Aug. 06, 2024
Praziquantel (PZQ) provides an effective treatment against monogenean parasitic infestations in finfish. However, its use as an in-feed treatment is challenging due to palatability issues. In this study, five formulations of PZQ beads (1&#;4 mm) were developed using marine-based polymers, with allicin added as a flavouring agent. All formulations attained PZQ loading rates &#;74% w/w, and the beads were successfully incorporated into fish feed pellets at an active dietary inclusion level of 10 g/kg. When tested for palatability and digestibility in small yellowtail kingfish, the PZQ-loaded beads produced with alginate-chitosan, alginate-Cremophor ® RH40, and agar as carriers resulted in high consumption rates of 99&#;100% with no digesta or evidence of beads in the gastrointestinal tract (GIT) of fish fed with diets containing either formulation. Two formulations produced using chitosan-based carriers resulted in lower consumption rates of 68&#;75%, with undigested and partly digested beads found in the fish GIT 3 h post feeding. The PZQ-loaded alginate-chitosan and agar beads also showed good palatability in large (&#;2 kg) yellowtail kingfish infected with gill parasites and were efficacious in removing the parasites from the fish, achieving >90% reduction in mean abundance relative to control fish (p < 0.001). The two effective formulations were stable upon storage at ambient temperature for up to 18 months, showing residual drug content >90% compared with baseline levels. Overall, the palatability, efficacy and stability data collected from this study suggest that these two PZQ particulate formulations have potential applications as in-feed anti-parasitic medications for the yellowtail kingfish farming industry.
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This study aimed to develop effective taste masked particulate PZQ formulations capable of being incorporated into fish feed to provide in-feed anti-parasitic medications for cultured yellowtail kingfish (Seriola lalandi). The beads were formulated to control PZQ availability by withholding drug release in seawater to minimise detection, thus encouraging feeding by the fish, and once consumed by the fish, were digestible in the gastrointestinal tract (GIT) to provide a bolus drug dose. Beads were fabricated using sodium alginate, agar, and chitosan, applied individually and in combinations, as carriers. The polymers are generally regarded as safe (GRAS) materials derived from marine sources [ 27 ], and have been used as vehicles for drug delivery [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ] and binders for fish feed [ 36 , 37 , 38 ]. Garlic derivatives, including allicin, were applied as flavours based on literature evidence [ 25 ].
The palatability issues of PZQ in Seriola have been well described [ 4 , 18 , 19 ]. In an effort to address this issue, Partridge et al. () tested microencapsulated PZQ, which improved palatability, but reduced bioavailability compared with pure PZQ [ 4 ]. Incorporation of PZQ into solid lipid nanoparticles with and without a chitosan coating also failed to achieve acceptable palatability and bioavailability [ 11 ]. Similarly, delivery of only the R-(&#;) enantiomer of PZQ in feed failed to improve its palatability relative to racemic PZQ or the S-(+) enantiomer [ 12 ]. Pilmer () evaluated various taste masking agents with only limited improvements to palatability [ 25 ]. In , Forwood et al. effectively masked the flavour of PZQ to yellowtail kingfish using moist pellets; however, these have the disadvantage of having to be prepared fresh (at sea) just prior to use [ 26 ].
Praziquantel (PZQ) is a highly efficacious anthelmintic agent [ 8 , 9 ] against a wide range of platyhelminth parasites, including monogeneans. It is effective when administered to infected fish via injection, bathing, or feeding [ 4 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Injection, however, is impractical in commercially farmed fish, and bathing presents challenges similar to those described above for hydrogen peroxide, with the added disadvantage of discharging relatively large quantities of a pharmacologically active agent into the environment. Feeding can potentially deliver PZQ into a large number of fish in a stress-free manner; however, the bitter taste of PZQ is a major constraint to effective delivery [ 4 , 11 , 12 , 15 , 18 , 19 ]. Significant formulation research has been conducted on masking the bitterness of PZQ to improve its palatability but with little success, particularly in Seriola species. This group represents the fourth most valuable cultured marine fish industry in the world [ 20 ], predominantly from Japan, and with rapid expansion into new regions, including Australia, New Zealand, South Africa, Europe, and the Americas [ 21 ]. In these regions, the various Seriola species are infected with monogenean parasites (Benedenia seriolae, Zeuxapta seriolae, and Neobenedenia melleni), the management of which contributes up to 20% of the cost of production [ 22 , 23 , 24 ], at a cost of circa $0.5 billion in Japan alone [ 1 ].
The economic cost of parasitism in cultured fish has been estimated at circa $US10 billion annually [ 1 ]. Monogenean ectoparasites are a highly diverse subset of this group that affect both marine and freshwater fish. Between and species have been described with many causing considerable economic impacts on fish farming industries globally [ 2 , 3 ]. Wild fish are commonly infected with monogeneans with little impact on their wellbeing; however, the parasites&#; direct lifecycles and other ecological and reproductive features make confined (i.e., cultured) fish highly susceptible to uncharacteristically high levels of infection [ 3 ]. Left untreated, these parasites can cause growth impairment, anaemia, and secondary bacterial infections, leading to high morbidity and mortality [ 4 ]. A common treatment for infected fish in sea cages involves bathing in hydrogen peroxide [ 5 , 6 ], an expensive, labour-intensive, time-consuming, and weather-dependent method that can affect fish health [ 7 ] and pose significant occupational health risks for farm staff.
At the end of the 6-day trial period, fish fed the unmedicated control diet had a mean abundance of 55 ± 13 Zeuxapta gill parasites per fish. There was a significant reduction in parasite abundance in the three PZQ treatment groups relative to this unmedicated control (p < 0.001; ). Fish fed diets containing Formulation B and Formulation C had parasite reductions of 93 ± 2% and 94 ± 3%, respectively, both higher than that seen in the positive PZQ control, where a 73 ± 4% reduction in parasite abundance was observed. The percentage reduction in parasites relative to the negative control was significantly higher in fish fed Formulation B and Formulation C than the positive control (p < 0.001). Gut dissections showed no whole beads retained in either the stomach or gut of the fish. However, there was evidence of white mucous, and it is not known whether this was from the PZQ beads or just a normal digestive function.
For the palatability and efficacy trial in parasite infected fish, the fish ate significantly less of the positive PZQ (powder) control treatment (17 ± 4%) than that of the unmedicated control treatment (79 ± 6%) (p < 0.001, a). However, there was no significant difference between the intake of fish fed the unmedicated control and those fed with Formulation B (61 ± 5%) or Formulation C (62 ± 5%). b shows the feed intake by day, demonstrating that the fish in all the tanks fed poorly on the second day, and that the feed intake subsequently increased for all treatment groups over the following days, with the exception of the PZQ control treatment group, which consistently showed low feed intakes. The intake of diets containing Formulation B and Formulation C increased with time, mirroring the trend of the unmedicated control group.
