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Contracaecum sp. infection in Hoplias malabaricus (moncholo) from rivers and marshes of Colombia
Veterinary Parasitology 140 (2006) 90–97 www.elsevier.com/locate/vetpar
Contracaecum sp. infection in Hoplias malabaricus (moncholo) from rivers and marshes of Colombia ´ vila, Jorge Gu¨ette-Ferna´ndez, Jesu´s Olivero-Verbel *, Rosa Baldiris-A Amparo Benavides-Alvarez, Jairo Mercado-Camargo, Barbara Arroyo-Salgado Environmental and Computational Chemistry Group, University of Cartagena, Cartagena, Colombia Received 25 May 2005; received in revised form 2 March 2006; accepted 8 March 2006
Abstract Nematode infection indices were recorded in Hoplias malabaricus captured in six different rivers and a marsh belonging to the North Coast Basin of Colombia, and from the Amazon River, during February 2003–December 2004. Preliminary morphological analysis of nematodes indicated the presence of Contracaecum sp. Parasites were mostly found in the intestinal mesenteries and a very low percentage in muscle. Parasite prevalence in all sampling locations at the north of Colombia was 100%, whereas in the Amazon River it was 6.12%. The mean intensity in the different stations were as follows: Magdalena River at the City of Magangue´ (58.92 7.59), Magdalena river at the city of Zambrano (128.9 7.08), Sinu´ River (53.88 4.92), Dique Channel (207.3 59.52), Cauca River (77.26 9.35), Atrato River (21.11 2.6), San Jorge River (39.5 7.13), and Totumo Marsh (62.5 6.38). In average, all specimens of Hoplias malabaricus from the north coast basin of Colombia were infected with a mean intensity of 77.82 4.81 (1–466 parasites per host) whereas in fish from the Amazon River this value was significantly lower (intensity 1.0 0.0). Size and weight correlated significantly with parasite intensity in fish collected from sampling locations at the north of Colombia (R = 0.240, P < 0.001 and R = 0.199, P = 0.008, respectively). Moreover, a significant, but low and negative correlation was found between condition factor and parasite intensity (R = 0.159, P = 0.034), suggesting a possible impact of parasites on fish health. These results suggest, for the first time, that the parasitism in Moncholo is a widespread phenomenon in Colombian rivers and could represent a risk factor for human consumers. # 2006 Published by Elsevier B.V. Keywords: Freshwater fish; Nematode; Prevalence; Intensity; Human infection
1. Introduction
* Corresponding author at: Environmental and Computational Chemistry Group, Department of Chemistry, University of Cartagena, Cartagena, Colombia. Tel.: +57 5 6698179/6698180; fax: +57 5 6698323. E-mail address: [email protected] (J. Olivero-Verbel). 0304-4017/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.vetpar.2006.03.014
Parasites are an essential part of the aquatic community. Their presence becomes evident after a massive development, causing health effects including oxidative stress (Marcogliese et al., 2005), tissue damage, immunesuppression, and endocrine
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disruption (Jobling and Tyler, 2003), among others, and sometimes even leading to the mass mortality of infested hosts. These parasitic events are often the results of biotic or abiotic changes in the environment. In particular they have been proposed as pollution indicators by heavy metals and hydrocarbons (Schludermann et al., 2003; Williams and MacKenzie, 2003), and their presence can be the result of organic enrichment of sediments by domestic sewage (Marcogliese and Cone, 2001), although factors such as fish population size can also determine parasite infection rates (Bagge et al., 2004). Once nematodes have infected fish, the overall quality of this food decreases enormously, not only because of the possibility of human infection by eating not properly cooked fish (Sakanari and McKerrow, 1989), but also because there is no safe preparation that could completely avoid a risk for human health when consuming parasitized fish (Skirnisson, 2006; Toro et al., 2004; Audicana et al., 2002). In fact, several pathological conditions such as anthropozoonosis, allergy events, central nervous system damage, and anaphylactic reactions (Walker and Zunt, 2005; Lopez-Serrano et al., 2000; Audicana et al., 1995) could occur in some individuals whose main diet is composed of infected fish, as those living along waterbodies that receive untreated wastewater (Ensink et al., 2005). The problem might become severe when fish diversity has decreased to a point where few species are the only available because of their low price and abundance, this is the case of Hoplias malabaricus, commonly called Moncholo. Hoplias malabaricus is a tropical species found in most rivers basins of Central and South Ame´rica, from Costa Rica to Argentina (Zara´te et al., 1989). This species can breathe air by using their swimbladders as respiratory organs. This allows them to live in extremely muddy waters. Its diverse habitats varies from free flowing clear water streams, well up into the valleys, to slow turbid waters, as well as water courses, irrigation and drainage ditches, and ponds on the plains (Kenny, 1995). It usually rests in vegetation during the day and is active at night (Bussing, 1987). Adults feed on fish; juveniles feed on crustacean and insect larvae (Galvis et al., 1997), shrimps and other small invertebrates (Planquette et al., 1996). In Colombia, Hoplias malabaricus is one of the most abundant fish species found in river basins except
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the high Cauca River (Galvis et al., 1997). It represents a valuable resource not only because of its high presence during the whole year, but also because it is part of traditional dishes, such as the smoky Moncholo. Reports of parasite nematodes in Colombian fish species are scarce and therefore the goal of the present investigation was to investigate the occurrence of the nematode infection in Hoplias malabaricus captured in different habitats.
