Diseases of Opakapaka

Hawai'i Institute of Marine Biology

Introduction

Opakapaka (Pristipomoides filamentosus), a highly valued commercial deep-water snapper is under investigation at the University of Hawaii through support of Department of Land and Natural Resources. The purpose of this endeavor is to develop aquaculture methods for enhancement of wild stocks of this fish and related species (e.g., ehu, onaga) as well as development of this species of commercial aquaculture.

 The usual scenario for investigations or identification of serious diseases in aquaculture is that as the culture of a fish species expands, devastating economic and biological losses due to "new" or "unknown" diseases follow, and then research is conducted to identify their cause and develop methods for their control. In contrast, the purpose of this study was to provide a proactive approach to identify potential health problems that may be encountered in opakapaka before large-scale culture of this species is underway.

A few diseases have already been recognized as potential problems for the culture of captive opakapaka. Before this study, we observed infections by Cryptocaryon irritans, a common parasitic ciliate that is well recognized as a serious pathogen in warm water mariculture. An idiopathic eye disease presenting as gas emboli around the globe has also been a common problem in captive opakapaka at this facility. Certainly these represent only two of many other health problems that will be encountered as successful breeding and rearing of this species is achieved, and subsequent captive production is increased. Therefore, Hawaii Sea Grant, through their Program Development, supported this investigation to more thoroughly elucidate present and potential disease problems in opakapaka held in captivity.

Materials and Methods

A total of 143 moribund and healthy opakapaka were examined in July-August 2001. Fish represented those held in seawater netpens, and flow through circular aquaria kept at the Hawai'i Institute of Marine Biology at Coconut Island, Oahu. All fish had been previously captured by hook and line, and were held in captivity for up to about 45 days. Wild caught fish were included in the study, which examined immediately after capture or after being held in captivity for a maximum of 3 days.

Fish were euthanized with an overdose of MS-222, immediately bled from the caudal vein, and blood smears prepared. Wet mount preparations of the gills and skin scrapings were examined by light microscopy. Then the fish were dissected and examined. Spleen and kidney imprints and blood smears were prepared, and were later stained with Dif Quick. Representative organs - eyes, gills, skin with muscle, heart, liver, kidney, spleen, and intestine were preserved in Davidson's solutions. Histological sections were prepared and stained with hematoxylin and eosin at Oregon State University's Veterinary Diagnostic Laboratory, and Drs. Kent and Heidel examined the slides. Routine virology screening was conducted on frozen spleen and kidney using EPC and CHSE-214 cells maintained at 17 C (Ganzhorn and LaPatra 1994). Tissues were diluted 1:10 and 1:100 and blind passed after 2 wk culture.

Results and Discussion

Results from necropsies and histological evaluations are summarized in Table 1.

 Table 1. Prevalence of major diseases or infections in opakapaka held at Coconut Island. Number infected or affected/number examined (%).

Disease or Infection	Total	Tanks	Netpens	Fresh caught
Exophthalmia*	35/128(27)	10/47(21)	25/70(36)	0/11
Swim bladder inflammation*	23/128 (18)	13/47(28)	10/70(14)	0/11
Cyst of unknown origins	2/143 (1)	0/62	1/70 (1)	1/11(9)
Epitheliocystis	1/43 (1)	1/62 (2)	0/70	0/11
Goussia (surface type)	6/143 (5)	3/62(5)	3/70(4)	0/11
Cryptocaryon irritans	35/143 (24)	35/62(56)	0/70	0/11
IchthyophonusÝ	4/143(3)	3/62(5)	1/70(1)	0/11
Didymozoid digenean	31/143 (22)	9/62(14)	15/70(21)	7/11(64)
Monogenea (e.g., Diplectanum)*	11/128(9)	8/47(17)	3/70 (4)	0/11
Caligid Copepods*	34/128(27)	34/47(72)	0/70	0/11
Encysted helminthes ý	44/143(31)	21/62(34)	16/70(23)	7/11(64)

*P group not included in analysis as samples evaluated primarily by histology, and swim bladder or eyes were often absent in collection
Ý One fish exhibited intact Ichthyophonus parasites, while three had degenerative structures consistent with that organism.
ý Based on both macroscopic observations of white nodules in viscera and histology. Note that presences of macroscopic "white nodules" not always reported, and thus prevalence is probably higher.

 Swim bladder Infections and Inflammation. Several moribund fish exhibited septic swim bladders. At necropsy, the swim bladder wall was opaque, and the swim bladder often contained an opaque, viscous to caseous, whitish or yellow exudate (Fig. 1). Histologically, the lesions were characterized by severe, chronic inflammation (Fig. 2).
 

