301 Bulgarian Journal of Agricultural Science, 12 (2006), 301-309 National Centre for Agrarian Sciences New Tendencies for Therapy Against Parasitic Crustacean in Fish N. ANEV National Agency of Fisheries and Aquaculture, BG - 4000 Plovdiv, Bulgaria Abstract ANEV, N., 2006. New tendencies for therapy against parasitic Crustacean in farms. Bulg. J. Agric. Sci., 12: 301-309 Parasitic crustaceans that are most frequently found in rearing of freshwater species cause diseases such as Lerneosis and Argulosis on freshwater that are widely spread both in Europe and North America and Asia as well. Susceptible to diseases are carp (Cyprinus carpio), silver (Carassius auratus gibelio) and gold (Carasius carasius), white (Hypophthalmichthys molitrix) and bright big-head carp (Aristichthis nobilis), white grass carp (Ctenopharingodon idella), bream (Abramis brama), arched trout (Salmo gairdneri irideus), etc. Key words: parasitic crustaceans, lerneosis, argulosis Introduction Over the last couple of years the disease Lerneosis has been established both in natural and artificial s of this country. Unlike Lerneosis, Argulosis is a more frequently found disease that has been registered long enough. (Маrgaritov, 1969, 1972; Каranikolov and Radev, 1978). Those causing the infections with parasites are respectively the species: Lernaea cyprinacea and Argulus foliaceus. The first case of clinically manifested Lerneosis in carp (Cyprinus carpio L.) in Bulgaria was described on 11.10.1993 in the village of Koniovo, Nova Zagora district from Daskalov and Ivanov (1994). The development of the parasitic cray goes through metamorphosis (Grabda et al., 1963). From the eggs in the ovary sacks of sexually mature anchor worms, nauplioses are being hatched, the latter going out and freely swimming in the water environment. After some time (depending on water temperature) they are transformed into meta-naupliuses and later into copepod larvae from І, ІІ, ІІІ and ІV phase having parasites, at the end being differentiated in terms of sex. The temperature optimum for the development of parasites is 25-28 C, thus ensuing that the most intensive infection with parasites is supposed to be expected in summer months. Argulus crustaceans are also a hot-liking parasite. At an optimal temperature of 25-28 С it gives birth to three generations. Under favorable conditions one female Argulus and its generation could bring
302 about up to two million parasites in summer. In autumn when the temperature drops Argulus remains on the, envelopes itself in mucus and falls into anabiosis. And in such a way it is wintering (Schaperclaus, 1979). Under the current market conditions manufacturers have turned to three-summer period of fattening of carp, which brings about favorable conditions for the transfer of these invasions via the stocking material. The curative preventive baths with various disinfectants are not efficient enough when the stocking material is being treated in March - April, since during that time Lernaea are still incised in the under skin and it is difficult to be destroyed by disinfectants (Аnev and Boyadgiev, 2003). Shariff and Roberts (1989) carry out investigations on the peculiarities of the diagnostics and the specific features of the prophylactics and treatment of Lerneosis. Due to the setting up of new centers of Lerneosis and argulosis in the country over the last few years we have set ourselves the goal to investigate the efficiency of some preventive treatments, at the same time offering some innovative tendencies for fighting parasitic cray. Мaterial and Methods N. Anev We carried out the observations in the economic farms on the territory of Plovdiv and Bourgas district in the period 1999-2004. The examined farms are shown in Table 1. The diagnostics was performed on the basis of a clinically and laboratory-microscope research relying on samples of live with manifested clinical behavior wounds along the body with parasites in them. Check catches were performed in the above farms, the total number of the examined, the number of the infected with parasites and the quantity of parasites spread on them being registered. The basic physical and chemical parameters of water such as Т ( C), рн, dissolved oxygen were registered using the combined field oxy - рн meter JENWEY (9010, 3200).Tests were carried out in two main aspects: I. Fighting adult forms of parasites in May and June. II. Fighting nauplius forms of parasites in July and August. To fight adult forms of parasites we Таble 1 Examined economic farms Area, da Маximum volume, l Average depth, m Fishery ponds in the village of Manole 60 60000000 1.0 Каra dere 220 330000000 1.5 Gichita 566 849000000 1.5 Тоpra Hisar 90 108000000 1.2 Banski dol 435 652500000 1.5 Selski giol 500 700000000 1.4 Devet mogili 300 380000000 1.6
