Austrodiplostomum compactum (Lutz, 1928) (Digenea, Diplostomidae) in the eyes of fishes from Paraná river, Brazil

Parasitological indexes of Austrodiplostomum compactum (Digenea, Diplostomidae) in fishes from the Paraná River, Presidente Epitácio region, state of São Paulo, Brazil, and their relationship with climate and water quality are evaluated. Fifty-one specimens of Plagioscion squamosissimus, 39 Geophagus surinamensis, 27 Hoplias malabaricus and 23 Cichla sp. were collected between June 2007 and June 2008. Water quality and rainfall indexes were measured monthly. P. squamosissimus had the highest parasite rate, with 98% total prevalence, intensity of infection varying between 1 and 255 and parasite mean abundance totaling 40.5 ± 9.9. H. malabaricus had the next parasite rate, with 66.6% total prevalence, parasite intensity between 2 and 184 and mean abundance totaling 22.4 ± 20.9. Cichla sp. had a total prevalence of 52.1%, intensity between 1 and 21, and mean abundance 4.3 ± 2.9. Finally, G. surinamensis had a total prevalence of 46.1%, intensity between 1 and 53 and mean abundance 7.1 ± 8.8. A survey of diplostomid infection in Brazil and the role of piscivore fish as an important host have also been discussed.


Introduction
Metacercariae of Diplostomum and Austrodiplostomum are frequently reported parasitizing freshwater fishes which are the second intermediate hosts in the parasite's life-cycle (RINTAMAKI-KINNUNEN et al., 2004;SEPPALA et al., 2004).Whereas the adult stage inhabits the digestive tract of piscivore birds, its larval cercariae stage may be found in aquatic mollusks.The parasite has a geographically wide range with reports from over 125 fish species (EIRAS, 1994).
Current research reports on the occurrence of Austrodiplostomum compactum (Lutz, 1928) in four fish species from the Paraná river, as well as its ecological aspects with regard to parasitological indexes, rainfall and water quality.(2002).Geophagus surinamensis, H. malabaricus and Cichla sp. were not captured during the summer of 2007.Parasite specimens were fixed in 5% formaldehyde, washed in distilled water, stained in carmine or diaphanized in beechwood creosote and mounted on permanent slides using Canada balsam.The terms prevalence, mean intensity of infection and mean parasite abundance were attributed according to Bush et al. (1997).

Current
The water quality was monthly measured.Water temperature and dissolved oxygen were measured with digital oxymeter YSI 5512; ammonia, pH, and alkalinity were calculated with Merk's colorimetric method.Rainfall indexes were obtained from the meteorological station of the Universidade do Oeste Paulista (UNOESTE) in Presidente Prudente, São Paulo State, Brazil.
Collected data underwent statistical tests to compare rainfall, air temperature and water quality between seasons; to determine differences between the intensity of infection and abundance of parasites in fish between the seasons; to verify which fish species was more parasitized during the whole period and the possible differences in host weight and length for each fish species examined according to season.ANOVA and Bartlett's test for variance homogeneity were used, with significance for all statistical analyses set at α = 0.05.

