Abstract: Background and Objective: Tilapia hatchery sector at Kafrelsheikh was suffered from increased mortality in newly hatched fry that results in severe economic losses, the present research was conducted to determine the possible causes of fry mortality affecting two Oreochromis niloticus hatcheries at Kafrelsheikh governorate. Materials and Methods: A total number of 100 tilapia fry from two affected hatcheries were randomly collected for clinical and parasitological examination, another 150 naturally infected tilapia fry was used in the treatment trial using Viroscan or formalin-malachite green combination for determination of their efficacy in controlling of such infection. Water temperature, dissolved oxygen, ammonia, pH and salinity were also determined. Results: The cumulative mortality was estimated by 14 and 10% during 21-day post hatch. Temperature, dissolved oxygen, pH and salinity were suitable for Oreochromis niloticus fry rearing while, ammonia level was on the borderline. The main clinical signs observed on diseased fry were darkening color, tail erosion and swimming near the water surface. Microscopic examination revealed infestation with Gyrodactylus and Trichodina. The total prevalence of parasitic infestation was 92 and 84%, the mean intensity was 25.97±7.28 and 17.3±3.92 parasite/fry for Gyrodactylus, 33.92±8.59 and 13±3.51 for Trichodina in first and second hatchery, respectively. Gyrodactylus infestation controlled with long bath treatment using formalin-malachite green combination (50+0.2 ppm)/litter for 4 h while either the recommended dose (25+0.1 ppm) or Viroscan at (100 and 50 ppm) was ineffective. Trichodina resist all treatments after 4 h of application. All the applied treatments were safe for the fry but the high concentration of formalin-malachite green causes limited mortality (3.33%). Conclusion: Gyrodactylus sp. and Trichodina sp. were the direct causes of mortality in affected hatcheries, Formalin-malachite green mixture (50+0.2 ppm) was effective in controlling Gyrodactylus infestation also they considered safe at this concentration.
INTRODUCTION
Egypt is the second global tilapia producer, in 2014 the Egyptian production reached 867557 metric ton1. Fish fry (seeds) are the main element in aquaculture, tilapia hatcheries at Kafrelsheikh district produce about 291.300 million units that represents 88% of the total Egyptian production2.
External parasites have a potential risk in pisciculture, they have direct effect on growth and survival of affected fish3, it also play a role as a predisposing cause for bacterial infection through skin abrasions induced by these parasites and consider a route of entry for bacterial fish pathogens. Fry are considered more susceptible to external parasitic infestation than adult fish.
The Gyrodactylus species are among the monogenean flatworms parasitizing a great variety of brackish and freshwater fish, they develop directly without any intermediate hosts4-6. Gyrodactylus is provided with two large central hooks and 16 marginal booklets in their opisthaptor, these hooks cause severe irritation and damage to infested fish skin, gills and fins that adversely affect fish health7.
Trichodinids are the most common ciliate parasites present on the skin of pond-reared fish, it can cause severe economic losses in aquaculture8. Heavy infection with Trichodina sp., particularly in small fish has caused gigantic financial losses9,10. Infected fish are lethargic, generate excessive mucus and become off-feed eventually which results in considerable deaths11.
Formalin and malachite green are extensively used as biocides in the aquaculture industry, they are effective against ectoparasites.
Formalin is effective parasiticide against most of external fish parasites including monogenean trematodes and protozoa12. The antiparasitic effect of formalin is achieved through denaturation of the ectoparasite protein, this mechanism occurs as a result of its reaction with aromatic rings and thiol group and other active groups of the protein molecules13.
Malachite green is a triphenylmethane dye, it has a potent antiparasitic activity and so it is widely used as bath treatment in aquaculture. The mechanism of malachite green action is achieved through destruction of respiratory chain proteins of bacteria, fungus and small parasites resulted in pathogen death14.