2. Materials and methods 2.1. Specimen collection In total, 227 specimens of Hoplias malabaricus of 32.15 0.3 cm total length and 346.64 9.5 g mean weight were collected from five rivers and one marsh (Fig. 1) at the North Coast Basin of Colombia: Magdalena River, specifically in Magangue´ (88580 N and 748310 W) and Zambrano (98450 N and 748480 W), Sinu´ River (98140 N and 758500 W), Cauca River (88060 N and 748450 W), San Jorge River (88460 N and 748520 W), the Dique Channel (108220 N and 758080 W), Atrato River (58 510 N and 768420 W), and Totumo marsh (758160 W and 108440 N). In addition, 49 specimens were collected from the Amazon River in the city of Leticia (698530 N and 48140 W). Sampling was carried out at independent campaigns during February 2003–December 2004. Fish were collected by local fishermen and transported on ice to the lab where necropsies were performed immediately. 2.2. Morphometric analysis and larvae identification Morphometric analysis was performed for each fish. Total lengths and weights were measured to the nearest millimeter and decigram, respectively. Examination also included inspection of macroscopic external lesions and other anatomical abnormalities. Each necropsy was performed cutting from vent to pelvic girdle and consisted of a complete examination of the internal organs, including the mesenteries. Only nematode parasites were searched and counted. Parasite prevalence and intensity were calculated according to Margolis et al. (1982) and Bush et al.
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Fig. 1. Map of sampling sites in Colombia. M1, Magdalena River (Magangue´); M2, Magdalena River (Zambrano); S, Sinu´ River; C, Cauca River; J, San Jorge River; D, Dique Channel; T, Totumo marsh; At, Atrato River; Am, Amazonas River.
(1997). The robustness of each fish was measured using the condition factor (FC) and the hepato-somatic index (HSI) (Ashfield et al., 1998). Preliminary parasite identification, using 287 specimens, was based on general keys of Anderson (2000), and on morphological features suggested by Berland (1989) and Martins et al. (2005). 2.3. Data analysis Data are presented as mean standard errors. All the data were evaluated for normality and variance homogeneity using Kolmogorrov–Smirnov and Bartlett tests, respectively. In the absence of normality, data were log-transformed. An ANOVA was used to evaluate differences among means for different sampling stations. When normality was not achieved, ANOVA was replaced by a Kruskal–Wallis test. Pearson correlation was used to determine associations between parasite intensity and morphometric parameters. For all statistical analysis, the criterion for significance was P < 0.05.
3. Results Morphological identification of all nematode larvae specimens (n = 287) found in Hoplias malabaricus showed the two typical features of Contracaecum species (Nematoda: Anisakidae): the presence of a prominent intestinal caecum extending from the ventriculus to close proximity to the nerve ring, and a ventricular appendix that faces to the posterior extremity. The nematode also has lips with a cephalic tooth between them and a well defined nerve ring near the cephalic end, characters also defined by other authors (Nadler and Hudspeth, 1998; Martins et al., 2005) (see Fig. 2). These larval nematodes were usually found on large numbers on the intestinal mesenterium, with a very small proportion within the muscle, possibly after postdeath migration. Morphometric parameters obtained for all Hoplias malabaricus specimens collected in the sampling locations are shown in Table 1. Greatest and lowest condition factors were found in fish obtained from
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Fig. 2. Morphological features of parasites found in Hoplias malabaricus.
