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Toxicity of aqueous aluminium to the ectoparasitic monogenean Gyrodactylus salaris
Aquaculture 250 (2005) 616 – 620 www.elsevier.com/locate/aqua-online
Toxicity of aqueous aluminium to the ectoparasitic monogenean Gyrodactylus salaris Arnulf Solenga,*, Antonio B.S. Pole´ob, Tor A. Bakkec a
Norwegian Institute of Public Health, Division of Infectious Disease Control, Department of Pest Control, P.O. Box 4404 Nydalen, NO-0403 Oslo, Norway b Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, NO-0316 Oslo, Norway c Natural History Museum, Department of Zoology, University of Oslo, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway Received 3 March 2005; received in revised form 3 May 2005; accepted 4 May 2005
Abstract Both laboratory and field experiments have demonstrated that aqueous aluminium can act as a paraciticide to the monogenean Gyrodactylus salaris Malmberg, 1957 infecting freshwater salmonids. However, the reproductive conditions of gyrodactylids surviving to cessation of an aluminium exposure is unknown. Therefore, Atlantic salmon (Salmo salar L.) parr infected with G. salaris was experimentally exposed to elevated concentrations of aqueous aluminium for more than 1 month. During this period, the infection increased the first week before it peaked and started to steadily decline approaching elimination. When almost all parasite specimens were eliminated, the water quality was adjusted to normal aluminium-poor freshwater. During the next 3 weeks, the surviving G. salaris were shown to clearly resume their reproduction. The experiment demonstrates the potential of the gyrodactylids to reproduce after cessation of an aluminium exposure. Thus, the as-yet unknown mechanism behind the toxic effects of aluminium on G. salaris survival and reproduction might to some extent be reversible. The present study gives further support for this metal as a parasiticide to G. salaris but demonstrates at the same time that in order to eliminate the parasites totally, the aluminium treatment must eradicate all G. salaris from the skin of the infected hosts. Furthermore, the potential development of resistance to aluminium should also be studied. D 2005 Elsevier B.V. All rights reserved. Keywords: Acidification; Aluminium; Monogenea; Parasiticide; Rotenone
1. Introduction
* Corresponding author. Tel.: +47 22 04 24 80; fax: +47 22 04 25 31. E-mail address: [email protected] (A. Soleng). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.05.006
Finding species-specific biocides is a major goal in the treatment of pests and diseases. In Norway, chemical treatments with rotenone have frequently been used in attempts to eradicate the pathogenic monoge-
A. Soleng et al. / Aquaculture 250 (2005) 616–620
nean Gyrodactylus salaris Malmberg, 1957 ectoparasitic on Atlantic salmon (Salmo salar L.) by eradication of all salmon in the infected population (Johnsen and Jensen, 1991; Mo, 1994; Johnsen et al., 1999). Rotenone is, however, an indiscriminate poison, killing all fish species within the treated river system. In addition, rotenone has negative effects on the aquatic biota in general by killing all gill-breathing inverte¨ berg, 1961; Morrison, 1977). brates (Lindahl and O Several of the rotenone treatments in Norway have also failed (Mo, 1994; Haukebø et al., 2000) probably due to the hydrological and topological complexity of the larger infected river systems. The use of rotenone is therefore controversial and debated, and research for effective and environmentally safer alternatives is needed. Soleng et al. (1996) reported the first promising results suggesting that aluminium treatment could complement the controversial rotenone treatments used against G. salaris. However, earlier field observations have also shown a devastating effect of acidification and aqueous aluminium on several species of freshwater fish and invertebrates (e.g., Havens and Heath, 1989; Muniz, 1991; Kullberg, 1992; Herrmann et al., 1993; Sparling and Lowe, 1996). Free-living freshwater invertebrates show a wide diversity of responses to aqueous aluminium, but it is generally assumed that they are less sensitive to aluminium than fish (Howells et al., 1994). Among tested fish, the Atlantic salmon is recognised as the most aluminiumsensitive species (Grande et al., 1978; Pole´o et al., 1997). The laboratory study of Soleng et al. (1999) demonstrates that aqueous aluminium indirectly can have a positive effect even on this aluminium-sensitive fish species. The reason is that heavily G. salarisinfected Atlantic salmon parr were disinfected after exposure to acidic water with elevated levels of aqueous aluminium with no clear damage noted on the salmon parr. Later studies have confirmed these remarkable results (Pole´o et al., 2004). However, every treatment of pests and diseases should cause minimum stress to the environment and optimally eliminate the problem completely. Hence, the length of the aluminium exposure is an important factor that must be addressed properly if aluminium is to be used as a parasiticide. However, the fate of G. salaris surviving an exposure is still unknown. Therefore, the aim of the present study was to elucidate whether the toxic
617
effects of aluminium on exposed but living G. salaris are irreversible, thus allowing treatments to be ended before all parasites are eradicated from the hosts.
