INTRODUCTION
Many parasites are serious pathogens in intensive aquaculture in Japan (Ogawa and Yokoyama, Reference Ogawa and Yokoyama1998). Neobenedenia girellae, a capsalid monogenean, is one of them because of its ability to cause high mortality in host fishes (Ogawa et al. Reference Ogawa, Bondad-Reantaso, Fukudome and Wakabayashi1995) and its broad host specificity (Bondad-Reantaso et al. Reference Bondad-Reantaso, Ogawa, Fukudome and Wakabayashi1995). Some commercially important, cultured fishes, such as yellowtail, Seriola quinqueradiata (Carangidae), amberjack, Seriola dumerili (Carangidae), tiger puffer, Takifugu rubripes (Tetraodontidae), Japanese flounder, Paralichthys olivaceus (Paralichthyidae), and spotted halibut, Verasper variegatus (Pleuronectidae), become infected with this monogenean (Ogawa and Yokoyama, Reference Ogawa and Yokoyama1998; Hirazawa et al. Reference Hirazawa, Mitsuboshi, Hirata and Shirasu2004).
Bondad-Reantaso et al. (Reference Bondad-Reantaso, Ogawa, Fukudome and Wakabayashi1995) described the life-cycle of this parasite. At a water temperature of 25°C, free-swimming oncomiracidia (body length: ∼200 μm) hatch from eggs after 4 days, attach predominantly to the fins and then migrate from the fins to the skin surface as they grow. The maturation of N. girellae (body length: ∼2·1 mm) takes 10 days from larval attachment to the host. The life-span of N. girellae oncomiracidia has not yet been investigated, but the typical life-span of a monogenean oncomiracidium is usually 24–48 h (Whittington et al. Reference Whittington, Chisholm and Rohde2000). Active feeding by large populations of capsalid monogeneans on mucus and epithelial cells of the host fish can cause haemorrhage, inflammation and mucus hyperproduction (Paperna, Reference Paperna1991). Heavily infected fish may stop feeding, and then their body colour darkens, they swim erratically and rub against the net, which may result in dermal ulceration and subsequent bacterial invasion (Woo et al. Reference Woo, Bruno and Lim2002).
To prevent infestation by N. girellae, a freshwater dip of 3–5 min is often used to dislodge the parasites from the host fishes (Leong, Reference Leong1997), but long-term treatments cause damage to cultured fish (Roberts and Powell, Reference Roberts and Powell2003a, Reference Roberts and Powellb). The treatment requires additional labour and causes stress to the fish (Kim and Choi, Reference Kim and Choi1998). An effective treatment with medicated feed may be more practical than bath treatments. When administered orally, praziquantel is an effective chemotherapeutic compound against skin monogeneans (Okabe, Reference Okabe2000; Hirazawa et al. Reference Hirazawa, Mitsuboshi, Hirata and Shirasu2004) which feed on epithelium and against gill monogeneans (Hirazawa et al. Reference Hirazawa, Ohtaka and Hata2000; Kim et al. Reference Kim, Lee, Kwon and Cho2001) which feed on blood. The recommended treatment against N. girellae on S. quinqueradiata is an oral dose of praziquantel at 150 mg per kg body weight per day for 3 successive days (Okabe, Reference Okabe2000). However, the appetite of fish tends to decrease, and fish vomit when fed pellets medicated with praziquantel (Hirazawa et al. Reference Hirazawa, Mitsuboshi, Hirata and Shirasu2004). In addition, praziquantel administered orally is much harder to deploy to epithelial tissues for uptake by epithelial feeders (Okabe, Reference Okabe2000). Therefore, in addition to chemical treatment, biological control measures with no unfavourable effects on the environment should be incorporated into the control strategy. However, much remains to be studied of the biology of N. girellae.
N. girellae has 2 pairs of eye spots, and phototactic responses are known to occur only in oncomiracidia (Yoshinaga et al. Reference Yoshinaga, Nagakura, Ogawa and Wakabayashi2000). However, information on the response of N. girellae oncomiracidia to brightness has been limited, and the response of oncomiracidia to black-and-white contrast has not been investigated. Information on the response to black-and-white contrast may be useful to develop an effective control method. The present study investigated whether brightness influences N. girellae oncomiracidia infection in P. olivaceus, when the fish are exposed to oncomiracidia in the same aquarium with areas of different brightness. The response of oncomiracidia to black-and-white contrast was investigated in challenge trials using host fish mucus and whole live fish.
