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Sea lice infections of wild fishes near ranched southern bluefin tuna (Thunnus maccoyii) in South Australia
Aquaculture 320 (2011) 178–182
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Sea lice infections of wild fishes near ranched southern bluefin tuna (Thunnus maccoyii) in South Australia C.J. Hayward a,b,⁎,1, I. Svane a, S.K. Lachimpadi a, N. Itoh c, N.J. Bott d, B.F. Nowak b a
South Australian Research and Development Institute, Lincoln Marine Science Centre, P.O. Box 1511, Port Lincoln, South Australia 5606, Australia National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Locked Bag 1-370, Launceston, Tasmania 7250, Australia Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori Amamiya-machi, Aoba-ku, Sendai 981-8555 Miyagi, Japan d South Australian Research and Development Institute-Aquatic Sciences, P.O. Box 120, Henley Beach, South Australia 5022, Australia b c
a r t i c l e
i n f o
Article history: Received 10 June 2010 Received in revised form 15 October 2010 Accepted 28 October 2010 Available online 4 November 2010 Keywords: Sea lice Aquaculture Tuna Caligus chiastos Thamnaconus degeni Degen's leatherjacket
a b s t r a c t In contrast with sea lice infestations of other farmed fishes, attached larval stages of sea lice on ranched southern bluefin tuna (Thunnus maccoyii) are rarely detected. In this study, we monitored sea lice on ranched T. maccoyii and surveyed wild fishes adjacent to ranching sea cages over a 3-month period in early 2009. Prevalence of the adult Caligus chiastos on tuna within a day of arrival at the ranching site was 10%; prevalence then increased significantly and peaked almost 25 days later to 75%; by harvest (after a further 18–28 days), prevalence decreased significantly to 0%. We collected and examined a total of 502 wild fishes outside T. maccoyii sea cages, comprising 307 Degen's leatherjackets (Thamnaconus degeni), 136 yellowtail horse mackerel (Trachurus novaezelandiae), 31 sand trevally (Pseudocaranx wrighti), 10 West Australian salmon (Arripis truttacea), 6 Port Jackson sharks (Heterodontus portusjacksoni), and a single blue mackerel (Scomber australasicus) and pilchard (Sardinops sagax); we also examined an additional 10 pilchards that were collected from the centre of Spencer Gulf and stored fresh in a T. maccoyii feed bin. Of these potential hosts, we identified adult C. chiastos only from Degen's leatherjackets; of the many larvae also occurring on this host, molecular comparison of five specimens analysing cytochrome C oxidase I region of mitochondrial DNA and five specimens analysing partial D1–D2 domains of 28S rDNA confirmed that these were C. chiastos. In contrast with the decline in infections of C. chiastos on ranched T. maccoyii near the end of March, on Degen's leatherjackets there was a significant increase in prevalence and abundance over the study period, with a peak prevalence of 97.14% and a mean abundance reaching 11.17 lice per fish near the end of April. The percentage of chalimus larvae on Degen's leatherjackets increased over the study period, ranging from 0% near the start of sampling to over 93% on the final sample date. We also recorded additional copepod infestations, including Orbitacolax williamsi on Degen's leatherjackets, Caligus sp. on sand trevally, and Dissonus nudiventris on Port Jackson sharks. We conclude that Degen's leatherjacket, which is a major scavenger of excess tuna feed, is likely to contribute to sea lice infestations of T. maccoyii. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Sea lice are a significant pathogen in marine finfish farming (Rosenberg, 2008). Fish farmed in sea cages are likely to be infested by parasites from wild fishes and because of their high concentration can in turn become a source of parasites (Costello, 2009). Epizootics of sea lice (predominantly Caligus chiastos) have recently been observed to occur on southern bluefin tuna (Thunnus maccoyii) ranched off Port Lincoln, South Australia (Hayward et al., 2008, 2009). This species has
⁎ Corresponding author. South Australian Research and Development Institute, Lincoln Marine Science Centre, P.O. Box 1511, Port Lincoln, South Australia 5606, Australia. E-mail address: [email protected] (C.J. Hayward). 1 Tohoku University Institute for International Education, 41 Kawauchi Aoba-ku, Sendai, Miyagi-ken 980-8576, Japan and Tohoku University Graduate School of Agricultural Science, 1-1 Tsutsumidori Amamiya-machi, Aoba-ku, Sendai, Miyagi 981-8555, Japan. 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.10.039
also been recorded on a number of other aquacultured species of fishes, including mulloway (Argyrosomus japonicus) and yellowtail kingfish (Seriola lalandi) in Australia (Hayward et al., 2007), and John's snapper (Lutjanus johni) in Malaysia (Venmathi Maran et al., 2009). On T. maccoyii, the numbers of these lice have been demonstrated to be correlated with two indicators of stress — plasma cortisol and glucose — as well as with low condition index and gross eye damage (Hayward et al., 2008, 2009, 2010). In contrast with sea lice infestations of other farmed fishes, attached larval stages of C. chiastos on ranched T. maccoyii are rarely detected. For example, of over 5400 individual lice collected from ranched tuna in 2008, only three (0.06%) were larval stages; the remainder were adult females and males of predominantly C. chiastos (Hayward et al., submitted for publication). This indicates that infected wild fishes attracted to tuna sea cages must be the source of infections of mobile, adult C. chiastos. In this study, we therefore aimed to monitor numbers of C. chiastos on
C.J. Hayward et al. / Aquaculture 320 (2011) 178–182 Table 1 Dates of monitoring sea lice burdens on ranched southern bluefin tuna (Thunnus maccoyii) off Port Lincoln in early 2009 (LCF, length to caudal fork). Dates of sampling
Sea cage no.
No. of tuna
Tuna mean LCF (cm)
01 17 26 12 16 23
‘tow’ 1–4 3, 4 3 3 3
40 8 4 3 30 30
93.1 97.6 107.8 115.3 107.7 111.3
Feb Feb Feb Mar Mar Mar
(78–105) (92–107) (100–115) (115–116) (89–140) (103–126)
ranched tuna from transfer to the pens at the beginning of the season through to harvest, and to sample wild fishes around ranching sea cages over the same period, to determine which species are likely to be the main host(s) of chalimus stages.