2. Materials and methods Hatchery-reared Atlantic salmon (S. salar) parr (age 0+) of the River Lierelva stock (Buskerud County, Norway), were used in the experiments in the laboratory at the Natural History Museum, Department of Zoology, University of Oslo. The parr ranged from 0.7 to 2.5 g in weight (mean 1.5 g) and 4.3 to 6.7 cm in length (mean 5.5 cm). In the laboratory, the fish were kept in fish tanks (100 100 cm, water level 30 cm), and acclimated at 12 8C for at least 4 weeks prior to the experiments. The fish tanks received activated charcoal-filtered and dechlorinated Oslo tap water. The experimental fish had not previously been exposed to any gyrodactylids and were routinely disinfected (with formalin and salt) against ectoparasites and fungus in the hatchery. The G. salaris used in the experiments originated from naturally infected salmon parr caught in the River Lierelva. Wild-caught G. salaris-infected fish were kept in fish tanks in the laboratory for at least 4 weeks before they were used to infect the experimental fish. After the acclimation period and prior to the experiment, the experimental fish were infected with G. salaris. This was done by placing 15 individuals of the experimental fish in small aerated infection chambers (38 27 cm and 14 cm deep) for 24 h together with fins removed from anaesthetized (0.04% chlorobutanol) and killed naturally infected parr. The number of G. salaris on the experimental fish was measured after this infection procedure, and the fish was then transferred to the experimental exposure tanks. Three test media were prepared: acidic aluminium-enriched water; 45 Ag Al/l, pH 5.3 by the addition of an acidic Al(NO3)3 stock solution to the laboratory water (Oslo tap water). Acidic aluminium-poor water (pH 5.3) was prepared by the addition of HNO3. Untreated tap water (pH 6.4) was used as control. Three flowthrough systems were used, one for each medium. Each experimental system consisted of a mixing tank (75 55 cm and 40 cm deep) and an exposure tank (100 100 cm and 30 cm deep) where the experimental fish were kept during the experiments. The aluminium stock solution and HNO3
A. Soleng et al. / Aquaculture 250 (2005) 616–620
were added to the laboratory water entering the mixing tank. The water flow into each exposure tank was approximately 1 l/min. The water was well aerated, and the water flow provided at least 68 l of water per gram fish per day. Within each exposure tank, water was circulated by aquarium power filters (Fluval 403 without filter medium). The experiments were performed at 12.0 8C (range 11.4–12.6 8C) at constant dim illumination, and the fish were sheltered by nontransparent lids covering half of each tank. The fish were fed unmedicated pellet food (Ewos) during the acclimation periods but deprived of nutrition during the experiments. In each exposure tank, water temperature and flow, pH and chemical dosage were measured daily during the experiments. The total amount of aluminium was determined five times during the experiment. Aqueous aluminium in all test media was analysed as described in Soleng et al. (1999). Growth and survival of infrapopulations of G. salaris were measured by counting the G. salaris specimens on the external skin and fins of the fish. This was performed under a stereomicroscope, on anaesthetized (0.04 % chlorobutanol) fish placed in a Petri dish (see Bakke et al., 1991; Soleng and Bakke, 1997). The Petri dishes contained water from the same exposure tank as the fish. The reproductive rate (r) of G. salaris on grouped fish was calculated from the equation: N t = N 0ert where N t = the number of parasites recorded at time t, and N 0 = the number of parasites recorded at time 0.