Totumo marsh (1.266 0.04) and Magdalena river (0.830 0.1), respectively. In terms of hepatosomatic index, fish caught at the Dique channel and the Atrato River had the greatest and lowest mean values, respectively. Parasite prevalence and intensity for all sampled stations are shown in Figs. 3 and 4, respectively. A total of 13852 nematodes were recovered from 227 fish collected from the north coast of Colombia. Prevalence was 100% for all tested waterbodies except for Amazon River (6.12%). In average, moncholos from the north coast were infected with a mean intensity of 77.82 4.81 parasites per specimen (1–466 parasites per host), whereas in the Amazon River this value was 1.0 0.0. Maximum intensity values were found in fish collected at the Dique channel (207.3 59.5 parasites/fish), a waterbody that connects the Magdalena River with Cartagena Bay. Statistical differences were found for morphometric parameters such as total length, weight, hepatosomatic index and condition factor for H. Malabaricus collected from different sampling sites (P < 0.05). Correlation analyses for morphometric and parasitic parameters are shown in Table 2. Significant and positive correlations were detected between parasitic intensity and fish length and weight. However, this was absent with hepatosomatic index. The same
parasitic parameter correlated negatively but significantly with condition factor.
4. Discussion Fish are an important source of protein in Colombia, however, their habitats are being polluted by both industrial and sewage waters without treatment. Although all the sampled rivers at the north of Colombia receive untreated urban waters, pollution could not be directly related to the high prevalences found. In fact, based on the condition factor, the most robust fish were those collected at Totumo Marsh, a low polluted waterbody. Of great interest was the finding that all parasite infection indexes were lower in the Amazon River than in the other sampling sites (Leticia, Colombia). The statistical differences observed for morphometric and parasitic parameters in fishes collected in the sampling locations may, at least in part, be due to the season of the year, differences in environmental conditions that depend on water quality, and the presence of intermediate hosts, among other aspects (Williams and MacKenzie, 2003). In Colombia, most important cities discharge their sewage and industrial waters into the Magdalena
Statistically different between stations, Anova, p < 0.05.
Fig. 3. Prevalence of parasite infection in Hoplias malabaricus from different freshwater waterbodies in Colombia.
*
Total Totumo Marsh (T) San Jorge River (J) Dique Channel Atrato River (D) (At) Cauca River (C) Sinu´ River (S) Magdalena Magdalena River (1) M1 River (2) M2 Parameter
Table 1 Morphometric indexes of Hoplias malabaricus collected from Colombian rivers and marshes
Fish number 13 45 24 23 7 18 28 20 227 49 31.7 0.4 28.5 0.9 34.6 0.5 35.0 3.0 34.0 0.5 30.0 0.5 28.4 0.5 32.2 0.3 34.2 0.98 Length (cm)* 34.6 2.1 Weight (g) * 309.9 24.7 299.3 7.9 182.7 12.7 288.6 9.6 394.7 61.5 419.76 23.6 315.17 11.9 291.7 13.3 346.6 9.5 434.5 29.8 0.830 0.1 0.957 0.03 0.837 0.1 1.06 0.1 0.921 0.1 1.062 0.02 1.170 0.03 1.266 0.04 1.02 0.02 1.027 0.03 Condition factor* Hepatosomatic 1.1 0.002 1.4 0.017 1.1 0.0005 1.6 0.001 1.8 0.002 0.8 0.001 1.2 0.001 1.1 0.002 1.4 0.0011 2.0 0.004 index *
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River basin, and few actions are taken to control or minimize these events. At the same time, there is not any concern about the impact of these effluents on the fish populations, although most low-income people living along the rivers derive their meals from this source. The most popular fish in this country has been the Bocachico (Prochilodus magdalenae), however due to its intensive catching, pollution and destruction of habitats, it is facing extinction. As a consequence, other fish species are taking an important role as food sources. Among them is the Hoplias malabaricus, a predatory fish whose nature makes this species suitable for being an intermediary host of parasites. In fact, several studies have shown the presence of several parasite species in this fish, for instance, Spirocamallanus hilarii (Nematoda, Camallanidae) (Ramallo, 1997), Henneguya malabarica sp. nov. (Myxozoa, Myxobolidae), Tetrauronema desaequalis n. sp. (Azevedo and Matos,
Fig. 4. Intensity of parasite infection in Hoplias malabaricus from different freshwater waterbodies in Colombia.