Results and discussion
No significant differences in rainfall indexes between seasons were observed.However, temperature rates were high (p < 0.05) in the spring and summer 2007.The highest water temperatures (p < 0.05) occurred in the spring 2007, summer 2007 and autumn 2008.High alkalinity rates were reported in the winter and spring 2007 and autumn 2008, but no difference in pH and dissolved oxygen occurred during the seasons (Table 1).
Mean highest rainfall during the studied period occurred in the winter 2007, spring 2007 and autumn 2008.Oscillations occurred in water alkalinity and pH during the period under analysis.The highest temperature rates were registered between spring 2007 and autumn 2008, although there were considerable variations in all months.Dissolved oxygen levels also varied during the year (Table 1).
Metacercariae at different stages of development were collected from the crystalline of host´s eyes in all fish species.
Parasites from G. surinamensis were reported only between autumn and winter 2008.Although no fish was collected in the summer 2008, mean infection intensities were low, varying between 12 and 18.2 parasites, and mean abundance varying between 10.4 and 18.2 (Table 3).
No significant difference (p > 0.05) occurred between host weight and total length for all fish species between the seasons.Santos et al. ( 2002) registered a positive relationship between mean infection intensity and rainfall, associated with high temperatures.Current study agrees with previous one in which all fish species were reported with the highest infection intensity during the months with the mean highest rainfall and which coincided with the highest temperatures.
In contrast, analyzing P. squamosissimus from Nova Avanhandava reservoir in the state of São Paulo, Brazil, Paes et al. (2010) did not report any influence either of aquatic parameters or of rainfall on the parasitic fauna.Since transmission is a major determinant of parasite fitness, perhaps other factors, such as local host population dynamics, fish feeding habits, strategies in cercarial release from first intermediate hosts and also the type of fresh water environments (lotic or lentic environment), would be primarily influencing success in parasite transmission.
There is a wide range of fish species collected from Brazil which are parasitized by metacercariae of A. compactum.Fifty percent of these fish species, namely, A. osteomystax, C. monoculus, C. ocellaris, S. pappaterra, H. malabaricus and P. squamosissimus, registered over 50% prevalence rates.The latter species achieved indexes up to 100% (Table 6).Plagioscion squamosissimus, also with mean high infection intensity, was the most parasitized fish species reported not only in current study but also in Santos et al. (2002), Martins et al. (2002) and Paes et al. (2010).Santos et al. (2002)  Although no significant difference in mean intensity and abundance of metacercariae throughout the seasons has been reported in current research, P. squamosissimus examined in spring and autumn had the highest parasitic load when compared to findings by Paes et al. (2010) in the same fish species in the Nova Avanhandava reservoir.
According to Lyholt and Buchmann (1996) and Hakalahti et al. (2006), infection of fishes by Diplostomum spathaceum (Rudolphi, 1819) metacercariae is temperature dependent.Hoglund and Thulin (1990) observed in their study that the maximum temperature (up to 23°C) coincided with the period of reduced recruitment of Diplostomum baeri Dubois 1937 metacercariae in perch Perca fluviatilis Linnaeus 1758.The highest prevalence and mean intensity observed in this study for P. squamosissimus, G. surinamensis and H. malabaricus were registered at water temperatures between 21.4 and 29.0°C.Such data may be explained by data from Hakalahti et al. (2006), according to whom extended summer temperatures would alter the dynamic population of cercariae and consequently the fish infection by metacercariae.
Due to the fact that water temperature at the collection site analyzed in current research was higher than that in Hakalahti et al. (2006), infection behavior at low temperatures cannot be evaluated.Martins et al. (2002) registered the positive influence of high temperatures on A. compactum infection in P. squamosissimus.In fact, their suggestion may be confirmed in current study for P. squamosissimus, a fish collected during the whole period under analysis.According to Berrie (1960), Diplostomum cercariae emerge in waters only at temperatures above 10°C.Lyholt and Buchmann (1996) registered that daily shedding of D. spathaceum cercariae in Lymnaea stagnalis (Linnaeus, 1758) reached a maximum of 58,000 cercariae snail -1 day -1 at 20°C and a maximum of 10,000 cercariae snail -1 day -1 at 10°C.These authors also concluded that cercariae were 4 to 5 times more infective at 15°C than at 7°C, reinforcing the impact of temperature on the parasite's life-cycle.Although adaptations in temperature margins would be expected to suit local conditions, it seems that parasite biology is dependent on high temperatures, as suggested by Santos et al. (2002), Martins et al. (2002) and Hakalahti et al. (2006).
Luque and Poulin ( 2004) compared 50 teleost fish species from the coast of Brazil to evaluate the effects of host traits on the richness and abundance of larval helminths and reported that among all the potential correlates, host body length was positively correlated with helminth larvae abundance.Machado et al. (2005) registered a positive correlation between C. britskii size and the prevalence of A. compactum and attributed this fact to the possibility of larger fish being easier targets for cercariae.The parasite load in P. squamosissimus increased with fish length, as reported by Paes et al. (2010).These authors also attributed the cumulative process to a greater body surface available for cercariae infection.According to Pojmanska (1994), there is evidence on the positive correlation between parasite number and fish size, arguing that accumulation is due to the metacercariae's long life.On the other hand, Valtonen and Gibson (1997) concluded that the diplostomid metacercariae infection in fishes from northern Finland provided little or no association between the prevalence of infection and host-size, even though there was a distinct accumulation in the number of parasites.
With regard to the above, Burrough (1978), analyzing the population biology of D. spathaceum in roach Rutilus rutilus and rudd Scardinius erythrophthalmus, concluded that parasite accumulation occurred up to the time fish reached 130 mm.Unfortunately, it was not possible to compare our results with those of the latter author due to the lack of analyzed fishes measuring less than 130 mm.This fact did not depend on fishing tactics since the animals were captured with hook and net, similar to that for collection species.No distinct accumulation was reported and thereafter intensity ranged between all levels.The Brazilian records for diplostomid metacercariae parasitosis show different sites of parasite infection in the host's body, despite an apparent preference for the fish's eyes, including aqueous humor and crystalline (Table 6) 6).According to Eiras (1994), reports by several authors suggest that Diplostomum species have their own location preferences.Bortz et al. (1988) verified that parasites collected from the hosts' retina and crystalline corresponded to populations with different epidemiological characteristics and thus different parasite species.Alternatively, the parasite infection site may be related to fish species.Whatever the involved in parasite's preferences, more studies identifying correctly the parasite location and also a revision of the group systematics are necessary to elucidate this question.The taxonomy, especially regarding larval stages, is still unclear and several species, considered adults in nature, have never actually been observed as larvae and vice-versa (HOGLUND; THULIN, 1990).

