Perulernaea gamitanae (Copepoda: Lernaeidae) parasitizing tambaqui (Colossoma macropomum) (Characidae) and the hybrids tambacu and tambatinga, cultured in northern Brazil

The infestation rate in Colossoma macropomum, hybrid tambacu (C. macropomum x Piaractus mesopotamicus) and hybrid tambatinga (C. macropomum x Piaractus brachypomum) with Perulernaea gamitanae Thatcher and Paredes, 1985 from two fish farms in Amapá State, Brazil was studied. Lernaeid parasites (n=2887) were collected mainly on the tongue and the mouth cavity and also on cartilage of gill arches and filaments. Inflammation and fibrous nodules were observed on the attachment sites of the parasites. The infestation rate varied according to the fish farm and host. The prevalence of P. gamitanae was of 100% in hosts from one fish farm and was lower in the other fish farm. Higher intensity of P. gamitanae occurred in hybrids tambacu and tambatinga, but despite the high prevalence its intensity was moderate. This is the first report on epidemiology of P. gamitanae in cultured fishes from Brazilian Amazonia, and the occurrence of this crustacean parasite in two new hosts, the hybrids tambacu and tambatinga.


INTRODUCTION
Problems with diseases caused by lernaeid parasites have increased due to epizootics on farmed (Shariff et al., 1986;Martins et al., 2000;Kim et al., 2002;Kir, 2007) and wild fishes (Öktener et al., 2008) in cases of heavy infestations, mainly in small fish.In Brazil, Lernaea cyprinacea is the most common species of lernaeid, which has spread to several farms (Gabrielli and Orsi, 2000;Martins et al., 2000;Martins et al., 2002;Mabilia and Souza, 2006) and natural environments (Magalhães, 2006) due to its specificity.Lernaeidae pertaining to the genus Lamproglena are emerging parasites with pathogenic potential that have been recently reported to occur in cultured fish in the Southeastern (Alves et al., 2000;Martins et al., 2002;Lizama et al., 2007) and Southern Brazil (Ghiraldelli et al., 2006), as well as in wild fish (Azevedo et al., 2007).Up to date, L. cyprinacea and Lamproglena species have not been found in Amazonian fishe, while other Lernaeidae species are restricted to Amazonian hosts (Thatcher, 1991;Thatcher, 1998;Thatcher, 2006).
Perulernaea gamitanae Thatcher and Paredes, 1985 was described in tambaqui Colossoma macropomum collected in the Amazon River in Iquitos, Peru, and subsequently, in the same host, in the Brazilian portion of the same river (Thatcher, 1991;Thatcher, 2006).The morphology of nauplius and copepodite stages of P. gamitanae was studied from eggs of adult post-metamorphic females obtained from the tambaqui C. macropomum (Benetton and Malta, 1999).Recently, Fischer et al. (2003) (MPA, 2010).The intensification of C. macropomum and hybrid tambacu and tambatinga culture has contributed to the occurrence and emerging parasites, which may compromise fish health and productivity.The present study investigated infection rates by P. gamitanae in C. macropomum, hybrids tambacu and tambatinga from two fish farms in the State of Amapá, and also, extended the evaluation of this parasite on other hosts, describing the its abundance in tambacu and tambatinga for the first time.
Fish were measured and necropsied by taking the mouth and gills for the collection and quantification of parasites.All parasites were collected, counted and fixed in 70% alcohol solution.The identification of the parasites was carried out as suggested by Thatcher and Paredes (1985) and Thatcher (2006).Parasitic indexes (prevalence, mean intensity, variation of intensity, mean abundance) were calculated according to Bush et al. (1997) in order to evaluate fish infestation rate.
Following the parasitological analysis and collection of the parasites, all ponds were submitted to indefinite bath treatment with 100g/1000m 3 of Diflubenzuron (Dimilin®) in single application.During fish collection, water physical-chemical variables were analyzed.Temperature and dissolved oxygen (OD) were determined through digital equipment (YSI-Model-55) and pHpH was also determined through digital equipment (pH-meter WTW -D812 model).