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2.2. Sampling of wild fishes beside T. maccoyii ranching sea cages Wild fishes were sampled from outside these four tuna ranching sea cages using a gill net and two fish traps. Table 2 lists the dates of collection, the species, and their numbers. With the exception of Port Jackson sharks (which were examined freshly without placement in plastic bags), all fish were placed individually as quickly as possible after catching into ziplock plastic bags; 100% ethanol was added, and the bags were sealed. The bagged fishes were stored on ice until they were returned to the laboratory, where they were transferred to a −20°C freezer until examination. The external surfaces of these fishes and the contents of the bags were examined under a dissection microscope; all lice detected were collected and stored in 100% ethanol. Adult lice were identified to species under low- and highpower microscopes; samples of larval lice were identified using molecular methods (see below).
2. Materials and methods 2.3. Data analysis 2.1. Sampling of T. maccoyii Two samples each of 10 T. maccoyii from different schools caught in the Great Australian Bight by a tuna ranching company were examined for sea lice in late January 2009. A tow cage containing T. maccoyii caught in the Great Australian Bight (33°47.81S 132°145.617E) on 19 January 2009 by a second tuna ranching company arrived at the ranching zone on the evening of 31 January 2009. On the morning of 1 February 2009, a sample of 40 of these tuna were weighed and measured; these tuna were also examined for sea lice and returned to the tow cage. These tuna were then transferred into four ranching sea cages (each of 40 m diameter), two on the same day and the other two the following day. Feeding of the tuna commenced on 2 February 2009 (three sea cages) and 3 February (one sea cage). Samples of tuna from these four sea cages were examined on several dates, up until the time of harvest; Table 1 lists the dates and sample sizes in this study. All lice visible to the naked eye were collected as soon as possible at the time of capture of tuna; any additional lice remaining on tuna surfaces were then detected using a technique described in Hayward et al. (2010), in which wetted fingers were gently moved over all the external skin surfaces of each tuna, to feel for characteristic hard ‘bumps’ (indicating the presence of sea lice obscured by tuna mucus and, in rare cases, attached chalimi). All lice were collected and preserved in ethanol and identified later in the laboratory using a dissecting microscope.
Parasite infections were characterised, for each species of host, by prevalence (the number of host infections as a proportion of the population at risk) and mean abundance (the average number of parasites in all hosts; Bush et al., 1997). Sterne's exact 95% confidence intervals were calculated for prevalence, and 95% bootstrap confidence intervals (with 2000 replications) were calculated for mean abundances, using the statistical package ‘Quantitative Parasitology 3.0’ (Reiczigel and Rózsa, 2005). Prevalences and mean abundance for each species for each sample date were compared pairwised with other sample dates. Given the high total number of pairwise comparisons, an alpha level of 0.01 was regarded as significant for these statistics. Spearman's rank correlation coefficient was calculated for the relationship between Caligus counts and host length using the VassarStats online statistical calculator (http://www.faculty.vassar. edu/lowry/VassarStats.html); a Mann–Whitney U test was used to compare the total numbers of sea lice on male and female hosts. 2.4. Molecular comparisons of parasites Genomic DNA extraction from individual parasites (an adult male, an adult female and five chalimus larvae) was performed with QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer's protocol. Polymerase chain reaction to amplify the mitochondrial COI region was conducted mainly according to Øines and Heuch (2005). Briefly, 5 μl of the extracted DNA and 75 μl of water
Table 2 Numbers of wild species collected from beside tuna ranching sea cages in early 2009 and their external (skin and fin) parasites (excluding Caligus chiastos from Thamnaconus degeni). Species abundance
n
Date
LCF (cm): mean (range)
Parasites
Prevalence (%)
Mean
Thamnaconus degeni
28 19 55 23 101 44 35 30 1 6 10 1 1 22 71 26 15 3 8 2
14 Feb 20 Feb 12 Mar 24 Mar 01 Apr 08 Apr 21 Apr 04 Feb 14 Feb 05 Feb 11 Feb 14 Feb 14 Feb 14 Feb 20 Feb 24 Mar 08 Apr 21 Apr 20 Feb 21 Apr
11.55 (8.8–13.7) 10.99 (8.5–13.5) 10.22 (7.4–13.5) 11.46 (8.5–13.7) 10.15 (7.0–14.2) 10.59 (8.1–13.2) 10.15 (7.4–12.8) 16.9 (16.0–18.5) 8.5 57.6 (48.0–61.5) 14.5 (13.8–15.0) 23.4 23.5 21.6 (17.5–23.9) 21.4(19.5–25.0) 21.0 (18.5–24.5) 21.1(19.6–23.4) 20.7(20.5–20.9) 16.8 (16.0–18.0) 23.0 (22.4–23.6)
Orbitacolax williamsi O. williamsi O. williamsi O. williamsi O. williamsi O. williamsi O. williamsi Caligus sp. Caligus sp. Dissonus nudiventris – – – – – – – – – –
17.9 31.6 52.7 73.9 70.3 86.4 60.0 50.0 100 100 – – – – – – – – – –
0.21 0.32 0.84 1.30 1.41 1.73 1.31 0.83 1.00 3.67 – – – – – – – – – –
Pseudocaranx wrighti Heterodontus portusjacksoni Sardinops sagax Scomber australasicus Trachurus novaezelandiae
Arripis truttacea