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Table 2 Correlations between morphometric indexes and parasitic intensity calculated for fish from the north coast rivers and marshes of Colombia Parameters
Length
Total length Weight Condition factor Hepatosomatic index Parasitic intensity
1 0.762 0.462 0.162 0.240
(P < 0.0001) (P < 0.0001) P = (0.014) (P < 0.001)
Weight
Condition factor
Hepatosomatic index
Parasitic intensity
1 0.137 (P = 0.039) 0.196 (P = 0.003) 0.199 (P = 0.008)
1 0.029 (P = 0.627) 0.159 (P = 0.034)
1 0.006 (P = 0.935)
1
1996a,b), and Contracaecum sp. larvaes (Nematoda: Anisakidae) (Martins et al., 2005), among others. To the best of our knowledge, this is the first report of Anisakidae larvae in this Colombian fish. It is really dramatic that all eight freshwater sampling stations at the North Coast Colombian basin had parasite prevalences of 100%, whereas at the Amazon River in Leticia, this was only 6.12%. This high prevalence of parasites in Hoplias malabaricus has also been observed from occidental marshlands in the State of Maranhao (100%), but it is greater than those reported for Parana´ River, Presidente Epita´co, State of Sao Paulo, Brazil (Martins et al., 2005). Moreover, the mean intensity found in this work (77.82 8.11) is greater than that reported by some authors in several ecosystems of Brazil (9.5–49.1) (Luque and Alves, 2001; Kohn et al., 1988; Machado et al., 2000). Taken together, these findings might suggest that these high parasite prevalences and intensities could be part of a harmful epidemic that has started in Hoplias malabaricus living in these ecosystems. It should be pointed out that the three stations with the highest parasitic intensity were those located at the end of Magdalena River, the most important waterbody in Colombia. This polluted river receives chemical and microbiological contaminants from a high percentage of the total population of Colombia, in particular from major cities such as Bogota´, Cali, Medellı´n, Bucaramanga and Barranquilla, among others. On the other hand, particular physicochemical, microbiological, geochemical or ecological characteristics of the sampling sites could not be neglected as responsible factors for the observed data. For instance, fish from Totumo marsh, despite the fact that it is a low polluted environment, have parasitic intensity greater than those observed in rivers with urban sewage inputs. These findings in Hoplias malabaricus at this site could be the result of increased salinity due to its proximity to the sea or to the presence of these
nematodes in their diet, the Lisa, Mugil incilis (Olivero-Verbel et al., 2005). Another important factor triggering these massive parasitic events might be related to the presence of one or several abundant and appropriate intermediary species, where these parasites were opportunistic in their colonization of new hosts (Poulin, 2005), and favored the nematode life cycle, leading to make difficult or impossible the on site control of the epidemic event. Although the Contracaecum sp. larvae found in Colombian Hoplias malabaricus had almost the same morphological features than those reported by Martins et al. (2005) in the same fish species in Brazil, it is intriguing that the average length for Colombian parasites (Fig. 5) is almost 9 mm lower than those published by them. This might be in part due to the number of parasites measured by those authors (20) compared with ours (11,142). In addition, although Colombian parasites are similar in total length to those described by Moravec et al. (1985), their wide range of sizes could suggest the presence of other sibling species. Other fish species have also been reported parasitized by Contracaecum sp. larvae, such as Serrasalmus spilopleura (80%) (Hamann, 1999), Aphanopus carbo (69.5%), Scomber japonicus (62.5%) (Costa
Fig. 5. Size distribution for parasites found in Hoplias malabaricus.
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et al., 2003), Platichthys flesus (El-Darsh and Whitfield, 1999), Merluccius hubbsi (Herreras et al., 2000), Cichlasoma (Nandopsis) urophthalmus (Bergmann and Motta, 2004), Salminus maxillosus (Ramallo and Torres, 1995), and Clarias gariepinus (Burchell) (Barson, 2004), indicating that this parasite is found widespread and infecting important commercial species. The positive and significant relationship between parasitic intensity and length and weight observed for Hoplias malabaricus might be the result of time of exposure-dependent process. This has been observed also for other parasites infecting different fish species such as Spatulifer maringaensis and Nupelia portoriquensis in Sorubim lima (Takemoto and Pavanelli, 2000). On the other hand, fish condition factor correlated negatively with parasitic intensity, suggesting that parasites might alter fish physiology functions related to growth and development. In terms of parasite preference for visceral organs, the presence of larvae on the intestinal mesenteriun might limit its zoonotic potencial. However, if fish are not properly frozen or filleted after capture, nematodes may migrate into the flesh (Wharton et al., 1999) and represent a risk for human parasitosis. Although it is clear that the relationships between parasite/host/ecology might impact the high prevalences and intensities recorded here, these results are alarming and must force new research about the origin of this fish susceptibility for parasite infection and its link to environmental conditions.
Acknowledgments We thank to the fishermen from sampling sites for their help in the capture of the fish. Also, we appreciate the effort of Dr. Santiago Duque, Biologist from the Universidad Nacional de Colombia, Bogota´, Colombia.
