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Introduction
The yellowtail Seriola dorsalis (Gill 1863) is a coastal pelagic species found in subtropical and temperate regions. It is distributed along the Pacific coast of California (USA) and along the Baja California Peninsula and in the Gulf of California (Mexico) (Martinez-Takeshita et al. 2015). In Mexico, yellowtail catches in 2016 comprised nearly 455 t, with an approximate value of 611,000 USD (SEPESCABC 2017). Due to its high market value, adaptation to captivity (Poortenaar et al. 2001), rapid growth (Moran et al. 2009), and resistance to handling, this species is highly prized in recreational fisheries and is considered the target for aquaculture expansion in Mexico (commonly sold as hamachi or baja hiramasa in the seafood industry; Martinez-Takeshita et al. 2015). Currently, yellowtail commercial aquaculture operations are carried out mainly in the Baja California Peninsula, Mexico.
Marine fish culture, both in hatchery and grow-out systems, is prone to parasitic infestations, which lead to fish health impairment and generally to high mortality rates (Nakada 2002). The dinoflagellate Amyloodinium ocellatum causes one of the most serious diseases in fish species, amyloodiniosis, which inflicts the greatest damage on commercial fish aquaculture (Fajer-Avila et al. 2012). In the culture of the genus Seriola, the recurrent mortality outbreaks reported worldwide have been caused by ectoparasites, such as the protozoan A. ocellatum and the monogeneans Zeuxapta seriolae and Benedenia seriolae (Aiello and D’Alba 1986, Thoney and Hargis 1991, Montero et al. 2004, Reyes-Becerril et al. 2015).
The parasite A. ocellatum has a direct life cycle and it can easily multiply and disperse itself in confined areas, reaching very high infestation intensities that lead to mass mortality in fish cultures a few days from the outbreak. This parasite can also cause injuries in the gill tissue with several pathologies, such as lamellar fusion, high number of mucous cells, hyperplasia, and decreased gas exchange (Fajer-Avila et al. 2012), and clear changes in blood parameters. Monitoring fish health using blood analysis as a direct or inferential indicator is important and recommended. A blood analysis provides relevant information about the tolerance to a stressor agent and the health condition of fish under a parasitic outbreak (Del Rio-Zaragoza et al. 2008, Del Rio-Zaragoza et al. 2011).
In recent years, commercial aquaculture of yellowtail in Baja California has undergone numerous parasitic infection cases. Fortunately, no mass mortality events have been reported in this area (Reyes-Becerril et al. 2015). The present work thus aimed to evaluate the effects of parasitic infection (A. ocellatum) in juvenile yellowtail in terms of mortality, gill histology, and blood parameters.
Material and methods
Fish and experimental system
Juvenile S. dorsalis were obtained from Ocean Baja Labs (Eréndira, Baja California) through the Centro de Investigación Científica y de Educación Superior de Ensenada. Organisms (n = 530) were transported to our facilities at the Universidad Autónoma de Baja California (UABC). Upon arrival, fish (n = 30; 69.53 ± 9.68 g, grand mean ± SD) were stocked in a 10-m3 tank with water exchange rate at approximately 10% per hour. Ten fish were examined and no signs of disease or parasites were observed. Fish were acclimated for 2 weeks in this tank. Two experiments were performed at different times to evaluate the effect of A. ocellatum on the gill tissue and examine blood parameter changes in infected fish.
In the 2 experiments, fish were randomly distributed in twelve 500-L tanks in a closed recirculating aquaculture system (RAS) connected to a PolyGeiser biofilter (0.17 m3). Filtered seawater passed through a 40-W ultraviolet light. The RAS was equipped with an 1,100-L water reservoir and a 6,000-W titanium heater. Seawater was delivered to the tanks with a 1/3 HP water pump, with daily water renewal set to 5%.