Currently, Viroscan is one of the most frequently used disinfectants in tilapia farms and hatcheries at Kafrelsheikh, it used to reduce fish mortality caused by bacterial infection and is to reduce fry mortality caused by parasitic infestation at a dose of 0.5 ppm.
Many tilapia hatcheries in Kafrelsheikh suffered from increased mortality. So, the aims of the present study were to determine the direct cause of mortality and to estimate the efficacy of Viroscan in termination of such infestation in comparison with traditional method using formalin and malachite green and to study the safety of used chemicals on treated fry.
MATERIALS AND METHODS
Study area: Samples were taken from two tilapia hatcheries suffered from fry mortality in Torombat seven, Rhyead district, Kafrelsheikh governorate northern Egypt. The studied hatcheries lie between longitudes 30.9564° East and latitudes 31.3842° North, this study was conducted in April and May, 2016.
Fish: A total number of one hundred tilapia fry were randomly sampled, fifty fry was microscopically examined from each hatchery and another 150 infested fry were collected for experimental treatment trial, examined fry were ranged between 10-15 mm in the total length.
Clinical examination: Clinical examination was performed as described by Noga12.
Parasitological examination: Parasitological examination was performed using binocular microscope, each five fry were picked up to a glass Petri dish contain pond water and methane-sulfonate (MS-222) anesthetic at a concentration of 50 mg L–1 as recorded by Popovic et al.15 for preventing fry movement during the microscopical examination, types and number of the present parasites were recorded.
Experimental treatment trial: This trial was conducted to determine the efficacy of formalin and malachite green combination or Viroscan in controlling Gyrodactylus infestation.
Chemicals used in the treatment trial: Formalin 37% solution ADWIC, Co., Egypt, Malachite green Alpha chemika, India and Viroscan® disinfectant, composition: Alkyl dimethyl benzyl ammonium chloride 240 g L–1, glutaraldehyde 107.25 g L–1, isopropanol 146.25 g L–1 and pine oil 20 g L–1, SLANT CHEM, Co.
Experiment design: One hundred and fifty infested Oreochromis niloticus (tilapia) fry were used for experimental treatment trial, fry was divided randomly into five equal groups, in triplicates, each group contain 10 infested fry, each group was kept in 20 L glass aquaria supplied with hatchery water with continuous aeration using air pump Boyo®, fry grouping and the used chemicals doses are presented in Table 1.
| Table 1: | Fry grouping, chemicals used in treatment trial and their concentrations |
Fry was grossly observed for clinical signs and mortality they were also, microscopically examined for the survival and activity of Gyroductylus every 30 min during the first hour then hourly.
Tested water parameters
Water temperature: Water temperature was measured using floating thermometer according to the method number 2550 described by APHA16.
Dissolved oxygen: Dissolved oxygen was determined according to the method number 4500 described by APHA16 depending on the electrometric method using membrane electrodes that based on the rate of diffusion of molecular oxygen across a membrane. Dissolved oxygen was determined using portable dissolved oxygen meter for aquaculture (HANNA, Italy).
Ammonia: The NH3 was determined using low range ammonia portable photometer (HANNA, Italy) based on Nessler method as described by the manufacturer instructions.
pH: The activity of the hydrogen ions was determined according to the method number 4500 H+ described by APHA16 depending on electrometric method by potentiometric measurement using a standard hydrogen electrode and a reference electrode. It was measured using pH meter (HANNA, Italy).
Salinity: Salinity was measured using refractometer as described by Zhao et al.17.
Statistical analysis: One sample T-test was used to determine the mean intensity of parasitic infestation per fry expressed as mean±standard deviation using MedCalc for windows, version 17.2.2, (MedCalc Software, bvba, Ostend, Belgium, https://www.medcalc.org; 2017).