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were mixed with 0.75 μl of 100 μM of each primer (WOBCOI-F and WOBCOI-R), 0.5 μl of Takara Ex Taq DNA polymerase, 10 μl of 10 × PCR buffer and 8 μl of dNTP (Takara, Kyoto, Japan). The PCR conditions consisted of an initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 45 °C for 30 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 2 min. The PCR products were separated and visualized by electrophoresis on 1.5% agarose gel containing SYBR Safe DNA gel stain (Molecular Probes, Eugene, OR, USA) and the DNA was extracted and purified from the band with approximate 600-bp length using QIAquick Gel Extraction Kit (Qiagen Inc.). The partial D1–D2 domains of 28S rDNA was PCR amplified in 25 μl reactions with Bioline BioMix Red PCR Mastermix (2×) (12.5 μl) and PCR primers (final concentration, 10 pmol/μl): forward (5'-TAG GTC GAC CCG CTG AAY TTA AGC A-3') and reverse (5'-CTT GGT CCG TGT TTC AAG ACG GG-3'), with the remaining volume made up with RNAase-free H2O, on a MJ Research PTC-225 Peltier thermocycler using the following thermocycling conditions: 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 30 s, with a final extension step of 72 °C for 10 min. PCR products were subjected to 1.5% Tris-acetate-EDTA (TAE) Agarose gel electrophoresis, stained with GelRed (Biotium) and visualized on a GelDoc (BioRad). Positive PCR products were purified using QIAquick PCR purification kit (QIAGEN), and concentration of products was estimated by fluorometry (Wallac) using Quant-iT™ PicoGreen® (Invitrogen). The purified PCR products were quantified and sequenced. 3. Results No sea lice were detected on either of the two samples of 10 T. maccoyii from one tuna ranching company examined in the Great Australian Bight in late January. Of the T. maccoyii sampled within 24 h of arrival at the tuna ranching zone by another ranching company, 10% were infected with adult female and male C. chiastos; prevalence then increased significantly and peaked almost 25 days later at 75%; by the end of the ranching period, prevalence decreased significantly to 0% (Fig. 1). There were no significant differences in the mean abundances of sea lice on ranched tuna over the ranching period (Fig. 2). No chalimus larvae were detected on ranched T. maccoyii. Among wild fishes sampled, we detected adult C. chiastos only on the external surfaces of one species, namely Degen's leatherjacket
Fig. 1. Prevalence (± 95% Sterne's exact CI) of Caligus chiastos on ranched southern bluefin tuna and wild Degen's leatherjackets in early 2009. Letters group prevalences that do not differ significantly.
Fig. 2. Mean abundance (± 95% Bootstrap CI) of Caligus chiastos on ranched southern bluefin tuna and wild Degen's leatherjackets in early 2009. Letters group mean abundances that do not differ significantly. There are no significant differences in abundances of C. chiastos on tuna.
(Thamnaconus degeni). This species was also host to numerous caligid larvae, the proportion of which increased over the study period up to over 93% (Fig. 3). For the CO1 region, seven DNA sequences (for one adult female, one adult male and five chalimi larvae collected from Degen's leatherjackets) with 561-bp length were identified. The BLAST search in NCBI (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) revealed that the identified sequences were homologous to the COI region of C. chiastos (EF452644, EF452645 and EF452646), and the range of identity was 97–99% with E value of 0.00. For partial D1–D2 28S rDNA, five DNA sequences (from chalimi larvae collected from Degen's leatherjackets) with 602-bp length were identified. The BLAST search in NCBI revealed that the sequences were 100% identical to the partial D1–D2 28S rDNA region of C. chiastos (EU118303, EU118304, and EU118305) with E value of 0.00. These results indicate that the larvae collected from T. degeni were conspecific with C. chiastos. Burdens of sea lice were significantly correlated with size of the leatherjackets (rs,303 df = 0.2425, p = 0.0017), and significantly more sea lice were found on the (larger) males than the (smaller) females (Mann–Whitney U test, p b 0.05). A second, unidentified species of Caligus was also recovered from another host species, sand trevally, P. wrighti. Hosts examined in this study and the respective identities of their parasites are listed in Table 2.
Fig. 3. Ratio of adult male, female, and chalimus larvae of Caligus chiastos on wild Degen's leatherjackets collected beside ranched southern bluefin tuna sea cages in early 2009.
C.J. Hayward et al. / Aquaculture 320 (2011) 178–182
4. Discussion Excess feed from tuna ranched off Port Lincoln provides feeding opportunities for a range of wild fishes, where the most important scavenger has been identified to be Degen's leatherjacket, T. degeni (see Svane and Barnett, 2008). Floating sea cages allow the movements of smaller scavengers and therefore increase the likelihood of pathogen transfer between wild and farmed fish (Costello, 2009). The present study confirms that larval C. chiastos occur on the external surfaces of at least one wild species of teleost found in the tuna ranching environment. In contrast with the epizootic increase and decline in prevalence on T. maccoyii by the time of harvest, on Degen's leatherjackets, there was a significant increase in prevalence (and abundance) over the 3-month study period (Figs. 1 and 2). At the tuna sea cages, more than 100 Degen's leatherjacket can be observed at bait within minutes over an area of 1 m2 (Svane and Barnett, 2008). This fish species was also the most common species caught in a prawn trawl research survey in the same area (Currie et al., 2009). T. degeni was found to represent an even higher percentage of the total biomass (over 20%) than the commercial target species (king prawn, Melicertus latisulcatus; over 14% of biomass; Currie et al., 2009). Degen's leatherjacket scavenge bait during day, whereas other known scavengers around tuna farms, the nonparasitic isopods of the genus Natatolana and unidentified amphipods, dominate at night (Svane and Barnett, 2008). Because nocturnal crustaceans feeding on excess baitfish at tuna ranches do not carry parasitic sea lice, we conclude that Degen's leatherjackets are likely to contribute to sea lice infestations of ranched T. maccoyii. As with C. chiastos infections of ranched tuna, anecdotal evidence suggests that infections of Caligus elongatus on Atlantic salmon farmed in Europe similarly occur in the form of a sudden pulse of adult lice (Heuch et al., 2007). In contrast with the results of the present study, in which only one species of wild fish (a tetraodontiform fish, T. degeni) was found to be infected with larval and adult C. chiastos, in the case of C. elongatus, Heuch et al. (2007) documented a number of wild fish species to be