Figure 1. Swim bladder inflammation and bacterial infections. A. Intact swim bladder before dissection. Note that the swim bladder wall is opaque. B. Dissected swim bladder revealing yellow, caseous exudate (E).

Fig. 2. Low magnification of swim bladder showing severe chronic inflammation. Fig 3. Masses of bacteria and associated chronic inflammation in swim bladder.

Masses of presumptive Gram-negative bacilli were observed in most lesions, and one fish exhibited a fungal infection of the swim bladder (Fig 4). Two fish exhibited intracellular cocci in macrophages in these lesions suggestive of Piscirickettsia. Further tests are underway to verify the identity of this putative rickettsia-like infection. Piscirickettsia salmonis and related organisms have caused severe disease in aquaculture species, including pen-reared salmon, white sea bass, and tilapia (Fryer and Lannan 1996). Infections of the swim bladder were likely due to iatrogenic causes - i.e., associated with introduction of contaminated hypodermic needles during de-gassing of swim bladders immediately after capture or due to a tract from the skin surface being created by the needle, and thus allowing bacteria to infect the swim bladder. Considering the severity of the swim bladder bacterial infections, it was remarkable that this did not progress to septicemia. Based on our experiences, such progression would be expected with similar infections in other fish species. This suggests that opakapaka held in captivity have a very competent immune system, which is capable of containing severe infections.

Fig 4. Intracellular piscirickettsia-like organisms (arrows) in macrophages.

Fig 5. Fungus infection of swim bladder. A. low magnification showing granulomas (G). B. Fungal hyphae in lesion.

Figure 6. Opakapaka with bilateral exophthalmia. A. Note bulging eyes. B, C. Histological sections of eyes. B. Gas bubbles (B) within hemorrhage (H) and chronic inflammation (I) in retrobulbar vascular plexus. C. Chronic inflammation with multinucleate giant cells (arrows) and eosinophilic granule cells in eye with panophthalmitis.

Eye Lesions. Many fish exhibited eye lesions, generally characterized by exophthalmia (pop-eye), with gas bubbles in or around the eye (Fig 6). Representative samples were evaluated by histology. Eyes are very difficult to process for histology, and thus notations on eye lesions do not occur for every fish with macroscopic eye damage (Table 1). In general, the eyes exhibited gas bubbles (emphysema) and hemorrhage in the retrobulbar vascular plexus (Fig. 6). Many of these eyes subsequently developed chronic inflammatory lesions. As with the swim bladder lesions, the underlying cause is probably iatrogenic - i.e., due to gas bubble disease induced by transporting the fish to the surface after capture in deep waters. However, spontaneous exophthalmia associated with gas bubbles in the eyes was recently reported in the West Australian dhufish (Glaucosoma hebraicum) that were reared from birth in captivity as well as in wild-caught fish (Stephens et al. 2001). Moreover, we have seen an identical condition in ling cod (Ophidon elongatus) that were hatched in captivity. Stephens et al. (2001) concluded that the likely cause of the lesions in captive-reared dhufish (and some other deep water marine fishes) was a reduced ability to adapt to rapid changes in activity patterns or water quality differences, even if hatched in captivity. For opakapaka, rearing fish in surface water netpens or tanks would represent a situation with lower water pressure and warmer water temperature than their normal environment.

Figure 7. Epitheliocystis, caused by a chlamydia-like microorganism, in the gill of opakapaka. Figure 8 (right). Ichthyophonus in opakapaka. A. Inflammation and fibroplasia around intact organism (arrows). Note fungus-like appearance of parasite. B. Possible degenerated organism (arrow) in lesion.

Epitheliocystis. One fish exhibited a lesion consistent with infection by epitheliocystis, a chlamydia-like infection (Fig. 7). Epitheliocystis is common in wild marine fishes, and has occasionally caused disease in aquaculture (Lannan et al. 1999). The disease is particularly problematic in cultured bream (Sparus aurata) (cf. Paperna 1977) and sea bass (Dicentrachus labrax) (cf. Crespo et al. 2001). Ichthyophonus. One fish exhibited infection by Ichthyophonus sp. (Fig. 8). In addition, three other exhibited degenerative lesions that we suspect were resolved Ichthyophonus infections. This protistan parasite is a common in wild marine fishes and occasionally is problematic in fish culture (McVicar 1999). Previously the parasite has been considered a fungus, but molecular systematics suggest that it is related to the choanoflagellates (including Dermocystidium and the rosette agent of salmon). The life cycle is direct, and fish can contract the infection by water-borne spores, or feeding on infected fish and crustaceans. The infection is cosmopolitan, and has been reported from cold and warm water marine fishes. The lesions were minor in opakapaka, but this infection should be monitored, as it is has the potential to spread in culture situations and can be highly pathogenic.