A diet was formulated to contain 46% crude protein and 16% lipids as previously tested (Guerra-Olvera and Viana 2015, Rombenso et al. 2016). This diet was manufactured at the Laboratorio de Investigación y Desarrollo de Alimentos para Acuacultura (LINDEAACUA-UABC) under an extrusion process for floating feed (3 mm), in which an EXTRU-TECH E325 extruder was used and feed was dried in a horizontal air dryer (EXTRUTECH, USA). During both trials fish were hand fed 3 times a day (8:00, 13:00, and 18:00) until apparent satiation. Water temperature was maintained at 23.9 ± 1.6 ºC, dissolved oxygen at 6.0 ± 0.5 mg·L-1, salinity at 34.6 ± 0.4, and pH at 8.1 ± 0.3.
Parasitic induction
In the first experiment, juvenile yellowtails (n = 96) were randomly distributed in 12 tanks (8 fish per tank) that had been previously exposed to a natural outbreak of A. ocellatum. Organisms were acclimated in the experimental system for 10 d at 18 ºC. Then, as a stress factor, water temperature was increased to 24 ± 1 ºC (1 ºC per day) to stimulate the development of the parasitic outbreak. Once water temperature reached 24 ºC, it was kept constant throughout the experiment. External signs of disease and fish mortality were monitored daily. Gills of dying fish were sampled at the end of the experiment.
Gill analysis
Gills from the right side of each dying fish were removed (n = 12) and placed on Petri dishes with sea water. Amyloodinium ocellatum was identified following the description by Lom and Dykova (1992). Parasites were counted using a stereomicroscope (Meiji, EMZ-TR; Tokyo, Japan) at ≥20×. Prevalence (%) and mean intensity of infection were determined as described by Bush et al. (1997).
Gills from the left side of each dying fish were removed and fixed in 10% buffered formalin. The second gill arch was then removed from the whole left gill, then dehydrated in graded ethanol series following standard histological procedures, and finally embedded in paraffin. Gills were stained with haematoxylin and eosin and mounted in acrylic resin of low viscosity (Cytoseal 60 Richard-Allan Scientific; Kalamazoo, MI, USA) for light microscopy.
Blood parameters
In the second experiment 132 juvenile yellowtails without parasites were randomly distributed in the 12-tank system (11 fish per tank), which was previously exposed to a natural outbreak of A. ocellatum. Fish were closely monitored, and blood samples were collected from dying fish. Fish from the same batch, but stocked in an independent RAS, were maintained under similar culture conditions to be used as control (healthy fish). No signs of disease or parasites were observed in this control group, and blood samples were also taken from this group.
Blood sample collection
Healthy fish (n = 23; 24.87 ± 4.37 g, 12.97 ± 0.88 cm) and infected fish (n = 12; 38.41 ± 10.62 g, 17.28 ± 1.54 cm) were carefully handled to minimize stress. Fish were anesthetized with 0.5 mL·L-1 of 2-phenoxyethanol (Sigma; St. Louis, MO, USA) following UABC protocols. In less than 3 min, blood samples were collected from the caudal vein using 1 mL non-anticoagulant tuberculin syringes (BD PlastipakTM, Mexico). Blood aliquots were placed into 2 tubes immediately after sampling. The first tube had no anticoagulant and was used for the hematocrit test (HCT). The remaining blood was centrifuged for 10 min and the serum was stored at -20 ºC for analysis of total protein, albumin, globulin, glucose, and triglyceride concentrations. The second tube had K2EDTA (BD Microtainer; Franklin Lakes, NJ, USA) to prevent coagulation and was used for hemoglobin concentration, total red blood cell (RBC) count, and total white blood cell (WBC) count.