With regards to digestibility, there was no evidence of beads found in the digestive tract in either small or large fish fed the Formulation A treatment ( a). However, undigested and partly digested beads of the Formulation E treatment were found throughout the entire length of the intestinal tract in the large fish ( b). For the other three treatments, apart from a single Formulation D bead in the stomach, there was no digesta or evidence of beads in the intestinal tracts of fish fed with diets containing Formulations B, C, or D ( c&#;e).
As there was no evidence of fish size affecting PZQ palatability, only small fish were subsequently used for assessing the palatability of Formulations B, C, and D. The fish ate &#;99% of all treatments ( a), with no significant difference between treatments (p = 0.28, one-way ANOVA). However, whilst the fish ate their full ration at each feeding, the time taken to consume the ration was significantly different between all formulations and the unmedicated control (0.64 ± 0.07 min) (p < 0.02). The times taken to eat a complete ration of diets containing Formulation B and Formulation C were 1.15 ± 0.14 min and 1.16 ± 0.14 min (n = 4), respectively, while Formulation D took the longest at 1.42 ± 0.04 min (n = 3) ( b). On the basis of the slower time taken to consume the diet containing Formulation D, it was not progressed to the efficacy trials.
Analysis by two-way ANOVA showed no effect of fish size (p = 0.77) or treatment (p = 0.07) on overall consumption of the medicated feeds incorporating Formulations A and E, both of which contained chitosan only in the bead matrix. Large fish fed the unmedicated control treatment consumed 79 ± 14% of the ration, while those fed Formulation A ate on average 74 ± 15%, and fish fed Formulation E ate 80 ± 11% of the ration ( a). The small fish fed the unmedicated control diet ate their entire ration, but only ate 62 ± 11% and 71 ± 18% of Formulation A and Formulation E treatment rations, respectively. There was no significant effect of fish size on the time taken to consume the fixed ration (p = 0.212); however, there was a significant difference between the treatments (p < 0.01; b). On those occasions when the fish ate the entire medicated ration, the time to do so was circa 3 min. Fish offered the unmedicated control treatment consumed their ration in circa 1.5 min. The large yellowtail kingfish fed with Formulation A did not eat an entire ration of feed during the 5-day trial and the small fish only ate the full ration of Formulation A on one occasion.
DSC analysis was conducted for Formulations B and C, and the thermograms were compared with the DSC thermograms of PZQ and the corresponding blank beads in . PZQ (3.4 mg) exhibited a sharp melting endotherm with onset at 138.9 °C and peak temperature of 141.99 °C. The enthalpy for melting was 99.870 J/g. PZQ melting peak for Formulation B (3.8 mg) had onset at 138.35 °C and peak temperature at 141.50 °C. Its enthalpy of 72.607 J/g was 72.70% that of pure PZQ, and corresponded to the drug loading of Formulation B. PZQ peak in Formulation C (3.4 mg) had onset at 138.03 °C and peak temperature at 141.93 °C. Its enthalpy of 71.645 J/g was 71.74% that of pure PZQ, which again corresponded closely to the drug loading of Formulation C. Thus, it may be concluded from the respective DSC thermograms that PZQ retained its crystalline characteristics, and did not interact with the matrix materials in Formulations B and C.
Formulation B and Formulation C beads did not disintegrate even after 3 h incubation in 500 mL of seawater, and only 6.51 ± 0.59% and 1.36 ± 0.71% of the drug loads from the respective beads were leached into the seawater at 3 h ( a). Simulation of the bead passage from seawater into the fish GIT showed undetectable drug release after 5 min in seawater from both formulations. The Formulation B beads remained intact after a further 60 min incubation in SGF, releasing only 2.3 ± 0.4% of the drug load; however, bead disintegration was noted in the SIF accompanied by the release of 84.7 ± 2.9% of the drug load at 185 min ( b). In contrast, the beads of Formulation C remained intact not only in seawater, but also in the SGF and SIF, with low levels of drug release measured over the entire dissolution period. Cumulative percent drug release from the Formulation C beads was only 3.87 ± 0.24% at 185 min.
Dissolution profile of PZQ for Formulation B and Formulation C were studied by simulating the passage of the beads from seawater into the fish GIT. This simulation could not be performed for the pure drug powder using the basket apparatus; instead, the dissolution profile of the pure drug was determined separately in each medium. Dissolution of PZQ was not detectable after 5 min in seawater; however, 56.7 ± 4.1% of the drug powder had dissolved after 3 h in 500 mL of seawater ( a), which was comparable to the 53.7 ± 0.9% dissolution obtained after 1 h in 100 mL of SGF ( b). Incubation for 2 h in SIF led to the dissolution of 78.2 ± 4.8% of the drug powder ( b). There was incomplete dissolution of the PZQ powder under the simulated GIT conditions, although the drug:media ratio (7&#;13 mg in 100 mL) was below the solubility of PZQ in water (approximately 36 mg/100 mL) [ 39 ].
Qualitative results of the bead disintegration in seawater and 0.1 M HCl are shown in . As shown in the figure, none of the beads from the five formulations disintegrated in seawater, as the medium remained clear and intact beads were still present after 5 h of incubation. Beads from Formulations A and E, prepared with chitosan only as carrier, showed complete disintegration in 0.1 M HCl to yield white suspensions with bead fragments. Formulation B turned the 0.1 M HCl solution slightly turbid after 5 h, suggesting evidence of bead disintegration; however, the disintegration was incomplete as the beads did not show any breakdown of structure ( b). No disintegration of the beads was observed for Formulations C and D in 0.1 M HCl.
All formulations achieved a drug loading of at least 74% w/w PZQ in the dry beads ( ). Increasing the batch size by 10-fold did not affect the efficiency of drug loading in Formulations B, C, and D ( ). The formulations had mean dry bead diameters ranging from 1.36 to 3.03 mm ( ). Formulation B had the largest bead diameter due to the incorporation of insoluble chitosan particles in the alginate matrix for these beads.