MATERIALS AND METHODS
Fish rearing
Rearing methods for V. variegatus (see Hirazawa et al. Reference Hirazawa, Hara, Mitsuboshi, Okazaki and Hata1999) and P. olivaceus (see Kumai, Reference Kumai2000) were used to establish the flow rates, aeration and feeding rates. Aerated tanks (of 100, 500 or 1000 litre capacity) containing V. variegatus or P. olivaceus were supplied with seawater previously filtered through sand and irradiated with ultraviolet (UV) light (∼50 000 μw⋅s/cm2 for a Flonlizer 4 l unit; Chiyoda Kohan, Ltd, Japan). The mean salinity, pH and chemical oxygen demand of seawater used in our laboratory was ∼34 parts per million, 8·1 and 1·0 mg/l, respectively, throughout the year. The fish were fed a commercial extruded 2 mm pellet diet (Nippon Suisan Kaisha Ltd, Japan) twice a day. The daily feeding rate was 3% of body weight.
Uninfected fish
P. olivaceus (500 individuals) weighing approximately 5 g each were obtained from a local juvenile flounder producer in Oita Prefecture. They were maintained in a 1000 litre tank. V. variegatus (500 individuals), weighing approximately 8 g each and hatched in our laboratory, were maintained in a 500 litre tank. The skin surface of 10 fish of each species, sampled randomly, was examined under a microscope to confirm that the fish were not infected with N. girellae or other skin parasites before each experiment. Their gills were also examined using a microscope to confirm that the fish were not infected by gill parasites.
Oncomiracidia of N. girellae
The source of N. girellae and its propagation on V. variegatus has been described previously (Hirazawa et al. Reference Hirazawa, Mitsuboshi, Hirata and Shirasu2004; Hatanaka et al. Reference Hatanaka, Umeda, Yamashita and Hirazawa2005). Eggs of N. girellae have filaments that can easily entangle with each other, and many such egg masses can further entangle with a net cage (Ogawa and Yokoyama, Reference Ogawa and Yokoyama1998). We collected the eggs efficiently by putting 5-cm-square nylon nets (mesh size 5 mm) into a 100 litre tank, in which V. variegatus infected with the parasites were maintained. Eggs entangled in the nets were incubated in a 300 ml plastic beaker containing filtered seawater (20°C). The filtered seawater was exchanged every day during incubation. Within 12 h of hatching, oncomiracidia were used for the experiments.
Assessment of response by N. girellae oncomiracidia to brightness in a challenge trial
The experiment was conducted twice (Experiments I and II). Thirty uninfected specimens of P. olivaceus (mean weight: 38·6 g in Exp. I; mean weight: 47·2 g in Exp. II) were transferred to one tank of 200 litre capacity in each experiment. The fish were acclimatized for 1 week. The tanks were aerated and supplied with sand-filtered and UV-irradiated seawater. The fish were fed, and the temperature was maintained at 25±1°C (using a heating device) throughout the experiment. A tank (inside dimension: length 96·5×width 30·0×height 20·0 cm) was divided into 3 areas with nylon nets (mesh size was 5 mm) (Fig. 1). Then 32 litres of seawater at 25°C containing ∼18 000 N. girellae oncomiracidia were put into the tank. The tank was aerated for 3 min. The aeration supply to the tank was stopped, the tank was covered with a shading lid (length 33×width 33×height 0·5 cm; vinyl chloride), and then the tank was rested for 30 min. The brightness of each of the 3 areas in the tank was ∼1·3 lux, 41·3 lux and 138·0 lux, respectively, measured using a lux meter (Fine lux meter FLX-1330, Tokyo Asahi Kikai Ltd, Japan); 3 locations in each area were measured and the values were averaged. The acclimatized fish were divided into 3 groups: low brightness group (1·3 lux), middle brightness group (41·3 lux), high brightness group (138·0 lux). The shading lid of the square tank was opened, each group was transferred to each area and the tank was covered again. The fish were exposed to the oncomiracidia for 1 h. After the exposure to oncomiracidia, individual groups of fish were transferred to separate 100 litre tanks of the same brightness (∼160 lux). The fish were kept alive for a further 9 days after infection. At the end of the experiment, each fish was dipped separately in fresh water for 10 min to dislodge the parasites from the host (Leong, Reference Leong1997). The total number of dislodged N. girellae per fish was counted under a stereomicroscope. Analysis of variance (one-way ANOVA) followed by Fishers PLSD post hoc test was used to identify the statistical difference in the numbers of parasites between the groups. Significance was accepted at P<0·05. The data were analysed using the Statcel statistical software package (OMS, Inc. Japan).