Blood parameter analysis
HCT was measured using a blood sample in a heparinized 2/3 filled capillary tube (LEEX Equipment, Mexico). The tube was sealed and placed in a micro-hematocrit centrifuge at 7,000 rpm (Premiere XC-3012, Mexico) for 10 min. The packed cell volume was measured using a hematocrit reader and reported as a percentage (Del Rio-Zaragoza et al. 2008). Hemoglobin in erythrocytes was determined using a HemoCue Hb 201 analyzer following the manufacturer’s instructions (HemoCue AB; Angelholm, Sweden). The Natt and Herrick’s (1952) method was used for total RBC and WBC-plus-thrombocyte counts. Cells were counted using a Neubauer hemacytometer under an optical microscope (Zeiss, PrimoStar; Göttingen, Germany). Mean corpuscular volume, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated by using standard formulas with the HCT, RBC, and hemoglobin data. Total protein, albumin, globulin, glucose, and triglycerides were determined from blood serum using colorimetric kit assays (MexLab Group; Jalisco, Mexico) following the manufacturer’s instructions. Globulin was obtained by subtracting the value of albumin from total protein.
Statistical analyses
All measurements were done in triplicate for each fish. The normality test and t-test (P < 0.05) were applied to all data using SigmaStat 4 software (Systat Software; San Jose, CA, USA). All data reported in percentages were arcsine transformed before statistical comparisons. A non-parametric test (Mann-Whitney rank sum test) was used for data that failed to show normality.
Results
Fish mortality (16.7%) due to parasitic infection began 3 d after temperature reached 24 ºC. Mortality exceeded 50% a day later, and by the fifth day, mortality was more than 80% (Fig. 1a). The analyzed yellowtails showed 100% parasitic prevalence with mean intensity of 766 ± 500 parasites per fish throughout the experiment (Fig. 1b). Histological analyses indicated attachment of A. ocellatum to the gill, and gill tissue reactions were characterized by inflammation of vascular structures, epithelial detachment, proliferation of mucous cells (Fig. 2a), hyperplasia, fusion of secondary lamellae (Fig. 2 b, c), and telangiectasia (Fig. 2d).
Figure 1 (a) Percent cumulative mortality of juvenile yellowtail Seriola dorsalis during 8 d of exposure to Amyloodinium ocellatum and (b) mean parasitic intensity of A. ocellatum on right side S. dorsalis gills.
Figure 2 Histopathology of the second gill arch of juvenile yellowtail Seriola dorsalis associated with Amyloodinium ocellatum. (a) Gill lamellae with inflammation of vascular structures, proliferation of mucous cells, and hyperplasia in the distal parts of gill filaments; (b) gill lamellae with proliferative epithelial cells causing lamellar fusion; (c) gill filament with severe acute fusion of secondary gill lamellae; (d) gill filaments with fusion of secondary gill lamellae and telangiectasia. Arrow heads indicate the presence of A. ocellatum attached to the gill of the host. Haematoxylin and eosin stain. Images at 100× (a) and 400× (b, c, d).
Blood parameters of infected fish revealed 100% parasitic prevalence, with mean intensity of 882.2 ± 265.1 parasites per fish. Counts for RBC, hemoglobin concentration, MCHC, glucose concentration, and triglycerides were significantly higher (P < 0.05) in infected fish relative to healthy fish. In addition, mean corpuscular volume, total protein, albumin, and globulin were significantly lower (P < 0.05) in infected fish than in healthy fish. No significant differences were found in HCT, MCH, and WBC counts (P > 0.05) between healthy and infected fish. However, in the WBC counts an increase of 1.52 × 103 mm3 was observed in the infected fish group (Table 1).