Though widely regarded to be an efficacious drug for the treatment of fish infected with monogenean parasites, praziquantel (PZQ) is currently constrained in its use as an in-feed treatment because of its bitter taste. In this study, we successfully developed two PZQ particulate formulations using a combination of alginate and chitosan (Formulation B) and agar alone (Formulation C) as the bead matrix, both also containing allicin powder as a flavouring agent. The beads of Formulations B and C were shown not to disintegrate in seawater and could be incorporated into fish feed pellets with high palatability for small and large yellowtail kingfish. Unlike the preparation of praziquantel nanoparticles using chitosan-N-arginine and alginate [40,41], the manufacturing processes for these beads are less complex and can easily be up-scaled, and the beads are stable to storage at ambient temperature. More importantly, they were demonstrated to be efficacious against gill parasites, indicating that the incorporated PZQ was released from the beads in vivo for bioactivity in the fish. Prior research has demonstrated that microencapsulating PZQ can improve its palatability to yellowtail kingfish [4]; however, these microcapsules were damaged by the heat and/or pressure of the extrusion process used to manufacture the fish diets, resulting in leaching of PZQ into the medicated feed and a subsequent reduction in palatability. Surface coating fish feed post-extrusion with solid lipid nanoparticles containing PZQ has also been trialled unsuccessfully to improve PZQ palatability to yellowtail kingfish. A major advantage of the beads produced in the current study was the high loading rate (>74%) compared to the aforementioned microcapsules (40%) [4] and solid lipid nanoparticles (<10%) [11]. Such low loading rates necessitate much higher inclusion levels of these particles to achieve the same active dietary inclusion level of PZQ and was hypothesised to contribute to the lack of palatability improvement.
In this study, alginate, chitosan, and agar were used to formulate the bead matrix as they are extracted from marine sources [27], and alginate and agar are commonly used as binders for fish feed [36,37]. Preliminary formulations that used alginate alone as the bead matrix (i.e., without chitosan) resulted in improved palatability of PZQ compared to unencapsulated PZQ; however, the alginate beads were not digestible in vivo, and intact beads were found along the length of the dissected GIT of treated fish (data not shown). The poor digestibility was attributed to the poor solubility of alginate and PZQ in the acidic conditions of the fish stomach [42]. As the polycationic chitosan dissolves under acidic conditions [43] and chitin is known to be digestible by fish [44], chitosan alone was used to prepare the PZQ-loaded beads in Formulations A and E. The chitosan beads showed better digestibility in the fish gut; however, palatability was reduced, which could be attributed to PZQ being released from the beads into seawater. Prolonging the complexation time between chitosan and the tripolyphosphate (TPP) ions from 3 h (Formulation A) to overnight (Formulation E) did not resolve the palatability; instead, it reduced the bead digestibility in vivo. It was concluded that the acidic solvent used to dissolve the chitosan polymer might have a residual acidic (sour) taste, or the premature disintegration of the chitosan beads prior to reaching the fish stomach might have adversely affected palatability.
To make the palatable PZQ-loaded alginate beads more digestible in the fish gut, chitosan and Cremophor® RH40 were trialled as additives. Microparticles having a mix of alginate and chitosan have been fabricated by several methods, including co-dissolving sodium alginate and chitosan in an acidic solvent and extruding this solution into a calcium chloride solution [45]; extruding an aqueous solution of sodium alginate into a solution containing chitosan and calcium chloride dissolved in an acidic solvent [28]; incubating freshly formed calcium alginate beads in a chitosan solution [33]; or extruding a chitosan solution into a TPP solution containing sodium alginate [34]. Beads fabricated using these methods aim to produce an external layer of crosslinked alginate-chitosan, which is not favoured for PZQ as the crosslinked polymer would adversely affect digestibility in vivo. Instead, the PZQ-loaded alginate-chitosan beads (Formulation B) were prepared by mixing a powder comprising PZQ, allicin, and chitosan into the alginate solution and extruding the suspension into a calcium chloride solution. The chitosan powder served as a wicking agent, which upon dissolution in the acidic gastric fluid in the fish would increase porosity and capillary action in the beads to enhance bead digestibility and PZQ release. Conversely, Cremophor® RH40 (Formulation D) was used as a surfactant solubiliser, allowing not only smaller beads to be made, but also enhancing the wettability and digestibility of the beads in vivo. Formulations B and D did not disintegrate in seawater, nor did they disintegrate completely in 0.1 M HCl; however, the initially floating beads were observed to sink after 5 h of incubation in the acidic medium, indicating water intake. Formulation B showed some evidence of disintegration, turning the medium slightly turbid, and the in vitro dissolution study suggested that Formulation B beads disintegrated to allow PZQ release in the fish GIT. Both formulations, when incorporated into fish feed, were palatable and digestible for the yellowtail kingfish. Formulation D was less palatable than Formulation B, based on the time taken for the fish to completely eat the given feed ration, and this is attributed to Cremophor® RH40 being a surfactant; it could have facilitated the dissolution and leaching of PZQ from the beads into the seawater, leading to the detection of the drug by the fish.
Agar used in Formulation C was digestible by the yellowtail kingfish, which is not surprising, as it is a marine product that is also widely used as a binder for fish food [37]. Agar has been used to provide sustained drug release [35], but it is not as widely used as alginate and chitosan for drug delivery, possibly because heat is required for its manufacture, making it inaccessible to heat labile drugs. PZQ is, however, very heat stable [46]. In this study, the PZQ-loaded agar beads (Formulation C) showed similar palatability, in vivo digestibility, and efficacy to Formulation B. Unlike Formulation B, the Formulation C beads did not disintegrate in seawater, SGF, or SIF, and the in vitro dissolution data showing low levels of PZQ release were not reflective of the in vivo digestibility and efficacy data. The dissolution media were adjusted for pH, surface activity, and enzymes (pepsin and trypsin) to represent the fish gastrointestinal fluids; however, the compositional content of the fish gut is not known with certainty, and it may well be that in vivo, the Formulation C beads were digested in the fish GIT by bacteria or other enzymes.
In the efficacy trial, fish in the PZQ control treatment group received an average PZQ dose over the 6 days of 21 mg/kg, which was adequate to provide a 73% reduction in parasite abundance. This appears to be in contrast to Forwood et al. [26] who found no reduction in gill parasites when yellowtail kingfish were intubated daily for 3 days with moist pellets containing 30 mg/kg of PZQ. The apparent discrepancy can be explained by the decreasing intake of the PZQ control diet over time i.e., whilst the average dose over 6 days was 21 mg/kg, the fish ate a much higher percentage of their ration on day 1, equivalent to a dose of 49 mg/kg. It is, therefore, likely that most of the parasites in this treatment were dislodged on the first day.
