Technical and economic feasibility of integrating seahorse culture in shrimp/oyster farms

Integrated multi‐trophic aquaculture systems (IMTA) are building ecosystems designed to produce aquatic organisms with less environmental impact. We have developed a simple system and a management strategy for introducing seahorse (Hippocampus spp.) culture in shrimp/oyster farms to increase economic sustainability. The system includes broodstock, nursery and grow‐out phases of Hippocampus reidi G. culture. We performed a test in a 42.4‐ha marine shrimp/oyster farm, which showed that this system is technically feasible. Then, we performed a partial budget analysis including cost‐return, cash flow and financial feasibility analysis. More than 12 000 marketable size seahorses may be produced in 402 m² annually. Initial investment was only US$ 21 103.00. The benefit–cost ratio was ~US$ 20.00 for each US$ 1.00 spent, internal rate of return was 131% and payback period <2 years. In conclusion, the introduction of seahorse in shrimp/oyster IMTA system is technically feasible, profitable, resilient and shows high liquidity. This system may be easily established in developing countries and has potential to provide seahorses to supply different markets (live specimens for the marine aquarium trade and dried specimens for traditional Chinese medicine and the curio trade). In addition, it can indirectly contribute to preserve natural populations.     

Effects of Anesthetic Disposal on the Physiological and Behavioral Responses of the Lined Seahorses,Hippocampus erectus

Anesthetic disposal for fish often results in physiological and behavioral responses. There is limited information involved on the metabolic and behavioral responses of the seahorses. The lined seahorse, Hippocampus erectus, is of great interest in the sciences because of its unusual S‐shaped morphology and male pregnancy behavior. This study found that the lined seahorse could be effectively anesthetized into Stage II at the concentrations of 10 mg/L clove oil or 20 mg/L MS‐222 based on the comparison of six different treatments. The ventilation frequency, oxygen consumption, and ammonia‐N excretion of the seahorses were decreased significantly as soon as they were exposed to the anesthetic agents. A higher O : N ratio was found in seahorses anesthetized by clove oil as compared to those exposed to MS‐222. After the anesthetic treatment, the feeding frequencies of recovered seahorses were low at the beginning and then increased during the 7‐d culture. Moreover, there was no incremental difference of wet weights among the tested seahorses (P = 0.534). The findings indicate that the anesthetic process has few effects on the feeding and short‐term growth of seahorses in spite of the occurrence of physiological and behavioral responses.     

Rearing the spotted seahorse Hippocampus kuda by feeding live and frozen copepods collected from shrimp ponds

This study evaluated the use of live and frozen copepods collected from shrimp ponds for rearing juveniles of the spotted seahorse Hippocampus kuda. Protein and HUFA contents in frozen copepods were all higher than in Artemia nauplii, the conventional live food for seahorse juveniles. The results of this study showed that copepods can be used as feed for rearing seahorse fry and juvenile. The spotted seahorse showed obvious preference for live copepods and rarely fed on dead copepods on the tank bottom. Furthermore, the combination of frozen copepods and live Artemia nauplii resulted in highest growth and highest survival of the experimental seahorses. Further research on possible effects of DHA:EPA ratio on survival and growth of young H. kuda is recommended.     

The administration of Lactobacillus plantarum directly to food: An effective way to improve the survival and growth of juvenile lined seahorses (Hippocampus erectus)

Previously, we found Lactobacillus plantarum had probiotic effects on lined seahorse (Hippocampus erectus) juveniles by the addition Lactobacillus directly into rearing seawater containing juveniles and their food (i.e. copepods). However, how the juveniles consumed Lactobacillus, directly from water or from copepods in whose guts Lactobacillus may have accumulated by ingestion, is still unclear. To clarify this, three experiments were successively carried out in this study. In the first experiment, the manner by which juveniles consumed Lactobacillus, namely, directly from food, was discovered via a 2 × 2 factorial design with two factors of water and food, and two levels with and without L. plantarum. In the second experiment, a 5 × 4 factorial design with five levels of L. plantarum doses (D: 5, 10, 20, 40 and 80 × 10⁸ cfu/L) and four levels of enrichment time (T: 0.5, 1.0, 1.5 and 2.0 hr) was conducted to analyse lactic acid bacteria (LAB) contents in the enriched copepods under different treatments. In the third experiment, six kinds of Lactobacillus‐enriched copepods chosen from the second experiment that carried significantly different LAB contents were fed to juveniles, and it was found that the copepods containing LAB greater than 5 log₁₀colonies/g had the most improved effects for survival and growth. In conclusion, the effective manner of L. plantarum administration in seahorses is by the addition to their food. Additionally, D40 * T1.0, whose LAB content was more than 5 log₁₀colonies/g, is generally the most economic copepod treatment for improving survival and growth in H. erectus rearing.     