infected. However, these authors found that the likely reservoir host of C. elongatus, as indicated by the highest numbers of adults and relatively high numbers of chalimi, was a scorpaeniform fish, lumpfish (Cyclopterus lumpus). We found no C. chiastos on 20 wild T. maccoyii in this study, and nor have there been any published reports of this sea louse on wild T. maccoyii in the literature. Nevertheless, within 24 h of arriving near the ranching site in Spencer Gulf (on 1 February 2009), tuna stock were already infected with adult C. chiastos, even though they had not been fed any baitfish up to this time. This may indicate that leatherjackets (and perhaps other lice-infected kleptivores [wild fishes consuming uneaten feed]) were already attracted to tow cages before any baitfish had been fed. The six Port Jackson sharks examined in this study were not infected with any C. chiastos. This species was, however, reported by Kabata (1965) as among those elasmobranchs likely to have been the host of male C. chiastos collected from another location in South Australia, Port Willunga. (Kabata identified the material as Caligus rapax, a name which has since been synonymised with C. elongatus. However, the appearance of the striations of the maxillule (as illustrated by Kabata in Fig. 1F) indicates that the material is in fact C. chiastos.) We therefore suspect that larger sample sizes of Port Jackson sharks (and perhaps collection at later times in the season, when C. chiastos were significantly more abundant on Degen's leatherjackets) and examination of other elasmobranchs known to occur in the tuna ranching environment may reveal that they are also other common hosts of C. chiastos in South Australian waters. Although it remains a controversial issue, sea lice originating from farmed fishes are regarded by a number of authors as potentially threatening to wild species (for example, see Krkošek et al., 2007; Costello, 2009; Frazer, 2009). However, in our case — and in contrast
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with other species of sea lice infecting farmed fishes — the farmed host of the sea louse C. chiastos, southern bluefin tuna, carries, almost exclusively, mobile adult stages of the parasite, and furthermore the wild fish which we document to host numerous larval and adults of the same species of sea louse (Degen's leatherjacket) are not related to this farmed host. Furthermore, our epidemiological data indicate that after the numbers of lice significantly declined on ranched tuna, they continued to increase on the leatherjackets, indicating that infected ranched tuna were likely not acting as reservoir hosts for wild leatherjackets. The reason for the decline of epizootics of C. chiastos on ranched tuna is not known. Hayward et al. (2008) speculated that it may have been due simply to decreasing ambient water temperatures during the culture period, as such a drop would have undoubtedly reduced the growth and reproductive rate of these sea lice. However, in the present study, decreasing temperature did not prevent a significant increase in numbers of lice on Degen's leatherjacket. This being the case, we now speculate that tuna, or the leatherjackets, or both may alter their swimming behavior as the water cools, reducing their proximity to each other, which in turn leads to reduced transmission of postchalimus stages from wild leatherjackets to ranched tuna. The species of sea louse we detected on sand trevally, Caligus sp., did not match the description of the only other species of Caligus previously reported from this host in the literature: C. kurochkini Kazachenko, 1975. However, this species was described from the gills rather than from the skin and fins of this host. In contrast to farmed Atlantic salmon, the impact of sea lice on farmed southern bluefin tuna is largely unknown, although recently it has been shown that sea lice loads are positively correlated with two stress indicators (plasma cortisol and glucose) and negatively correlated with gross eye damage and condition index (Hayward et al., 2008, 2009, 2010). However, in an expanding farming situation with high densities of tuna build-up of infested scavengers are likely to increase the rate of tuna infestations with a subsequent reduction in fish quality and health (Costello, 2009). Tuna sea-pen facilities are set in a complex ecosystem where the increased expansion of populations of scavengers is likely to affect ecosystem structure and function (Rosenberg, 2008; Svane and Barnett, 2008; Krkošek et al., 2007). It is therefore important for management to not only consider production and quality, but also environmental cost and balance. Some possible practical management strategies which may reduce the numbers of adult sea lice that ranched tuna may acquire include: reducing the amount of feed fed to ranched tuna; the trialling of manufactured feeds; and the relocation of tuna lease sites to deeper waters with greater clearance from the substrate. Acknowledgements We thank Sekol Farm Tuna for allowing us to sample wild fishes on their lease site and for permitting us to sample T. maccoyii for sea lice. We thank David Allan of Marnikol for his examination of T. maccoyii caught in the Great Australian Bight for sea lice. CJH and NJB were supported by Marine Innovation South Australia (MISA), an initiative of the South Australian Government. References Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Par. 83, 575–583. Costello, M.J., 2009. How sea lice from salmon farms may cause wild salmonid declines in Europe and North America and be a threat to fishes elsewhere. Proc. Royal Soc. B 276, 3385–3394. Currie, D.R., Dixon, C.D., Roberts, S.D., Hooper, G.E., Sorokin, S.J., Ward, T.M., 2009. Fishery-independent by-catch survey to inform risk assessment of the Spencer Gulf Prawn Trawl Fishery. Report to PIRSA Fisheries. South Australian Research and Development Institute (Aquatic Sciences), Adelaide, Australia. Frazer, L.N., 2009. Sea-cage aquaculture, sea lice, and declines of wild fish. Conserv. Biol. 276, 3385–3394.