RESULTS
The recorded cumulative mortality (during first 21-day post hatch) was 14 and 10% in the first and second hatchery, respectively. Affected fry appeared dark in color (Fig. 1a), swim near the water surface, easily caught (lost escape reflex) and were smaller in size than the normal one of the same age. Microscopic examination of affected fry revealed the presence of tail erosions (Fig. 1b), Gyrodactylus attached to eroded fry tail (Fig. 1c), nearly all fry had mixed infestation with both Gyrodactylus and Trichodina (Fig. 1c, 2d).
There was not any evidence observed during the microscopical examination of affected fry indicating the presence of bacterial infection as skin ulceration, hemorrhage or scale desquamation.
The parasitological examination revealed the presence of mixed infestation with Gyrodactylus and Trichodina and very rarely to be alone.
Gyrodactylus was 440-510 μm in length, prohaptor has 2 cephalic lopes and was 42 μm in diameter, opisthaptor diameter was 64 μm in diameter and has one pair of central hooks (Fig. 1c, d).
Trichodina was cub shaped 40-50 μm in diameter and was 30 μm in height (lateral view), with an obvious denticulate ring and adhesive disk (Fig. 2a, b).
The total prevalence of parasitic infestation in the first hatchery was 92% (46 out of 50) the prevalence of infestation with Gyrodactylus and Trichodina was 86 and 78%, respectively, the intensity was 25.97±7.28, 33.92±8.59 and the range was 12-37 and 17-46 parasite per fry for Gyrodactylus and Trichodina, respectively (Table 2-4).
The total prevalence of parasitic infestation in the second hatchery was 84% (42 out of 50) the prevalence of infestation with Gyrodactylus and Trichodina was 80 and 70%, respectively, the intensity was 17.3±3.92, 13±3.51 and the range was 9-23 and 5-18 parasite per fry for Gyrodactylus and Trichodina, respectively (Table 2-4).
Treatment trial revealed the efficacy of formalin-malachite green at a concentration of (50+0.2 ppm) in partial control of Gyrodactylus infestation as it was effective in decreasing monogenean parasite population to 50% (kill 50% of Gyrodactylus) and decrease the vitality of remaining portion after 4 h of application, while application of formalin-malachite green at a concentration of (25+0.1 ppm) have a limited effect restricted in decreasing parasite motility after 3 h of application and all parasites remain live after 4 h post application.
| Fig. 1(a-d): | (a) Oreochromis niloticus fry suffered from tail erosion disease with obvious dark body coloration, (b) Diseased fry tail with marked erosion, X = 40, (c) Diseased fry tail with mixed infestation, Gyrodactylus (black arrow) and Trichodina (blue arrow), X = 100 and (d) Gyrodactylus attached to fry tail with opisthaptor carry 16 marginal hooklets and two large central hooks, X = 400 |
| Table 2: | Total prevalence of parasitic infestation in monosex Oreochromis niloticus fry |
| Table 3: | Prevalence of Gyrodactylus and Trichodina infestation in monosex Oreochromis niloticus fry |
| Table 4: | Intensity of Gyrodactylus spp. and Trichodina spp. infestation in monosex Oreochromis niloticus fry |
Treatment with viroscan at a concentration of 100 or 50 ppm was also not effective in Gyrodactylus eradication as it is decreasing parasite motility 2 and 4 h after application but not kill any parasite (Table 5).
All treatment was ineffective in termination of Trichodina infestation, the protozoan remains viable, motile and attached to infested fry during treatment even after 4 h of application.