Nutrient removal efficiency of Hydropuntia cornea in an integrated closed recirculation system with pink shrimp Farfantepenaeus brasiliensis

In this study, we have tested the effect of seaweed stocking density in an experimental seaweed biofilter using the economically important red seaweed Hydropuntia cornea integrated with the cultivation of the pink shrimp Farfantepenaeus brasiliensis. Nutrient removal efficiency was evaluated in relation to seaweed stocking density (2.5, 4, 6 and 8 g fw L^?1). Total ammonia nitrogen (TAN) was the main nitrogen source excreted by F. brasiliensis, with concentrations ranging from 41.6 to 65 μM of NH₄⁺‐N. H. cornea specific growth rates ranged from 0.8 ± 0.2 to 1.4 ± 0.5% day^?1 with lowest growth rates at higher seaweed stocking density (8 g fw L^?1). Nutrient removal was positively correlated with the cultivation densities in the system. TAN removal efficiency increased from 61 to 88.5% with increasing seaweed stocking density. Changes in the chemical composition of the seaweed were analysed and correlated with nutrient enrichment from shrimp effluent. The red seaweed H. cornea can be cultured and used to remove nutrients from shrimp effluents in an integrated multi‐trophic aquaculture system applied to a closed recirculation system. Recirculation through seaweed biofilters in land‐based intensive aquaculture farms can also be a tool to increase recirculation practices and establish full recirculation aquaculture systems (RAS) with all their known associated benefits.     

Colonization of Aeromonas salmonicida subsp. masoucida strains in Atlantic salmon (Salmo salar L.) during infection

Aeromonas salmonicida subsp. masoucida (ASM) is classified as atypical A. salmonicida and brought huge economic damages to the local salmonid aquaculture in China. An ASM strain named AS‐C4 was used to investigate the colonization of ASM in Atlantic salmon (Salmo salar L.) by an immersion challenge with the control group (T0, no AS‐C4), group T1 (2.67 × 10⁴ CFU/ml AS‐C4) and group T2 (2.67 × 10⁷ CFU/ml AS‐C4). The numbers of AS‐C4 copies in different fish tissues (gill, intestine, skin, blood, muscle, spleen, liver and kidney) were determined at different time points post challenge using the quantitative real‐time PCR (qRT‐PCR). AS‐C4 were detected in the gill and intestine as early as 0 hr after the challenge both in T1 and T2 groups, suggesting that the gill and intestine were probably the portals of entry of AS‐C4 into salmon. Although AS‐C4 could not be detected in the skin until 24 hr after the challenge in T1 group, it could be detected in the skin as early as 0 hr after the challenge in T2 group, indicating that the skin may also be a portal of entry of AS‐C4 into salmon. AS‐C4 was immediately detected in the blood within 3 hr after it entered the host, suggesting that AS‐C4 successfully invaded the bloodstream of fish. After AS‐C4 colonized the host, it colonized the internal tissues, such as the spleen, liver, kidney and muscle. The results of this study will contribute to the understanding of the pathogenesis of the ASM strains and give a broader understanding of the infection route of ASM in it's host, providing more information for the development of new therapeutic strategies to protect against this pathogen in aquaculture.     

Biochemical composition of wild and cultured seahorses (Hippocampus kuda Bleeker)

The seahorse has been used for thousands of years as an important traditional Chinese medicine (TCM) in China. Many species of wild seahorse have become endangered, but demand for them continues to grow rapidly. Therefore, it is urgent to cultivate seahorses to relieve the strain on natural populations and meet the market demand for this TCM. In this study, medicinal components of wild and cultured yellow seahorses (Hippocampus kuda Bleeker) were analysed and compared. Different parts of the body (cortex and bone), male and female specimens, and seahorses of different ages were analysed. Crude protein, crude fat, trace elements, calcium, amino acid, fatty acid, steroid and cholesterol content were measured. The results showed that moisture content accounted for 70% of wet weight and ash content increased with growth. Crude protein and fat contents were higher in cultured seahorses than in wild specimens, and the content of Zn in cultured seahorses (93.26 ± 10.44%) was significantly higher than that of same‐aged wild seahorses (74.20 ± 3.83%). Amino acid and fatty acid contents of cultured seahorses also were higher than those of wild seahorses. These findings show that cultured seahorses can also be used in TCMs and that 1‐year‐old cultured seahorses have the best properties.     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