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Hayward, C.J., Bott, N.J., Itoh, N., Iwashita, M., Okihiro, M., Nowak, B.F., 2007. Three species of parasites emerging on the gills of mulloway, Argyrosomus japonicus (Temminck and Schlegel, 1843), cultured in Australia. Aquacult. 265, 27–40. Hayward, C.J., Aiken, H.M., Nowak, B.F., 2008. An epizootic of Caligus chiastos (Copepoda, Caligidae) on Southern Bluefin Tuna (Thunnus maccoyii) farmed in Australia. Dis. Aquat. Organ. 79, 57–63. Hayward, C.J., Bott, N.J., Nowak, B.F., 2009. Seasonal epizootics of sea lice (Caligus spp.) on Southern Bluefin Tuna (Thunnus maccoyii) in a long-term farming trial. J. Fish Dis 32, 101–106. Hayward, C.J., Ellis, D., Foote, D., Wilkinson, R.J., Crosbie, P.B.B., Nowak, B.F., 2010. Concurrent epizootic hyperinfections of sea lice (predominantly Caligus chiastos) and blood flukes (Cardicola forsteri) in ranched Southern Bluefin tuna. Vet. Par. 173, 107–115. Heuch, P.A., Øines, Ø., Knutsen, J.A., Schram, T.A., 2007. Infection of wild fishes by the parasitic copepod Caligus elongatus on the south east coast of Norway. Dis. Aquat. Organ. 77, 149–158. Kabata, Z., 1965. Copepods parasitic on Australian fishes. IV. Genus Caligus (Caligidae). Ann. Mag. Nat. Hist. 13, 109–126.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Sea lice infections of wild fishes near ranched southern bluefin tuna (Thunnus maccoyii) in South Australia C.J. Hayward a,b,⁎,1, I. Svane a, S.K. Lachimpadi a, N. Itoh c, N.J. Bott d, B.F. Nowak b a
South Australian Research and Development Institute, Lincoln Marine Science Centre, P.O. Box 1511, Port Lincoln, South Australia 5606, Australia National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Locked Bag 1-370, Launceston, Tasmania 7250, Australia Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori Amamiya-machi, Aoba-ku, Sendai 981-8555 Miyagi, Japan d South Australian Research and Development Institute-Aquatic Sciences, P.O. Box 120, Henley Beach, South Australia 5022, Australia b c
a r t i c l e
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Article history: Received 10 June 2010 Received in revised form 15 October 2010 Accepted 28 October 2010 Available online 4 November 2010 Keywords: Sea lice Aquaculture Tuna Caligus chiastos Thamnaconus degeni Degen's leatherjacket
a b s t r a c t In contrast with sea lice infestations of other farmed fishes, attached larval stages of sea lice on ranched southern bluefin tuna (Thunnus maccoyii) are rarely detected. In this study, we monitored sea lice on ranched T. maccoyii and surveyed wild fishes adjacent to ranching sea cages over a 3-month period in early 2009. Prevalence of the adult Caligus chiastos on tuna within a day of arrival at the ranching site was 10%; prevalence then increased significantly and peaked almost 25 days later to 75%; by harvest (after a further 18–28 days), prevalence decreased significantly to 0%. We collected and examined a total of 502 wild fishes outside T. maccoyii sea cages, comprising 307 Degen's leatherjackets (Thamnaconus degeni), 136 yellowtail horse mackerel (Trachurus novaezelandiae), 31 sand trevally (Pseudocaranx wrighti), 10 West Australian salmon (Arripis truttacea), 6 Port Jackson sharks (Heterodontus portusjacksoni), and a single blue mackerel (Scomber australasicus) and pilchard (Sardinops sagax); we also examined an additional 10 pilchards that were collected from the centre of Spencer Gulf and stored fresh in a T. maccoyii feed bin. Of these potential hosts, we identified adult C. chiastos only from Degen's leatherjackets; of the many larvae also occurring on this host, molecular comparison of five specimens analysing cytochrome C oxidase I region of mitochondrial DNA and five specimens analysing partial D1–D2 domains of 28S rDNA confirmed that these were C. chiastos. In contrast with the decline in infections of C. chiastos on ranched T. maccoyii near the end of March, on Degen's leatherjackets there was a significant increase in prevalence and abundance over the study period, with a peak prevalence of 97.14% and a mean abundance reaching 11.17 lice per fish near the end of April. The percentage of chalimus larvae on Degen's leatherjackets increased over the study period, ranging from 0% near the start of sampling to over 93% on the final sample date. We also recorded additional copepod infestations, including Orbitacolax williamsi on Degen's leatherjackets, Caligus sp. on sand trevally, and Dissonus nudiventris on Port Jackson sharks. We conclude that Degen's leatherjacket, which is a major scavenger of excess tuna feed, is likely to contribute to sea lice infestations of T. maccoyii. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Sea lice are a significant pathogen in marine finfish farming (Rosenberg, 2008). Fish farmed in sea cages are likely to be infested by parasites from wild fishes and because of their high concentration can in turn become a source of parasites (Costello, 2009). Epizootics of sea lice (predominantly Caligus chiastos) have recently been observed to occur on southern bluefin tuna (Thunnus maccoyii) ranched off Port Lincoln, South Australia (Hayward et al., 2008, 2009). This species has
⁎ Corresponding author. South Australian Research and Development Institute, Lincoln Marine Science Centre, P.O. Box 1511, Port Lincoln, South Australia 5606, Australia. E-mail address: [email protected] (C.J. Hayward). 1 Tohoku University Institute for International Education, 41 Kawauchi Aoba-ku, Sendai, Miyagi-ken 980-8576, Japan and Tohoku University Graduate School of Agricultural Science, 1-1 Tsutsumidori Amamiya-machi, Aoba-ku, Sendai, Miyagi 981-8555, Japan. 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.10.039
also been recorded on a number of other aquacultured species of fishes, including mulloway (Argyrosomus japonicus) and yellowtail kingfish (Seriola lalandi) in Australia (Hayward et al., 2007), and John's snapper (Lutjanus johni) in Malaysia (Venmathi Maran et al., 2009). On T. maccoyii, the numbers of these lice have been demonstrated to be correlated with two indicators of stress — plasma cortisol and glucose — as well as with low condition index and gross eye damage (Hayward et al., 2008, 2009, 2010). In contrast with sea lice infestations of other farmed fishes, attached larval stages of C. chiastos on ranched T. maccoyii are rarely detected. For example, of over 5400 individual lice collected from ranched tuna in 2008, only three (0.06%) were larval stages; the remainder were adult females and males of predominantly C. chiastos (Hayward et al., submitted for publication). This indicates that infected wild fishes attracted to tuna sea cages must be the source of infections of mobile, adult C. chiastos. In this study, we therefore aimed to monitor numbers of C. chiastos on
C.J. Hayward et al. / Aquaculture 320 (2011) 178–182 Table 1 Dates of monitoring sea lice burdens on ranched southern bluefin tuna (Thunnus maccoyii) off Port Lincoln in early 2009 (LCF, length to caudal fork). Dates of sampling
Sea cage no.