Conclusion
Hoglund and Thulin (1990) state that fishes harboring more than 40 diplostomid metacercariae on their eyes are largely parasitized.In the present study, P. squamosissimus and H. malabaricus may therefore be considered the main host resources used by the parasite within the context of the river under analysis.It may be concluded that these fish species may not only be the most susceptible fish species but also good indicators of Austrodiplostomum infection.
research was undertaken at the Aquaculture Center of the Universidade do Oeste Paulista (UNOESTE), on the shores of the Paraná river, near the town of Presidente Epitácio, state of São Paulo (21 o 45′ 48″ S; 52 o 06′ 56″ W).Fifty-one specimens of P. squamosissimus, 39 G. surinamensis, 27 H. malabaricus and 23 Cichla sp. were collected with gill nets and hooks, between June 2007 and June 2008, comprising the winter of 2007 thru the winter of 2008, for parasitological exam undertaken according to Santos et al.

*
Different letters indicate significant difference (p < 0.05) between the seasons.

Table 1 .
Rainfall, air temperature and water quality mean rates measured at the shores of the Paraná river, near Presidente Epitácio, São Paulo State, Brazil.

Table 2 .
Parasitological indexes (± standard error) and biometry of Plagioscion squamosissimus parasitized by Austrodiplostomum compactum from Paraná river, region of Presidente Epitácio, São Paulo State, Brazil.Mean values and variation, between parentheses, of fish weight and length, infected fish/examined fish (IF/IE), prevalence, total number of parasites (TNP), mean intensity and range, between parenthesis, and mean abundance.

Table 3 .
Parasitological indexes (± standard error) and biometry of Geophagus surinamensis parasitized by Austrodiplostomum compactum from the Paraná river, region of Presidente Epitácio, São Paulo State, Brazil.Mean values and variation, between parentheses, of fish weight and length, infected fish/examined fish (IF/IE), prevalence, total number of parasites (TNP), mean intensity and range, between parentheses, and mean abundance.*Differentletters indicate significant difference between months (p < 0.05).

Table 4 .
Parasitological indexes(± standard error) and biometry of Hoplias malabaricus parasitized by Austrodiplostomum compactum from Paraná river, region of Presidente Epitácio, São Paulo State, Brazil.Mean values and variation, between parentheses, of fish weight and length, infected fish/examined fish (IF/IE), prevalence, total number of parasites (TNP), mean intensity and range, between parentheses, and mean abundance.

Table 5 .
Parasitological indexes (± standard error) and biometry of Cichla sp.parasitized by Austrodiplostomum compactum from Paraná river, region of Presidente Epitácio, São Paulo State, Brazil.Mean values and variation, between parenthesis, of fish weight and length, infected fish/examined fish (IF/IE), prevalence, total number of parasites (TNP), mean intensity and range, between parentheses, and mean abundance.
reported the highest parasitological indexes in March 2001 for fish species, with infection intensities ranging from 4 to 137 parasites per host eye, while Martins et al. (2002) reported the highest infection intensities during late winter 2000 and late summer 2001.

Table 6 .
Parasitological indexes and sites of infection of Austrodiplostomum compactum collected in fishes from Brazil.