Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 58

Seasonal occurrence of Caligus spinosus and Parabrachiella seriolae (Copepoda) parasitic on cage-cultured yellowtail (Seriola quinqueradiata) at a fish farm in western Japan

E. R. Cruz-Lacierda1*, A. Yamamoto1 and K. Nagasawa2

1 Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima 890-0056, Japan; 2 Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan

Abstract The occurrence of Caligus spinosus Yamaguti, 1939 (Caligidae) and Parabrachiella seriolae (Yamaguti and Yamasu, 1960) (Lernaeopodidae) parasitic on yellowtail, Seriola quinqueradiata Temminck and Schlegel, 1845, cultured at a fish farm in Nagashima, Kagoshima Prefecture, Japan, was exam- ined. Both parasites were absent on wild-caught juvenile yellowtail (“mojako”) (length=6.5-13cm; weight=5.4-16g) at the time of stocking in net cages. Prevalence and median intensity of C. spinosus infection on the gill rakers were strongly and positively correlated with seawater temperature. Most of the C. spinosus population was adult females (45-100%). Prevalence of P. seriolae found aached at the base of the pectoral fin showed a high and positive correlation with seawater temperature. Most of the P. seriolae population comprised of adult females (78%). No gross pathological changes or host mortality were associated with the presence of either C. spinosus or P. seriolae.

Introduction The yellowtail or Japanese amberjack (Seriola and lernaeopodids (Ogawa and Yokoyama, quinqueradiata), greater amberjack (S. dumerili), 1998). Caligids, known as sea lice, are aached and goldstriped amberjack (S. lalandi) are cul- to the skin and gills of host fish and can cause tured in Japan with the yellowtail as the most skin lesions, fish mortality, and production valuable species. Aquaculture of yellowtail in losses in several farmed fish species (Pike and Japan is based on wild-caught juveniles (<15 Wadsworth, 1999). Caligus spinosus Yamaguti, mm long), called “mojako” (FAO, 2005-2010). 1939 is parasitic on the gill rakers of both wild The grow-out phase is conducted in floating and cultured S. quinqueradiata (Ho et al., 2001). net cages and “mojako” stocked in April-May Large-scale mortality due to C. spinosus infec- reach 1.0-1.5 kg by December and 2-3 kg aVer tion has been recorded by Fujita et al. (1968). a one year culture period. The lernaeopodid copepod Parabrachiella seri- olae (Yamaguti and Yamasu, 1960) originally One problem identified in yellowtail culture is described as Brachiella seriolae Yamaguti and infection by parasitic copepods such as caligids Yamasu, 1960, is aached at the base of pectoral

* Corresponding author’s email: erlinda@fish.kagoshima-u.ac.jp, [email protected] Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 59

fins, oral cavity and buccal folds of Seriola spp. oral and opercular cavities were immediately (Ono, 1984; Sheppard, 2004; Johnson et al., 2004; examined for parasites. The gills were excised, Hutson et al., 2007). There has never been any and either examined immediately in seawater report of P. seriolae causing disease in farmed or under a dissection microscope, or placed in wild fish in Japan or elsewhere (Johnson et al., labeled containers and frozen at -20°C until 2004). As studies on these parasites are limited, thawing before examination. The parasites were the occurrence of two copepods parasitic on counted and fixed in 70% ethanol. Terminolo- yellowtail cultured at a fish farm in western gies describing the infection follow Bush et al. Japan was conducted to determine whether (1997). Infection intensities were highly skewed the infection is seasonal. shown by large standard deviation (SD) in some points, thus median intensity was included as Materials and methods a measure of central tendency. Scheffe post hoc Collection of yellowtail and examination for multiple comparison test was used to determine copepods differences between the monthly temperature Yellowtail samples were collected from measurements while Pearson coefficient test a single fish stock and a single net cage was done to determine correlation between (32°13’57.85”N and 130°10’52.31”E) belong- infection and water temperature and between ing to Azuma-Cho Fishery Cooperative (ACFC) size of fish and intensity of infection using SPSS in Nagashima Island, Kagoshima Prefecture, version 15. Japan. The island is a dense farming area with about 300 yellowtail cages along an area of The above study was repeated in the next 116 km2 with an average annual production farming season at the same yellowtail farm in of 12,000 tons. Two examination periods were Nagashima, Kagoshima, particularly to examine done, from June 2006 to May 2007 and from caligids on the body surface at the time of stock- May to October 2007. ing of “mojako” on May 15, 2007, and approxi- mately every month thereaVer until August 21, In June 15, 2006, following the stocking of 2007. The same examination protocol as de- “mojako”, 10 fish were collected once a month scribed above was followed with 50 fish samples and at approximately monthly intervals (Table per collection period. The examination was 1) until May 15, 2007. Sample size was limited continued for two more months, until October to 10 fish because of the high value of yellowtail 30, 2007, but the sample size was reduced to 10 and the destructive nature of examination. Fish fish per sampling, with the aim of collecting were collected randomly by hand net, separately additional infection data for P. seriolae. placed in a plastic bag and transported on ice to the laboratory of the Faculty of Fisheries, Daily records of seawater temperature were Kagoshima University. Larger fish (>200 g) were supplied by the ACFC, measured by wireless immediately spiked through the head before system every hour for 24 hours at 1.0 meter placing them separately in plastic bags. The depth on the cage site. The daily 24-hour read- total length (TL) and body weight (BW) of the ings were pooled to calculate the monthly mean fish were recorded and the body surface, nasal, water temperature. Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 60