No. of tuna
Tuna mean LCF (cm)
01 17 26 12 16 23
‘tow’ 1–4 3, 4 3 3 3
40 8 4 3 30 30
93.1 97.6 107.8 115.3 107.7 111.3
Feb Feb Feb Mar Mar Mar
(78–105) (92–107) (100–115) (115–116) (89–140) (103–126)
ranched tuna from transfer to the pens at the beginning of the season through to harvest, and to sample wild fishes around ranching sea cages over the same period, to determine which species are likely to be the main host(s) of chalimus stages.
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2.2. Sampling of wild fishes beside T. maccoyii ranching sea cages Wild fishes were sampled from outside these four tuna ranching sea cages using a gill net and two fish traps. Table 2 lists the dates of collection, the species, and their numbers. With the exception of Port Jackson sharks (which were examined freshly without placement in plastic bags), all fish were placed individually as quickly as possible after catching into ziplock plastic bags; 100% ethanol was added, and the bags were sealed. The bagged fishes were stored on ice until they were returned to the laboratory, where they were transferred to a −20°C freezer until examination. The external surfaces of these fishes and the contents of the bags were examined under a dissection microscope; all lice detected were collected and stored in 100% ethanol. Adult lice were identified to species under low- and highpower microscopes; samples of larval lice were identified using molecular methods (see below).
2. Materials and methods 2.3. Data analysis 2.1. Sampling of T. maccoyii Two samples each of 10 T. maccoyii from different schools caught in the Great Australian Bight by a tuna ranching company were examined for sea lice in late January 2009. A tow cage containing T. maccoyii caught in the Great Australian Bight (33°47.81S 132°145.617E) on 19 January 2009 by a second tuna ranching company arrived at the ranching zone on the evening of 31 January 2009. On the morning of 1 February 2009, a sample of 40 of these tuna were weighed and measured; these tuna were also examined for sea lice and returned to the tow cage. These tuna were then transferred into four ranching sea cages (each of 40 m diameter), two on the same day and the other two the following day. Feeding of the tuna commenced on 2 February 2009 (three sea cages) and 3 February (one sea cage). Samples of tuna from these four sea cages were examined on several dates, up until the time of harvest; Table 1 lists the dates and sample sizes in this study. All lice visible to the naked eye were collected as soon as possible at the time of capture of tuna; any additional lice remaining on tuna surfaces were then detected using a technique described in Hayward et al. (2010), in which wetted fingers were gently moved over all the external skin surfaces of each tuna, to feel for characteristic hard ‘bumps’ (indicating the presence of sea lice obscured by tuna mucus and, in rare cases, attached chalimi). All lice were collected and preserved in ethanol and identified later in the laboratory using a dissecting microscope.
Parasite infections were characterised, for each species of host, by prevalence (the number of host infections as a proportion of the population at risk) and mean abundance (the average number of parasites in all hosts; Bush et al., 1997). Sterne's exact 95% confidence intervals were calculated for prevalence, and 95% bootstrap confidence intervals (with 2000 replications) were calculated for mean abundances, using the statistical package ‘Quantitative Parasitology 3.0’ (Reiczigel and Rózsa, 2005). Prevalences and mean abundance for each species for each sample date were compared pairwised with other sample dates. Given the high total number of pairwise comparisons, an alpha level of 0.01 was regarded as significant for these statistics. Spearman's rank correlation coefficient was calculated for the relationship between Caligus counts and host length using the VassarStats online statistical calculator (http://www.faculty.vassar. edu/lowry/VassarStats.html); a Mann–Whitney U test was used to compare the total numbers of sea lice on male and female hosts. 2.4. Molecular comparisons of parasites Genomic DNA extraction from individual parasites (an adult male, an adult female and five chalimus larvae) was performed with QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer's protocol. Polymerase chain reaction to amplify the mitochondrial COI region was conducted mainly according to Øines and Heuch (2005). Briefly, 5 μl of the extracted DNA and 75 μl of water
Table 2 Numbers of wild species collected from beside tuna ranching sea cages in early 2009 and their external (skin and fin) parasites (excluding Caligus chiastos from Thamnaconus degeni). Species abundance
n
Date
LCF (cm): mean (range)
Parasites
Prevalence (%)
Mean
Thamnaconus degeni
28 19 55 23 101 44 35 30 1 6 10 1 1 22 71 26 15 3 8 2
14 Feb 20 Feb 12 Mar 24 Mar 01 Apr 08 Apr 21 Apr 04 Feb 14 Feb 05 Feb 11 Feb 14 Feb 14 Feb 14 Feb 20 Feb 24 Mar 08 Apr 21 Apr 20 Feb 21 Apr