New Tendencies for Therapy Against Parasitic Crustacean in Fish 303 used the following methods and combinations of medicines: 1. Secondary treatment at an interval of 15 days in May with the phosphor organic medicine Neguvon 0.3 g/m 3 water and 50 кg/dka caustic lime placed 3-4 days prior the first and second treatment. 2. Preventive baths with potassium permanganate 1:50 000 in May at a treatment period of 30 minutes, the temperature of water up to 15 0 С. To fight nauplius forms of parasites we used: 1. Secondary treatment at an interval of 15 days in July with the phosphor organic medicine Neguvon 0.25 g/m 3 water and 50 кg/dka caustic lime placed 3-4 days prior the first and second treatment. 2. Secondary treatment at an interval of 15 days in July and August with the NEW medicine Piriproxifen 0.0002 g/ m 3 water. Check catches (50 pcs. of ) on the second and seventh month following the last treatment were made to verify the effect from the treatments. Two ponds, 20 dka each, were left for the check. Results and Discussion The appearance of parasitic cray spread on carp, gold and white grass carp were established in the ery farms in the village of Manole (A) Plovdiv district on 15.06.2001, where the disease Lerneosis was ascertained, caused by the species Lernaea cyprinacea. On 22.06.2001 in Kara dere (B) in Bourgas district we found out the disease Lerneosis, caused by the species Lernaea cyprinacea and parasitic cray from the Argulus foliaceus species causing the argulosis spread on carp. Anchor worms were established in Gichita (C) and in Тоpra Hisar (D), Plovdiv district on 28.06.2001. On 18.06.2002 in Banski dol (Е), Plovdiv district during the check catch the presence of argulus crustaceans from the Argulus foliaceus species was estab- Таble 2 Results from check catches for tracing out the infection of with anchor worms (Lernaea ) in the village of Manole Каra dere Gichita Тоpra Hisar Total number of examined 200 185 92.5% Number of infected 78 Extension ЕI 78% Intensity II 15 25 Т, C water рн water Contents of oxygen 28 7.5 3.5 25 7.8 3.1 150 25 16.6% 3 8 5.5 27 7.0 4.2 50 36 72% 11 18 14.5 28 7.2 4.0 20 10 22 16
304 N. Anev Таble 3 Results from check catches for tracing out the infection of with argulus crustaceans Каra dere Banski dol Selski giol Devet mogili Total number of examined Number of infected Extension ЕI Intensity II Т, C water рн water Contents of oxygen 55 55% 15 20 28 6.9 3.4 17.5 85 85% 75 75% 80 80% 15 25 20 15 25 20 15 30 22.5 29 6.8 3.2 30 7.2 3.6 29 6.8 3.2 lished. Arguluses were also found in Каra dere, Selski giol (F) and Devet mogili (J) at the beginning of July 2004. Data on the check catches are shown in Table 2 and Table 3. In all cases when diseases were first established the clinic behavior was vivid and the diagnosis was made comparatively easy. On the place of the incision the first skin redden appeared small papules that later grow into prurygo-papules and in case of a secondary infection they are frequently filled with suppurative exuded, which subsequently are inclined to form ulcers with a central humid necrosis as in Aeromonosis on carp (Aeromonosis cyprinorum) (Figure 1 Figure 4). Fig. 1. Parasitic cray from Crustacean family: Lernaea cyprinacea, Argulus foliaceus (in the middle) Fig. 2. Primary skin redden developed into hemorrhagic-papule, filled with blood exuded
New Tendencies for Therapy Against Parasitic Crustacean in Fish 305 Fig. 3. Humid necrosis as a result of infection with Lernaea cyprinacea Fig. 4. Secondary infection as a result of a high degree infection with Lernaea cyprinacea The finding and the separation of female parasites located sub-dermally in cases of Lerneosis and upon the body in cases of argulosis, as well as the microscope investigation made proving unquestionable. In the above ery farms, where our observations have lasted for several years, it was found out that the appearance of parasitic cray was followed by the presence of Aeromonosis spread on carp (Boyadjiev and Anev, 2003). The same spring in the ery farms