Identification of Copepods 30% and 10% in February and March 2007. By Caligus spinosus were categorized as chalimus, the end of observation period in May 2007, pre-adult (males and females combined), adult prevalence had started to increase again. female with or without egg sacs, and adult male based on Izawa (1969). Parabrachiella seriolae Although the overall intensity of C. spinosus on were identified as immature females, and adult the gill rakers of yellowtail was low with large females with or without egg sacs, and with or SDs indicating an aggregated distribution, the without an adult male following Yamaguti and highest median intensity at 7.3 in August 2006 Yamasu (1960) and Ho and Do (1984). was strongly and positively correlated with water temperature (r=0.717, p=0.009). There Results was no correlation between mean and median Seawater temperature intensity of C. spinosus on the body surface, and During the June 2006 to May 2007 period, the mean intensity on the gill rakers of yellowtail seawater temperature was significantly highest and water temperature. in August and September, 2006 at 26.5°C (± 0.8) and 26.1 (± 0.4), and significantly lowest in Feb- In the May-August 2007 period, no caligids were ruary 2007 at 14.6°C (± 0.2) (Table 1). In May to detected when yellowtails were newly stocked. October 2007 period, the water temperature was A similar seasonal paern was observed in the significantly highest in August and September, monthly prevalence of C. spinosus on the gill 2007 at 26.2°C (± 1.1) and 27.6°C (± 0.3). rakers of yellowtail at 100% in August with high and positive correlation with temperature Seasonal occurrence and maturation of C. (r=0.950; p=0.05) but not in the mean and median spinosus intensity of infection. Contrary to the previous Table 1 shows that no caligid was detected in observation, C. spinosus was not detected on the June 2006, when the fish were newly stocked body surface of yellowtail. into the fish cage. Prevalence of C. spinosus on the body surface of yellowtail increased to 60% Life stages of C. spinosus found on the body one month aVer stocking in July 2006, then surface consisted of pre-adult males and females, declined to 10% in August 2006, and was not and mating pairs (an adult male guarding a pre- observed thereaVer, except in April 2007 at 10%. adult female). Individuals of C. spinosus found Pearson correlation showed that prevalence of on the gills were chalimus, pre-adults, adult C. spinosus on the body surface of yellowtail was females with or without egg sacs, and adult not significantly correlated with water tempera- males. The majority of the C. spinosus during ture. On the other hand, a distinctive seasonal the entire study period were adult females (45.2- paern in the prevalence of C. spinosus on the 100%), either with or without egg sacs, which gill rakers was seen, indicating a strong and were always found aached to the gill rakers positive correlation with water temperature of the first gill. Chalimus larvae aached to (r=0.734; p=0.007). Prevalence increased to 70% the gill filaments comprised <10% of the total one month aVer stocking, peaking at 100% in C. spinosus population and were detected in August, then started to taper off declining to September 2006 only and in July and August Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 61