The average daily dose of PZQ received by fish in the Formulation B and Formulation C treatment groups was more than three times higher than those in the positive control treatment. This dose yielded a 93% and 94% reduction in gill parasites, respectively. Whilst Forwood et al. demonstrated that it is possible for a single dose of 50 mg/kg to eliminate >97% of gill parasites, in a commercial context, medicated feeds are offered over a longer period of time (3&#;7 days) to overcome the variation in intake between individual fish on a day-to-day basis [26]. Complete parasite elimination was not achieved with either formulation since not all fish fed equally. Indeed, Forwood et al. highlighted that competitive feeding is much stronger in a larger (commercial) population of fish, which may facilitate a more uniform intake [26].
An added advantage is that Formulation B and Formulation C are stable to store at ambient temperature for up to 18 months, while the storage stability of other formulations is not often reported. One final attraction of Formulation B for fish farmers is that chitosan as a supplement for fish feed has been found to reduce mortality and improve the growth performance of cultured marine fish [47].
Data are available on request.
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Key words:
Aquaculture, control, disease, fish, parasite
Finfish aquaculture in freshwater and marine environments is continuously expanding globally, and the potential for a substantial further increase is well documented. The industry is supplying fish products for human consumption to the same extent as capture fisheries, and new fish species for domestication are still being selected by the industry. The challenge faced by all aquacultured species, classical and novel, is the range of pathogens associated with each new fish type. A fish host in its natural environment carries a series of more or less specific parasites (specialists and generalists). Some of these show a marked ability to propagate in aquaculture settings. They may then elicit disease when infection intensities in the confined aquaculture environment reach high levels. In addition, the risk of transmission of parasites from aquaculture enterprises to wild fish stocks adds to the parasitic challenge. Control programmes of various kinds are needed and these may include chemotherapeutants and medicines as the farmer's first and convenient choice, but mechanical, biological, immunological and genetic control methods are available solutions. New methods are still to be developed by scrutinizing the life cycle of each particular parasite species and pin-pointing the vulnerable stage to be targeted. As parasites exhibit a huge potential for adaptation to environmental changes, one must realize that only one approach rarely is sufficient. The present work therefore elaborates on and advocates for implementation of integrated control strategies for diseases caused by protozoan and metazoan parasites.
Finfish aquaculture in freshwater and marine environments is continuously expanding globally (FAO, ) and the potential for a substantial further increase is well documented in the marine environment (Gentry et al., ). Freshwater aquaculture of fish dominates at present in Asia and particularly in China and Indonesia, but expansion of this branch in Europe and the Americas is possible through application of recirculation technology, which receives attention and increased investment capital. The aquaculture industry is supplying fish products for human consumption to the same extent as capture fisheries, and new fish species for domestication are still being selected by the industry (FAO, ). The challenge faced by all aquacultured species, classical and novel, is the range of pathogens associated with each new fish type. A fish host in its natural environment carries a series of more or less specific parasites (specialists and generalists) (Woo et al., ). Even if disease-free fish are used for stocking, disease problems may arise in a fish production system, provided the farm becomes exposed to pathogens (water intake from external sites). Parasite stages from wild fish populations can infect and propagate on the aquacultured fish (spill-over from the environment). As some of these organisms show a marked ability to propagate in aquaculture settings, they may elicit disease as infection intensities increase in the confined aquaculture environment. This will challenge the health and welfare of the fish and the economy of the aquaculture enterprises (Shinn et al., ). The risk of transmission of parasites from the aquaculture enterprises to wild fish stocks (back-spill) adds to the need for initiation of control programmes in order to protect the original endemic fish populations. Chemotherapeutants and medicines may be the farmer's first and convenient choice but mechanical, biological, immuno-prophylactic and genetic control methods are available as sustainable solutions. The present study outlines control possibilities for various parasitic groups of importance including oomycetes (Saprolegnia), protozoans (amoebae, flagellates, ciliates) and metazoans (myxozoans, monogeneans, digeneans, cestodes, nematodes, crustaceans) and advocates for an integrated control strategy due to the remarkable adaptivity of parasites. As background for the assessments and recommendations, a selection of relevant published scientific articles were included. Those studies are elaborating on various methods used to control parasitic infections in aquacultured fish with focus on chemotherapeutants, biocides, herbal extracts, medicines, mechanical methods and immunoprophylactic methods including vaccination. Combination of the methodologies may be considered if an integrated control strategy is to be implemented. The different legislation on usage of biocides and medicines in different countries may complicate their application as many of the compounds may be licensed in some countries but not in others.
A series of chemicals with a problematic toxicity profile are well known in aquaculture suffering from ectoparasitic infections. The organic dye malachite green was previously applied in even low concentrations for elimination of oomycetes (e.g. Saprolegnia) from fish eggs and fish larvae. It also effectively kills parasitic ciliates such as Ichthyophthirius multifiliis and flagellates such as Ichthyobodo, Piscinoodinium and Amyloodinium. However, concerns on the toxicity of malachite green were raised early (Alderman, ). Studies have shown that the compound and its metabolite leucomalachite green are carcinogenic and genotoxic (EFSA, ). Following the ban of malachite green several decades ago, other chemicals with some, but lower, efficacy were used in increasing amounts (Rintamäki et al., ). A range of insecticides (malathion and parathion) were previously used to eradicate crustacean parasites (Kabata, ), but the environmental issues, including toxicity to fish and workers, limit their application.
Ectoparasites on freshwater fish may be eliminated by immersion of the infected host into high NaCl concentrations. Similarly, marine parasites succumb when exposed to freshwater dependent on their ability to adjust to the change of salinity. Parasites as free-living invertebrates may show different tolerance to changing salinities. Thus, some are euryhaline and others stenohaline. The osmotic stress induced by a change of salinity may kill a range of protozoans (amoebae, flagellates, ciliates) and metazoans (monogeneans). Freshwater treatments are regularly applied to reduce populations of marine amoebae such as Neoparamoeba perurans on gills causing amoebic gill disease (AGD) in maricultured Atlantic salmon (Nowak, ). The treatment of white spot disease caused by trophonts of the freshwater ciliate Ichthyopthirius multifiliis is more complicated. The parasite is in principle not an ectoparasite, due to its location in the epidermis, where it is covered by a hyperplasic epithelium. It is therefore protected against osmotic stress in the host tissue. In order to eliminate this parasite in a fish farm system, the free-living stages (tomonts, tomocysts, theronts) must be targeted. This may be achieved by sustaining a high (10 ppt) concentration over 10 days at temperatures over 20°C, whereby all trophonts in the fish surface will have sufficient time to escape into the fish tank water. The high salinity will prevent development of the tomont, via the tomocyst stage, into infective theronts, whereby the life cycle is broken and the parasite population exhausted (Li and Buchmann, ). The corresponding marine species Cryptocaryon irritans may be controlled by a similar strategy but by use of seawater diluted (up to 1:3) with distilled water (Cheung et al., ).