in the village of Manole there appeared Aeromonosis spread on carp. When pictures were taken of the farm we understood that in 2000 year the farm had been invaded by Anchor worms. According to Boyadjiev and Anev (2003) parasites from Crustacea class represent a vector of distribution of Aeromonosis and Spring viremia spread among carp. Data in Table 4 show that the best results to fight the nauplius forms of Lernaea are achieved during the secondary treatment of the water area with Piriproxifen a medicine that inhibits the formation of chitin structures of parasites. The inhibitors of the chitin synthesis (HIS) (from the group of acillurea drugs) are growth regulators that are harmless for men and. HIS destroy the harmful copepod larvae of parasitic crustaceans in the early phases of their development without damaging the useful aqua fauna. The acting mechanism constitutes in blocking the enzyme complex responsible for the formation of chitin in the cuticle of the body shell of embryos in eggs and larvae as a result of which they stop to eat and die at the moment of their withering. Embryos in eggs stop their development and do not hatch. The medicine shows the so-called retarded effects. Larvae that have been in contact with the minimal efficient concentration of Piriproxifen become adults that stay sterile or hatch an insignificant number of eggs. From the data in Table 5 it can be seen that good results for fighting arguloses are achieved when the water area is treated both with Neguvon, as well as with Piriproxifen. According to data of Pell Gar International (manufacturer of medicines) Piriproxifen is much less toxic com-
306 N. Anev Таble 4 Tested medicines and their efficiency in fighting Lernaea For fighting adult forms For fighting nauplius forms Potassium HIS Neguvon Neguvon 0.3 g/m 3 permanganate 0.25 g/m 3 Piriproxifen 1 : 50000 0.0002 g/m3 A + + + + + + + + + B + + + + + + + + C + + + + + + + + D + + + + + + + + Effect from treatment poor /+/; satisfactory /++/; good /+++/; very good /++++/; excellent /+++++/ Таble 5 Tested medicines and their efficiency in fighting argulosis disease For fighting adult forms Neguvon 0.3 g/m 3 Potassium permanganate 1 : 50000 For fighting nauplius forms Neguvon 0.25 g/m 3 HIS Piriproxifen 0.0002 g/m3 B + + + + + + + + + + + E + + + + + + + + + + + F + + + + + + + + + + J + + - + + + + + + + Effect from treatment poor /+/; satisfactory /++/; good /+++/; very good /++++/; excellent /+++++/ pared to chemicals such as Trichlorfon, Chlorofos, Neguvon that are forbidden for use both in Bulgaria and in the countries members of the European Union. Ecotoxycallity of Piriproxifen for arched trout is more than 325 mg/l. Ecotoxicallity of Neguvon for arched trout is 0.7 mg/l. After the treatments with HIS that were carried out, parasitic cray were established only in individual. When check catches were performed in the ery farms in the village of Manole at the end of October 2001 it was found out that only two pieces out of the totally examined pcs of were infected with parasites, namely containing 2-3 pcs of cysts in the abdominal area. We assumed that this finding could be also attributed to the fact that parasites from the so-called winter generation winter either in the shape of cysts or deeply planted in the skin of the. In this process of penetration around the place of penetration we established that a reactive process was present as a result of the secondary micro-flora which is possible to penetrate from the
New Tendencies for Therapy Against Parasitic Crustacean in Fish 307 environment together with the parasite. As a result the so-called cysts (small reddened papules with a central necrosis) are formed Figure 2. When pcs of were caught in autumn (in October) in the check pond we registered the infection of 92 pcs with an exuberance of cysts around the abdominal area. In spring (in May) next year a check catch was also performed in the check ponds finding no specimen without Lernaea along the body out of 50 pcs of carp stocking material (infection from 22 tо 35 pcs of parasitic cray). Very good results were achieved in Кara dere. During the check catch of stocking material out of pcs of small carp we noticed infection of 9 pcs with two to three cysts in the abdominal area. In Gichita 7 pcs with three to five cysts on them were found out of pcs. Data from the check catches after the treatments and from the check pond are