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Contracaecum sp. infection in Hoplias malabaricus (moncholo) from rivers and marshes of Colombia ´ vila, Jorge Gu¨ette-Ferna´ndez, Jesu´s Olivero-Verbel *, Rosa Baldiris-A Amparo Benavides-Alvarez, Jairo Mercado-Camargo, Barbara Arroyo-Salgado Environmental and Computational Chemistry Group, University of Cartagena, Cartagena, Colombia Received 25 May 2005; received in revised form 2 March 2006; accepted 8 March 2006
Abstract Nematode infection indices were recorded in Hoplias malabaricus captured in six different rivers and a marsh belonging to the North Coast Basin of Colombia, and from the Amazon River, during February 2003–December 2004. Preliminary morphological analysis of nematodes indicated the presence of Contracaecum sp. Parasites were mostly found in the intestinal mesenteries and a very low percentage in muscle. Parasite prevalence in all sampling locations at the north of Colombia was 100%, whereas in the Amazon River it was 6.12%. The mean intensity in the different stations were as follows: Magdalena River at the City of Magangue´ (58.92 7.59), Magdalena river at the city of Zambrano (128.9 7.08), Sinu´ River (53.88 4.92), Dique Channel (207.3 59.52), Cauca River (77.26 9.35), Atrato River (21.11 2.6), San Jorge River (39.5 7.13), and Totumo Marsh (62.5 6.38). In average, all specimens of Hoplias malabaricus from the north coast basin of Colombia were infected with a mean intensity of 77.82 4.81 (1–466 parasites per host) whereas in fish from the Amazon River this value was significantly lower (intensity 1.0 0.0). Size and weight correlated significantly with parasite intensity in fish collected from sampling locations at the north of Colombia (R = 0.240, P < 0.001 and R = 0.199, P = 0.008, respectively). Moreover, a significant, but low and negative correlation was found between condition factor and parasite intensity (R = 0.159, P = 0.034), suggesting a possible impact of parasites on fish health. These results suggest, for the first time, that the parasitism in Moncholo is a widespread phenomenon in Colombian rivers and could represent a risk factor for human consumers. # 2006 Published by Elsevier B.V. Keywords: Freshwater fish; Nematode; Prevalence; Intensity; Human infection
1. Introduction
* Corresponding author at: Environmental and Computational Chemistry Group, Department of Chemistry, University of Cartagena, Cartagena, Colombia. Tel.: +57 5 6698179/6698180; fax: +57 5 6698323. E-mail address: [email protected] (J. Olivero-Verbel). 0304-4017/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.vetpar.2006.03.014
Parasites are an essential part of the aquatic community. Their presence becomes evident after a massive development, causing health effects including oxidative stress (Marcogliese et al., 2005), tissue damage, immunesuppression, and endocrine
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disruption (Jobling and Tyler, 2003), among others, and sometimes even leading to the mass mortality of infested hosts. These parasitic events are often the results of biotic or abiotic changes in the environment. In particular they have been proposed as pollution indicators by heavy metals and hydrocarbons (Schludermann et al., 2003; Williams and MacKenzie, 2003), and their presence can be the result of organic enrichment of sediments by domestic sewage (Marcogliese and Cone, 2001), although factors such as fish population size can also determine parasite infection rates (Bagge et al., 2004). Once nematodes have infected fish, the overall quality of this food decreases enormously, not only because of the possibility of human infection by eating not properly cooked fish (Sakanari and McKerrow, 1989), but also because there is no safe preparation that could completely avoid a risk for human health when consuming parasitized fish (Skirnisson, 2006; Toro et al., 2004; Audicana et al., 2002). In fact, several pathological conditions such as anthropozoonosis, allergy events, central nervous system damage, and anaphylactic reactions (Walker and Zunt, 2005; Lopez-Serrano et al., 2000; Audicana et al., 1995) could occur in some individuals whose main diet is composed of infected fish, as those living along waterbodies that receive untreated wastewater (Ensink et al., 2005). The problem might become severe when fish diversity has decreased to a point where few species are the only available because of their low price and abundance, this is the case of Hoplias malabaricus, commonly called Moncholo. Hoplias malabaricus is a tropical species found in most rivers basins of Central and South Ame´rica, from Costa Rica to Argentina (Zara´te et al., 1989). This species can breathe air by using their swimbladders as respiratory organs. This allows them to live in extremely muddy waters. Its diverse habitats varies from free flowing clear water streams, well up into the valleys, to slow turbid waters, as well as water courses, irrigation and drainage ditches, and ponds on the plains (Kenny, 1995). It usually rests in vegetation during the day and is active at night (Bussing, 1987). Adults feed on fish; juveniles feed on crustacean and insect larvae (Galvis et al., 1997), shrimps and other small invertebrates (Planquette et al., 1996). In Colombia, Hoplias malabaricus is one of the most abundant fish species found in river basins except
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the high Cauca River (Galvis et al., 1997). It represents a valuable resource not only because of its high presence during the whole year, but also because it is part of traditional dishes, such as the smoky Moncholo. Reports of parasite nematodes in Colombian fish species are scarce and therefore the goal of the present investigation was to investigate the occurrence of the nematode infection in Hoplias malabaricus captured in different habitats.