Mean Intensity C. spinosus C. seriolae Parabrachiella Parabrachiella (%) Prevalence d Scheffe post hoc multiple comparison Median 00 0 0 00 0 0 0 0 10 1 Intensity ) in June 2006 2007 to May a net at cage in 0 0.6 0.6 ± 0 0 0 0 0 ± ± 0 0 0 10 2 ± ± ± 0.5 0 0.5 0 0 26 1.0 1.0 1.3 1.3 1.2 SD Seriola quinqueradiata ± 0 Caligus spinosus 4 Mean Intensity ± significantly lowest in 2007 ( Feb. b c on yellowtail ( yellowtail on median intensity (Pearson of correlation p = 0.009) coefficient, r = 0.717, d

0 90 00300 3.4 ± 1.8 0 3.5 0 0 10 100 6.0 ± 0 12.6 ± 11.9 0 7.3 0 0 Prevalence (%) body s gills body s gills body s gills a a b ) ° Parabrachiella seriolae Parabrachiella SD (C and and ± 26.1 ± 0.4 14.6 ± 0.2 Seawater** Temperature Temperature SD ± (g) Caligus spinosus Caligus BW BW SD ± (cm) TL TL Seriola quinqueradiata* Monthly occurrence of date 5-Feb-07 44.6 ± 1.4 1,097 ± 160 27-Jul-06 18.6 ± 0.9 60.6 ± 8 24.4 ± 1.1 60 90 2.2 ± 0.8 2.7 ± 1.4 1.5 1.5 0 0 11-Jan-07 43.3 ± 1.5 1,053 ± 130 15.3 ± 0.6 0 50 0 15-Jun-06 7.5 ± 0.5 9.7 ± 0.9 21.0 ± 0.8 0 0 0 0 0 0 0 0 31-Oct-06 40.2 ± 1.5 743.5 ± 87 24.7 ± 0.5 0 50 0 2.2 ± 0.8 0 1.0 0 0 21-Sep-06 33.0 ± 2.3 412.3 ± 38 23-Dec-06 42.8 ± 1.9 1,103 ± 166 17.9 ± 0.9 0 30 0 17-Apr-07 43.7 ± 2.1 1,109 ± 210 17.1 ± 0.2 10 10 Sampling 16-Mar-07 44.1 ± 0.9 1,053 ± 104 15.4 ± 1.1 0 10 0 Seawater temperature were significantly highest and in Sept. Aug. 2006; and 24-Aug-06 27.5 ± 0.8 204.6 ± 28 26.5 ± 0.8 30-Nov-06 41.6 ± 1.1 900.1 ± 62 21.9 ± 1.3 0 60 0 1.7 ± 0.8 0 1.0 0 0 Prevalence (Pearson correlation coefficient, r = 0.734, p = 0.007); and 15-May-07 48.5 ± 2.6 1,273 ± 122 18.7 ± 0.8 0 30 0 infection on the gill rakers were strongly and positively correlated with seawater temperature. test, p=0.05). test, *mean of 10 fish.*mean of 10 **mean monthly seawater temperature taken meter water 1.0 at depth. TL, standard total body weight; SD, length; deviation; BW, body body surface s., a c Nagashima, Kagoshima. Table 1. Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 62