11.55 (8.8–13.7) 10.99 (8.5–13.5) 10.22 (7.4–13.5) 11.46 (8.5–13.7) 10.15 (7.0–14.2) 10.59 (8.1–13.2) 10.15 (7.4–12.8) 16.9 (16.0–18.5) 8.5 57.6 (48.0–61.5) 14.5 (13.8–15.0) 23.4 23.5 21.6 (17.5–23.9) 21.4(19.5–25.0) 21.0 (18.5–24.5) 21.1(19.6–23.4) 20.7(20.5–20.9) 16.8 (16.0–18.0) 23.0 (22.4–23.6)
Orbitacolax williamsi O. williamsi O. williamsi O. williamsi O. williamsi O. williamsi O. williamsi Caligus sp. Caligus sp. Dissonus nudiventris – – – – – – – – – –
17.9 31.6 52.7 73.9 70.3 86.4 60.0 50.0 100 100 – – – – – – – – – –
0.21 0.32 0.84 1.30 1.41 1.73 1.31 0.83 1.00 3.67 – – – – – – – – – –
Pseudocaranx wrighti Heterodontus portusjacksoni Sardinops sagax Scomber australasicus Trachurus novaezelandiae
Arripis truttacea
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were mixed with 0.75 μl of 100 μM of each primer (WOBCOI-F and WOBCOI-R), 0.5 μl of Takara Ex Taq DNA polymerase, 10 μl of 10 × PCR buffer and 8 μl of dNTP (Takara, Kyoto, Japan). The PCR conditions consisted of an initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 45 °C for 30 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 2 min. The PCR products were separated and visualized by electrophoresis on 1.5% agarose gel containing SYBR Safe DNA gel stain (Molecular Probes, Eugene, OR, USA) and the DNA was extracted and purified from the band with approximate 600-bp length using QIAquick Gel Extraction Kit (Qiagen Inc.). The partial D1–D2 domains of 28S rDNA was PCR amplified in 25 μl reactions with Bioline BioMix Red PCR Mastermix (2×) (12.5 μl) and PCR primers (final concentration, 10 pmol/μl): forward (5'-TAG GTC GAC CCG CTG AAY TTA AGC A-3') and reverse (5'-CTT GGT CCG TGT TTC AAG ACG GG-3'), with the remaining volume made up with RNAase-free H2O, on a MJ Research PTC-225 Peltier thermocycler using the following thermocycling conditions: 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 30 s, with a final extension step of 72 °C for 10 min. PCR products were subjected to 1.5% Tris-acetate-EDTA (TAE) Agarose gel electrophoresis, stained with GelRed (Biotium) and visualized on a GelDoc (BioRad). Positive PCR products were purified using QIAquick PCR purification kit (QIAGEN), and concentration of products was estimated by fluorometry (Wallac) using Quant-iT™ PicoGreen® (Invitrogen). The purified PCR products were quantified and sequenced. 3. Results No sea lice were detected on either of the two samples of 10 T. maccoyii from one tuna ranching company examined in the Great Australian Bight in late January. Of the T. maccoyii sampled within 24 h of arrival at the tuna ranching zone by another ranching company, 10% were infected with adult female and male C. chiastos; prevalence then increased significantly and peaked almost 25 days later at 75%; by the end of the ranching period, prevalence decreased significantly to 0% (Fig. 1). There were no significant differences in the mean abundances of sea lice on ranched tuna over the ranching period (Fig. 2). No chalimus larvae were detected on ranched T. maccoyii. Among wild fishes sampled, we detected adult C. chiastos only on the external surfaces of one species, namely Degen's leatherjacket
Fig. 1. Prevalence (± 95% Sterne's exact CI) of Caligus chiastos on ranched southern bluefin tuna and wild Degen's leatherjackets in early 2009. Letters group prevalences that do not differ significantly.
Fig. 2. Mean abundance (± 95% Bootstrap CI) of Caligus chiastos on ranched southern bluefin tuna and wild Degen's leatherjackets in early 2009. Letters group mean abundances that do not differ significantly. There are no significant differences in abundances of C. chiastos on tuna.
(Thamnaconus degeni). This species was also host to numerous caligid larvae, the proportion of which increased over the study period up to over 93% (Fig. 3). For the CO1 region, seven DNA sequences (for one adult female, one adult male and five chalimi larvae collected from Degen's leatherjackets) with 561-bp length were identified. The BLAST search in NCBI (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) revealed that the identified sequences were homologous to the COI region of C. chiastos (EF452644, EF452645 and EF452646), and the range of identity was 97–99% with E value of 0.00. For partial D1–D2 28S rDNA, five DNA sequences (from chalimi larvae collected from Degen's leatherjackets) with 602-bp length were identified. The BLAST search in NCBI revealed that the sequences were 100% identical to the partial D1–D2 28S rDNA region of C. chiastos (EU118303, EU118304, and EU118305) with E value of 0.00. These results indicate that the larvae collected from T. degeni were conspecific with C. chiastos. Burdens of sea lice were significantly correlated with size of the leatherjackets (rs,303 df = 0.2425, p = 0.0017), and significantly more sea lice were found on the (larger) males than the (smaller) females (Mann–Whitney U test, p b 0.05). A second, unidentified species of Caligus was also recovered from another host species, sand trevally, P. wrighti. Hosts examined in this study and the respective identities of their parasites are listed in Table 2.
Fig. 3. Ratio of adult male, female, and chalimus larvae of Caligus chiastos on wild Degen's leatherjackets collected beside ranched southern bluefin tuna sea cages in early 2009.