2. Materials and methods 2.1. Specimen collection In total, 227 specimens of Hoplias malabaricus of 32.15 0.3 cm total length and 346.64 9.5 g mean weight were collected from five rivers and one marsh (Fig. 1) at the North Coast Basin of Colombia: Magdalena River, specifically in Magangue´ (88580 N and 748310 W) and Zambrano (98450 N and 748480 W), Sinu´ River (98140 N and 758500 W), Cauca River (88060 N and 748450 W), San Jorge River (88460 N and 748520 W), the Dique Channel (108220 N and 758080 W), Atrato River (58 510 N and 768420 W), and Totumo marsh (758160 W and 108440 N). In addition, 49 specimens were collected from the Amazon River in the city of Leticia (698530 N and 48140 W). Sampling was carried out at independent campaigns during February 2003–December 2004. Fish were collected by local fishermen and transported on ice to the lab where necropsies were performed immediately. 2.2. Morphometric analysis and larvae identification Morphometric analysis was performed for each fish. Total lengths and weights were measured to the nearest millimeter and decigram, respectively. Examination also included inspection of macroscopic external lesions and other anatomical abnormalities. Each necropsy was performed cutting from vent to pelvic girdle and consisted of a complete examination of the internal organs, including the mesenteries. Only nematode parasites were searched and counted. Parasite prevalence and intensity were calculated according to Margolis et al. (1982) and Bush et al.
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Fig. 1. Map of sampling sites in Colombia. M1, Magdalena River (Magangue´); M2, Magdalena River (Zambrano); S, Sinu´ River; C, Cauca River; J, San Jorge River; D, Dique Channel; T, Totumo marsh; At, Atrato River; Am, Amazonas River.
(1997). The robustness of each fish was measured using the condition factor (FC) and the hepato-somatic index (HSI) (Ashfield et al., 1998). Preliminary parasite identification, using 287 specimens, was based on general keys of Anderson (2000), and on morphological features suggested by Berland (1989) and Martins et al. (2005). 2.3. Data analysis Data are presented as mean standard errors. All the data were evaluated for normality and variance homogeneity using Kolmogorrov–Smirnov and Bartlett tests, respectively. In the absence of normality, data were log-transformed. An ANOVA was used to evaluate differences among means for different sampling stations. When normality was not achieved, ANOVA was replaced by a Kruskal–Wallis test. Pearson correlation was used to determine associations between parasite intensity and morphometric parameters. For all statistical analysis, the criterion for significance was P < 0.05.
3. Results Morphological identification of all nematode larvae specimens (n = 287) found in Hoplias malabaricus showed the two typical features of Contracaecum species (Nematoda: Anisakidae): the presence of a prominent intestinal caecum extending from the ventriculus to close proximity to the nerve ring, and a ventricular appendix that faces to the posterior extremity. The nematode also has lips with a cephalic tooth between them and a well defined nerve ring near the cephalic end, characters also defined by other authors (Nadler and Hudspeth, 1998; Martins et al., 2005) (see Fig. 2). These larval nematodes were usually found on large numbers on the intestinal mesenterium, with a very small proportion within the muscle, possibly after postdeath migration. Morphometric parameters obtained for all Hoplias malabaricus specimens collected in the sampling locations are shown in Table 1. Greatest and lowest condition factors were found in fish obtained from
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Fig. 2. Morphological features of parasites found in Hoplias malabaricus.