3. Results In normal freshwater (pH 6.4) and acidic aluminium-poor water (pH 5.3), the G. salaris infrapopulational growth was exponential with a population growth rate (r) of, respectively, 0.143 and 0.103 during the experiment. However, negative effect on the parasite infrapopulations in the grouped salmon parr subject to an elevated aluminium concentration was apparent (Fig. 1). Initially, after a short period (up to 1 week) of infrapopulational growth, an obvious effect of the aluminium exposure appeared as the intensity of all the infrapopulations started to continuously decline. Approximately 30 days after the negative effect of the elevated aluminium concentration appeared, and
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Time (days) Fig. 1. Course of infection of grouped G. salaris on Atlantic salmon (S. salar) parr at 12.0 8C. (n) Untreated control water at pH 6.4; (o) acidified aluminium-poor water at pH 5.3; (.) acidified aluminium-enriched water at pH 5.3, 45 Ag Al/l. The arrow indicates the end of the period for addition of the acidic aluminium stock solution. The prevalence was 100% in all experiments throughout. The number of fish (n) was 15 in each exposure tank at day 0 (changes in n are indicated by numbers close to data points). Error bars are S.E. of the arithmetic mean. Note log10 scale.
when the G. salaris infrapoulations were almost eliminated, the addition of acid and aluminium was stopped. Water quality then returned to normal freshwater (pH 6.4) within hours. Three weeks thereafter (day 58), the examination of the infrapopulations demonstrated that the intensity of the G. salaris infection had increased clearly. The population growth rate during this period was calculated to 0.104, similar to the group exposed to aluminium-poor water at pH 5.3, and slightly less than the freshwater control group. Hence, it seems that the recovery of the reproductive function of surviving G. salaris after cessation of an aluminium exposure is rapidly resumed but somewhat delayed compared to the natural situation. Some salmon host died (see Fig. 1 for details) in all three exposures during the experiment, and consequently, this cannot be addressed to aluminium toxicity.
4. Discussion The present results confirm the detrimental effect of aluminium in G. salaris previously reported by
A. Soleng et al. / Aquaculture 250 (2005) 616–620
Soleng et al. (1996, 1999) and Pole´o et al. (2004). However, in the previously published studies, infected fish were exposed continuously to elevated aluminium levels until elimination of all parasites from the hosts. Soleng et al. (1999) examined more closely detached G. salaris specimens exposed to 52 Ag Al/l at pH 5.9. These specimens had been off the host for less than 24 h and were all found to be opaque and torpid, and did not resume normal colour and activity after transfer to optimal water conditions. Hence, Soleng et al. (1999) suggested that following exposure to aluminium, G. salaris die on the host or shortly after detachment. In the present study, however, the aluminium exposure ended when the abundance of G. salaris reached a very low level but before total detachment and elimination. Thus, some parasites were still attached and, hence, influenced also by the host surface microenvironment. The present study demonstrate that G. salaris infrapopulations which have been exposed for prolonged periods (up to 37 days) of elevated aluminium concentrations still have the ability to recover and start reproduction when the water quality returns to normal. However, it is worth noting that the decline observed during the experiments could also have been because mortality exceeds natality rather than to a complete cessation of reproduction. Nevertheless, the as-yet unknown mechanism of aluminium toxicity in G. salaris seems to be reversible. Soleng et al. (1999) reported that it was not possible to determine whether the rapid elimination of G. salaris from the fish was due to a direct effect of aluminium on the parasite and/or an indirect effect of aluminium through effects on the microenvironment of the host. In a later study, Grimsmo (2000) pre-exposed Atlantic salmon to elevated aqueous aluminium levels before being infected with G. salaris and found no difference in the subsequent course of infection compared to control fish, thus suggesting a direct effect of aluminium on the parasite. The reasons for the differences in individual parasite mortality during an aluminium exposure, similar to observations after exposure to increased salinity (Soleng and Bakke, 1997; Soleng et al., 1998), are unknown, but genetic heterogeneity in water chemical resistance cannot be excluded. It is earlier reported that monogeneans can develop resistance to different chemotherapeutica such as mebendazole and organophosphates (Goven et al., 1980; Buchmann and Roep-
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storff, 1992). For example, it was recently reported that resistance had developed in the gill monogenean parasite, Pseudodactylogyrus, to all the benzimidazole anthelminthics on a specific eel farm due to the intensive use of mebendazole (Waller and Buchmann, 2001). Thus, there is a need to study the potential of both acclimation and acclimatization of G. salaris to aluminium if this metal is to be used as a parasiticide. In the present study, G. salaris was exposed to elevated but not immediately deadly aluminium concentrations. During the exposure to aluminium, the infection course increased initially for a week similar to the findings of Soleng et al. (1999), indicating a time lag in the toxic effects of aluminium at these low concentrations. However, a clear decline of all infrapopulations appeared thereafter. Minor increases of aqueous metal concentrations as, e.g., aluminium and probably zinc (Pole´o et al., 2004) may have negative effects on the reproductive growth rate of such ectoparasites. Hence, specific water chemistry factors might prevent and/or decrease development and reproduction of gyrodactylids after a transitory colonization and establishment or keep the growth of the parasite infrapopulations below the level of pathogenic host effects. For example, epidemics of G. salaris are not reported from the Baltic area, even though Baltic salmon parr may be relatively susceptible (Bakke et al., 2002, 2004). Besides the potential of differences in virulence between parasite strains (see Hansen et al., 2003), the absence of epidemics may be due to macroenvironmental factors linked to water chemistry (see Soleng et al., 1999) and water temperature (see Jansen and Bakke, 1991). In conclusion, the results confirm the importance of water chemistry for ectoparasite survival and reproduction. Hence, there is an urgent need for studies on the influence of slight increases in toxic heavy metals on the reproductive rate of gyrodactylids. In addition, management of infected wild salmon by extermination of G. salaris by restricted concentrations of aluminium as a parasiticide must persist until all pathogenic gyrodactylids are eliminated from the hosts surfaces as they may have the ability to resume their reproductive power after the exposure. However, the present study confirms the potential of aluminium as a parasiticide for gyrodactylids at concentrations and exposure periods well tolerated by the Atlantic salmon host.