Fig. 1. Outline of the protocol to assess the response of Neobenedenia girellae oncomiracidia to brightness.
Assessment of response by N. girellae oncomiracidia to black-and-white contrast in host fish mucus trials
A DC8SC glass slide (Neuro Probe, Inc., USA) has 8 recessed circular areas of 6·4 mm in diameter and 32 mm2 in area. The slide outside the recessed areas and 5 recessed areas were painted white or black using permanent markers and the remaining 3 recessed areas were painted the opposite colour (Fig. 2). Skin mucus (50 μl) from P. olivaceus was added to each of 6 (3 areas of each colour) of the 8 recessed areas. The glass slide was incubated for 3 h at 30°C to dry the mucus to prevent from sloughing off the slide in seawater. The glass slide was put into a tissue culture dish (diameter 9 cm), containing ∼3000 oncomiracidia within 12 h of hatching in 50 ml of seawater, and was incubated at 25°C for 1 h. Then, the glass slide was removed from the dish and the attached mucus-treated oncomiracidia in each recessed area were counted under a light microscope. The oncomiracidia counted shed their ciliated epidermal cells and attached to the mucus with the haptor unfolded. The experiment was conducted 3 times (Exps I, II and III) and mucus from different fish and different batches of parasites were used in Exps I, II and III. The data were tested using a t-test to find significant differences between the white and black groups. Significance was accepted at P<0·05. All analyses were conducted using the Statcel statistical software package.
Fig. 2. Outline of the protocol to assess the response of Neobenedenia girellae oncomiracidia to black-and-white contrast.
Assessment of response by N. girellae oncomiracidia to black-and-white contrast in challenge trials
Uninfected V. variegatus (mean weight: 18·8 g) were randomly divided into 2 groups, each group consisting of 10 ‘normal’ (colour of their uppermost ocular side was dark) and 10 ‘mal-coloured’ (colour of their uppermost ocular side was light) fish (Fig. 3). Mal-coloured fish (pigment abnormalities) in hatchery-reared juveniles of flatfish are often observed (Venizelos and Benetti, Reference Venizelos and Benetti1999). These abnormal fish suffer from thyroid hormone deficiency (Okada et al. Reference Okada, Morita and Tagawa2005) and dietary deficiencies (Kanazawa, Reference Kanazawa1993). One group was kept in a 100 litre tank with a white bottom (Fig. 3A), and the other group was kept in a 100 litre tank with a black bottom (Fig. 3B). Fish were acclimatized for a week. The tanks were aerated and supplied with seawater. The fish were fed the diet, and the temperature was maintained at 25±1°C throughout the experiment. After acclimatization, ∼3000 oncomiracidia were put into each tank within 12 h of hatching. The seawater supply was discontinued for 1 h to assist in establishing the infection, and the aeration supply to the tank was stopped for 1 h. The experiment continued for 9 days after the exposure to oncomiracidia. At the end of the experiment, each fish was dipped in fresh water for 10 min to dislodge the parasites from the host and the total number of dislodged N. girellae per fish was counted. The data were tested using the t-test to find significant differences between normal and mal-coloured fish in each group. Significant differences were accepted at P<0·05. All analyses were made using the Statcel statistical software package.
Fig. 3. Verasper variegatus. (A) White background. (B) Black background. Dark-coloured V. variegatus on their uppermost ocular side are normal fish (N). Light-coloured V. variegatus on their uppermost ocular side are mal-coloured fish (M).
RESULTS
Assessment of response by N. girellae oncomiracidia to brightness in a challenge trial
We investigated whether brightness influences the infection by N. girellae oncomiracidia when P. olivaceus were exposed to larvae in the same aquarium with areas of different brightness. The number of parasites on fish reared in 138 lux, the brightest condition, was significantly higher than on fish of the other 2 test groups, indicating that the infection was influenced by brightness (Fig. 4).
Fig. 4. Infection levels of Neobenedenia girellae on Paralichthys olivaceus exposed to oncomiracidia in the same aquarium that had areas with different brightness. (A) Experiment I. (B) Experiment II. Values are means and standard deviations. The values with different superscripts are significantly different at P<0·05.
Assessment of response by N. girellae oncomiracidia to black-and-white contrast in host fish mucus trials
The response of N. girellae oncomiracidia to P. olivaceus mucus added to a glass slide that had black-and-white contrast (see Fig. 2) was examined. Glass slides with a white background had markedly more N. girellae oncomiracidia attached to the black areas compared with white areas (Fig. 5A), and the number of oncomiracidia attached to black areas was significantly higher than for white areas in Exps I–III.