Cryptocaryon irritans. Gill infections associated with prominent epithelial hyperplasia were observed in moribund fish held in tanks (Fig. 9). This ciliate is a well-recognized and serious pathogen of warm water marine aquaculture and aquaria (Dickerson and Dawe 1999). It is difficult to treat with routine bath chemotherapeutics as it has an off-host stage and the on-host stage (trophont) embeds under the skin and gill epithelium (Fig. 9). Moreover, the use of external baths to treat parasites in large, extensive aquaculture operations, such as netpens, is usually impractical due to costs and problems associated with environmental contamination. This ciliate already is clearly recognized as a potential cause of high mortalities in tank-held opakapaka at Coconut Island. Interestingly, infections in netpen-held opakapaka appear to be absent (Table 1), possibly due to flushing of the off-host stages of the parasite from the pens and relatively low density of fish in netpens compared to tanks. Many fish from tanks exhibited early Cryptocaryon infections in which the parasite was present but was not associated with significant pathological changes. However, moribund fish from the group held in tanks showed lesions consistent with the disease - e.g., severe, diffuse epithelial hyperplasia of the gills (Fig. 9). One of us (A. Moriwake) conducted treatment experiments (Table 2), and found that transferring fish to a new tank every 3 days for 3 cycles was effective for controlling infections.     Figure 9 (right). Gill infected with Cryptocaryon. A.Wet mount. Note that the parasite occurs under the epithelium. B. Histological section showing severe epithelial hyperplasia associated with parasites (arrows).

Four parasites were found on the gills and skin of captive opakapaka. This included a Caligus sp. and two types of trematodes on newly caught fish, and a Cryptocaryon sp. ("marine ich") occurring repeatedly in the hatchery. Several experiments were conducted to control or eliminate parasites. Three to four individuals were used in each experiment. All fish were examined for parasites prior to treatment and 3 days after treatment with the exception of the last two experiments in which fish were examined every 3 days for 3 cycles. Fish that died during the course of the experiment were examined upon death.

Table 2. Treatment Protocols to Control External Parasites on Juvenile Opakapaka (Prepared by Aaron Moriwake)
(Summary of trials conducted on opakapaka for parasite treatment)

Parasite	Treatment	Duration	Effective	Survival Day 1 (%)	Survival Day 3 (%)
Caligus	Formalin 25 ppm	60 minutes	YES	100	100
Caligus	Formalin 50 ppm	60 minutes	YES	100	100
Caligus	Formalin 75 ppm	30 minutes	YES	100	33
Caligus	Formalin 100 ppm	15 minutes	YES	100	100
Caligus	Freshwater bath	5 minutes	YES	100	100
Monogeneans	Formalin 200 ppm	15 minutes	NO	100	100
Monogeneans	Formalin 740 ppm	5 minutes	NO	67	*
Monogeneans	Formalin 740 ppm	5 minutes	NO	67	*
Monogeneans	Freshwater bath	15 minutes	NO	100	75
Monogeneans	Freshwater bath	10 minutes	NO	100	100
Cryptocaryon	H202 150 ppm	30 minutes	NO	67	0
Cryptocaryon	H202 150 ppm	15 minutes	NO	67	0
Cryptocaryon	Formalin 250 ppm	10 minutes	NO	100	80
Cryptocaryon	Freshwater bath 3 day cycle	5 minutes	YES	100	100
Cryptocaryon	Transfer 3 day cycle		YES	100	100

* Fish were sacrificed for microscopic examination.

Goussia spp. (Coccidia). We observed a coccidian species, most likely a Goussia sp., infecting the brush boarder of the intestinal epithelium. Many fish were infected, with two moribund fish showing severe infections (Fig 10). Fish with heavy infections exhibited enteritis and atrophy of the infected epithelium. The gut epithelium is a primary site of infection by coccidia, including those infecting fish (Molnár 1999). Infections at the surface of epithelial cells, as seen here, are similar to Cryptospordium infections of mammals, and this type of Goussia infections in fish may likewise be pathogenic. For example, Goussia (=Epieimeria) anguillae is a pathogen of eels (Lacey and Williams 1983), and we described chronic mortality associated with a wasting syndrome in opaleye (Girella nigricans) due to G. girellae held at the Scripps Aquarium, San Diego (Kent et al. 1988). Therefore, this infection in opakapaka should be considered as a potential problem in the future and should be monitored. A second Goussia sp. was observed in one fish (Fig 10B). In this case, the organism developed deep within the epithelium and occasionally in the lamina propria. Also in contrast to the other Goussia species from opakapaka, sporulated oocysts were frequently observed. Although a different species, the infection and pathogenic changes were suggestive of Goussia carpelli infections in carp and goldfish. In this infection, "yellow bodies" are formed around the oocysts and the parasite causes atrophy of the gut and a wasting syndrome (Kent and Hedrick 1985).   Fig. 10 (right). Coccidians in opakapaka. A. Intestinal epithelium-surface type found in many fish. B. A second species occurred deep within the epithelium and lamina propria. Arrow = three sporulated oocysts in the epithelium.