Administration of formalin directly to fish tank water containing live infected fish is currently used in conventional farms and even in some recirculated systems (Madsen et al., ; Noga, ). Such bath treatments with the chemical in concentrations around 20&#;50 mg L&#;1 remove epibionts (sessile ciliates, flagellates, amoebae) (Buchmann and Bresciani, ; Noga, ) including Amyloodinium (Noga, ), Ichthyobodo (Jaafar et al., ) from fish surfaces, monogeneans from fish skin (Buchmann and Kristensson, ), gills (Buchmann, ) and kill infective free-living stages of e.g. Ichthyophthirius and Diplostomum (Larsen et al., ) in the fish tank water. In addition, it reduces the bacterial concentration and the infective stages of various pathogens. The bath treatment initiates a stress response in the fish, which can be measured as a surge of plasma cortisol (Jørgensen and Buchmann, ) and a general upregulation of proinflammatory cytokines in skin and gills (Mathiessen et al., b). As the compound is allergenic and carcinogenic it is considered a human health hazard. Chloramine T has been widely used as bath treatment against similar ectoparasites but due to lack of approval by authorities the application is restrained (Lasee, ).
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Another widely used compound is copper sulphate with corresponding lethal effects on ectoparasites and or external infective stages (Lasee, ; Noga, ). It has documented toxic effects on Ichthyophthirius, Ichthyobodo, Amyloodinium and the crustacean parasite Argulus. Potassium permanganate has been used for similar purposes (Lasee, ; Straus and Griffin, ; Noga, ). Environmental concerns due to its effect on free-living organisms, including algae, may constrain approval, licensing and thereby usage in aquaculture facilities.
Hydrogen peroxide, sodium percarbonate and peracetic acid are potent oxidizing agents, which are widely applied in aquaculture as replacement for malachite green and formalin (Rach et al., ; Meinelt et al., ; Straus and Meinelt, ; Bruzio and Buchmann, ; Jaafar et al., ). The compounds are used for bathing of infected fish and interact with and effectively kill various ectoparasites and external stages such as theronts of Ichthyophthirius and Ichthyobodo necator. It is also applied in Mediterranean mariculture enterprises, including seabream aquacultures, suffering from gill infections caused by the monogenean Sparicotyle chrysophrii (Sitjà-Bobadilla et al., ). Further, hydrogen peroxide has been used for treatment of Japanese tiger puffer (Tagifugu rubriceps) suffering from infections of the branchial cavity wall with the diclidophorid gill monogenean Heterobothrium okamotoi (Ogawa and Yokoyama, ). The compound has also been widely applied to remove salmon lice from the surface of Atlantic salmon, but efficacy has decreased over time due to selection of partly H2O2-resistant parasite strains (Helgesen et al., ).
Extensive work has been conducted on the usage of plant extracts to control fish diseases, including those caused by bacterial infections (Zheng et al., ; Diler et al., ) and ectoparasites (Madsen et al., ; Tedesco et al., ). The volatile molecules in Allium, Thymus, Origanum and Coriander were found to have a short-term effect both in vitro and in vivo when screened for effects against Ichthyophthirius in rainbow trout (Mathiessen et al., a), and correspondingly Origanum extracts were found lethal to Trichodina and Ichthyobodo (Mizuno et al., ). In feed application of Chinese herbal medicines such as ginger (Zingiber officinale) also showed a significant reducing effect on I. multifiliis infection in grasscarp (Lin et al., ). The number of potential parasiticides in plants is high and a range of studies have documented effects of 18 compounds against the parasitic dinoflagellate Amyloodinium ocellatum (Tedesco et al., ). Several others may have a potential for future licensing, but unfortunately a large part of the tested substances exhibited toxic effects in cell cultures. Functional feeds (containing plant extracts, organic acids and yeast constituents) for gilthead seabream were shown to partly counteract the pathology induced by Enteromyxum leei (Palenzuela et al., ). Besides the direct toxic effect of the molecules on the parasites and a possible immunostimulatory effect on the host (Lin et al., ; Mathiessen et al., b), it is worthwhile to consider alternative mechanisms exerted by the herbal extracts, as they may disrupt the host-seeking behaviour of certain parasites and thereby prevent infections (O'Shea et al., ).
Unicellular parasites, such as ciliates, are sensitive to a surfactant released by the bacterium Pseudomonas H6. In vitro exposure of Ichthyophthirius theronts, tomonts and tomocysts demonstrated a full lethal effect of the compound, even when used in low concentrations (Al-Jubury et al., ). Follow-up in vivo work showed that the compound in a concentration of 10 mg L&#;1 in a fish tank with a high concentration of infective theronts effectively prevented infection of rainbow trout (Li et al., ). The low toxic effect of the surfactant on trout (Mathiessen et al., b) and to other ecosystem organisms (cyanobacteria, green algae, crustaceans and zebrafish) (Korbut et al., ) suggests that the product should be further assessed for a possible future application as a parasiticide in aquaculture.
The classical approach to parasite control is to apply various medicines as antiparasitic agents (Picon-Camacho et al., ), but a set of rules and legislation must be observed when parasitic infections in fish are to be treated with medicines. This applies for both preliminary investigational and validation studies before licensing and when administrating the licensed products (Sommerville et al., ). Before initiating treatments at farm level, a specific diagnosis should be stated and a prescription made by a veterinarian. The drug should be licensed in the particular country in which the treatment is planned. Several medicines with a known antiparasitic effect have been banned in animal production of various reasons.
The group of drugs banned include, e.g. for the group of nitro-imidazoles, such as metronidazole, secnidazole and dimetridazole, although they are highly effective against flagellates (Spironucleus vortens) (Sangmaneedet and Smith, ) and ciliates (Ichthyophthirius) (Tokşen and Nemli, ). Usage of these drugs for production animals was banned in the European community for decades due to lack of needed documentation (safety, residual levels). Drugs which are licensed for one host species may in several countries be applied also to treat corresponding infections in other hosts, provided that sufficient documentation for efficacy against the disease and low toxicity to the host are available. In that case the prescription is given according to cascade rules.
