Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 55 The morphology and life cycle of the Gill Monogenean ( lantauensis) on Orange-Spoed ( coioides) cultured in the Philippines

G. Erazo-Pagador1* and E. R. Cruz-Lacierda2

1 Southeast Asian Fisheries Development Center, Aquaculture Department Tigbauan, Iloilo, 5021 Philippines; 2 Faculty of Fisheries, Kagoshima University, Shimoarata, 4-50-20 Kagoshima City, 890- 0056, Japan

Abstract The morphology of Pseudorhabdosynochus lantauensis is described. It is the most numerous parasite recovered from the gills of cultured orange-spoed grouper Epinephelus coioides, in the Philippines and is smaller compared with similar reported in Hong Kong, Malaysia and Indonesia. This species is characterized by having an overlapping dorsal bar and short copulatory organ.

The life cycle of the gill monogenean is also described. One mature parasite lays at least 10–22 /day. Eggs are oval (0.021– 0.120 mm) with a spiral filament aached to one end. Eggs hatch into free-swimming larvae (oncomiracidia) within 2-6 d at 300C and 30 ppt. The rate of hatching of various eggs is highly variable, that is, some eggs hatch in 2 d while others take 6 d. The oncomiracidium can aach to the grouper within 8 h. Upon aachment, the oncomiracidium metamorphoses to an adult parasite in 4-7 d. The parasite becomes fully mature and delivers eggs in 7 d. Without a host, the life span of oncomiracidium is only 4-8 h. The life cycle is complete in 13-20 days (eggs to oncomiracidium 2–6 d; oncomiracidium to adult parasite 4-7 d; adult to fully mature and delivery parasite 7 d). This is the first report on the morphology and life cycle of P. lantauensis from the Philippines.

Introduction The orange-spotted grouper, Epinephelus severe mortalities have been encountered coioides, is one of the most economically due to parasitic infections. The diplectanid important marine fish cultured in Southeast monogenean Pseudorhabdosynochus lantauensis Asia. It is a high value marine food fish and is common occurring at 100% prevalence. The commonly cultured intensively in floating net site of aachment is mostly the gill filaments cages (Leong, 1997). near the gill arch area (Cruz-Lacierda, pers. comm.). In the Philippines, E. coioides are cultured in earthen ponds and floating cages. With Diplectanid monogeneans have also been the intensification of aquaculture industry, reported from serranid fish, especially

* Corresponding author’s e-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 56

Epinephelus spp. Leong and Wong (1988, were mounted in ammonium picrate to study 1995) reported P. epinepheli as the most the reproductive organs and other sclerotized abundant parasite found in cultured and parts like , hamuli, haptoral bars wild greasy grouper (E. malabaricus) in and marginal hooklets. Malaysia. Pseudorhabdosynochus lantauensis, P. coioides and Diplectanum grouperi were also Measurements were made in micrometers found parasitizing several species of grouper (μm); the mean is followed by the range within from Malaysia, Hong Kong and Indonesia parentheses. Drawings were made with the (Beverley-Burton and Suriano, 1981; Hla Bu aid of the Image Analysis System (Image- et al., 1999). Pro Plus Imaging SoVware v. 3.01). Based on published description, morphology of the This paper describes the morphology and present specimens was compared with that life cycle of the monogenean P. lantauensis on of Hla Bu et al. (1999) and Beverly-Burton and the gills of E. coioides. This is the first report Suriano (1981). Voucher specimens have been on the morphology of P. lantauensis from the deposited in the National Science Museum, Philippines. Tokyo, Japan (NSMT-Pl 5414, 5415, 5416).