Monogenea. Gill and skin infections by monogenea worms are well-recognized problems in marine and freshwater fishes held in captivity. Two species of monogenea were observed; Diplectanum opakapaka (Fig. 11A) and Pseudodiscocotyla opakapaka (Fig. 11B). Infections by both monogeneans in the present study were light and not associated with significant gill lesions. Another Diplectanum species, D. latesi, has caused mortality in captive sea bass (Lates calcarifer) (cf. Rajendran et al. 2000). Monogeneans can usually be easily eradicated with baths containing formalin or organophosphates, but the use of these compounds in netpens or other large-scale operations may be impractical. Furthermore, we have found formalin to be ineffective for treating these infections on opakapaka (Table 2). We found the best way to control large monogeneans (presumably members of the family Capsalidae) on the skin was to pick them off individually using forceps. A treatment of 15 minutes in freshwater appear to be close to the maximum amount of time the fish can survive in freshwater.

Figure 11. Monogeneans from the gills of opakapaka. A. Diplectanum opakapaka.
B. Pseudodiscocotyla opakapaka. 0 = opisthaptor (posterior attachment organ).

Digenea. Trematodes of the family Didymozoidae are unusual digeneans in that the adult stages are extraintestinal in marine fishes (whereas most other adult digeneans occur in the gut lumen). A large (up to about 100 mm), elongate worm superficially resembling a nematode was often observed in the gill chamber, heart, or near the anterior kidney (Fig 12). We identified the worm as Metanematobothrioides opakapaka. Histological sections of the infected tissue revealed little tissue reaction. The most severe lesion was moderate, chronic epicarditis of the heart in one fish (Fig 12B). Furthermore, as these worms require an intermediate host and do not replicate in fish, they will not likely be serious problems in the culture of opakapaka.

Figure 12. Didymozoid digenean trematode Metanematobothrioides opakapaka from visceral organs. A. Whole worm dissected from tissues. B. Histological sections of worms in pericardium of heart. Arrow = inflammation surrounding worms (D). C. Cross sections of worms (D) in viscera near kidney.

Cestodes. Larval cestodes were observed in the intestinal lumen of some fish, and were not associated with severe pathological changes (Fig 13).

Figure 13. Larval cestode in intestine of opakapaka. Infections were associated with little tissue damage. G = coccidium.

Copepods. Two types of copepods were observed; a Caligus sp. (Fig 14A), which moves freely on the skin, and an attached lernaeopodid (a member of the family Lernaeopodidae) (Fig 14B). The latter was observed on a wild-caught fish. Photographs of the lernaeopodid were shown to Dr. Z. Kabata, Pacific Biological Station, Nanaimo, British Columbia (the world's foremost authority on parasitic copepods) and he stated that these specimens likely represent a new genus. Caligid copepods can be extremely problematic in netpen culture - i.e., Lepeophtheirus salmonis is one of the most important problems caused by infectious agents in the netpen farming of salmonids in Europe (Boxshall and DeFaye 1993). Dr Z. Kabata also examined the caligids, and the following is from a pers. comm. that suggests that these specimens may represent a new species. "I have examined closely your specimens of Caligus from Hawaii. The closest I can come up with is Caligus brevis Shiino, 1954 and C. oviceps Shiino, 1952(these two are distinguishable with some difficulty). However, both of them differ from your specimens in the shape of the sternal furca. This is the only significant difference I could find. Unfortunately, Shiino's descriptions lack adequate detail to make any definite differences meaningful. The hosts are quite unrelated, too. (Another record of C.brevis, from New Zealand, is incorrect, it deals with a species distinct from C.brevis). Under the circumstances there is a possibility that it is a new species. The only way to clinch it is to check the type material of brevis, which is in Japan in custody of Kunihiko Izawa." The use of both formalin (between 25 - 100 ppm) and freshwater bath were effective in eliminating Caligus (Table 2). Fish appear to be stressed (head bobbing out of the water) at the higher doses (75 and 100 ppm) of formalin. After one minute in the freshwater bath, no parasites were observed on the fish.