灰海马染色体制备及核型分析

为了解灰海马的细胞遗传学特征,便于今后开展灰海马的种质评价与鉴定、规模化人工繁育、亲缘关系研究及人工选育等工作,实验以雌雄灰海马的背鳍为材料,采用秋水仙素浸泡和常规热滴片法制备染色体标本。借助Photoshop图像软件,将同源染色体配对、拼贴,做出染色体核型图。采用Image J软件的自定义曲线测量功能,以着丝粒的中心位置为起点,顺着染色体弯曲的形态,到染色体臂末端为终点,测量线段长度,得出图片中染色体的臂长,根据相同放大倍数下标尺的测量值,换算出染色体的实际臂长。根据臂比值,将染色体进行配对、分类后,得出灰海马的染色体核型公式。结果显示,灰海马的背鳍组织可作为其染色体制备的理想材料,实验选用的染色体制备方法能获得图像清晰、形态良好的细胞分裂相。此法简单、效果好,解决了海龙科鱼类染色体制备的难题;灰海马具有22对染色体,二倍体染色体数目2n=44,雄鱼的染色体核型公式为2n=2sm+20st+22t,雌鱼的染色体核型公式为2n=1m+2sm+20st+21t,雌鱼存在性染色体异型的现象,因此灰海马的性染色体为ZW/ZZ型。     

Study on an integrated eco‐aquaculture system for rearing the yellow seahorse,Hippocampus kuda Bleeker

Seahorse aquaculture is challenged by insufficient feeding supply and lack of biological and nutritional knowledge, resulting in low survival rate and poor economical profitability. We report here an integrated eco‐aquaculture system in which the yellow seahorse and its natural prey were co‐cultured with fertilized water and seaweed in cement ponds. In the first stage, urea (10 g m^?3) and chicken manure (50 g m^?3) were used to fertilize the cultured water, 5–7 days later, rotifer and micro‐crustaceans were flourishing. Then, seahorse juveniles were stocked at 200 ind m^?3. After 2 weeks, seaweed Gracilaria lichevoides was transplanted into the ponds to regulate water quality, light and to provide holdfast attachment for seahorses. The optimal density of G. lichevoides was controlled between 0.5 and 2 kg m^?3. The introduction of seaweed provides the habitat for natural food of seahorse. Within the cluster of the seaweed, small crustacean density was over 450 individuals per 100 g of fresh weight. Initial size of seahorse juvenile was 1.03 ± 0.091 cm, After the 146‐day rearing period, seahorse survival rates were 70.8%, 57.7% and 42.5%, and body standard lengths were 11.33 cm, 10.84 cm and 10.04 cm in the integrated eco‐aquaculture system (GFA) and in monoculture systems of FA (fertilized + feeding) and BA (only feeding) respectively. This system incorporated traditional Chinese aquaculture technique in which feedings nature food organisms were cultivated by fertilization and ecological regulation. Results from these preliminary experiments suggest that the integrated system could be technically feasible, suitable and exemplary.     

Comparison of the Intestinal Bacterial Flora in Healthy and Intestinal‐diseased Seahorses Hippocampus trimaculatus,Hippocampus erectus,and Hippocampus spinosissimus

This investigation examined the intestinal microbial flora among healthy and intestinal‐diseased seahorses Hippocampus trimaculatus, Hippocampus erectus, and Hippocampus spinosissimus by utilizing polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) and densitometric analysis. Results demonstrated that 16 disparate DGGE bands belong to six major bacterial groups, which were Vibrionace, Enterobacteriacea, Rhodobacteraceae, Sphingomonadaceae, Flavobacteriaceae, and Alcaligenaceae. It was found that Vibrionaceae was the dominant population among the healthy and intestinal‐diseased seahorses. Vibrio ponticus strain XJ3 and Vibrio neptunius strain WT82, especially V. ponticus strain XJ3 of high abundance, were identified for the first time in seahorses and concluded to be intestinal disease pathogens because of their co‐existence in three intestinal‐diseased seahorse species and other reported fish or oyster. In comparison, uncultured bacterium clone Alcaligenaceae, Vibrio sp., uncultured bacterium clone Rhodobacteraceae, Serratia nematodiphila strain, and Serratia marcescens strain comprised the basic intestinal bacterial flora of all healthy seahorses. This study is the first to report the presence of S. nematodiphila in seahorses.     