Fig. 5. Response of Neobenedenia girellae oncomiracidia to mucus from Paralichthys olivaceus on black and white areas with a white or black background. (A) White background. (B) Black background. Values are means and standard deviations. Asterisks indicate a significant difference (*P<0·05, **P<0·01).
Glass slides with a black background had markedly more N. girellae oncomiracidia attached to the white areas compared with the black areas (Fig. 5B), and the number of oncomiracidia attached to white areas was significantly higher than for black areas in Exps I and II, although not significantly higher in Exp. III.
Assessment of response by N. girellae oncomiracidia to black-and-white contrast in challenge trials
The response of N. girellae oncomiracidia to dark-coloured V. variegatus versus light-coloured V. variegatus in a tank with a white or a black bottom was examined. In a tank with a white bottom, the number of parasites on dark-coloured fish was significantly larger than for light-coloured fish (Fig. 6A). Conversely, the number of parasites on mal-coloured light-coloured fish in a tank with a black bottom tended to be higher compared with normal dark-coloured fish (Fig. 6B), but the difference was not statistically significant (P=0·265).
Fig. 6. Response of Neobenedenia girellae oncomiracidia to dark-coloured Verasper variegatus and light-coloured Verasper variegatus with white or black background. (A) White background. (B) Black background. Values are means and standard deviations. Asterisk indicates a significant difference from the values between dark-coloured fish and light-coloured fish (*P<0·05). In a tank with black bottom (B), the number of parasites on light-coloured fish tended to be high compared with dark-coloured fish, but the difference between the 2 different coloured fishes was not statistically significant (P=0·265).
DISCUSSION
Many monogenean oncomiracidia have eyes, and their orientation with respect to light is likely to be important in host-finding, if the eyes have a phototactic function. There have been few studies of the light-orientated behaviour of monogenean larvae. Photo-positive behaviour has been shown for the oncomiracidia of Diplozoon paradoxum (see Bovet, Reference Bovet1967) and Discocotyle sagittata (see Paling, Reference Paling1969). On the other hand, Fournier (Reference Fournier1976) referred to photo-negative behaviour in the oncomiracidium of Euzetrema knoepfferi. Oncomiracidia of dactylogyrids have 2 phases of phototaxis (Bychowsky, Reference Bychowsky1957); during the first phase, the larvae are positively phototactic and the haptor is folded in such a way that the hooks do not protrude; in the second phase, the larvae are photo-negative with an unfolded haptor and protruding hooks. There is also evidence that larvae of other monogeneans, such as Discocotyle sagittata (see Paling, Reference Paling1969) and Entobdella soleae (see Kearn, Reference Kearn1980), display age-dependent photoresponses. N. girellae has 2 pairs of eye spots, and their phototactic role is known only for the oncomiracidia which are positively phototactic (Yoshinaga et al. Reference Yoshinaga, Nagakura, Ogawa and Wakabayashi2000). The current study provided evidence that brightness influences the infection of fish with N. girellae oncomiracidia, and showed that fish tend to harbour more N. girellae under brighter conditions. However, further studies are needed to assess the responses of N. girellae oncomiracidia to brightness according to their age. In this study, N. girellae oncomiracidia (within 12 h of hatching) were used to obtain sufficient numbers for use in the experiments. These groups comprised larvae of various ages between 0 and 12 h old, and these oncomiracidia may differ in their photoresponses.
In most cases in Japan, marine fish are cultured in floating off-shore net cages. The positive phototactic behaviour of N. girellae oncomiracidia may be used to prevent outbreaks in aquaculture systems. For instance, Seriola species are in some cases cultured in sunken cage systems, because floating off-shore net cages collapse occasionally during typhoons which hit Japan in summer. The sunken cage system may reduce the chances of fish being exposed to N. girellae oncomiracidia. In general, light intensity at the water surface is ∼15 000 lux during the day (Sakakura and Tsukamoto, Reference Sakakura and Tsukamoto1997) and attenuates according to depth. The light intensity at a depth of 29·5 m on the Pacific coast is one hundredth of that at the water surface (Hirano, Reference Hirano1998).
