# OYSTER PARASITISM BY

LABYRINTHOMYXA MARINA

IN FLORIDA

J. A. Quick, Jr. and J. G . Mackin

PROFESSIONAL PAPERS SERIES

Number Thirteen April 1971

FLORIDA DEPARTMENT OF NATURAL RESOURCES

MARINE RESEARCH LABORATORY ST. PETERSBURG, FLORIDAEffective July 1, 1969, agencies of the State of Florida underwent legislative reorganization and the Florida Board of Conservation was renamed the Florida Department of Natural Resources. Similarly, the former Division of Salt Water Fisheries became the Division of Marine
Resources.

Extra effort always goes into the preparation of our publications. Cost of production limits the number that can be printed. In order that the fullest possible use can be made of each copy, please be good enough to give this copy to your nearest library when you have finished with it.

# OYSTER PARASITISM BY

LABYRINTHOMYXA MARINA

IN FLORIDA

J. A. Quick, Jr. and J. G. Mackin*

Professional Papers Series Number Thirteen

Florida Department of Natural Resources Marine Research Laboratory St. Petersburg, Florida

* Department of Biology, Texas A. and M. University

## FLORIDA DEPARTMENT OF NATURAL RESOURCES

REUBIN O'D. ASKEW

Governor

RICHARD (DICK) STONE Secretary of State

ROBERT L. SHEVIN A ttorney General

FRED 0. DICKINSON, JR.

Comptroller

THOMAS D. O'MALLEY

Treasurer

DOYLE CONNER Commissioner of Agriculture

FLOYD T. CHRISTIAN Commissioner of Education

RANDOLPH HODGES Executive Director

HARMON W. SHIELDS Director, Div. of Marine Resources

11

## SCIENTIFIC BOARD OF ADVISORS Marine Research Laboratory Division of Marine Resources Florida Department of Natural Resources

Dr. A. F. Chestnut Institute of Fishery Research University of North Carolina Morehead City, North Carolina

Dr. Albert Collier Oceanographic Institute Florida State University Tallahassee, Florida Mr. Ronald C. Phillips Seattle Pacific College Department of Botany Seattle, Washington

Dr. Sammy Ray Texas A. & M. Marine Station Fort Crockett Galveston, Texas

Dr. John D. Costlow, Jr. Duke University Marine Laboratory Beaufort, North Carolina

Dr. L. B. Holthuis, Curator Rijksmuseum van Natuurlijke Historic Raamsteeg 2, Leiden The Netherlands Dr. C. Lavett Smith Assistant Curator Department of Ichthyology The American Museum of Natural History New York, New York

Dr. Franklin Sogandares-Bernal Zoology Department Tulane University New Orleans, Louisiana

Dr. Victor G. Springer Associate Curator Division of Fishes Smithsonian Institution U. S. National Museum Washington, D. C. Dr. Victor L. Loosanoff University of the Pacific Stockton, California

Dr. J. G. Mackin Dr. W. B. Wilson Department of Biology Texas A. & M. Marine Station Texas A. & M. University Fort Crockett College Station, Texas Galveston, Texas

iii

## MARINE RESEARCH LABORATORY

FLORIDA DEPARTMENT OF NATURAL RESOURCES

P. 0. Drawer F, St. Petersburg, Florida 33731

Robert M. Ingle, M.S. Chief, Bureau of Marine Science & Technology



### Edwin A. Joyce, Jr., M.S. Supervisor, Marine Research Laboratory

LABORATORY STAFF

ICHTHYOLOGY Robert W. Topp, M.S. Senior Fisheries Biologist Frank H. Hoff, Jr., B.A Marine Biologist Dion Powell, B.A. Marine Sciences Technician Robert F. Presley, B.S. Marine Biologist Kenneth R. Halscott Student Research Assistant

HISTOLOGY
Jean Williams Marine Sciences Technician Rena S. Barco Marine Sciences Technician Sandra L. Farrington Student Research Assistant

LARVAL FISHES
Bonnie M. Eldred Marine Biologist Charles R. Futch, B.S. Marine Biologist