indicated in Table 6 and Table 7. We found out that the extension of the invasion is below 9%, while the intensity on the average is below four. The following year in spring the degree of the invasion rate of was similar to that in autumn. Upon the microbiological investigation that we carried out on certain infected with Lernaea and/or with arguloses we established the presence of bacterial flora of Aeromonass sp. type in both kinds of infection with parasites. We relate the results achieved both with the after-effect of IHS, as well as with the immune response, which according to Shariff et al. (1986), Shariff and Roberts (1989), Merla (1961), Rogers (1968) is obtained in that have been maintaining contacts with parasitic cray from the Crustacea class. The established by us therapeutical and preventive effect of IHS - Piriproxifen could be of great importance. Таble 6 Results from check catches for tracing out the infection of with Lernaea after the treatments of the water area with Piriproxifen, as well as data from the check pond that has not been treated Gichita Fishery farms in the village of Manole Fishery farms in the village of Manole check pond Каra dere Total number of examined Number of infected 2 2% 92 9 7 Extension ЕI 92% 9% 7% Intensity II 2 3 2.5 22 35 28.5 2 3 2.5 3 5 4
308 N. Anev Таble 7 Results from check catches for tracing out the infection of with argulus crustaceans after the treatments of the water area with Piriproxifen, as well as data from the check pond that has not been treated Total number of examined Number of infected Extension ЕI Intensity II Devet mogili check pond Каra dere Selski giol Banski dol Devet mogili 86 1 7 1 3 86% 1% 7% 1% 3% 18 28 23 2 3 2.5 1 3 2 1 2 1.5 2 3 2.5 Conclusions The secondary application of IHS Piriproxifen equally spread on the water surface at a concentration of 0.0002 g/m 3 in July and August achieves the best curative and preventive effect in the fight against Lerneosis and Argulosis diseases. The IHS medicines are without any proven harmful after-effects on live organisms. They are the better alternative to fight parasitic crustaceans than the forbidden phosphor organic chemicals such as Trichlorfon, Chlorofos and Neguvon. References Аnev, N. and N. Boyadgiev, 2003. New directions in the fight against lerneosis spread among carp. Collection of Scientific Reports (International scientific conference 50 years Technical University of Forestry ), pp. -101. Boyadgiev, N. and N. Аnev, 2003. Aeromonosis (erythema) spread among carp and the fight against it. Collection of Scientific Reports (International Scientific Conference 50 years Technical University of Forestry ), pp. 94-96. Boyadgiev, N. and N. Аnev, 2003. Spring viremia spread among carp (Spring viremia of carp - SVC) a dangerous disease for farmed and freely reared. Collection of Scientific Reports (International Scientific Conference 50 years Technical University of Forestry ), pp. 97-99. Daskalov, H. and А. Ivanov, 1994. Lerneosis on carp contemporary problem of Bulgarian eries. Veterinary Collection, 6 (7): 25-26 (Bg). Grabda, J., 1963. Life cycle and morphogen-
New Tendencies for Therapy Against Parasitic Crustacean in Fish 309 esis of Lernaea cyprinacea L. Acta Parasitol. Polon., Warszawa, 11 (14): 169-200. Каranikolov, J. and D. Radev, 1978. Neguvon efficient means for fighting argulosis spread among carp. Fish Economy, 7: 23-24. Маrgaritov, N., 1972. Investigations on the efficiency of Neguvon against some external parasites spread among carp. Annual book of Sofia University, Department of Biology, 64 (1): 10-21. Merla, G., 1961. Zur Bekampfung der Karpfenlaus (Argulus) in der Teichwirtschaft. Dt. Fischerei Ztg., 8 (6): 179-182. Rogers, W. A., 1968. Biology and control of the anchor worm, Lernaea cyprinacea. FAO Fish. Rep. (Proc. of the World Symposium on Warm-Water Pond Fish Culture, Rome, Italy, May, 1966), 44 (5) (IX/ E 10): 393-398. Shariff, M., Z. Kabata and C. Sommerville, 1986. Host susceptibility to Lernaea cyprinacea L. and its treatment in a large aquarium system. Journal of Fish Diseases, 9: 393-401. Shariff, M. and R. J. Roberts, 1989. The experimental pathology of Lernaea polymorpha Yu, 1938 infection in nanve Aristichthys nobilis Richardson and a comparison with the lesion in naturally infected clinically resistant. Journal of Fish Diseases, 12 (5): 405-414. Schaperclaus, W., 1979. Fish krankheiten, Akademie-Verbang, Berlin, 1089 pp.