Toltrazuril (brand name Baycox®) is an anticoccidial which was mentioned as a parasiticide by Schmahl et al. () following experimental in vitro studies with various ciliate parasites ranging from Ichthyophtirius to Apiosoma and Trichodina. In vivo work documented a preventive effect, when used in-feed, against Ichthyophthirius as well (Jaafar and Buchmann, ). Trophonts in the skin were not affected by treatment. The extended half-life in the environment makes the usage questionable from an environmental point of view. Other anticoccidials such as amprolium and salinomycin may show effect against the myxozoan E. leei in maricultured bream (Golomazou et al., ), although the exact mode of action is still to be determined.
The aquaculture industry initiated the usage of various types of organophosphates including metrifonate, dichlorvos and azamethiphos at early time points. The mode of action is the inhibition of the acetylcholinesterase in the parasites, whereby the worms get paralysed. Low concentrations (<1 mg L&#;1) have been shown to limit infections with monogeneans such as Dactylogyrus in cyprinids (Kabata, ), Pseudodactylogyrus in eels (Chan and Wu, ), crustacean parasites (e.g. Lernaea and Argulus) in cyprinids and Lepeophtheirus in salmon farming. Early warnings against development of anthelmintic resistance in monogeneans were placed by Goven et al. () and the extensive usage of the compounds (such as azamethiphos and others) in salmonid mariculture led to fast and well-documented selection of resistant strains of salmon lice (Kaur et al., ).
Natural extracts of the plant Chrysanthemum containing pyrethroids have a strong effect on crustacean parasites such as Argulus and were used in classical Chinese fish farming as a parasiticide (Kabata, ). Pyrethroids, such as the compound deltamethrin, were also applied against salmon lice in salmonid mariculture but continuous administration induces selection of resistant strains (Bakke et al., ). The toxicity of the compound to fish calls for precaution when applying these substances.
The salmon industry has suffered from significant infections by salmon lice Lepeophtheirus salmonis since the early start in the late s and early s. The usage of hydrogen peroxide and organophosphates such as azamethiphos and metrifonate (brand names Neguvon and Nuvan) was preferred in the first decades of Norwegian mariculture. The emamectin benzoate (an avermectin product) was introduced in the late s and shown highly effective as convenient in-feed treatment (Stone et al., ). Other crustacean parasites such as Argulus could be controlled in a corresponding way (Hakalahti et al., ). This accelerated its use until widespread drug resistance appeared in salmon lice (Lees et al., ), whereafter the industry turned to other ways of control (cleaner fish, mechanical removal, flushing with high-temperature water).
Mebendazole belongs to the benzimidazole group, which has been used in human and veterinary medicine for decades. A solution of the compound was shown by Szekely and Molnar () to exert a strong and effective effect on the gill monogenean Pseudodactylogyrus parasitizing the European eel Anguilla anguilla. Following toxicological studies in the laboratory, it was also found effective as a bath in large-scale settings in recirculated eel farms (Buchmann and Bjerregaard, ). It was then regularly and extensively used in the aquaculture industry for years despite laboratory experiments warned about the risk for development of anthelmintic resistance (Buchmann et al., ). Consequently after 6 years, it could be demonstrated that a high degree of anthelmintic resistance occurred at farm level (Waller and Buchmann, ). Other benzimidazoles such as flubendazole and albendazole showed effects as well but due to their common mode of action (binding to tubulin monomers) cross resistance is expected to occur due to several common structures of the molecules. Problematic monogenean (Heterobothrium okamatoi) infections of maricultured tiger puffer in Japanese waters were previously treated by hydrogen peroxide bathing (Ogawa and Yokoyama, ), but a new compound, the pro-benzimidazole febantel, has been the drug of choice for the last decades (Hirazawa et al., ; Kimura et al., ). Elimination of external parasites, such as monogeneans, by use of anthelmintics is less complicated because the affected parasites are released from the host surface. However, endoparasites represent another problem. Benzimidazoles have seen a wide application in treatment of livestock nematode infections and fish nematodes such as Anguillicoloides (swimbladder nematode) in eels are susceptible as well. However, when treating fish with large nematode burdens in internal organs (such as the swim bladder) special attention should be placed on the risk of excessive antigen liberation from dying worms. The exposure of the host to high nematode antigen concentrations (in organs or systemically) may lead to an exacerbated immuno-pathological reaction.
Already four decades ago the anthelmintic praziquantel was found highly effective against the trematode Schistosoma and subsequently it was introduced in various aquaculture settings targeting monogeneans (Schmahl and Mehlhorn, ; Sitjà-Bobadilla et al., ), trematodes such as Diplostomum eyeflukes (Bylund and Sumari, ) and cestodes such as Bothriocephalus (Pool et al., ). The compound is still being applied although lower sensitivities of various parasite species have been reported.
Infections of fish with myxozoans are a major problem in both marine and freshwater. The infections are not readily treated but an antibiotic, termed fumagillin as it is isolated from Aspergillus fumigatus, has been shown to prevent development and cyst formation in the fish. Documentation was provided for Tetracapsuloides bryosalmonae (PKD agent) in salmonids (Hedrick et al., ), Myxidium giardi in eel (Szekely et al., ), Myxobolus spp. in common carp (Buchmann et al., ) and E. leei in maricultured sharpsnout bream (Golomazou et al., ).
Arthropods, such as insects and crustaceans (including parasites such as parasitic copepods, isopods and branchiurans), perform several moults during their life cycle, which leaves them vulnerable to compounds inhibiting their formation of a new exoskeleton made of chitin. The chitin synthesis inhibitors comprise several compounds, which have been successfully used against salmon lice infections in Atlantic salmon in Norwegian mariculture and against the isopod Ceratothoa oestroides infecting seabass, seabream and meagre in Mediterranean mariculture (Bouboulis et al., ; Colak et al., ). Diflubenzuron, hexaflumuron, lufenuron and teflubenzuron all inhibited the transition of the salmon louse from the nauplius to the copepodid stage. The inhibition was associated with a decreased expression of the chitin synthase 1 gene in hexaflumuron and diflubenzuron-treated larvae (Harðardottir et al., ). Environmental concerns with regard to free-living invertebrates and vertebrates may limit the use of the drugs.