RESULTS
Water quality variables in the ponds from farm one varied as follows: pH ranged from 6.8 to 7.8, temperature from 31.0 to 31.4 o C and dissolved oxygen 2.9 to 3.1mg/L.In farm two, pond water pH ranged from 6.4 to 6.8, temperature from 29.1 to 31.0 o C and dissolved oxygen 0.9 to 2.1mg/L.In all fish the Lernaeidae parasites were identified as Perulernaea gamitanae Thatcher and Paredes (1985) (Figure 1A).Parasites were collected mainly on the tongue and gills of the three fish species (Figure 1C-D), but in the C. macropomum parasites were also found on the cartilage of gill arches and filaments, and no hybrid tambatinga had gill infestation.Some specimens of hybrid tambacu also presented the parasites on the palate.In general, the sites of infestation on the gill filaments showed inflammation spots and fibrous nodules (Figure 1C), regardless of the presence or absence of the crustacean.The parasitism index of C. macropomum, and of hybrids tambacu and tambatinga of the collected fish from farm one and two are presented in Table 1 and Table 2, respectively.These parasites were collected from the mouth cavity and gills of hybrid tambatinga, but in C. macropomum the infestation occurred only in the mouth cavity (Table 2).
The prevalence of P. gamitanae was similar in hybrid tambatinga, but differences occurred in C. macropomum and in infestation rates on both fish farms.
The single indefinite bath treatment with 100g/1000m 3 of Diflubenzuron eliminated the parasites in gills and mouth of fish in both fish farms.

