Journal of Fish Diseases 1989, 12, 175-178 SHORT COMMUNICATION Uleerative myeosis: a serious menhaden disease of the southeastern eoastal fisheries of the United States M . J . D Y K S T R A , J . F . L E V I N E & E . J. NOG A School of Veterinary Medicine, North Carolina Suile Ufiiversity. Raleiiih. North Carolina, USA J. H. H A W K I N S North Carolina Division of Marine Fisheries, Washington, North Carolina, USA P. G E R D E S & W. J. H A R G I S J R Virginia Institute of Marine Science, Gloucester Point, Virginia, USA H. J. G R I E R Bureau of Marine Research, St Petersburg, Florida, USA D. T E S T R A K E Department of Biology, University of South Florida, Tampa, Florida, USA Atlantic menhaden, Brevoortia tyrannus (Latrobe), are of great economic significance to the East coast fisheries of the United States. Menhaden comprise over 50% of the yearly commercial catch (Cross, Peters & Schaff 1984). During the summer of 1984, large numbers of menhaden afflicted with severe skin ulcers ('red sore' disease) were collected by the North Carolina Division of Marine Fisheries from the Pamlico River estuary. Anecdotal reports during 1984—1986 suggested that the uleerative syndrome was a regional problem in the U.S., being observed on fish from Delaware through to Florida (Hargis 1985). Accordingly, in 1986, studies were initiated to determine if lesions observed on menhaden collected in states other than North Carolina also contained oomycete hyphae and fungal granulomas characteristic of this disease that was formally described as uleerative myeosis (Noga & Dykstra 1986). Estuarine trawl surveys conducted in the Pamlico River revealed that uleerative mycosis (UM) occurred sporadically between 1985 and spring 1987 but was most prevalent during May and June of 1985 and 1986. At times, 100% of the menhaden eollected in an individual trawl sample had UM. Attempts to isolated fungi from North Carolina menhaden with lesions were performed from April 1985 to May 1986 (Table 1). Fresh lesion material was plated onto Petri dishes containing corn meal agar (Difeo) supplemented with 1% yeast extract, 2% dextrose, 0-01% penicillin G and 0 01% streptomycin sulphate. Plates were examined immediately by light microscopy for the presence of fungal hyphae. Certain trawl samples were packed on ice for 8h prior to examination. The plates prepared for lesion evaluation were incubated at room temperature in ambient light for 72 h with examinations every 8-12h for evidenee of fungal growth. When viable aseptate hyphae were observed, hyphal tips were excized and transferred to fresh media for subsequent observation. Once the plates were deemed to contain pure axenic cultures, subcultures were prepared upon which sterile hemp seed halves and snakeskin (Scott 1961) were placed into which the fungus grew. After these substrates were invaded, they were placed in sporulation medium (SM) containing 0 055 g CaCK 6H2O, 0 019 g KCI (Griffin 1966), and 4 g NaCI. After 24-48h at room temperature, sporulation took place allowing identification of the fungal genera represented. Five Aphanomyees, two Saprolegnia and one Correspondence: Dr M. J. Dykstra, School of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA. 176 SL ,/. Dvkstnt ct al. tinidontitiod specimen (;i probjihlc Oumycctf which has never sporulated) were recovered. C^illiircs of 0 5'X. ot lesions vvilli hy[>hac seen in wcl mounts yielded Oomycctes. Ifi Novcmhcr l^'Nd, 108 young ol year (YOY) menhaden were collected in a series of trawl collcclions Ml the Kappah;uinock Kivcr in Virginia. Skin ulcerations were noted on 90% ofthe menhaden. Immodiale cxaniinalioti of the material from 74 infected (ish revealed hyphae in 83'/'.. of lhe lesions. Three Aphanotuyces and lour Suprolegnia sp. were ultimately isolated from those lish. A second survey was made in January 1987. Of lhe 126 menhaden that were collected, 3(i (29%) had UM and 44% of the lish lesions contained fungal hyphae. Two Aphatunfiyccs isolates were recovered from this survey (12-5% of the lesions that contained hyphae). Two trawl surveys of the St Johns River in Jacksonville, Florida, USA, were conducted on 22 and 23 January I9S7. Of the 41 menhaden caught, 10 had UM (24%). Five of seven fish had hyphae in their lesions (717o) and one unidentified water mould was isolated from one lesion. Two llounder. Paraliehlhys lelhostigrua Jordan and Gilbert, and a number of yellow mouth trout, Cynoscion regalis Bloch and Schneider, had deep skin ulcers. Examination of material removed from the C. regalis lesions consistently revealed oomyccte-like hyphae that were significantly broader (12-20 f.im) than those found in menhaden lesions. The predominant