Totumo marsh (1.266 0.04) and Magdalena river (0.830 0.1), respectively. In terms of hepatosomatic index, fish caught at the Dique channel and the Atrato River had the greatest and lowest mean values, respectively. Parasite prevalence and intensity for all sampled stations are shown in Figs. 3 and 4, respectively. A total of 13852 nematodes were recovered from 227 fish collected from the north coast of Colombia. Prevalence was 100% for all tested waterbodies except for Amazon River (6.12%). In average, moncholos from the north coast were infected with a mean intensity of 77.82 4.81 parasites per specimen (1–466 parasites per host), whereas in the Amazon River this value was 1.0 0.0. Maximum intensity values were found in fish collected at the Dique channel (207.3 59.5 parasites/fish), a waterbody that connects the Magdalena River with Cartagena Bay. Statistical differences were found for morphometric parameters such as total length, weight, hepatosomatic index and condition factor for H. Malabaricus collected from different sampling sites (P < 0.05). Correlation analyses for morphometric and parasitic parameters are shown in Table 2. Significant and positive correlations were detected between parasitic intensity and fish length and weight. However, this was absent with hepatosomatic index. The same
parasitic parameter correlated negatively but significantly with condition factor.
4. Discussion Fish are an important source of protein in Colombia, however, their habitats are being polluted by both industrial and sewage waters without treatment. Although all the sampled rivers at the north of Colombia receive untreated urban waters, pollution could not be directly related to the high prevalences found. In fact, based on the condition factor, the most robust fish were those collected at Totumo Marsh, a low polluted waterbody. Of great interest was the finding that all parasite infection indexes were lower in the Amazon River than in the other sampling sites (Leticia, Colombia). The statistical differences observed for morphometric and parasitic parameters in fishes collected in the sampling locations may, at least in part, be due to the season of the year, differences in environmental conditions that depend on water quality, and the presence of intermediate hosts, among other aspects (Williams and MacKenzie, 2003). In Colombia, most important cities discharge their sewage and industrial waters into the Magdalena
Statistically different between stations, Anova, p < 0.05.
Fig. 3. Prevalence of parasite infection in Hoplias malabaricus from different freshwater waterbodies in Colombia.
*
Total Totumo Marsh (T) San Jorge River (J) Dique Channel Atrato River (D) (At) Cauca River (C) Sinu´ River (S) Magdalena Magdalena River (1) M1 River (2) M2 Parameter
Table 1 Morphometric indexes of Hoplias malabaricus collected from Colombian rivers and marshes
Fish number 13 45 24 23 7 18 28 20 227 49 31.7 0.4 28.5 0.9 34.6 0.5 35.0 3.0 34.0 0.5 30.0 0.5 28.4 0.5 32.2 0.3 34.2 0.98 Length (cm)* 34.6 2.1 Weight (g) * 309.9 24.7 299.3 7.9 182.7 12.7 288.6 9.6 394.7 61.5 419.76 23.6 315.17 11.9 291.7 13.3 346.6 9.5 434.5 29.8 0.830 0.1 0.957 0.03 0.837 0.1 1.06 0.1 0.921 0.1 1.062 0.02 1.170 0.03 1.266 0.04 1.02 0.02 1.027 0.03 Condition factor* Hepatosomatic 1.1 0.002 1.4 0.017 1.1 0.0005 1.6 0.001 1.8 0.002 0.8 0.001 1.2 0.001 1.1 0.002 1.4 0.0011 2.0 0.004 index *
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River basin, and few actions are taken to control or minimize these events. At the same time, there is not any concern about the impact of these effluents on the fish populations, although most low-income people living along the rivers derive their meals from this source. The most popular fish in this country has been the Bocachico (Prochilodus magdalenae), however due to its intensive catching, pollution and destruction of habitats, it is facing extinction. As a consequence, other fish species are taking an important role as food sources. Among them is the Hoplias malabaricus, a predatory fish whose nature makes this species suitable for being an intermediary host of parasites. In fact, several studies have shown the presence of several parasite species in this fish, for instance, Spirocamallanus hilarii (Nematoda, Camallanidae) (Ramallo, 1997), Henneguya malabarica sp. nov. (Myxozoa, Myxobolidae), Tetrauronema desaequalis n. sp. (Azevedo and Matos,
Fig. 4. Intensity of parasite infection in Hoplias malabaricus from different freshwater waterbodies in Colombia.