Table 1 Blood parameters for healthy and infected (by Amyloodinium ocellatum) yellowtail Seriola dorsalis. Data are mean ± standard deviation.
| Parametersa | Healthy fish (n = 23) |
Infected fish (n = 12) |
| Hematocrit (%) | 51.91 ± 5.70 | 49.50 ± 6.84 |
| RBC (× 106 mm3) | 4.19 ± 0.79 | 5.22 ± 1.13* |
| Hemoglobin (g·dL-1) | 13.69 ± 2.01 | 16.02 ± 1.86* |
| MCV (fL) | 127.53 ± 25.70 | 99.21 ± 28.20* |
| MCH (pg) | 33.63 ± 7.69 | 32.11 ± 8.62 |
| MCHC (g·dL-1) | 26.75 ± 5.16 | 32.98 ± 6.09* |
| WBC (× 103 mm3) | 26.26 ± 9.74 | 27.78 ± 6.03 |
| Total protein (g·dL-1) | 4.42 ± 0.89 | 3.19 ± 0.42* |
| Albumin (g·dL-1) | 1.36 ± 0.34 | 1.13 ± 0.10* |
| Globulin (g·dL-1) | 3.06 ± 0.75 | 2.06 ± 0.38* |
| Glucose (mg·dL-1) | 64.25 ± 12.01 | 115.66 ± 14.52* |
| Triglycerides (mg·dL-1) | 48.61 ± 13.18 | 141.36 ± 45.43* |
aRBC, red blood cells; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cells.
*Significant differences (P < 0.05); n = number of fish analyzed
Discussion
Mexico has great potential for commercial mariculture operations and land-based hatchery production. However, mariculture operations are prone to parasitic infestations that can impair fish health, generally causing mortality. In this study we assessed the effect of a parasitic outbreak of A. ocellatum that resulted in mortality, induced gill damage, and changes in blood of juvenile yellowtail reared in experimental conditions. The A. ocellatum outbreaks found in our study have also been frequently observed in the bullseye puffer (Sphoeroides annulatus), spotted rose snapper (Lutjanus guttatus), Pacific red snapper (Lutjanus peru), yellow snapper (Lutjanus argentiventris), and mullet snapper (Lutjanus aratus), with nearly 100% mortality rate (Pérez-Urbiola et al. 2008, Fajer-Ávila et al. 2011). Rearing conditions beyond the optimum range for juvenile S. dorsalis, such as high temperatures (>24 ºC) and poor water quality, are likely to cause stress and are thus thought to promote protozoan outbreaks. In this context, our results highlighted a cumulative mortality rate of 100% 8 d postinfection, where highest mortality occurred between the fourth and the fifth day after detection of the parasite. Though mean intensity of A. ocellatum was lower than intensity on the third day, differences were not statistically significant. Similarly, in juvenile leopard groupers (Mycteroperca rosacea) and the juvenile red drum (Sciaenops ocellatum), proliferation of this dinoflagellate resulted in a cumulative mortality rate of 89.5% and 90.0%, respectively, 7 d post-infection, where highest mortality occurred after 48 h in both species (Li et al. 2005, Reyes-Becerril et al. 2008). A high mortality rate was also observed in seabream 2 d after Amyloodinium was detected (Soares et al. 2012).
Amyloodinium ocellatum infection in yellowtails was characterized by initial signs of appetite loss, followed by skin opacity with whitish spots. The fish started swimming sideways and rubbing themselves against the tank walls. Fast gill movement was also observed. Behavior of severely infected S. dorsalis during the present study was similar to that reported by Kuperman and Matey (1999), where fish rapidly gasped for air, swam spastically and constantly at the surface before sinking back to the bottom, jumped out of the water, and finally lost equilibrium and died. Similar cases have also been observed for the spotted rose snapper (Ontiveros-García 2008).
Our findings revealed severe infection in juvenile S. dorsalis, with hundreds of parasites per fish and 100% parasite prevalence in the gills of examined fish. The intensity of infection by A. ocellatum on gill filaments was high and similar to the numbers reported by Kuperman and Matey (1999) and Reyes-Becerril et al. (2008).