fungi isolated from all of the menhaden lesions examined belong in the genus Aphanomyees. The isolates displayed three distinct growth and morphological patterns: (1) isolates that grew vigorously, produced zoospores copiously and formed oospores {Aphanof7iyces isolate ATCC 62427); (2) isolates that produced asexual zoospores abundantly and grew vigorously, and (3) isolates that produced scant mycelium and few zoospores. Isolates of Saptolegnia sp. produced only asexual stages and grew vigorously. These asexual isolates cannot be identified to species with conventional taxonomic keys. The only sexual isolate (ATCC 62427) cannot be speciated positively because the final key choices involve identification of the host species and none are hsted as growing on fish (Scott 1961). Morphological characteristics, however, suggest that this isolate is Aphanomyces laevis de Bary. Oomycete hyphae found in the advanced lesions examined were primarily devoid of cytoplasmic contents. Indeed, the success rate for the isolation of living Oomycetes from lesions with standard techniques averaged about 10% from the sites studied. Accordingly, a series of experiments was initiated to determine if: (I) estuarine water was injurious to the fungi exposed at the surface of lesions; (2) menhaden flesh was a poor substrate for fungal growth; (3) other microorganisms found in mature lesions were detrimental to the fungi involved. Table 1. Frequency of oomycetes isolated from lesions In tncnhadon collected from the pamiico river. North Carolina, USA* Dutc 6 December 19X4 3 April 198S to Number of di.scased menhaden examined Wet mounts with hyphae (%) Oomycete fungi isolated (%) 40 Not done 9 Aphanomyces (23) 2 Saprolegnia (5) 164 123(75) 5 Aphanomyces (4) 27 May 19K6 ' Oi Atlantic menhailen, 6S7o had 2 Saprolegnia (1.6) 1 non-sporulating Ootnyccte (1) Aphanomyces. Vkerative tnveosis of menhaden 111 The first hypothesis was tested hy placing snakoskin substrates prc-infectcd with our test Aphanomyees isolate into sterile Jistilled water, SM, or non-sterile water samples collected from the Pamlico River estuary at three of our trawl sites (salinities of 0-{)%,,, 2- l%a and 3-9%.,). After 25 h of incubation at 23'^C, the plates were scored for vegetative growth and sporulation of the fungus. More sporulation and vegetative growth was visible in SM than in distilled water. However, the best growth and sporulation was found in all three of the non-sterile Pamlico River water samples. The second hypothesis was tested by inoculating a piece of aseptically removed menhaden musele tissue that had been placed on a sterile 2% water agar substrate in a Petri dish with the same Aphanomyees isolate used above. After 24h. the piece of original inoculum from the nutrient agar fungal culture was removed and placed on the surface of the water agar substrate. Following another 48h of growth at 23°C, the menhaden muscle was covered with substantially more growth than the original nutrient agar culture used as the inoculum. Aphanomyces growth was then compared on muscle tissue from menhaden, spot, Leiostomus xanthurtis Laeepede, croaker, Micropogonias undulattis L., flounder, gizzard shad, Dorosoma cepediamtm Lesueur, yellow mouth trout and lane snapper, Lutjanus synagris L, After 5 days at 23°C on 2% water agar plates, all the tissue supported similar amounts of vegetative fungal growth. Bacteria and protozoans were consistently found within fully developed lesions of menhaden, though no specific populations were repeatedly recovered (Noga & Dykstra 1986). When antibiotics were omitted from the fungal isolation medium, baeterial growth routinely prevented isolation of Aphanomvces sp. When fish muscle was contaminated by baeteria due to poor aseptic technique, Aphanomyces did not grow effectively. These studies have clearly shown that uleerative mycosis is a widespread disease of menhaden in estuaries in Florida, North Carolina and Virginia, USA. Several potentially different (and taxonomically uneertain) species of Aphationiyees and Saprolegnia from menhaden, i.e. with distinct reproductive phases and growth characteristics have been isolated. Previous studies have established that low salinity encourages the growth of the test Aphanomyces isolate ATCC 62427 (Dykstra et al. 1986). Due to laboratory studies of Aphatwmyees salinity tolerance (Dykstra et aL 1986), trawl sites in the Pamlico River studies were moved from areas of 10-15%o salinity to areas with 2-8%o and higher disease prevalence was noted in the lower salinity areas. Saprolegniales members are generally considered to be freshwater organisms, though they can tolerate moderate salinity under eertain laboratory