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Acknowledgements We thank the technical assistance of Kjersti Kvalsvik, Zoological Museum, University of Oslo, and the staff at DOFA (Drammen og Omegn Fiskeriadministrasjon) for providing fish for the experiment. This study was supported by the Norwegian Research Council (NFR 145861/720). References Bakke, T.A., Jansen, P.A., Hansen, L.P., 1991. Experimental transmission of Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes, Monogenea) from the Atlantic salmon (Salmo salar) to the European eel (Anguilla anguilla). Can. J. Zool. 69, 733 – 737. Bakke, T.A., Harris, P.D., Cable, J., 2002. Host specificity dynamics: observations on gyrodactylid monogeneans. Int. J. Parasitol. 32, 281 – 308. Bakke, T.A., Harris, P.D., Hansen, H., Cable, J., Hansen, L.P., 2004. Susceptibility of Baltic and East Atlantic salmon Salmo salar stocks to Gyrodactylus salaris (Monogenea). Dis. Aquat. Org. 58, 171 – 177. Buchmann, K., Roepstorff, A., 1992. Experimental selection of mebendazole-resistant gill monogeneans from the European eel, Anguilla anguilla L. J. Fish Dis. 15, 303 – 400. Goven, B.A., Gilbert, J.P., Gratzek, J.B., 1980. Apparent drug resistance to the organophosphate dimethyl (2,2,2-trichloro-1hydroxyethyl) phosphonate by monogenetic trematodes. J. Wildl. Dis. 16, 343 – 346. Grande, M., Muniz, I.P., Andersen, S., 1978. Relative tolerance of some salmonids to acid waters. Int. Ver. Theor. Angew. Limnol. Ver. 20, 2076 – 2084. Grimsmo, H., 2000. Aluminiums virkning pa˚ Gyrodactylus salaris infeksjon hos laks (Salmo salar): En direkte eller indirekte effekt? Cand Scient. thesis, University of Oslo (in Norwegian). Hansen, H., Bachmann, L., Bakke, T.A., 2003. Mitochondrial DNA variation of Gyrodactylus spp. (Monogenea, Gyrodactylidae) populations infecting Atlantic salmon, grayling and rainbow trout in Norway and Sweden. Int. J. Parasitol. 33, 1471 – 1478. Haukebø, T., Eide, O., Skjelstad, B., Bakkeli, G., Tønset, K., Stensli, J.H., 2000. Rotenonbehandlingen som tiltak mot lakseparasitten Gyrodactylus salaris. En gjennomgang av metodikk, utstyr og rutiner med forslag for forbedringer. Utredning for DN, 2000-2, 1–80 (in Norwegian). Havens, K.E., Heath, R.T., 1989. Acid and aluminium effects on freshwater zooplankton: an in situ mesocosm study. Environ. Pollut. 62, 195 – 211. Herrmann, J., Degerman, E., Gerhardt, A., Johansson, C., Lingdell, P.-E., Muniz, I.P., 1993. Acid stress effects on stream biology. Ambio 22, 298 – 307. Howells, G., Dalziel, T.R.K., Reader, J.P., Solbe´, J.F., 1994. Aluminium and freshwater fish water quality criteria. In: Howells,