All treatments were safe to Oreochromis niloticus fry, they were not having any adverse effect on treated fry and so considered generally safe but formalin-malachite green (50+0.2 ppm) caused 3.33% mortality after 4 h of application (Table 6).
| Fig. 2(a-d): | (a, b) Trichodina aboral surface surrounded by cilia with obvious adhesive disk and denticulate ring, X = 400 in a, 1000 in b, (c) Dead detached Gyrodactylus after treatment, X = 400 and (d) Mixed infestation with numerous Gyrodactylus and Trichodina, X = 40 |
| Table 5: | Effect of different treatments on Gyrodactylus spp. |
-ve: Parasite viability and activity (movement or motility) not affected, +(slight): All parasite remains live but motility of 40-50% of examined parasites decreased, ++(moderate): Majority of parasite remain live but the motility of 70-80% of examined parasites decreased, +++(severe): More than 50% of examined parasites contract without any motility (die) and the rest have sluggish movement | |
| Table 6: | Effect of different treatments on monosex Oreochromis niloticus fry |
| -ve: Fry activity not affected, +: Activity of 3 fry decreased and swim near aquarium floor, ++: One fry die (3.33%) | |
| Table 7: | Hatchery tested water parameters |
Tested hatchery water parameters were presented in Table 7, water temperature, dissolved oxygen, pH and salinity were in the acceptable rage suitable for normal growth of Oreochromis niloticus fry in both hatcheries. Ammonia level was slightly high in first and on borderline in the second one, generally acceptable ammonia level is less than 0.06 mg L–1.
DISCUSSION
The ectoparasitic diseases play an important role in the economic losses in farmed fish through direct mortality or decreased growth rate of fry or fingerlings18-20. The majority of fish ectoparasitic protozoa are commensals but some of them may produce serious diseases and mortality especially in smaller fishes21,22. Trichodina spp. are a group of dorso-ventrally flattened oval ciliated protozoan parasites of marine and freshwater species of finfish. Trichodina spp. can cause extensive fish mortality in aquaculture systems23. Gyrodactylus is an ectoparasite of potential significance to tilapia culture, clinical outbreaks of gyrodactylosis have been recorded in pond-reared tilapia worldwide24,25. Protozoans and monogenean parasites are frequently infesting cultured tilapia fry affecting fish growth26,27. The most frequent parasites infesting masculinized Oreochromis niloticus fry during first 22 day post hatch were Trichodina and Gyrodactylus cichlidarum (Monogenea)28.
The clinical signs including mortality, dark coloration and loss of condition that were observed on diseased fry could be attributed to heavy infestation with both Gyrodactylus and Trichodina, heavily infested fish do not eat well and lost their body conditions as described by Klinger and Floyd29, Soliman et al.30. Paperna31 recorded heavy infestation with trichodinids in cichlids fry 10-12 mm long which is similar to examined fry length, small fish and fry are especially susceptible to parasitism with Trichodina sp.32. Tail erosion is one of the most dominant clinical signs in this disease, this sign greatly affect fish swimming behavior and subsequently decrease fish speed and negatively affect fry competition for feed that subsequently resulted in underfeeding (small fry).
In the present work the recorded mortality was 14 and 10% in the first and second hatchery this is mainly due to increased prevalence (86 and 80%) for Gyrodactylus and (78 and 70%) for Trichodina. The incidence of parasitic infestation also followed the same trend (25.97±7.28 and 17.3±3.92) for Gyrodactylus and (33.92±8.59 and 13±3.51) for Trichodina in first and second hatchery, respectively. This trend is mainly due to the direct life cycle of both parasites that transmitted through direct contact and via water so increased mortality, prevalence and incidence follow the same manner (directly proportional to each other). These observations supported by Thoney and Hargis33, who reported, rapid propagation and direct transfer of monogenean parasites between fish.
Microscopic examination showed tail erosions, it could result from destruction induced by the attachment organs of infesting parasites i.e., the two central hooks and the marginal hooklets of opisthaptor in Gyrodactylus and denticulate ring that carry many sharp denticles in Trichodynia. These attachment organs mainly responsible for irritation and tearing of fry tail and fins.