DISCUSSION
For cultured fish in the Brazilian Amazon the infestation rates by lernaeids are still scant.In C. macropomum, hybrid tambatinga and hybrid tambacu collected from fish farm one (Amapá State) the high prevalence (100%) was similar to that reported by Benetton and Malta (1999) for C. macropomum (100%) cultured in Manaus.However, in C. macropomum and hybrid tambatinga collected from fish farm two, the prevalence was lower and ranged from 20% to 60%.On the other hand, prevalence and infection level by P. gamitanae were high compared to the infestation by P. gamitanae in C. macropomum from Solimões and Amazon Rivers (Fischer et al., 2003).This prevalence and infestation was also higher than those reported for L. cyprinacea, another Lernaeidae species that commonly parasitize C. macropomum (Békési, 1992;Martins and Romero, 1996;Martins et al., 2000) and hybrid tambacu (Martins and Romero, 1996;Martins et al., 2000;Gabrielli and Orsi, 2000;Schalch and Moraes, 2005) cultured in other regions of Brazil (Martins et al., 2000).Studies demonstrated that hybrid tambacu has shown high susceptibility to infestation by L. cyprinacea adults when compared to other cultured fish (Tavares-Dias et al., 2001).
The parasitized fish in fish farm one were possibly infested by P. gamitanae that entered the ponds through the water inlet from the Amazon River that was used to fill the ponds, Arq.Bras.Med.Vet.Zootec., v.63, n.4, p.988-995, 2011 infesting 14,000 C. macropomum, 10,000 hybrid tambacu and 7,000 hybrid tambatinga.
Shortly after the local transport (Macapá, AP) of the infested fish to farm two, 500 specimens of tambaqui and hybrid tambacu with mean weight of 1500g died, probably due to the low resistance of the fish infested by P. gamitanae.The parasite spread in the ponds and also infested the hybrid tambatinga.Hybrid tambatinga had higher rates of parasitism by P. gamitanae than C. macropomum, but this last species had only the oral cavity parasitized.P. gamitanae infestation was controlled with 100g/1000m 3 of Diflubenzuron indefinite bath treatment on both fish farms.
Parasites P. gamitanae caused inflammation on the place of attachment, especially in the gills, which could facilitate bacterial and mycotic infections, both relatively common in fish infested by the crustaceans.The species that cause hyperplasia and metaplasia in the gills of the host can reduce respiratory capacity (Thatcher, 1998).Besides, P. gamitanae can debilitate fish or cause their death when in confinement (Thatcher, 2006) due to apathy and anorexia.Three consecutive treatments with NaCl (4 to 6%) were applied to the pond to heal the wounds caused by parasites, which made the fish resume feeding, but such treatment did not eradicate the young and adult forms of P. gamitanae.Salt (NaCl) has been recommended for the control of several other parasites, as well as to avoid dissemination of parasites and stress in freshwater fish.However, the fish tolerance to salt, which varies depending on the fish species and age (Martins, 2004) must be considered.
During this same period, institutions of technical assistance to fish farmers in the states of Rondônia (Porto Velho and Ji-Paraná), Acre (Rio Branco, Bujari, Porto Acre and Senador Guiomard) and Amazonas (Manaus and Manacapuru) have also recorded infestations by P. gamitanae in tambaqui from several fish farms.
In Acre and Amazonas States, epizooties of juveniles tambaqui weighing 100 to 200g (Figure 1D) occurred due to the great infestation of P. gamitanae in the mouth, impeding fish from eating, causing massive mortality.However, in tambaqui with mean weight of 500g, infestation was mild, with no mortality, while in fish weighing between 2000 and 3000g infestation was mild to low, with no mortality either.In all cases, infestation took place mainly in the mouth and tongue.To control infestations by P. gamitanae, fish farmers also have applied 50 to 100g/1000m 3 of Diflubenzuron in ponds, which has eliminated all forms of this parasite.Copepodits of P. gamitanae survived up to seven days without a host (Benetton and Malta, 1999), thus the treatment would have to be repeated to eliminate all forms of the parasite.Martins (2004) suggested that any treatment with Diflubenzuron must be repeated three times in order to ensure its efficacy.Diflubenzuron is part of a group of insecticides which cause morphological alterations during the life cycle of the crustacean (Martins, 2004;Mabilia and Souza, 2006;Pelli et al., 2008) and was approved by the Environmental Protection Agency (EPA) in 1976 to be employed against plagues in cotton and soy cultures.In 1999, Diflubenzuron began to be used in Brazil to control infestations by crustacean ectoparasites (Martins, 2004).Currently, this product is used to control L. cyprinacea in 33.3% of fish farms in Rio Grande of Sul State (Mabilia and Souza, 2006), as well as in other fish farms in Brazil.
In order to be suitable for fish farming, a chemical product must be safe for fish, highly efficient and rapidly degraded, not interfering with the water, be of easy application and low cost (Cruz et al., 2009).Studies with juvenile rainbow trout Oncorhynchus mykiss and coho salmon Oncorhynchus kisutch (McKague and Pridmore, 1978), also Rhamdia quelen (Kreutz et al., 2008) and Zungaro zungaro (Pelli et al., 2008), have examined the toxicity of Diflubenzuron and they suggest that its mean lethal concentration for 96h exposure (LC 50 -96h) is greater than 150mg.L -1 .However, the concentration of Diflubenzuron recommended for direct application in ponds is 100 to 200g/1000m 3 of water, which corresponds to 0.1-0.2mg.L -1 (Martins, 2004).Hence, this product has low toxicity to fish (Burtle and Morrison, 1987;Moraes and Martins, 2004;Kreutz et al., 2008;Pelli et al., 2008;Schalch et al., 2009).Besides being effective in the treatment against crustacean parasites (Burtle and Morrison, 1987;Routh et al., 1993;Moraes and Martins, 2004;Schalch et al., 2009), Diflubenzuron is degraded in aerobic conditions (Routh et al., 1993;Pelli et al., 2008).This product shows potential of use in fish farms (Pelli et al., 2008), but still requires proper registration and regulation for its use as a chemotherapeutic for fish crustacean parasites.In Brazil, only two products are currently evaluated and approved by the Ministry of Agriculture, Livestock and Supply (MAPA) for use in aquaculture.Therefore, as development of fish farming is of great importance for the country, there is an imminent need of decision making regarding the legalization of chemical products employed in the treatment of fish diseases (Cruz et al., 2009), such as the Diflubenzuron.
As a result, besides the dispersion of lernaeid P. gamitanae among fish farms, the infestation may also take place from wild environment into fish farms, given that many tambaqui breeders are obtained in Amazonian rivers.Thus, parasites may be taken to production ponds in the Northern region, as well as other zones where fish is farmed.Consequently, Lernaeid P. gamitanae will probably not be restricted to the Northern region if it finds favorable conditions for development in other Brazilian regions.Thus, it is necessary to use caution when transporting fish, to perform quarantine and diagnosis before introducing fish into ponds, in order to prevent the dissemination of this crustacean in fish farms.P. gamitanae could pose a major concern for Amazonian aquaculture, jeopardizing the production of C. macropomum and its hybrids (tambacu and tambatinga), especially the earlier, which is the most cultivated species in the Amazon.Cautionary procedures must be reinforced, since massive infection can undermine the survival of fish juveniles as well as the depreciation of animals ready for commercialization, compromising business and consumption of such fish.

Table 1 .
Parasitological indexes of Perulernaea gamitanae in Colossoma macropomum and its two hybrids for fish farm one in Macapá (AP), February to April, 2009