G. (Ed.), Water Quality for Freshwater Fish. Further Advisory Criteria. Gordon and Breach Science Publishers, Great Britain, pp. 55 – 115. Jansen, P.A., Bakke, T.A., 1991. Temperature-dependent reproduction and survival of Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes: Monogenea) on Atlantic salmon (Salmo salar L.). Parasitology 102, 105 – 112. Johnsen, B.O., Jensen, A.J., 1991. The Gyrodactylus story in Norway. Aquaculture 98, 289 – 302. Johnsen, B.O., Møkkelgjerd, P.I., Jensen, A.J., 1999. The parasite Gyrodactylus salaris on salmon parr in Norwegian rivers, status report at the beginning of year 2000. NINA Oppdragsmelding 617, 1–129 (in Norwegian, English summary). Kullberg, A., 1992. Benthic macroinvertebrate community structure in 20 streams of varying pH and humic content. Environ. Pollut. 78, 103 – 106. ¨ berg, K.E., 1961. The effect of rotenone on respiLindahl, P.E., O ration and its point of attack. Exp. Cell Res. 23, 228 – 237. Morrison, B.R.S., 1977. The effects of rotenone on the invertebrate fauna of three hill streams in Scotland. Fish. Manage. 8, 128 – 139. Mo, T.A., 1994. Status of Gyrodactylus problems and research in Norway. In: Pike, A.W., Lewis, J.W. (Eds.), Parasitic Diseases in Fish. Samara, Tresaith, UK, pp. 43 – 58. Muniz, I.P., 1991. Freshwater acidification: its effects on species communities of freshwater microbes, plants and animals. Proc. R. Soc. Edinb. Sect. B Biol. 97, 227 – 254. Pole´o, A.B.S., Østbye, K., Øxnevad, S.A., Andersen, R.A., Heibo, E., Vøllestad, A.L., 1997. Toxicity of acid aluminium-rich water to seven freshwater fish species: a comparative laboratory study. Environ. Pollut. 96, 129 – 139. Pole´o, A.B.S., Schjolden, J., Hansen, H., Bakke, T.A., Mo, T.A., Rosseland, B.O., Lydersen, E., 2004. The effect of various metals on Gyrodactylus salaris (Platyhelminthes, Monogenea) infections in Atlantic salmon (Salmo salar). Parasitology 128, 169 – 177. Soleng, A., Bakke, T.A., 1997. Salinity tolerance of Gyrodactylus salaris (Platyhelminthes, Monogenea): laboratory studies. Can. J. Fish. Aquat. Sci. 54, 1837 – 1845. Soleng, A., Poleo, A.B.S., Bakke, T.A., Alstad, N.W., 1996. Aluminium toxicity to Gyrodactylus salaris (Monogenea). Parassitologia 38, 65. Soleng, A., Bakke, T.A., Hansen, L.P., 1998. Potential for dispersal of Gyrodactylus salaris (Platyhelminthes, Monogenea) by searunning stages of the Atlantic salmon (Salmo salar): field and laboratory studies. Can. J. Fish. Aquat. Sci. 55, 507 – 514. Soleng, A., Pole´o, A.B.S., Alstad, N.E.W., Bakke, T.A., 1999. Aqueous aluminium eliminates Gyrodactylus salaris (Platyhelminthes Monogenea) infections in Atlantic salmon. Parasitology 119, 19 – 25. Sparling, D.W., Lowe, T.P., 1996. Environmental hazards of aluminium to plants, invertebrates, fish, and wildlife. Rev. Environ. Contam. Toxicol. 145, 1 – 127. Waller, P.J., Buchmann, K., 2001. Anthelminthic resistance and parasite control in commercial eel farms: consequences for producers. Vet. Rec. 23, 783 – 784.