The most direct way to limit development of a parasite infection in a host population is to block the life cycle. For certain parasites with free-living infective stages this can be achieved by continuous mechanical filtration of fish tank water. Various mesh sizes of filter screens may be selected to fit the size of the parasite. Thus, tomonts of Ichthyophthirius released from the skin of fish generally have a diameter of several hundred micrometres and will be trapped by filters, even with a mesh size of 80 μm (Heinecke and Buchmann, ). Whenever a tomont is trapped by the filter and removed from the system, then the cyst formation is prevented, and thereby also production of up to infective theronts within the next 36 h, dependent on temperature. Infective cercariae of eye flukes may correspondingly be trapped by use of mechanical filters (Larsen et al., ). Filtration of tank water in eel farms using 40 μm filters is able to remove eggs and oncomiracidia of the gill parasitic monogenean Pseudodactylogyrus anguillae and Pseudodactylogyrus bini (Buchmann, ).
The branchiuran parasite Argulus reproduces by laying egg clusters on submerged objects including aquatic plants, branches and roots. Following hatching of Argulus eggs, the larvae develop into the infective stage and attach to the fish. This reproductive strategy can be utilized for control. Regular immersion into an infected pond of wooden slats, lattices and bundles of branches to which the female parasite attaches egg clusters provides an opportunity to kill the eggs. This is achieved simply by removing these egg traps from the ponds with few days intervals. New traps are replacing the recovered ones and will be used for parasite oviposition. In this way the reproductive potential of the parasites can be, at least partly, exhausted leading to a decreased infection pressure in the ponds (Kabata, ). Submerging boards of various materials into water bodies with problematic Argulus foliaceus infections on rainbow trout have been used to reduce the infection level of Argulus in rainbow trout lakes (Gault et al., ). Egg clusters could be harvested regularly when recovering the boards and pulling them ashore. Thereby the overall infection pressure fell and the prevalence and mean intensity decreased 6- to 9-fold.
Due to the decreasing sensitivity of salmon lice to the different biocides, chemotherapeutants and medicines, which occurred after extensive usage in mariculture farms, alternative control methods had to be developed. These included mechanical removal by heat treatment, brushing or flushing with freshwater. The technique necessitates capture and handling of large salmon, which challenges the health and welfare of the fish (Østevik et al., ). Other approaches in action are based on laser technology targeting salmon lice on the fish surface. Automated camera systems placed in the water are able to scan passing fish in the netpen and if a salmon louse is detected the laser entity emits a pulse of high energy light towards the louse aiming at killing the parasite in situ. Although potentially lethal to the louse, recent controlled full-scale tests could not document a decrease in the mean number of parasites in laser-exposed netpens (Bui et al., ).
New design of netpens applies the use of barriers preventing entrance of infective parasite stages into the section with fish. The upper water layers are preferred by the infective copepodids of the salmon louse. In salmon, mariculture skirts or tarpaulin may be placed around the upper part of the netcages in order to reduce contact between fish and parasites and thereby infections. Thus, the so-called snorkel netcage farms have been developed in order to minimize the attachment of sealice copepodids on maricultured Atlantic salmon. The fish are kept in submerged netpen compartments in the deeper water layers, zones which have reduced abundance of infective louse stages due to the surface-seeking behaviour of copepodids. A lower mean intensity of infection was observed in these cages (Geitung et al., ).
Eyefluke infections in fish are caused by infective cercariae, released from intermediate host snails, penetrating the surfaces of fish skin, fins or gills (Duan et al., ). If these cercariae cannot be eliminated by chemicals or mechanical filtration of water (Larsen et al., ) it is possible to remove the intermediate host snails simply by collecting snails from ponds. As each snail may produce 58 000 cercariae per day this procedure may effectively limit the infection pressure in a pond (Lyholt and Buchmann, ).
Natural marine and freshwater ecosystems exhibit a wealth of symbiotic relationships between fish infected with ectoparasites. In tropical fish farming, mosquito fish Gambusia feed on the branchiuran parasite Argulus during their free-swimming activity (Kabata, ) and smaller fish can easily recognize ectoparasites on other often larger fish and pick them of the host skin (Bjordal, ; Cowell et al., ). This basic biological function is being applied by the industry by stocking salmon netpens with cleanerfish. Various species of wrasse have been applied during the latest three decades for removal of salmon lice from salmon skin (Bjordal, ; Groner et al., ; Imsland et al., ). Their effect is low during wintertime whereas another cleanerfish, lumpsucker Cyclopterus lumpus, has a superior performance at low temperatures. A huge industry has been established in order to produce lumpsucker, a species with appetite for salmon louse attached to salmon skin (Groner et al., ; Imsland et al., ). Even this sustainable approach is challenged by the high adaptability of salmon lice. Forms with a lower degree of pigmentation, and thereby a lower chance of being recognized and eaten by cleaner fish, have appeared. Both environmental and genetic factors may influence this change of pigmentation (Hamre et al., ), but these less visible lice are likely to decrease the efficacy of cleaner fish. Various fish species predating on snails (Ben-Ami and Heller, ) may be considered a supplement for control of digenean parasites using snails as intermediate hosts. By eliminating snails by predation these fish may contribute to a lowered infection level.
In addition to cleaner fish removing ectoparasites from the surface of infected production fish, a series of other solutions, based on predation by invertebrates, for parasite control exist. The filtration of huge water masses by blue mussels Mytilus edulis can be used for trapping the pelagic larval stages (copepodids) of salmon lice L. salmonis (Bartsch et al., ). Free-living copepods such as Cyclops predate on fish parasitizing Diplostomum cercariae (Bulaev, ) and oncomiracidia of Pseudodactylogyrus monogeneans (Buchmann, ). Likewise free-living turbellarians (Stephanostomum sp.) ingest freshly delivered eggs of Pseudodactylogyrus whereby the infection level for fish decreases (Buchmann, ).
Stimulation of the immune system of the teleost host by adding various immunestimulants to the feed is a strategy applied by aquaculturists to a wide extent. Slight decreases of infection levels may be recorded following this type of feeding both with regard to protozoans such as Ichthyophthirius (Xueqin et al., ) and metazoans, such as L. salmonis (Poley et al., ). Effects of caprylic acid in combination with iron and mannan (a potential immunostimulant) in feed for seabream in Mediterranean mariculture against monogenean infections (Sparicotyle chrysophrii) were recorded by Rigos et al. (), but the exact mode of action needs to be investigated. However, the efficacy of in-feed immunostimulants for protection of fish against pathogens is generally very low when compared to the effect of vaccination.