Outbreaks of Streptococcosis associated with Streptococcus iniae in Siberian sturgeon (Acipenser baerii) in China

Streptococcus iniae has emerged as an important fish pathogen over the past few decades causing high losses in aquaculture farms all over the world. At least 27 species of fish have been documented to be infected by S. iniae, including cultured and wild populations. In August and October 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhages and nodules showing on epicardium occurred on the Acipenser baerii farms in Ya'an country, China. Histological examination revealed a multisystemic, necrotising inflammatory response that was particularly marked in liver, kidney, heart and brain. Mass mortality (>40%) was observed in infected fish and two Gram‐positive cocci (Ab130920 and Ab131025) were obtained from kidneys and livers of diseased fish. Experimental infections with these two isolates resulted in marked symptoms in the sturgeons similar to those observed in natural outbreaks, and the LD₅₀ values of the two isolates were 5.1 × 10⁵ and 6.4 × 10⁵ cfu per fish respectively. The two microorganisms were identified as S. iniae through physiological and biochemical tests, 16S rRNA and lctO gene sequence analysis. Both two isolates showed a similar antibiotic susceptibility, which were sensitive to ampicillin, amoxicillin, cefazolin, amikacin, deoxycycline, florfenicol, azithromycin, ciprofloxacin, vancomycin and resistant to streptomycin, gentamicin, kanamycin, norfloxacin and sinomin (SMZ/TMP). Multiplex PCR assay for virulence genes showed both isolates possessed six main virulence genes: simA, scpI, pdi, pgm, cpsD and sagA genes. These results indicated that S. iniae could act as a pathogen of farmed A. baerii. This is the first report of S. iniae infection associated with mass mortality in A. baerii.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Indigenous AHL‐degrading bacterium Bacillus firmus sw40 affects virulence of pathogenic Aeromonas hydrophila and disease resistance of gibel carp

Interrupting quorum sensing represents a novel anti‐infective strategy to combat bacterial pathogen, and biodegradation of quorum sensing signal AHLs has been proved to be an efficient way to control pathogenic Gram‐negative bacteria in aquaculture. In this study, the effect of Bacillus firmus sw40 as efficient AHL‐degrading strain on virulence of fish pathogen Aeromonas hydrophila and disease resistance of gibel carp Carassius auratus gibelio was investigated. The results demonstrated that in vitro the B. firmus sw40 extracellular production (ECP) was able to significantly decrease protease production, haemolytic activity and biofilm formation in A. hydrophila. Dietary administration of B. firmus sw40 (10⁹ CFU/g) for 4 weeks significantly reduced the inflammatory cytokines TNF‐1a, TNF‐2a and IFN‐γ genes expression, antioxidant parameter MDA and GSH levels in serum and increased antioxidant enzyme SOD activity. Besides, B. firmus sw40 could significantly increase the survival of gibel carp with pathogenic A. hydrophila infection.     

A study on bacteria associated with the intestinal tract of farmed yellow seahorse,Hippocampus kuda (Bleeker, 1852): characterization and extracellular enzymes

The microenvironment of bacteria associated with the gastrointestinal tract of an animal influences the host in many ways, including the metabolism of several nutrients. Isolation, molecular characterization and enzymatic activities of culturable bacteria associated with the gastrointestinal tract of hatchery‐reared oceanic yellow seahorse, Hippocampus kuda, were investigated in the present study. 16S rRNA sequencing of bacterial isolates yielded 10 different bacterial genera belonging to two major groups: proteobacteria (62.9%) and firmicutes (37.03%). The predominant microbial flora belonged to the genera Vibrio spp., Enterovibrio sp. and Bacillus spp. Burkholderia cenocepacia, hitherto unreported from the gastrointestinal tract of marine fish, was retrieved from the seahorse gut, albeit in small numbers. Enzymatic studies indicated that the bacterial isolates possess the ability to degrade lipids, cellulose, xylan, starch and proteins. Lipase activity was exhibited by a majority of the isolates (70.4%), indicating that the bacterial flora associated with the intestinal tract of seahorses is capable of digesting foods rich in lipids. The possible implications for the role of these bacteria in the digestion and physiological processes are discussed. To our knowledge, this is the first time that the characterization and enzymatic activities of bacteria associated with the intestinal tract of farmed seahorses have been reported.     