Toxicity of aqueous aluminium to the ectoparasitic monogenean Gyrodactylus salaris Arnulf Solenga,*, Antonio B.S. Pole´ob, Tor A. Bakkec a
Norwegian Institute of Public Health, Division of Infectious Disease Control, Department of Pest Control, P.O. Box 4404 Nydalen, NO-0403 Oslo, Norway b Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, NO-0316 Oslo, Norway c Natural History Museum, Department of Zoology, University of Oslo, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway Received 3 March 2005; received in revised form 3 May 2005; accepted 4 May 2005
Abstract Both laboratory and field experiments have demonstrated that aqueous aluminium can act as a paraciticide to the monogenean Gyrodactylus salaris Malmberg, 1957 infecting freshwater salmonids. However, the reproductive conditions of gyrodactylids surviving to cessation of an aluminium exposure is unknown. Therefore, Atlantic salmon (Salmo salar L.) parr infected with G. salaris was experimentally exposed to elevated concentrations of aqueous aluminium for more than 1 month. During this period, the infection increased the first week before it peaked and started to steadily decline approaching elimination. When almost all parasite specimens were eliminated, the water quality was adjusted to normal aluminium-poor freshwater. During the next 3 weeks, the surviving G. salaris were shown to clearly resume their reproduction. The experiment demonstrates the potential of the gyrodactylids to reproduce after cessation of an aluminium exposure. Thus, the as-yet unknown mechanism behind the toxic effects of aluminium on G. salaris survival and reproduction might to some extent be reversible. The present study gives further support for this metal as a parasiticide to G. salaris but demonstrates at the same time that in order to eliminate the parasites totally, the aluminium treatment must eradicate all G. salaris from the skin of the infected hosts. Furthermore, the potential development of resistance to aluminium should also be studied. D 2005 Elsevier B.V. All rights reserved. Keywords: Acidification; Aluminium; Monogenea; Parasiticide; Rotenone
1. Introduction
* Corresponding author. Tel.: +47 22 04 24 80; fax: +47 22 04 25 31. E-mail address: [email protected] (A. Soleng). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.05.006
Finding species-specific biocides is a major goal in the treatment of pests and diseases. In Norway, chemical treatments with rotenone have frequently been used in attempts to eradicate the pathogenic monoge-
A. Soleng et al. / Aquaculture 250 (2005) 616–620
nean Gyrodactylus salaris Malmberg, 1957 ectoparasitic on Atlantic salmon (Salmo salar L.) by eradication of all salmon in the infected population (Johnsen and Jensen, 1991; Mo, 1994; Johnsen et al., 1999). Rotenone is, however, an indiscriminate poison, killing all fish species within the treated river system. In addition, rotenone has negative effects on the aquatic biota in general by killing all gill-breathing inverte¨ berg, 1961; Morrison, 1977). brates (Lindahl and O Several of the rotenone treatments in Norway have also failed (Mo, 1994; Haukebø et al., 2000) probably due to the hydrological and topological complexity of the larger infected river systems. The use of rotenone is therefore controversial and debated, and research for effective and environmentally safer alternatives is needed. Soleng et al. (1996) reported the first promising results suggesting that aluminium treatment could complement the controversial rotenone treatments used against G. salaris. However, earlier field observations have also shown a devastating effect of acidification and aqueous aluminium on several species of freshwater fish and invertebrates (e.g., Havens and Heath, 1989; Muniz, 1991; Kullberg, 1992; Herrmann et al., 1993; Sparling and Lowe, 1996). Free-living freshwater invertebrates show a wide diversity of responses to aqueous aluminium, but it is generally assumed that they are less sensitive to aluminium than fish (Howells et al., 1994). Among tested fish, the Atlantic salmon is recognised as the most aluminiumsensitive species (Grande et al., 1978; Pole´o et al., 1997). The laboratory study of Soleng et al. (1999) demonstrates that aqueous aluminium indirectly can have a positive effect even on this aluminium-sensitive fish species. The reason is that heavily G. salarisinfected Atlantic salmon parr were disinfected after exposure to acidic water with elevated levels of aqueous aluminium with no clear damage noted on the salmon parr. Later studies have confirmed these remarkable results (Pole´o et al., 2004). However, every treatment of pests and diseases should cause minimum stress to the environment and optimally eliminate the problem completely. Hence, the length of the aluminium exposure is an important factor that must be addressed properly if aluminium is to be used as a parasiticide. However, the fate of G. salaris surviving an exposure is still unknown. Therefore, the aim of the present study was to elucidate whether the toxic
617
effects of aluminium on exposed but living G. salaris are irreversible, thus allowing treatments to be ended before all parasites are eradicated from the hosts.