Materials and methods Life cycle Morphology The life cycle of P. lantauensis on grouper The monogeneans were collected from was studied. Adult parasites were collected (n=250; 3-150 g in body weight) from from the gills of live grouper. Eggs laid by a floating cage in Roxas City, west central the parasite were incubated in a water bath Philippines from April 1999 to December 2000. at 300C with 30 ppt and were observed every Fish were brought alive to Southeast Asian 4 h until eggs were produced. If the parasites Fisheries Development Center Aquaculture did not produce eggs within 3 consecutive Department (SEAFDEC AQD) laboratory days, the experiment was terminated. Egg and examined within 24 h of collection. The development was observed at 12 h intervals total length (TL, mm) and body weight (BW, using a stereomicroscope to monitor g) of each fish were recorded. The fish were embryonic development until the larval or the sacrificed and gills were removed and placed oncomiracidial stage. separately in Petri dishes. The monogeneans were scraped from the gills and pipeed One hundred grouper juveniles (BW=2-8 g, to a slide. Freshly collected parasites were TL=58-82 mm) were used for experimental flaened between a slide glass and a cover infection studies. They were hatchery-reared slip, fixed in alcohol-formalin-acetic acid and uninfected, and held in two units of 250 L (AFA), stained with either alum carmine or aquaria (salinity=30-32 ppt; temperature=28- Semichone’s acetocarmine, dehydrated in 330C). Stocking density was 50 fish per a graded series of ethanol, cleared in xylol aquarium. The juveniles were fed with trash and mounted in Eukit (mounting medium). fish daily. Alternatively, fresh monogeneans specimens Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 57

Experimental infection of fish was carried out outer curve of blade, inner length 28 (25-30), aVer a 2-week acclimatization period. Prior to with equally well developed superficial and the experiment, the naïve grouper were treated deep roots, both pairs of hamuli with sharply with 100 ppm formalin for 30 min to ensure recurved hooks at tip. Dorsal bar (Figure 1e) 64 that they were free of parasites. About 80 (50-70) long with rounded lateral extremities newly hatched oncomiracidia were introduced and inflated rounded medial extremity; into the aquarium. Three fish were sampled at ventral bar (Figure 1d) 79 (70-90) long with a 1, 2, 4, 8 and 24 h and at 3, 7 and 14 d post- transverse groove; 14 marginal hooks (Figure infection (p.i.) to determine its host-parasite 1f), 10 long. Squamodiscs (Figure 1g) size 36 relationship. Newly-hatched oncomiracidia (30–40) by 34 (30–40) with 10 rows of scales were also kept in a separate aquarium to all of which with open loops, form a narrow study the growth and development of the horse-shoe shape, anteriorly and posteriorly monogeneans without a host. almost horizontal.

Results Testis oval 39 (30-40) long by 33 (30–40) Morphology wide, in posterior half of body; vas deferens Pseudorhabdosynochus lantauensis (Figure 1a). arising anteriorly and passing directly to form seminal vesicle; muscular ejaculatory duct Description (based on 10 specimens) Total opening into the posterior region of the penis length 463 (150-620), greatest body width bulb with prostatic reservoir. Male copulatory at -testis region, 107 (90-180), tapering organ (Figure 1h) sclerotized, proximal region anteriorly to a flat head region. Tegument reniform, 34 (30–40) long and divided by with anteriorly directed scales in posterior transverse partitions into four compartments; body region; scales are easily lost in preserved distal region consisting of a curved tube, 25 specimens. Two pairs of eyespots, anterior (20–30) long. pair smaller and wider apart, posterior pair well-developed and more distinct. Mouth Ovary oval shaped, 48 (40–50) long by 34 (30– ventral leading directly to . Pharynx 50) wide, located anterior to testis, distal region 30 (30) long by 27 (20-30) wide. curved dorsoventrally around right intestinal caecum. (Figure 1i) sclerotized, an with typical diplectanid, sclerotized elongated funnel shape, opening ventrally to components, 2 pairs of hamuli; 3 transverse leV of genital pore canal with 2 distal loops. bars and 2 squamodiscs (1 ventral and 1 dorsal). For dorsal hamuli (Figure 1b), outer length 37 Life cycle (37-43) long from tip of outer root to curve of The egg of P. lantauensis is an oval-like shape, blade, inner length 18 (15-30) from tip of inner but elongated with a long spiral filament root to curve of blade. The modified inner and aached to one end (Figure 2b). It measures outer roots merged into a broad thickened 0.021-0.120 mm from a sample of 30 eggs. The base. For ventral hamuli (Figure 1c), outer eggs have adhesive filamentous extensions length 37 (30-40) long from tip of deep root to on either end which help in aachment to Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 58