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4. Discussion Excess feed from tuna ranched off Port Lincoln provides feeding opportunities for a range of wild fishes, where the most important scavenger has been identified to be Degen's leatherjacket, T. degeni (see Svane and Barnett, 2008). Floating sea cages allow the movements of smaller scavengers and therefore increase the likelihood of pathogen transfer between wild and farmed fish (Costello, 2009). The present study confirms that larval C. chiastos occur on the external surfaces of at least one wild species of teleost found in the tuna ranching environment. In contrast with the epizootic increase and decline in prevalence on T. maccoyii by the time of harvest, on Degen's leatherjackets, there was a significant increase in prevalence (and abundance) over the 3-month study period (Figs. 1 and 2). At the tuna sea cages, more than 100 Degen's leatherjacket can be observed at bait within minutes over an area of 1 m2 (Svane and Barnett, 2008). This fish species was also the most common species caught in a prawn trawl research survey in the same area (Currie et al., 2009). T. degeni was found to represent an even higher percentage of the total biomass (over 20%) than the commercial target species (king prawn, Melicertus latisulcatus; over 14% of biomass; Currie et al., 2009). Degen's leatherjacket scavenge bait during day, whereas other known scavengers around tuna farms, the nonparasitic isopods of the genus Natatolana and unidentified amphipods, dominate at night (Svane and Barnett, 2008). Because nocturnal crustaceans feeding on excess baitfish at tuna ranches do not carry parasitic sea lice, we conclude that Degen's leatherjackets are likely to contribute to sea lice infestations of ranched T. maccoyii. As with C. chiastos infections of ranched tuna, anecdotal evidence suggests that infections of Caligus elongatus on Atlantic salmon farmed in Europe similarly occur in the form of a sudden pulse of adult lice (Heuch et al., 2007). In contrast with the results of the present study, in which only one species of wild fish (a tetraodontiform fish, T. degeni) was found to be infected with larval and adult C. chiastos, in the case of C. elongatus, Heuch et al. (2007) documented a number of wild fish species to be infected. However, these authors found that the likely reservoir host of C. elongatus, as indicated by the highest numbers of adults and relatively high numbers of chalimi, was a scorpaeniform fish, lumpfish (Cyclopterus lumpus). We found no C. chiastos on 20 wild T. maccoyii in this study, and nor have there been any published reports of this sea louse on wild T. maccoyii in the literature. Nevertheless, within 24 h of arriving near the ranching site in Spencer Gulf (on 1 February 2009), tuna stock were already infected with adult C. chiastos, even though they had not been fed any baitfish up to this time. This may indicate that leatherjackets (and perhaps other lice-infected kleptivores [wild fishes consuming uneaten feed]) were already attracted to tow cages before any baitfish had been fed. The six Port Jackson sharks examined in this study were not infected with any C. chiastos. This species was, however, reported by Kabata (1965) as among those elasmobranchs likely to have been the host of male C. chiastos collected from another location in South Australia, Port Willunga. (Kabata identified the material as Caligus rapax, a name which has since been synonymised with C. elongatus. However, the appearance of the striations of the maxillule (as illustrated by Kabata in Fig. 1F) indicates that the material is in fact C. chiastos.) We therefore suspect that larger sample sizes of Port Jackson sharks (and perhaps collection at later times in the season, when C. chiastos were significantly more abundant on Degen's leatherjackets) and examination of other elasmobranchs known to occur in the tuna ranching environment may reveal that they are also other common hosts of C. chiastos in South Australian waters. Although it remains a controversial issue, sea lice originating from farmed fishes are regarded by a number of authors as potentially threatening to wild species (for example, see Krkošek et al., 2007; Costello, 2009; Frazer, 2009). However, in our case — and in contrast
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with other species of sea lice infecting farmed fishes — the farmed host of the sea louse C. chiastos, southern bluefin tuna, carries, almost exclusively, mobile adult stages of the parasite, and furthermore the wild fish which we document to host numerous larval and adults of the same species of sea louse (Degen's leatherjacket) are not related to this farmed host. Furthermore, our epidemiological data indicate that after the numbers of lice significantly declined on ranched tuna, they continued to increase on the leatherjackets, indicating that infected ranched tuna were likely not acting as reservoir hosts for wild leatherjackets. The reason for the decline of epizootics of C. chiastos on ranched tuna is not known. Hayward et al. (2008) speculated that it may have been due simply to decreasing ambient water temperatures during the culture period, as such a drop would have undoubtedly reduced the growth and reproductive rate of these sea lice. However, in the present study, decreasing temperature did not prevent a significant increase in numbers of lice on Degen's leatherjacket. This being the case, we now speculate that tuna, or the leatherjackets, or both may alter their swimming behavior as the water cools, reducing their proximity to each other, which in turn leads to reduced transmission of postchalimus stages from wild leatherjackets to ranched tuna. The species of sea louse we detected on sand trevally, Caligus sp., did not match the description of the only other species of Caligus previously reported from this host in the literature: C. kurochkini Kazachenko, 1975. However, this species was described from the gills rather than from the skin and fins of this host. In contrast to farmed Atlantic salmon, the impact of sea lice on farmed southern bluefin tuna is largely unknown, although recently it has been shown that sea lice loads are positively correlated with two stress indicators (plasma cortisol and glucose) and negatively correlated with gross eye damage and condition index (Hayward et al., 2008, 2009, 2010). However, in an expanding farming situation with high densities of tuna build-up of infested scavengers are likely to increase the rate of tuna infestations with a subsequent reduction in fish quality and health (Costello, 2009). Tuna sea-pen facilities are set in a complex ecosystem where the increased expansion of populations of scavengers is likely to affect ecosystem structure and function (Rosenberg, 2008; Svane and Barnett, 2008; Krkošek et al., 2007). It is therefore important for management to not only consider production and quality, but also environmental cost and balance. Some possible practical management strategies which may reduce the numbers of adult sea lice that ranched tuna may acquire include: reducing the amount of feed fed to ranched tuna; the trialling of manufactured feeds; and the relocation of tuna lease sites to deeper waters with greater clearance from the substrate. Acknowledgements We thank Sekol Farm Tuna for allowing us to sample wild fishes on their lease site and for permitting us to sample T. maccoyii for sea lice. We thank David Allan of Marnikol for his examination of T. maccoyii caught in the Great Australian Bight for sea lice. CJH and NJB were supported by Marine Innovation South Australia (MISA), an initiative of the South Australian Government. References Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Par. 83, 575–583. Costello, M.J., 2009. How sea lice from salmon farms may cause wild salmonid declines in Europe and North America and be a threat to fishes elsewhere. Proc. Royal Soc. B 276, 3385–3394. Currie, D.R., Dixon, C.D., Roberts, S.D., Hooper, G.E., Sorokin, S.J., Ward, T.M., 2009. Fishery-independent by-catch survey to inform risk assessment of the Spencer Gulf Prawn Trawl Fishery. Report to PIRSA Fisheries. South Australian Research and Development Institute (Aquatic Sciences), Adelaide, Australia. Frazer, L.N., 2009. Sea-cage aquaculture, sea lice, and declines of wild fish. Conserv. Biol. 276, 3385–3394.
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