2. Materials and methods Hatchery-reared Atlantic salmon (S. salar) parr (age 0+) of the River Lierelva stock (Buskerud County, Norway), were used in the experiments in the laboratory at the Natural History Museum, Department of Zoology, University of Oslo. The parr ranged from 0.7 to 2.5 g in weight (mean 1.5 g) and 4.3 to 6.7 cm in length (mean 5.5 cm). In the laboratory, the fish were kept in fish tanks (100 100 cm, water level 30 cm), and acclimated at 12 8C for at least 4 weeks prior to the experiments. The fish tanks received activated charcoal-filtered and dechlorinated Oslo tap water. The experimental fish had not previously been exposed to any gyrodactylids and were routinely disinfected (with formalin and salt) against ectoparasites and fungus in the hatchery. The G. salaris used in the experiments originated from naturally infected salmon parr caught in the River Lierelva. Wild-caught G. salaris-infected fish were kept in fish tanks in the laboratory for at least 4 weeks before they were used to infect the experimental fish. After the acclimation period and prior to the experiment, the experimental fish were infected with G. salaris. This was done by placing 15 individuals of the experimental fish in small aerated infection chambers (38 27 cm and 14 cm deep) for 24 h together with fins removed from anaesthetized (0.04% chlorobutanol) and killed naturally infected parr. The number of G. salaris on the experimental fish was measured after this infection procedure, and the fish was then transferred to the experimental exposure tanks. Three test media were prepared: acidic aluminium-enriched water; 45 Ag Al/l, pH 5.3 by the addition of an acidic Al(NO3)3 stock solution to the laboratory water (Oslo tap water). Acidic aluminium-poor water (pH 5.3) was prepared by the addition of HNO3. Untreated tap water (pH 6.4) was used as control. Three flowthrough systems were used, one for each medium. Each experimental system consisted of a mixing tank (75 55 cm and 40 cm deep) and an exposure tank (100 100 cm and 30 cm deep) where the experimental fish were kept during the experiments. The aluminium stock solution and HNO3
A. Soleng et al. / Aquaculture 250 (2005) 616–620
were added to the laboratory water entering the mixing tank. The water flow into each exposure tank was approximately 1 l/min. The water was well aerated, and the water flow provided at least 68 l of water per gram fish per day. Within each exposure tank, water was circulated by aquarium power filters (Fluval 403 without filter medium). The experiments were performed at 12.0 8C (range 11.4–12.6 8C) at constant dim illumination, and the fish were sheltered by nontransparent lids covering half of each tank. The fish were fed unmedicated pellet food (Ewos) during the acclimation periods but deprived of nutrition during the experiments. In each exposure tank, water temperature and flow, pH and chemical dosage were measured daily during the experiments. The total amount of aluminium was determined five times during the experiment. Aqueous aluminium in all test media was analysed as described in Soleng et al. (1999). Growth and survival of infrapopulations of G. salaris were measured by counting the G. salaris specimens on the external skin and fins of the fish. This was performed under a stereomicroscope, on anaesthetized (0.04 % chlorobutanol) fish placed in a Petri dish (see Bakke et al., 1991; Soleng and Bakke, 1997). The Petri dishes contained water from the same exposure tank as the fish. The reproductive rate (r) of G. salaris on grouped fish was calculated from the equation: N t = N 0ert where N t = the number of parasites recorded at time t, and N 0 = the number of parasites recorded at time 0.
3. Results In normal freshwater (pH 6.4) and acidic aluminium-poor water (pH 5.3), the G. salaris infrapopulational growth was exponential with a population growth rate (r) of, respectively, 0.143 and 0.103 during the experiment. However, negative effect on the parasite infrapopulations in the grouped salmon parr subject to an elevated aluminium concentration was apparent (Fig. 1). Initially, after a short period (up to 1 week) of infrapopulational growth, an obvious effect of the aluminium exposure appeared as the intensity of all the infrapopulations started to continuously decline. Approximately 30 days after the negative effect of the elevated aluminium concentration appeared, and
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Time (days) Fig. 1. Course of infection of grouped G. salaris on Atlantic salmon (S. salar) parr at 12.0 8C. (n) Untreated control water at pH 6.4; (o) acidified aluminium-poor water at pH 5.3; (.) acidified aluminium-enriched water at pH 5.3, 45 Ag Al/l. The arrow indicates the end of the period for addition of the acidic aluminium stock solution. The prevalence was 100% in all experiments throughout. The number of fish (n) was 15 in each exposure tank at day 0 (changes in n are indicated by numbers close to data points). Error bars are S.E. of the arithmetic mean. Note log10 scale.
when the G. salaris infrapoulations were almost eliminated, the addition of acid and aluminium was stopped. Water quality then returned to normal freshwater (pH 6.4) within hours. Three weeks thereafter (day 58), the examination of the infrapopulations demonstrated that the intensity of the G. salaris infection had increased clearly. The population growth rate during this period was calculated to 0.104, similar to the group exposed to aluminium-poor water at pH 5.3, and slightly less than the freshwater control group. Hence, it seems that the recovery of the reproductive function of surviving G. salaris after cessation of an aluminium exposure is rapidly resumed but somewhat delayed compared to the natural situation. Some salmon host died (see Fig. 1 for details) in all three exposures during the experiment, and consequently, this cannot be addressed to aluminium toxicity.