Successful host-finding and attachment are crucial for parasite survival. Some studies have shown that monogenean eggs and oncomiracidia respond to environmental stimuli (i.e. light, gravity, mechanical disturbance, water currents, chemical substances and mucus pH), indicating that such responses increase the probability of successful host-finding (Ktari, Reference Ktari1969; Paling, Reference Paling1969; Kearn, Reference Kearn1974, Reference Kearn1980; Whittington and Kearn, Reference Whittington and Kearn1986, Reference Whittington and Kearn1989; Yoshinaga et al. Reference Yoshinaga, Nagakura, Ogawa and Wakabayashi2000; Buchmann and Lindenstrøm, Reference Buchmann and Lindenstrøm2002; Hirazawa et al. Reference Hirazawa, Oshima, Mitsuboshi and Yamashita2003). The response by N. girellae oncomiracidia to black-and-white contrast detected in the present study showed that more N. girellae oncomiracidia attached to black areas and dark-coloured fish in a white background, and, conversely, more N. girellae oncomiracidia tended to attach to white areas and light-coloured fish on a black background. These results show that black-and-white contrast may be important in host-finding by N. girellae oncomiracidia, which react more strongly to black objects on a white background than white objects on a black background. These findings may be applied to aquaculture to prevent or reduce N. girellae infection. In the case of P. olivaceus, which is mostly farmed in land-based tanks with running seawater, a rearing tank with a similar colour to the fish, in order to reduce black-and-white contrast, may suppress the chances of the fish encountering N. girellae. Usually, flatfish are able to change their pigmentation, and become camouflaged to resemble their surroundings. The camouflage is useful in deceiving enemies and prey, and may also suppress the chances of the fish encountering N. girellae. However, most fish species darken their bodies when infected with pathogens (Woo et al. Reference Woo, Bruno and Lim2002). This is the case in N. girellae infection, which often relates to relatively large numbers of worms (Hirazawa, unpublished observation).
N. girellae has been introduced into Japan through imported, wild S. dumerili fingerlings (Bondad-Reantaso et al. Reference Bondad-Reantaso, Ogawa, Fukudome and Wakabayashi1995) and is now recognized on some other cultured marine fishes. N. girellae eggs are able to hatch at 18–30°C but they do not hatch at 15°C (Bondad-Reantaso et al. Reference Bondad-Reantaso, Ogawa, Fukudome and Wakabayashi1995). These observations on hatching show that this parasite has adapted to high water temperature. Infection by N. girellae among fishes in southwest Japan substantially decrease in the winter months but the infection persists even in those months in subtropical areas, such as the Okinawa and Ogasawara Islands (Bondad-Reantaso et al. Reference Bondad-Reantaso, Ogawa, Fukudome and Wakabayashi1995). S. dumerili is distributed in these subtropical areas but P. olivaceus and V. variegatus are not. These observations suggest that N. girellae is not a natural parasite of P. olivaceus and V. variegatus. The susceptibility of V. variegatus to N. girellae is low compared with P. olivaceus (see Hirazawa et al. Reference Hirazawa, Mitsuboshi, Hirata and Shirasu2004) but the growth of the parasite on V. variegatus is similar to that on P. olivaceus. Differences in susceptibility among other fish species to N. girellae infection have not been investigated. The host species differences in susceptibility to this parasite may affect the behaviour of N. girellae oncomiracidia in relation to the black-and-white contrast. In this study, P. olivaceus mucus and live V. variegatus were used for challenge trials because the aim of this study was to assess N. girellae oncomiracidia behaviour to the black-and-white contrast.
The ocellus in many free-living and parasitic platyhelminths consists of a pigment cell, rhabdomeres and microvillus-like out-growths of dendrites connected to a sensory cell outside of the receptor (Kearn and Baker, Reference Kearn and Baker1973; Fournier and Combes, Reference Fournier and Combes1978; Kearn, Reference Kearn1978). These structures may change the position of the receptors relative to the adjacent tissues and the incoming light and, thus, may modify phototactic reaction (Rohde and Watson, Reference Rohde and Watson1991). Photoreceptors may be responsible for directed swimming toward the light, i.e. they may mediate phototactic responses that lead the larva into a habitat in which infection is possible (Rohde and Watson, Reference Rohde and Watson1991).
In this study, brightness influenced the N. girellae oncomiracidial infection in fish, the brighter conditions resulting in fish with greater numbers of N. girellae. The response by N. girellae oncomiracidia to black-and-white contrast suggests that black-and-white contrast may be important for host-finding by N. girellae oncomiracidia. Further studies are needed to assess the responses of N. girellae oncomiracidia of different ages to brightness and black-and-white contrast. Similarly, to determine whether these features may be used in different aquaculture systems, such as sunken cages or fish-skin-coloured aquaria to prevent infection. The apparent simplicity of the positive phototactic behaviour and the response to black-and-white contrast may lead to the development of practical and inexpensive approaches for the control of N. girellae outbreaks in fish farms.