Figure 1. Pseudorhabdosynochus lantauensis. a. Entire ventral view b. Dorsal hamuli c. ventral hamulus d. Ventral bar e. Dorsal bar f. Marginal hooklet g. h. Copulatory organ i. Vagina Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 59

Figure 2. The life cycle of P. lantauensis parasitizing the gills of E. coioides.

(a) mature fluke on gill lamellae (b) egg (c) free-swimming oncomiracidium (d) migrating post-oncomiracidium Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 60 substrates. The rate of egg production was to harbor parasites in their gills with 10-22 eggs/worm/d at 300C. On the first day and squamodisc present. The parasites were of incubation, dense granules were observed still immature and were transparent when inside the egg with a clear area formed at observed under the microscope since the one end. Two black eyespots and developing reproductive system had not yet developed. marginal hooks were observed on the The immature parasites metamorphosed to developing embryo aVer 2 d of incubation. mature adult (Figure 2a) in 4-7 d. The mature There was a twitching or wriggling movement adult parasites were filled with granular of the embryo from one end to another in vitellaria; male reproductive system was fully the egg capsules until the head of the developed while the female reproductive popped out of the egg’s opercular opening. system was not. They become fully mature The operculum generally breaks a day before and gravid in 7 d. The adult parasites were hatching took place. identified as P. lantauensis, having the same characteristics as described above. Eggs kept at water temperature of 300C in a water bath hatched within 2-6 d. When Discussion hatched, the larva (oncomiracidium) (Figure This is the first report on the morphology 2c) was ciliated anteriorly, centrally and of P. lantauensis from the Philippines. The posteriorly. It has two pairs of well developed morphology of the specimens in the present eyespots. The pharynx was posterior to the study was compared with descriptions from eyes. The eyespots help in their orientation other papers (Beverly-Burton and Suriano, and in locating the host. AVer hatching, 1981; Hla Bu et al., 1999). oncomiracidia were extremely active and were observed to swim in random fashion, spiraling Our specimens all possessed an overlapping and proceeding in straight lines for short dorsal bar which was similar to that described periods as observed under a stereomicroscope by Hla Bu et al. (1999). The specimens of P. with transmied light. The oncomiracidium lantaeunsis described by Beverly-Burton and was observed to swim continuously for up to Suriano (1981) was found to have longer 4-8 h. The larva dies if it fails to find a host copulatory organ, ventral bar, dorsal bar, within that time. squamodisc and outer lengths of ventral hamuli and dorsal hamuli than the specimens Grouper observed at 1, 2 and 4 h p.i. had no in the present study (Table 1). These differences parasitic larvae on the skin, mouth or gills. may be due to the geographical location of the At 8 and 24 h p. i. five fish harbored larvae fish hosts and the methods used in measuring aached to the skin (post-oncomiracidium) fresh and fixed specimens (Hla Bu et al., but no larvae were observed in the mouth 1999). and gills. The larvae aached to their host by means of the haptor and promptly lose their Knowledge of parasite life cycle is important cilia; however, the squamodisc is still absent in determining prophylactic measure. The at this period. At 3 d p.i. fish were observed egg-laying rates of monogenean are possibly Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 61

Table 1. Comparative measurements of Pseudorhabdosynochus lantauensis from the gills of Epinephelus spp.