Chemical treatments are the most common method used in controlling fish diseases in aquaculture34. It is the most successful way in termination of parasitic infection35, it provides short-term control and is effective against attached parasite stages36. In the current research formalin-malachite green combination at the recommended concentration (25+0.1 ppm) appear to be ineffective or have a limited activity restricted only in decreasing the parasite motility, while duplicated concentration i.e. (50+0.2 ppm) is more effective against Gyrodactylus, Rowland et al.37 recorded the ineffective effect of formalin at 25 ppm against Gyrodactylus, while they observed a significant decrease in parasite numbers occurs at 40 ppm. Srivastava et al.38 and Schelkle et al.39 also recorded the effectiveness of formalin-malachite green combination against Gyrodactylus. Anti-parasitic effect of formalin is induced through denaturation of parasite protein together with irreversible uncoupling of oxidative phosphorylation achieved by malachite green.
Trichodina was resistant to all treatment that may be due to insufficient concentration of formalin used in current study, Smith23 recorded that effective concentration of formalin against Trichodina is 170-250 ppm for 1 h, Hassan21also, found that formalin long path (250 ppm) for 24 h is effective in termination of Trichodina infestation in Oreochromis niloticus.
Formalin-malachite green combination appears to be safe for fry, as only one fry dies after 4 h of application, Perera and Pathiratne 40 indicated the safety of formalin when used for therapeutic purposes at a dose of 50 ppm in tilapia fingerlings without any adverse effect on treated fish. Omoregie et al.41 determined the 96 h LC50 of malachite green in Oreochromis niloticus fingerlings by 0.427 ppm, that indicated its safety when used at 0.2 ppm.
Viroscan and structurally related compounds are the most commonly used disinfectant in controlling mortality at Oreochromis niloticus hatchery in Kafrelsheikh governorate so it was important to determine their efficacy against Gyrodactylus infestation. Both Viroscan concentrations (50 and 100 ppm) were ineffective in such disease termination even it not only appears safe but also well tolerated by Oreochromis niloticus fry. Viroscan (quaternary Ammonium Compounds) are active against bacteria, molds and some viruses by bind irreversibly to the negatively charged phospholipids and proteins of the cell membranes of microorganisms, impairing membrane permeability42. This explain ineffectiveness of Viroscan in controlling the parasitic infestation, despite this product contain glutaraldehyde which works similarly to formalin but its concentration, when calculated alone, was 10 ppm (when viroscan used at 100 ppm) which consider insufficient to affect Gyrodactylus.
Measured water parameters were within the acceptable limit for optimum fry rearing as mentioned by El-Sayed27 , so they are not incorporated in this disease condition. Ammonia level was slightly elevated or on the borderline which consider a stress factor predisposing to parasitic infestation, this coincides with the observations recorded by Jimenez-Garcia et al.28. Stressful environmental conditions decrease the resistance of fish to monogenean infections43.
The current research results regarding the clinical signs and microscopical examination indicating the absence of any evidence for bacterial infection together with acceptable hatchery water quality indicating the responsibility of parasitic infestation for the occurrence of the erosion disease affecting Oreochromis niloticus fry at examined hatcheries.
CONCLUSION
In conclusion Gyrodactylus sp. and Trichodina sp. were the direct causes of mortality in affected tilapia hatcheries, the mortality percent is directly proportional to the incidence of infestation. Formalin-malachite green mixture at (50+0.2 ppm) was effective in controlling Gyrodactylus infestation also they considered safe at this concentration while the lower concentration does not affect the parasite survival. Viroscan fails to kill Gyrodactylus after 4 h of exposure at the tested concatenations (100 and 50 ppm). The low concentration of Formalin-malachite green mixture and Viroscan should not be used at the tested concentration in controlling Gyrodactylus infestation in Oreochromis niloticus fry.
SIGNIFICANT STATEMENT
This article identified Gyrodactylus and Trichodina as the actual causes of the huge economic losses in tilapia hatcheries, it also, proves the inefficacy of the most commonly used drug in tilapia hatcheries (Viroscan) together with inefficacy of formalin-malachite green mixture at (25+0.1 ppm) while when it used at (50+0.2 ppm) it was effective and safe in controlling this disease.