Figure 14. Copepods from opakapaka. A. Caligus sp., a common infection in captive fish. B. Lerneopodid copepod from wild-caught opakapaka. Arrows = egg sacs at posterior end of female copepods

Fig. 15. Encysted parasites, etc. A. Metacercaria in kidney. B. Melanized granuloma.

Granulomas and Encysted Helminthes. Encysted metacercariae of trematodes were occasionally observed in tissue sections (Fig. 15A). In addition, many granulomas contained debris, which may represent degenerated parasites were observed (Fig 15B). These represent the whitish nodules seen in the mesenteries or around the liver at necropsy. As the contents of the cysts were very degenerated (encapsulated spheres with necrotic centers), specific identifications were not possible. They likely represent the metacercariae, and will probably not be a concern in the culture of this fish. Both wild and captive fish exhibited these lesions (Table 1), and they can be considered merely normal "background" infections of wild fish. A few fish exhibited granulomas more suggestive of Mycobacterium infections (fish tuberculosis), but acid-fast stains of these lesions did not reveal bacteria. Fatty Liver Disease. Hepatic lipodosis (fatty livers) was observed in several of the fish from diet studies. Figure 16 contrasts normal with fatty livers. The long-term effects of this condition are unknown, but this change is usually reversible. It is likely that these liver changes were due to inappropriate diets. It should be noted that most of the fish with fatty livers came from diet studies.

Fig. 16. Opakapaka livers. A. fatty liver, note hepatocytes filled with clear vacuoles representing fat. B. normal liver.

Cysts of Unknown Origin. These enigmatic structures (Fig. 17), also referred to as "unidentified fish objects" or "UFO's" are rather common in marine fishes (MacLean et al. 1987). We observed these in the gills of one fish and the kidney of another. They generally are associated only with local tissue reactions, including prominent cartilage metaplasia (Heidel et al. 2001). Some researchers have proposed that these might represent ectopic eggs, but they have also been observed in male fish. Therefore, at present, the consensus of most researchers is that they are degenerated microorganisms of unknown identity. Conclusion This study confirmed previous observations of aquaculturists that Cryptocaryon irritans may be a serious pathogen of opakapaka. Other parasites such as the coccidians (Goussia spp.), the monogeneans and the copepods should be monitored as related species are already recognized as causes of disease in marine aquaculture. These parasites are monoxenous (one host) parasites and thus may proliferate in or on fish in captivity. Interestingly, the external parasites (Cryptocaryon, monogeneans and copepods) were much more prevalent in fish held in tanks, suggesting that the netpen environment was more favorable for avoiding proliferation of these potential pathogens in captive fish.

Figure 17. Cyst of unknown origin surrounded by metaplastic cartilage [C] in kidney.

Bacterial infections and associated inflammatory changes in the swim bladder were very likely the result of the practice of de-gassing captured fish by insertion of a hypodermic needle into the swim bladder, and thus at this point would be considered as only opportunists.

Likewise, exophthalmia was consistently associated with gas emboli in the eyes, again probably associated with collecting procedures. However, other causes should be considered if this condition arises in captive-reared fish. Blood smears and kidney imprints were for the most part not informative, but they did confirm that fish fish did have exhibit septicemia or obvious blood parasites.

Further studies should entail continued routine diagnostics of moribund and dead fish. Cryptocaryon is already recognized as a health problem with this and other marine fishes held in captivity, and we recommend research on the control of this pathogen. Considering the difficulties with the use of external baths in netpens, we suggest that a long-term solution may be the development of a vaccine for this ciliate parasite. This approach shows promise as significant success for the development of a vaccine has been achieved for Ichthyophthirius multifiliis (Clark and Dickerson 1997), Cryptocaryon's freshwater cousin. Moreover, as with Ich, it is already recognized that fish that have recovered from Cryptocaryon infections exhibit resistance to reinfection (Dickerson and Dawe 1999).

Although we have not identified viral diseases in opakapaka to date, it is likely that some viral diseases will afflict this species as culture of this fish moves forward. Therefore, another area of research would be the development of specific cell lines from opakapaka and related fishes. Many fish viruses require rather specific cell lines, and thus have only been isolated using cell lines derived from the host species. Last, it would be worthwhile to describe the coccidians as they may cause problems in the future in this host or related fish species.

Acknowledgement. The Hawaii Sea Grant Program Development supported this study and Hawaii Department of Land and Natural Resources, Division of Aquatic Resources. We thank Dr. Whitlow Au for assistance with administration of the grant.

References

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