Amyloodinium ocellatum infect gill and skin epithelia. Rhizoids, which are root-like structures that parasites use to penetrate and grasp and that probably do not absorb nutrients, anchor the parasite to the host cells. The stomopode could be a source of digestive enzymes that are injected into host cells or could serve as a feeding tentacle that gathers cell fragments severed by the pulling motion of the rhizoids (Noga and Levy 2006, Soares et al. 2012). High infection levels were further associated to severe inflammation, epithelial detachment, hyperplasia, fusion of secondary lamellae, telangiectasia, and proliferation of mucus cells in the gills of S. dorsalis. Similar responses, except telangiectasia, have also been observed for L. guttatus (Ontiveros-García 2008). Meagre (Argyrosomus regius) and seabream (Sparus aurata) gills showed extensive areas with hyperplasia and necrosis of the epithelium (Soares et al. 2012). Del Rio-Zaragoza et al. (2010) observed that gill parasite infection by dactylogyrid monogeneans could stimulate mucus production in the host as a protective mechanism in response to parasitism, as observed for S. dorsalis with A. ocellatum in our study. However, production of excess mucus could cause hypoxia, inducing gill dysfunction in fish and, consequently, the death of the fish.
In the present study, a notorious physiological response was the increment in several blood parameters in the infected group, such as the number of erythrocytes, hemoglobin, and MCHC, likely associated with gas transport and absorption issues or hemoconcentration caused by gill damage, dehydration, or stress. Lower levels in these blood parameters indicate anemia or hemodilution due to osmoregulation impairment (Wedemeyer and McLeay 1981). These results are in agreement with previous reports on L. guttatus and M. rosacea (Ontiveros-García 2008, Reyes-Becerril et al. 2008). Mean corpuscular volume resulted in a low count in infected fish, which reflects the size of the red blood cells, expressed as the volume of erythrocytes. In the present study, cell size adjustment was observed, which indicated that as the number of parasites increased, the number of erythrocytes increased. However, the diameter of erythrocytes decreased, causing a compensatory response from erythrocytes. This response was possibly caused by higher oxygen demand from infected organisms, due to the likely deficient gas exchange as a consequence of the obstruction and epithelial damage by A. ocellatum trophozoites in S. dorsalis gill filaments (Ontiveros-García 2008). Glucose and triglyceride concentrations were significantly higher in infected fish than in healthy ones. In the case of glucose, hyperglycaemia in yellowtail likely lead to chronic stress. The effect of chronic stress by exposure to a continuous sublethal agent is a long-term effect that alters the immune response of the organism (Wendelaar-Bonga and Balm 1999). No differences were found in HCT, MCH, and WBC counts (P > 0.05) for juvenile S. dorsalis. However, in the WBC counts an increase of 1.52 × 103 mm3 was observed in the infected fish group. Similarly, a positive correlation between the level of infection by dactylogyrid monogeneans and the total number of leucocytes was also observed by Del Rio-Zaragoza et al. (2011), indicating that this is a protective response to parasitic stress. In addition, total protein, albumin, and globulin levels were significantly lower (P < 0.05) in infected fish than in healthy ones. The low total protein levels found in infected fish could be associated with the presence of A. ocellatum, and total protein could be used to evaluate the physiological state and general condition of the fish as previously suggested (De Pedro et al. 2005). The decrease in protein concentration has been attributed to illness (e.g., liver damage), decreased nutrient absorption, nutritional deficiency, starvation, and infectious diseases (Wedemeyer and McLeay 1981, Del Rio-Zaragoza et al. 2011).
Changes in blood parameters could offer a direct or indirect assessment of the health and physiological state of the organism. For this reason, evaluation of haematological and blood chemistry parameters could help detect diseases and sublethal conditions affecting fish under culture conditions (Del Rio-Zaragoza et al. 2010). The present findings demonstrated that A. ocellatum infection caused severe gill damage and thus affected gas exchange efficiency, resulting in blood parameters changes and, consequently, high mortality rates in a short-term period. This study is the first contribution to acknowledge the effect of A. ocellatum on juvenile yellowtail and provide valuable insights for future studies implementing strategies to prevent and control amyloodiniosis in this species, which is of high commercial value.
