251, 2015.
• OLIVOTTO, I.; AVELLA, M.A.; SAMPAOLESI, G. et al. Breeding and rearing the longsnout seahorse Hippocampus reidi: rearing and feeding studies. Aquaculture, v.283, p.92-96, 2008.
• OLIVOTTO, I.; TOKLE, N.E.; NOZZI, V.; COSSIGNANI, L.; CARNEVALI, O. Preserved copepods as a new technology for the marine ornamental fish aquaculture: a feeding study. Aquaculture, v.30, p.124-131, 2010.
• OLIVOTTO, I.; PLANAS, M.; SIMÕES, N. et al. Advances in breeding and rearing marine ornamentals. J. World Aquacult. Soc., v.42, p.135-166, 2011.
• PALMA, J.; BUREAU, D.P.; ANDRADE, J.P. Effect of different Artemia enrichments and feeding protocol for rearing juvenile long snout seahorse, Hippocampus guttulatus. Aquaculture, v.318, p.439-443, 2011.
• PALMA, J.; BUREAU, D.P.; ANDRADE, J.P. The effect of diet on ontogenic development of the digestive tract in juvenilereared long snout seahorse Hippocampus guttulatus. Fish Physiol. Biochem., v.40, p.739-750, 2014.
• PALMER, P.J.; BURKE, M.J.; PALMER, C.J.; BURKE, J.B. Developments in controlled green-water larval culture technologies for estuarine fishes in Queensland, Australia and elsewhere. Aquaculture, v.272, p.1-21, 2007.
• PLANAS, M.; OLIVOTTO, I.; GONZÁLEZ, M.J.; ROSARIA, L.; ZARANTONIELLO, M.A. Multidisciplinary Experimental Study on the Effects of Breeders Diet on Newborn Seahorses (Hippocampus guttulatus). Original Res., v.7, p.638, 2020.
• PAYNE, M.; RIPPINGALE, R. Rearing West Australian seahorse, Hippocampus subelongatus, juveniles on copepod nauplii and enriched Artemia. Aquaculture, v.188, p.353-361, 2000.
• RANDAZZO, B.; ROLLA, L.; OFELIO, C. et al. The influence of diet on the early development of two seahorse species (H. guttulatus and H. reidi): traditional and innovative approaches. Aquaculture, v.490, p.75-90, 2018.
• RICO-VILLA, B.; LE COZ, J.R.; MINGANT, C.; ROBERT, R. Influence of phytoplankton diet mixtures on microalgae consumption, larval development, and settlement of the Pacific oyster Crassostrea gigas (Thunberg). Aquaculture, v.256, p.377-388, 2006.
• SALES, R.; SOUZA-SANTOS, L.P. Production of concentrated inocula from the microalgae Nannochloropsis oculata. Aquacult. Int.l, v.28, p.1609-1620, 2020.
• SARGENT, J.R.; TOCHER, D.R.; BELL, J.G. The lipids. Fish Nutr., v.2003, p.181-257, 2002.
• SCHUBERT, P.; VOGT, L.; EDER, K.; HAUFFE, T.; WILKE, T. Effects of feed species and HUFA composition on survival and Grh of the longsnout seahorse (Hippocampus reidi). Marine Sci., v.3, p.53, 2016.
• SHAH, M.R.; ALAM, G.A.A.; SARKER, P. et al. Microalgae in aquafeeds for a sustainable aquaculture industry. J. Appl. Phycol., v.30, p.197-213, 2018.
• SILVEIRA, R.B.; SICCHA-RAMIREZ, R.; SILVA, J.S.; OLIVEIRA, C. Morphological and molecular evidence for the occurrence of three Hippocampus species (Teleostei: Syngnathidae) in Brazil. Zootaxa, v.3861, p.317-332, 2014.
• SORGELOOS, P.; DHERT, P.; CANDREVA, P. Use of the brine shrimp, Artemia spp., in marine fish larviculture. Aquaculture, v.200, p.147-159, 2001.