Aeromonas veronii caused disease and pathological changes in Asian swamp eel Monopterus albus

More and more diseases were occurred in the aquaculture of Asian swamp eel. Bacterial isolates were collected from the pathogenic organs of diseased Asian swamp eels. A dominant strain, Aeromonas veronii JL‐01, was isolated and identified from the liver and intestine tissue of diseased Asian swamp eels. The symptoms of fish infected artificially with A. veronii were the same as those observed under natural infection. The mortality rate was 40%–80% and infected Asian swamp eels died 2–3 days after infection. Drug sensitivity tests showed that the isolated bacterium was highly sensitive to seven kinds of antibiotics, including cephalosporins, aminoglycoside, quinolones, tetracyclines (except Tetracycline), pheniclos, nitrofurans and sulfonamides. The bacterium was resistant to penicillins (except Piperacillin), macrolides, polypeptides and lincomycin. The histological changes in Asian Swamp Eel induced by A. veronii infection mainly occurred in the liver, intestines and kidney. A. veronii showed strong pathogenicity towards the Asian swamp eel, causing tissue damage and death. Gentamycin, doxycycline and sulfamethoxazole could be used for preventing and treat this disease.     

Comparison of Intestinal Microbiota in Intestinal Tracts of the Wild and Cultured Seahorses,Hippocampus trimaculatus (Leach)

Seahorse aquaculture has been a focus for meeting the demand of Chinese traditional medicine and aquarium, and one of the bottlenecks for aquaculture is disease. The present study focused on the intestinal microbiota between wild and cultured seahorses. Results showed that the Alcaligenaceae and Enterobacteriaceae were the dominant intestinal flora in intestinal tracts of seahorses, and Achromobacter sp., Serratia marcescens, and Serratia sp. were the dominant intestinal flora. It was the first time that two strains (TPL14 and yb97) of Achromobacter xylosoxidans were found in cultured seahorses and even in fishes. Interestingly, Vibrio, which accounted for 19.35% of the intestinal bacterial communities, only occurred in the cultured seahorses, and it was strongly involved in the intestinal diseases in seahorse H. trimaculatus.     

Feeding selectivity of the seahorse,Hippocampus kuda (Bleeker), juveniles under laboratory conditions

This study examined the feeding selectivity of Hippocampus kuda juveniles under captive conditions and evaluates different food organisms that could be used to improve hatchery‐rearing of this species. Newly born H. kuda were reared for 10 days in 60‐L capacity tanks and fed rotifers (Brachionus rotundiformis), zooplankton (mostly Pseudodiaptomus annandalei and Acartia tsuensis) alone or both food sources. The size and amount of food ingested increased as seahorses grew. Selective feeding of seahorses appeared to change as they develop, preferring copepod adults over nauplii and rotifers. A. tsuensis was highly selected by juveniles over P. annandalei. Specific growth rate in terms of body weight (SGR‐BW, 15% day^–1) was the highest and mortality rate (9% at day 10) the lowest in seahorses fed a mixed food sources. Slowest growth rate (0.3% day^–1) and highest mortality rate (60% at day 7) were observed in seahorses fed rotifers alone. These results indicate that copepods are suitable food for seahorse juveniles, but a mixture of food organisms in the rearing tank environment enhances survivorship and growth of H. kuda, thus potentially providing a source of cultured rather than wild specimens for characterizing the life history of this threatened species.     

Analysis of the diet of the long‐snouted seahorse Hippocampus guttulatus by 18SrDNA amplification of prey in faeces

Breeding in captivity for research or exhibition (e.g. in aquaria) can replace the capture of wild specimens of endangered species and allow controlled reinforcement of wild populations. With this aim, diet analysis and establishing the adequate prey are determinant factors for breeding success. However, non‐invasive approaches such as faecal DNA analysis are advisable for analysing the diet of these species. Therefore, the aim of the present study was to demonstrate the usefulness of faecal DNA analysis by specific PCR amplification of prey DNA for assessing the diet of the seahorse Hippocampus guttulatus. In a comparison of the suitability of different genes (COI, 18SrRNA and 28SrRNA), 18SrRNA was found to be the most suitable for designing specific primers for the prey types fed to seahorses (Artemia, Palaemonetes and Mysidae). The technique was assessed in feeding experiments in which prey ingestion was recorded daily, and faeces were collected for DNA extraction and presence/absence PCR analysis. Amplification of the prey DNA in faeces was consistent with the sequence of prey supplied (prey eaten the day before was always detected). Differences in the time between feeding and detection in faeces suggested prey‐specific gut passage times, which were shorter for Palaemonetes than for Mysidae. This fact highlights the importance of feeding trials to avoid overestimating the consumption of prey with long gut retention when PCR‐based methods are used. This molecular technique is thus applicable for studying the feeding behaviour of captive seahorses and could be adapted for use in other marine species.