Today vaccination against both bacterial and viral diseases has proven to be the most sustainable ways to control fish disease in aquaculture enterprises. In Europe alone about 1.3 billion fish are successfully vaccinated annually against various infective diseases (Midtlyng, ). The marked immune responses established in fish, when infected by various protozoan and metazoan parasites, may lead to some level of protective immunity in fish surviving a natural infection. This was documented for the parasitic ciliates Ichthyophthirius (Buschkiel, ; Bauer, ; Hines and Spira, ; Sigh and Buchmann, ; Alishahi and Buchmann, ) and Philasterides (Lamas et al., ), for the flagellates Trypanosoma (Woo, ), Cryptobia (Jones and Woo, ) and Ichthyobodo (Chettri et al., ), for monogeneans such as Gyrodactylus (Lindenstrøm and Buchmann, ), Pseudodactylogyrus (Slotved and Buchmann, ) and the crustacean parasite Lernaea (Woo and Shariff, ). This suggests the existence of a potential for development of antiparasitic fish vaccines (Jørgensen et al., ), but up until now no such products are licensed for use in commercial aquaculture. Immunological protection of fish against various parasites is likely to be based on a plethora of cellular and humoral (innate and adaptive) elements, which are not so easily induced by a vaccine. So, although no antiparasitic vaccines are available for fish, it may be worthwhile to apply controlled (low to moderate) parasite infections in order to induce a protective response, which may supplement other control strategies. This response induced by a natural infection is likely to include the relevant immune reactions needed to control, at least partly, the host against reinfection.
The interface between mucosal immunity, gut microbiota and resistance towards parasites has been explored with some success. Stimulation of the gut microbiota by functional feed additives, such as sodium butyrate, may be a way to at least partly control infections with the myxozoan E. leei in maricultured seabream (Piazzon et al., ). It may also be speculated if feed additives such as caprylic acid and mannan exert their effects on the gill monogenean S. chrysophryi through a general systemic immune stimulation in seabream (Rigos et al., ).
The ability to resist a parasitic infection is genetically determined, which has been indicated for Ichthyophthirius and Myxobolus infections in rainbow trout (Hedrick et al., ; Avila et al., ). Breeding towards more disease-resistant fish is a possibility, and marker-assisted selective breeding is a tool which has been increasingly used in breeding programmes. Selective breeding of fish carrying certain desirable traits has been in use for decades also in fish aquaculture. The classical approach often takes several years before results are seen, because the generation time of fish can be several years. Several studies have discovered quantitative trait loci (QTL) for viral, bacterial and parasitic diseases. This was demonstrated for infectious pancreatic necrosis virus (Houston et al., ; Moen et al., ) and salmonid alpha virus (Aslam et al., ) in Atlantic salmon and for viral haemorrhagic septicaemia virus resistance in rainbow trout (Verrier et al., ). Studies have also described QTL associated with resistance in salmonids to bacterial infections caused by Piscirickettsia salmonis (Correa et al., ), Flavobacterium psychrophilum (Wiens et al., ; Vallejo et al., ) and Vibrio anguillarum (Du et al., ; Wang et al., ; Shao et al., ; Karami et al., ). In addition, the approach is useful for parasite&#;host systems as well. QTL for resistance towards AGD were described in Atlantic salmon (Robledo et al., ) and for the scuticociliate Philasterides dicentrachi infecting turbot (Rodriguez-Ramilo et al., ). QTL for resistance in rainbow trout against Myxobolus cerebralis was studied by Hedrick et al. () and later by Baerwald et al. (). Investigation of single-nucleotide polymorphism (SNP) markers indicated that some genes associated with resistance towards I. multifiliis are located on rainbow trout chromosomes 16 and 17 (Jaafar et al., ). Innate response genes in the Atlantic salmon were targeted by Gilbey et al. () focusing on gyrodactylid monogeneans (Gyrodactylus salaris). Likewise, Gharbi et al. () and Robledo et al. () searched for genes associated with resistance towards salmon lice in this host. Ozaki et al. () found corresponding host&#;parasite associations for the capsalid monogenean Benedenia seriolae infecting yellowtail Seriola quinqueradiata. Novel typing technology applying markers makes it easier to conduct genotyping. A microarray comprising 57 501 markers (SNP) was used to locate genes encoding resistance to V. anguillarum on chromosome 21 (Omy 21) (Karami et al., ), and genes associated with Ichthyophthirius resistance [chromosomes 16 and 17 (Omy 16 and 17)] (Jaafar et al., ). Genotyped breeders (females and males) carrying the SNPs associated with the favourable trait can easily be selected and used for production of a new generation of trout with a higher innate resistance to infection (Buchmann et al., ). The basis for this innate and heritable protection may be partly associated with immune factors in the host. However, it cannot be excluded that other elements, including chemoattraction of the parasite to the host, may be involved in better performance of QTL fish.
Parasites are in general highly adaptable to environmental changes and even a documented control method may in a few years show inferior. An example from the search on compounds with lethal effects on salmon lice is the successful introduction, documentation and victory of the avermectin emamectin benzoate against L. salmonis infections. However, its use decreased due to rapid selection of drug-resistant parasite strains. Similar processes apply for organophosphates and pyrethroids. Parasites possess a remarkable ability to adjust to environmental changes as strains with resistance to new conditions are readily selected. In order to secure the fish production from massive infestations of parasites it is worthwhile to introduce integrated control strategies involving several antiparasitic approaches. At present, control of Ichthyophthirius infections in freshwater trout farms involves mechanical filtration with micro-sieves (removing tomonts from the water phase), regular addition to fish pond water of biocides/auxiliary substances (hydrogen peroxide, peracetic acid or sodium percarbonate eliminating infective theronts), tolerance of a low initial parasite infection (which induces a relative and partly protective immune response) and use of breeds with an elevated level of natural resistance to infection (Jaafar et al., ; Buchmann et al., ).
The impressive ability of parasites to adapt to environmental changes challenges an effective and lasting control of parasitoses in aquaculture settings. Even novel compounds with high efficacy may be ineffective within a few years of constant usage at farm level. It is recommended that aquaculturists should combine various methods in an integrated control strategy. Alternation between different chemical, medical, biological and mechanical control methods may delay development of resistance. Continued research into basic biology and biochemistry of the parasites can lead to novel approaches replacing old and less effective methods. Up until now no effective control method based on hyperparasitism has been documented in aquaculture settings. Hyperparasites occur in natural ecosystems and future research should show if this could be a supplementary tool in fish parasite control.
The author is indebted to Drs Moonika H. Marana and Jakob Skov for providing the photograph for taken in a Danish mariculture farm.
Data are available on request.
K. B. devised, wrote and revised the paper.
The present study was supported by Innovation Fund Denmark by grant -B to the project TECHFISH.
None.
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