conditions (Te Strake 1959; Harrison & Jones 1974; Padgett 1984). Freshwater fish that become infected by members of the Saprolegniales, particularly Saprolegnia sp., become covered with a superficial, cottony mycelium. Although this superficial growth may cause osmoregulatory problems and some mortality, deep ulcers similar to those observed on fish affected with uleerative mycosis are not commonly assoeiated with members of this order of fungi. One of the most intriguing aspects of uleerative myeosis is the low rate of recovery {10% or less) of Oomycetes from the ulcers despite the fact that almost all ulcers contained copious quantities of broad aseptate hyphae characteristic of this group of fungi. Ultrastructural examination (Dykstra et al. 1986) of fish lesions demonstrated healthy-appearing oomycete hyphae at the centre of granulomas in muscle tissue. Studies comparing the growth and sporulation capabilities of the test Aphanomyces isolate in distilled water, SM augmented with 4%o saline and the three Pamlico River water samples demonstrated that the river water was the best medium for growth and sporulation. This 178 M. J. Pykstra el ;il. cloaily iiulic;ilotl ihal ostiKirinc wutcr was not inimicjible to growth of this isolate and may acltiiilly he slinnikitory. Fish llosh \v;is, iiulocd, a good stibstratc for the fungus. Various types of fish musde supported ihe growth o\' the Aphanomyces isolate. The oily, herring-like flesh of menhaden, the predominant speeies alTeeted by UM, provided v\o evident nutrient advantages when eonipared with Ihe llesh ol the other speeies examined. The poor growth eharaeteristies o( Aphanomyces in the presence of baeterial eontaminants suggests that potential eompetition from other microorganisms may be responsible for the predominance of dead hyphae within menhaden lesions. Another possibility that has not been tested yet is that the immune system of the fish may not prevent the uleerations caused by the fungi, but does suppress the fungus by the time the fully developed lesions whieh are usually encountered in trawl samples are studied. This work suggests that some factors(s) predispose menhaden to infeetion with Oomycetes from two genera that are normally associated with freshwater environments. The pathogenicity of the Oomycetes in this estuarine system may also be altered. This newly described disease of menhaden arose in epidemic proportions throughout the mid- and south Atlantic coast in the summer of 1984. High precipitation levels depressed salinity, particularly in the Pamlico River estuary system. During periods of high run-off from agricultural lands such as those that surround the Pamlico River, nutrient loading occurs and large amounts of detritus enter the system. These nutrient materials may serve as substrates for the normally saprophytic Oomycetes associated with UM. In addition, suspended organic materials, inorganics and low salinity may alter the response of menhaden to infection with the fungus. The manner in which these various environmental and host factors facilitate development of lesions in fish, however, awaits confirmation. Acknowledgments This work was supported by a grant from the Water Resources Research Institute of North Carolina (#7(X)54). References Cross F. A., Peters D. S. & Schaff W. E. (1984) Implications of waste disposal in coastal waters on fish populations. Special Technical Testing Pttblicotion 854, 3S4-389. American Society for Testing and Materials. Philadelphia. Dykstra M. J.. Noga E. J., Levine J. P., Moye D. W. & Hawkins J. H. (1986) Characterization ofthe Aphanomyces species involved with uleerative mycosis (UM) in menhaden. Mycologia 78, 664-672. Griffin D, H. (1966) Effect of electrolytes on differentiation in Achlya sp. Plant Physiology 41, 12M-1256. Hargis W. J., Jr (1985) Quantitative effects of marine diseases on fish and shellfish populations. Transactions of the North American Wildlife Natural Resources Conference 50, 608-640. Harrison J. L. & Jones E. B. G. (1974) The effect of salinity on sexual and asexual sporulation of members of the Saprolcgniaceae. Transactions of the British Mycolological Society 65, 389-394. Noga E. J, & Dykstra M. J. (19K6) Oomycete fungi are associated with ulcerative mycosis In menhaden, Brevoortia iyranntts (Lalrobe). Journal of Fish Diseases 9, 47—53. Padgett D. E. (19H4) Evidence for extreme salinity tolerance in saprolegniaceous fungi (Oomyectes). Mycolopa ^76, 372-375. Scott W. W, (1961) A monograph of the genus Aphanomyces. Virginia Agricultural Experiment Station Technical Bulletin 151, 1-95. Te Slrakc D. (1959) Esluarine distribution and saline tolerance of some Saprolcgniaceae. Phyton 12, 147-I5i'