Beverley-Burton Present study and Suriano, 1981 Total body (L x W) 463(150-620) x 107(90-180) 550(460-680) x 120(100-130) Haptor (L x W) 46(40-60) x 159(150-180) 60(50-70) x 201(175-225) Copulatory organ (L) (proximal) 34(30-40) 45(40-49) Copulatory organ (L) (distal) 25(20-30) 39(34-48) Testis (L x W) 39(30-40) x 33(30-40) - Ovary (L x W) 48(40-50) x 34(30-50) - Pharynx (L x W) 30(30) x 27(20-30) - Vagina (L) 29(20-40) - Ventral hamuli Inner length 28(25-30) - Outer length 35(30-40) 36(33-43) Dorsal hamuli Inner length 18(15-30 - Outer length 35(30-40) 36(35-41) Ventral bar (L) 79(70-90) 84(74-96) Dorsal bar (L) 56(50-70) 57(51-61) Squamodisc (L x W) 36(30-40) x 34(30-40) 37(30-42) Squamodisc rows 10 9-12 Marginal hooklet (L) 10 10(9-12) No. of specimens measured 10 10 Host E. coioides E. brunneus, E. fario Locality Philippines South Sea, Hongkong

Measurements in μm; range in parenthesis; L, length; W, width

temperature-dependent, however there were observed to be 10-22 eggs/parasite/day at only a few studies on this. Kearn (1985) 300C. Tinsley (1983) however noted that most recorded that Entobdella solea lays 40-60 eggs/ monogeneans deposit less than 100 eggs/ parasite/day at 200C. The rate of Benedenia parasites/day, depending on temperature and seriolae (Kearn et al., 1992 a) and Neobenedenia age of the parasite. girellae (Bondad-Reantaso et al., 1995) were 648 at 200C and 293-850 at 27-300C eggs/ Egg hatching is also temperature-controlled parasite/ day, respectively. Tsutsumi et al. phenomenon among monogeneans. Eggs of (2002) reported that Neoheterobothrium hirame P. lantauensis hatched into free-swimming produced 651 eggs/day at 200C. In the present larvae (oncomiracidium) within 2 to 6 d at study, the egg-laying rate of P. lantauensis was 300C temperature and 30 ppt salinity. The Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 62 embryonic development of P. lantauensis It has been postulated that the larvae of is similar to that of other dactylogyrid diplectanid and dactylogyrid gill parasites monogeneans (Paperna, 1963; Prost, 1963). aach initially to the skin surfaces of their host Kearn et al. (1992 b) reported that the eggs and may then migrate to their final aachment of Heteraxine heterocerca hatch at about 5 d site such as the gills. In the present study, it at 230C. Similar observation was made for B. was observed that the oncomiracidium of P. seriolae (Kearn et al.,1992 a). Other workers lantauensis first aach themselves to the skin to have reported longer periods of embryonic develop as post-oncomiracidium observed at development at lower temperatures (Kearn, 8 hr post infection then migrate to the gills and 1968; MacDonald, 1977; Cone 1979). developed into adult parasites. This invasion route of parasite was observed in Diplectanum There are different mechanisms of egg hatching aequans (Kearn, 1968) and in Urocleidus in monogenean parasites. nobilis adspectus (Cone and Burt, 1981). However, the would not hatch without the presence of host adult monogenean Heterobothrium okamotoi mucus (Shaharom-Harrison, 1986) and so does developed first on the gills and then becomes Acanthocotyle lobianchi (MacDonald, 1974). mature on the branchial cavity lining of tiger However, the eggs of P. lantauensis developed puffer, Takifugu rubripes (Ogawa and Inouye, and hatched without host stimulant as shown 1977). In addition, Kearn (1967) reported that in this study. the oncomiracidium of E. soleae first made contact with their host’s skin due to the In P. lantauensis, the ciliary action of the larva substances secreted by the fish’s epidermis. plays a part in dislodging the operculum. This was also observed in many other monogeneans Following attachment to the host, the (Prost, 1963; Kearn, 1974). The oncomiracidium oncomiracidium of P. lantauensis developed of P. lantauensis swims freely in straight lines to mature parasites in 14 d at 300C. Imada and in the water. The direction of swimming is Muroga (1978) reported that oncomiracidia of great importance in finding hosts. Paling of Pseudodactylogyrus microrchis developed to (1969) observed two behavioral phases of mature parasites in 6-7 d at 280C. oncomiracidia on Discocotyle saggitata. The first phase is the free-swimming phase where In this study, it was observed that in the absence the larvae move in straight lines in search of a of a host, the life span of the oncomiracidium host and show positive phototaxis. The second is only 4–8 h without undergoing further phase starts when the larvae lose response to development. The oncomiracidium of the light and move randomly to aach itself to a skin parasite, E. soleae lives for about 24 h host. This was also observed in the present (Kearn, 1967). Prost (1963) reported that D. study. The oncomiracidia may also have a anchoratus larvae die within about 10 d if they non-infective stage and this may be the reason do not find a host to parasitize. The survival why no larvae were found on fish examined 1 of the oncomiracidium is dependent on the h following infection. larvae finding and aaching themselves to their specific fish hosts and that selection Bull. Eur. Ass. Fish Pathol., 30(2) 2010, 63