2007. Also, the highest percentage of adult C. This indicates that juveniles caught in the wild spinosus was observed when chalimus larvae and stocked for farming in net cages were not were not detected. infected. Our result is in contrast with that of Ho et al. (2001), who found wild “mojako” The size of yellowtail showed a significant posi- carrying adult and larval stages of C. spinosus. tive linear correlation with intensity of C. spino- This could imply that occurrence of parasitic sus infection although this is weak as shown copepods on wild juveniles varies among years. by r=0.155, r2=0.024 with p-value=0.005 for the As mentioned, the inspected cage was in a dense length and r=0.161, r2=0.026 with p-value=0.008 farming area, thus the absence of copepods on for the weight. the “mojako” shortly aVer stocking in cages and the rapid increase in the prevalence of C. Seasonal occurrence of P. seriolae spinosus on the gills of yellowtail one month Parabrachiella seriolae was not detected on the aVer stocking indicates that the initial source of “mojako” that were newly stocked in June 2006, copepod infection is external, most likely from nor in the subsequent monthly examinations infected older yellowtail reared in the area. until it was detected in the last two months of the study at the base of the pectoral fin of one Although adult males guarding pre-adult fish each, with an intensity of 1.0-2.0 (Table 1). females of C. spinosus were observed, not even Similarly, P. seriolae was not detected on newly- a single adult female was detected on the body stocked “mojako” in May 2007. AVerwards, surface. No previous study has reported the prevalence increased to 20-26% in August-Sep- body surface as an aachment site for C. spino- tember 2007 and decreased to 10% by the end of sus; the gill rakers (Sheppard, 2004; Fujita et the study in October 2007. Mean intensity was al., 1968) and gill arches (Hutson et al., 2007) low at 1.0 (± 0) to 1.2 (± 0.4). There was a high are clearly the preferred aachment site. Also, and positive correlation between prevalence and the presence of cleaner fish in the area such as water temperature with r=0.832 at p=0.040 but wrasse feeding on sea lice aached to the body no correlation between intensity of infection and surface of infected fish cannot be discounted as temperature. The majority of P. seriolae during reported by Treasurer (1993) on Salmo salar. the whole study period were adult females with egg sacs (78.2%), either with (33%) or without Statistical analyses showed that prevalence and (67%) a dwarf male on the posterior end of the median intensity of infection of C. spinosus on female’s trunk. cultured yellowtail follow a seasonal paern. A similar increase in prevalence during summer Discussion and decline during winter has been observed Caligus spinosus and P. seriolae were not de- in C. elongatus on farmed Atlantic salmon Salmo tected on the “mojako” when they were initially salar in Scotland (Revie et al., 2002), and C. chi- stocked in the net cages. This may be due to the astos on farmed southern bluefin tuna Thunnus small sample size but even when the fish sample maccoyii in South Australia (Hayward et al., size was increased to fiVy neither copepod 2008) as well as in Lepeophtheirus salmonis on species could be detected on the “mojako.” wild sea trout S. trua (Schram et al., 1998). Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 63

This seasonal paern may indicate the growth Adults of C. spinosus were found on the gill of the population during periods of warmer tem- rakers of the first gill arch of yellowtail. This peratures and reduced growth and reproductive result is consistent with the findings of Shep- rates of the parasite in winter (Boxaspen, 2006). pard (2004) and Fujita et al. (1968). Chalimus Heuch et al. (2002) aributed this decline to both larvae were found aached to the gill filaments, low temperature and salinity during winter. suggesting that larval aachment occurs in this region and copepods later move to the gill A weak but significant positive linear correlation rakers for their final aachment. was seen in fish size and intensity of C. spinosus infection. Although intensity of ectoparasite Although the overall prevalence of P. seriolae was infection generally increases with an increase low, there was a high and positive correlation in host size because of the larger surface area with seawater temperature. However, there was available for aachment or longer exposure of no correlation between intensity of infection the host to the parasite (Jaworski and Holm, and temperature. Previous reports of P. seriolae 1992; Saksida et al., 2007), intensity can also de- infection noted aachment at the base of the crease with size, possibly due to host or parasite pectoral fin of S. quinqueradiata (Yamaguti and responses (Hogans and Trudeau, 1989). Yamasu, 1960; Ho and Do, 1984; Ono, 1984), as in this study. This indicates the site preference The majority of C. spinosus were adults, mostly of the species. Most of the recovered individu- ovigerous and non-ovigerous females. The pres- als were adult females with egg sacs and no ence of mobile stages of C. spinosus one month larval stages were observed. Recently, Hutson post-stocking further suggests that the fish et al. (2007) recorded this parasite on the buccal were infected soon aVer stocking and that the folds of wild-caught S. lalandi from eastern and parasites could have come from infected older southern Australia. yellowtail reared at nearby farms. Furthermore, chalimus larvae never exceeded 10% of the total Lernaeopodid copepods of the genus Parabra- sea lice population on the yellowtail examined. chiella are highly host specific and are exten- Similarly in S. trua, the highest abundance of sively adapted to parasitism (Piasecki et al., pre-adult and adult L. salmonis coincided with 2010). There is no record of this parasite causing the lowest prevalence of chalimus larvae (Tully disease in farmed fish in Japan or elsewhere et al., 1993). Schram et al. (1998) reported that the (Ono, 1984; Johnson et al., 2004). proportion of chalimus larvae never exceeded 15% of the total L. salmonis population. The In the present study, no gross pathological authors concluded that the absence of chalimus changes or mortality was associated with either larvae on the fish is an indication of very low C. spinosus or P. seriolae infections. Generally, sea transmission rates. This paern is consistent lice infections have been reported to affect the with some other host-parasite systems in which growth, fecundity, and survival of fish hosts juvenile stages in a parasite population are scarce (Boxaspen, 2006) and Fujita et al. (1968) reported except at certain seasons of the year (Pike and large scale mortality of yellowtail due to C. Wadsworth, 1999). spinosus infection. It is possible that infection Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 64