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Table 2 Correlations between morphometric indexes and parasitic intensity calculated for fish from the north coast rivers and marshes of Colombia Parameters
Length
Total length Weight Condition factor Hepatosomatic index Parasitic intensity
1 0.762 0.462 0.162 0.240
(P < 0.0001) (P < 0.0001) P = (0.014) (P < 0.001)
Weight
Condition factor
Hepatosomatic index
Parasitic intensity
1 0.137 (P = 0.039) 0.196 (P = 0.003) 0.199 (P = 0.008)
1 0.029 (P = 0.627) 0.159 (P = 0.034)
1 0.006 (P = 0.935)
1
1996a,b), and Contracaecum sp. larvaes (Nematoda: Anisakidae) (Martins et al., 2005), among others. To the best of our knowledge, this is the first report of Anisakidae larvae in this Colombian fish. It is really dramatic that all eight freshwater sampling stations at the North Coast Colombian basin had parasite prevalences of 100%, whereas at the Amazon River in Leticia, this was only 6.12%. This high prevalence of parasites in Hoplias malabaricus has also been observed from occidental marshlands in the State of Maranhao (100%), but it is greater than those reported for Parana´ River, Presidente Epita´co, State of Sao Paulo, Brazil (Martins et al., 2005). Moreover, the mean intensity found in this work (77.82 8.11) is greater than that reported by some authors in several ecosystems of Brazil (9.5–49.1) (Luque and Alves, 2001; Kohn et al., 1988; Machado et al., 2000). Taken together, these findings might suggest that these high parasite prevalences and intensities could be part of a harmful epidemic that has started in Hoplias malabaricus living in these ecosystems. It should be pointed out that the three stations with the highest parasitic intensity were those located at the end of Magdalena River, the most important waterbody in Colombia. This polluted river receives chemical and microbiological contaminants from a high percentage of the total population of Colombia, in particular from major cities such as Bogota´, Cali, Medellı´n, Bucaramanga and Barranquilla, among others. On the other hand, particular physicochemical, microbiological, geochemical or ecological characteristics of the sampling sites could not be neglected as responsible factors for the observed data. For instance, fish from Totumo marsh, despite the fact that it is a low polluted environment, have parasitic intensity greater than those observed in rivers with urban sewage inputs. These findings in Hoplias malabaricus at this site could be the result of increased salinity due to its proximity to the sea or to the presence of these
nematodes in their diet, the Lisa, Mugil incilis (Olivero-Verbel et al., 2005). Another important factor triggering these massive parasitic events might be related to the presence of one or several abundant and appropriate intermediary species, where these parasites were opportunistic in their colonization of new hosts (Poulin, 2005), and favored the nematode life cycle, leading to make difficult or impossible the on site control of the epidemic event. Although the Contracaecum sp. larvae found in Colombian Hoplias malabaricus had almost the same morphological features than those reported by Martins et al. (2005) in the same fish species in Brazil, it is intriguing that the average length for Colombian parasites (Fig. 5) is almost 9 mm lower than those published by them. This might be in part due to the number of parasites measured by those authors (20) compared with ours (11,142). In addition, although Colombian parasites are similar in total length to those described by Moravec et al. (1985), their wide range of sizes could suggest the presence of other sibling species. Other fish species have also been reported parasitized by Contracaecum sp. larvae, such as Serrasalmus spilopleura (80%) (Hamann, 1999), Aphanopus carbo (69.5%), Scomber japonicus (62.5%) (Costa
Fig. 5. Size distribution for parasites found in Hoplias malabaricus.
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et al., 2003), Platichthys flesus (El-Darsh and Whitfield, 1999), Merluccius hubbsi (Herreras et al., 2000), Cichlasoma (Nandopsis) urophthalmus (Bergmann and Motta, 2004), Salminus maxillosus (Ramallo and Torres, 1995), and Clarias gariepinus (Burchell) (Barson, 2004), indicating that this parasite is found widespread and infecting important commercial species. The positive and significant relationship between parasitic intensity and length and weight observed for Hoplias malabaricus might be the result of time of exposure-dependent process. This has been observed also for other parasites infecting different fish species such as Spatulifer maringaensis and Nupelia portoriquensis in Sorubim lima (Takemoto and Pavanelli, 2000). On the other hand, fish condition factor correlated negatively with parasitic intensity, suggesting that parasites might alter fish physiology functions related to growth and development. In terms of parasite preference for visceral organs, the presence of larvae on the intestinal mesenteriun might limit its zoonotic potencial. However, if fish are not properly frozen or filleted after capture, nematodes may migrate into the flesh (Wharton et al., 1999) and represent a risk for human parasitosis. Although it is clear that the relationships between parasite/host/ecology might impact the high prevalences and intensities recorded here, these results are alarming and must force new research about the origin of this fish susceptibility for parasite infection and its link to environmental conditions.
Acknowledgments We thank to the fishermen from sampling sites for their help in the capture of the fish. Also, we appreciate the effort of Dr. Santiago Duque, Biologist from the Universidad Nacional de Colombia, Bogota´, Colombia.
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