• SOUZA, A.P.L.; FERREIRA, T.; MOURIÑO, J.L.P. et al. Use of Artemia supplemented with exogenous digestive enzymes as sole live food increased survival and growth during the larviculture of the longsnout seahorse Hippocampus reidi. Aquacult. Nutr., v.26, p.964-977,2020.
• STUART, K.R.; DRAWBRIDGE, M. The effect of light intensity and green water on survival and growth of cultured larval California yellowtail (Seriola lalandi). Aquaculture, v.321, p.152-156, 2011.
• THE IUCN red list of threatened species. Available in: http://www.redlist.org Accessed in: 15 Feb. 2019.
  » http://www.redlist.org
• TOCHER, D.R. Fatty acid requirements in ontogeny of marine and freshwater fish. Aquacult. Res., v.41, p.717-732, 2010.
• VAN DER M.; TENSEN-MANGOR, T.J.; PIKOVA, J. The effect of green water and light intensity on survival, grow the and lipid composition in Atlantic cod (Gadus morhua) during intensive rearing. Aquaculture, v.265, p.206-277, 2007.
• VENDRUSCOLO, R.; DEPRÁ, M.C.; PINHEIRO, P.N. et al. Food potential of Scenedesmus obliquus biomasses obtained from photosynthetic cultivations associated with carbon dioxide mitigation. Food Res. Int., v.160, p.111590, 2022.
• VILLENEUVE, L.; GISBERT, E.; ZAMBONINO-INFANTE, J.L.; QUAZUGUEL, P.; CAHU, C.L. Effect of nature of dietary lipids on European sea bass morphogenesis: implication of retinoid receptors. Br. J. Nutr., v.94, p.877-884, 2005.
• VISENTAINER, J.V. Aspectos analíticos da resposta do detector de ionização em chama para ésteres de ácidos graxos em biodiesel e alimentos. Quím. Nova, v.35, p.274-279, 2012.
• WATANABE, T.; IZQUIERDO, M.S.; TAKEUCHI, T.; SATOH, S.; KITAJIMA, C. Comparison between eicosapentaenoic and docosahexaenoic acids in terms of essential fatty acid efficacy in larval red seabream. Nippon Suisan Gakkaishi, v.9, p.1635-1640, 1989.
• WANG, J.; SHU, X.; WANG, W.X. Micro-elemental retention in rotifers and their trophic transfer to marine fish larvae: influences of green algae enrichment. Aquaculture, v.499, p.374-380, 2019.
• WILLADINO, L.; SOUZA-SANTOS, L.P.; MELO, R.C.S. et al. Ingestion rate, survival, and growth of newly released seahorse Hippocampus reidi fed exclusively on cultured live food items. Aquaculture, v.360-361, p.10-16, 2012.
• WILSON, J.M.; CASTRO, L.F.C. Morphological diversity of the gastrointestinal tract in fishes. Fish Physiol., v.30, p.1-55, 2010.
• WOODS, C.M.C. Growth of cultured seahorses (Hippocampus abdominalis) in relation to feed ration. Aquacult. Int., v.13, p.305-314, 2005.
• ZAMBONINO, J.L.I.; CAHU, C.L. High dietary lipid levels enhance digestive tract maturation and improve dicentrarchus labrax larval development. J. Nutr., v.129, p.1195-1200, 1999.
• ZHANG, D.; LIN, T.; LIU, X. Comparison of growth, survival, and fatty acid composition of the lined seahorse, Hippocampus erectus, juveniles fed enriched Artemia and a calanoid copepod, Schmackeria dubia. J. World Aquacult. Soc., v.46, p.608-616, 2015.

Publication Dates

• Publication in this collection
  27 Jan 2025
• Date of issue
  Jan-Feb 2025

History

• Received
  01 May 2024
• Accepted
  17 June 2024