South Australia. Diseases of Aquatic Organisms may combine with pre-disposing factors to 79, 57-63. cause such effects, and further investigations Heuch PA, Knutsen JA, Knutsen H and Schram into the epidemiology of these parasites are T (2002). Salinity and temperature effects on warranted. sea lice over-wintering on sea trout (Salmo trua) in coastal areas of the Skagerrak. Acknowledgements Journal of the Marine Biological Association of the United Kingdom 82, 887-892. This study was funded by the Asia CORE Program of the Japan Society for the Promo- Ho JS and Do TT (1984). Three species of Lernaeopodidae (Copepoda) parasitic tion of Science (JSPS) to the Faculty of Fish- on fishes of Japan, with proposition of a eries, Kagoshima University, and partly by a new genus and discussion of Charopinopsis Grant-in-Aid for Scientific Research (B) (No. Yamaguti, 1963. Publications of the Seto Marine 18380116) from JSPS to one of the authors (KN). Biological Laboratory 29, 333-358. The assistance of Mr. Hitoshi Matsuo of the Fish Ho JS, Nagasawa K, Kim IH and Ogawa K (2001). Disease Center, ACFC, Nagashima, in facilitat- Occurrence of Caligus lalandei Barnard, 1948 (Copepoda: Siphonostomatoida) on ing the collection of yellowtail samples and Prof. amberjacks (Seriola spp.) in the western North Vicente Balinas of Division of Physical Sciences Pacific. Zoological Science 18, 423-431. and Mathematics, UP Visayas for the statistical Hogans WE and Trudeau DJ (1989). Caligus analyses are acknowledged. elongatus (Copepoda: Caligoida) from Atlantic salmon (Salmo salar) cultured in References marine waters of the lower Bay of Fundy. Boxaspen K (2006). A review of the biology and Canadian Journal of Zoology 67, 1080-1082. genetics of sea lice. ICES Journal of Marine Hutson KS, Ernst I, Mooney AJ and Whiington Science 63, 1304-1316. ID (2007). Metazoan parasite assemblages Bush AO, Lafferty KD, Lotz JM and Shostak of wild Seriola lalandi (Carangidae) from AW (1997). Parasitology meets ecology on its eastern and southern Australia. Parasitology own terms: Margolis et al. revisited. Journal International 56, 95-105. of Parasitology 83, 575-583. Izawa K (1969). Life history of Caligus spinosus FAO (2005-2010). Cultured Aquatic Species Yamaguti, 1939 obtained from cultured Information Programme. Seriola yellowtail, Seriola quinqueradiata T. & S. quinqueradiata. In: “FAO Fisheries and (Crustacea: Caligoida). Report of the Faculty Aquaculture Department” (online) (Text of Fisheries, Prefectural University of Mie 6, by PT Dhirendra). Rome. Updated 16 127-157. Feb. 2007 (cited 12 July 2010) (hp:www. Jaworski A and Holm JC (1992). Distribution fao.org/fishery/cultured species/Seriola_ and structure of the population of sea lice quinqueradiata/en). Lepeophtheirus salmonis Krøyer, on Atlantic Fujita S, Yoda M and Ugajin I (1968). Control of salmon Salmo salar L. under typical rearing an ectoparasitic copepod, Caligus spinosus conditions. Aquaculture Fish Management Yamaguti, on the cultured adult yellowtail. 23, 577-589. Fish Pathology 2, 122-127. (in Japanese) Johnson SC, Treasurer JW, Bravo S, Nagasawa K Hayward CJ, Aiken HM and Nowak BF (2008). and Kabata Z (2004). A review of the impact An epizootic of Caligus chiastos on farmed of parasitic copepods on marine aquaculture. southern bluefin tuna Thunnus maccoyii off Zoological Studies 43, 229-243. Bull. Eur. Ass. Fish Pathol., 31(2) 2011, 65

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