(: ) and description pressure is exerted on the oncomiracidia of C. hongkongeneses, sp. and C. lantauensis during the timing of hatching and on their n. sp. from Epinephelus spp. () in behaviour during their short free-swimming South China Sea. Canadian Journal of Zoology lives (Llewellyn, 1984). 59, 1276-1285. Bondad-Reantaso MG, Ogawa K, Fukudome Completion of the life cycle from hatching M. and Wakabayashi H (1995). Reproduction to egg-laying adult took 13-20 d at 300C. and growth of Neobenedenia girellae According to Bondad-Reantaso et al. (1995), (Monogenea: ), a skin parasite of cultured marine fishes of Japan. Fish Pathology 0 the life cycle of N. girellae is 15-17 d at 25 C. 30, 227-231. Benedenia seriolae reaches sexual maturity on day 14 at 220C (Kearn et al., 1992 a). Paperna Cone DK (1979). Hatching of Urocleidus (1963) reported that D. vastator takes 11-13 d adspectus Mueller, 1936 (Monogenea: Ancyrocephalinae). Canadian Journal of Zoology 0 to complete its life cycle at 22 C. 57, 1896-1904.

Monogeneans display a high degree of host Cone DK and Burt MDB (1981). The and aachment site specificity. This was invasion route of the gill parasite Urocleidus adspectus Mueller, 1936 (Monogenean : confirmed in the present study where only Ancyocephalinae). Canadian Journal of Zoology one host is required to complete an entire 59, 2166-2171. life cycle which occurred in the gills of the host. Moreover, it was also shown that the Hla Bu SS, Leong TS, Wong SY, Woo YSN and Foo RWT (1999). Three diplectanid infection level of this monogenean was very monogeneans from cultured grouper, high in grouper (Cruz-Lacierda, pers.comm) Epinephelus spp. Journal of Helminthology 73, studies on the aspects of parasite ecology, 301-312. pathology and control measures are therefore Imada R and Muroga K (978). warranted. Pseudodactylogyrus microrchis (Monogenea) on the gills of cultured eels – II. Oviposition, Acknowledgments hatching and development on the host. Bulletin The authors express their gratitude to of the Japanese Society of Scientific Fisheries 44, 571-576. Francisco Gernade, Susan Torrento and Fely Torreta for technical assistance, Prof. Kazuya Kearn GC (1967). Experiments on host-finding Nagasawa for technical guidance; and Dr. and host-specificity in the monogenean skin Elena Catap and Ms. Milagros Castaños for parasite Entobdella soleae. Parasitology 57, 585- 605. helpful criticism and editorial comments. This study was funded by the Government Kearn GC (1968). The development of of Japan, Trust Fund under the Regional Fish the adhesive organs of some diplectanid, Disease Project. tetraonchid and dactylogyrid gill parasite (Monogenea). Parasitology 58, 149-163.

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