4. Discussion The present results confirm the detrimental effect of aluminium in G. salaris previously reported by
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Soleng et al. (1996, 1999) and Pole´o et al. (2004). However, in the previously published studies, infected fish were exposed continuously to elevated aluminium levels until elimination of all parasites from the hosts. Soleng et al. (1999) examined more closely detached G. salaris specimens exposed to 52 Ag Al/l at pH 5.9. These specimens had been off the host for less than 24 h and were all found to be opaque and torpid, and did not resume normal colour and activity after transfer to optimal water conditions. Hence, Soleng et al. (1999) suggested that following exposure to aluminium, G. salaris die on the host or shortly after detachment. In the present study, however, the aluminium exposure ended when the abundance of G. salaris reached a very low level but before total detachment and elimination. Thus, some parasites were still attached and, hence, influenced also by the host surface microenvironment. The present study demonstrate that G. salaris infrapopulations which have been exposed for prolonged periods (up to 37 days) of elevated aluminium concentrations still have the ability to recover and start reproduction when the water quality returns to normal. However, it is worth noting that the decline observed during the experiments could also have been because mortality exceeds natality rather than to a complete cessation of reproduction. Nevertheless, the as-yet unknown mechanism of aluminium toxicity in G. salaris seems to be reversible. Soleng et al. (1999) reported that it was not possible to determine whether the rapid elimination of G. salaris from the fish was due to a direct effect of aluminium on the parasite and/or an indirect effect of aluminium through effects on the microenvironment of the host. In a later study, Grimsmo (2000) pre-exposed Atlantic salmon to elevated aqueous aluminium levels before being infected with G. salaris and found no difference in the subsequent course of infection compared to control fish, thus suggesting a direct effect of aluminium on the parasite. The reasons for the differences in individual parasite mortality during an aluminium exposure, similar to observations after exposure to increased salinity (Soleng and Bakke, 1997; Soleng et al., 1998), are unknown, but genetic heterogeneity in water chemical resistance cannot be excluded. It is earlier reported that monogeneans can develop resistance to different chemotherapeutica such as mebendazole and organophosphates (Goven et al., 1980; Buchmann and Roep-
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storff, 1992). For example, it was recently reported that resistance had developed in the gill monogenean parasite, Pseudodactylogyrus, to all the benzimidazole anthelminthics on a specific eel farm due to the intensive use of mebendazole (Waller and Buchmann, 2001). Thus, there is a need to study the potential of both acclimation and acclimatization of G. salaris to aluminium if this metal is to be used as a parasiticide. In the present study, G. salaris was exposed to elevated but not immediately deadly aluminium concentrations. During the exposure to aluminium, the infection course increased initially for a week similar to the findings of Soleng et al. (1999), indicating a time lag in the toxic effects of aluminium at these low concentrations. However, a clear decline of all infrapopulations appeared thereafter. Minor increases of aqueous metal concentrations as, e.g., aluminium and probably zinc (Pole´o et al., 2004) may have negative effects on the reproductive growth rate of such ectoparasites. Hence, specific water chemistry factors might prevent and/or decrease development and reproduction of gyrodactylids after a transitory colonization and establishment or keep the growth of the parasite infrapopulations below the level of pathogenic host effects. For example, epidemics of G. salaris are not reported from the Baltic area, even though Baltic salmon parr may be relatively susceptible (Bakke et al., 2002, 2004). Besides the potential of differences in virulence between parasite strains (see Hansen et al., 2003), the absence of epidemics may be due to macroenvironmental factors linked to water chemistry (see Soleng et al., 1999) and water temperature (see Jansen and Bakke, 1991). In conclusion, the results confirm the importance of water chemistry for ectoparasite survival and reproduction. Hence, there is an urgent need for studies on the influence of slight increases in toxic heavy metals on the reproductive rate of gyrodactylids. In addition, management of infected wild salmon by extermination of G. salaris by restricted concentrations of aluminium as a parasiticide must persist until all pathogenic gyrodactylids are eliminated from the hosts surfaces as they may have the ability to resume their reproductive power after the exposure. However, the present study confirms the potential of aluminium as a parasiticide for gyrodactylids at concentrations and exposure periods well tolerated by the Atlantic salmon host.
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