PELAGIC FISHES Dale S. Beaumariage, B.S. Marine Biologist

INVERTEBRATES William G. Lyons, B.A. Marine Sciences Technician David K. Camp, B.S. Marine Biologist Stephen P. Cobb, B.S. Marine Biologist Linda H. Lyons Marine Sciences Technician William D. Leever Student Research Assistant

STONE CRAB RESEARCH Thomas Savage, M.S. Marine Biologist Charles E. Kalman Marine Sciences Technician

PHYSALIA F. Stuart Kennedy, Jr., B.S Marine Biologist

OYSTER BIOLOGY Stanley W. Morey, B.S. Marine Biologist Walter K. Havens, B.S. Marine Sciences Technician Scott A. Willis, B.S. Marine Sciences Technician Janie M. Staley, S.S. Marine Sciences Technician

ivJack F. Van Breedveld Marine Sciences Technician

OYSTER PARASITES Joe A. Quick, Jr., B.S. Marine Biologist Melvin W. Bidwell, Jr. Student Research Assistant

CLAM BIOLOGY Mark F. Godcharles, B.A. Marine Biologist

MARICULTURE Carlton F. Rowell, B.A. Marine Biologist Charles E. Dugan, B.A. Marine Biologist

ORGANIC CHEMISTRY Mary Ann Burklew, B.A. Marine Biologist

STUART FIELD LABORATORY
Ross Witham Marine Sciences Technician

KEY WEST FIELD LABORATORY Sean J. Bollar, B.S. Marine Biologist Edward J. Little, Jr., B.S. Marine Biologist

PENSACOLA FIELD LABORATORY Robert M. Gallagher, B.S. Marine Biologist
ST. JOHNS FIELD LABORATORY (Sanford) Roy 0. Williams, B.S. Marine Biologist Gerard E. Bruger, B.S. Marine Biologist

APALACHICOLA FIELD LABORATORY James G. Mills, Jr., B.A. Marine Biologist
THERMAL ADDITION STUDIES (Crystal River) Churchill B. Grimes, B.A. Marine Biologist Joe A. Mountain, B.S. Marine Biologist

WEST PALM BEACH FIELD LABORATORY John W. Jolley, Jr., B.S. Marine Biologist

CHOCTAWHATCHEE BAY FIELD LABORATORY Edwin W. Irby, B.A Marine Biologist

RESEARCH VESSEL
Earl Girard Marine Captain Malcolm L. Hollinger, B.S. Marine Biologist Thomas A. Frakes, B.S. Marine Sciences Technician Walter C. Jaap, B.S. Marine Sciences Technician Terry R. Pulver, S.S. Marine Sciences Technician Andrew Kaloski, B.S. Marine Biologist

ADMINISTRATION
Alan H. Sellen Director of Administration Betty Accurso Office Supervisor Jane F. Biggs, B.A. Publications Heather Grothe Reports

### John J. Clopine, M.S.L.S. Librarian

Marjorie S. Clopine, M.S. Library Assistant Cheryl Medema Personnel Paula Newton Budget and Financial Marion W. Stance11 Purchasing Katherine B. Stoelzel,B.A. Archives Linda L. Gilmore Student Assistant Mary E. Miller Student Assistant Lana S. Tester Student Assistant

ENGINEERING
Marco A. Omeechevarria Phillip Ford, Jr. Vladimir Janek George Omeechevarria Student Assistant

TALLAHASSEE OFFICE William K. Whitfield, Jr., B.A. Administrative Assistant Hazel Jones Administrative Assistant Marjorie Core Records June Smith Secretary

APALACHICOLA Joseph Martina, Jr. Field Supervisor (Div. Oyster Culture)

vi

### CONTENTS

ABSTRACT 1 INTRODUCTION 1 METHODS AND MATERIALS 1
STATION DESCRIPTIONS 1 LABYRINTHOMYXA DISTRIBUTION STUDY 4 INVESTIGATIONS OF ETIOLOGY AND MORTALITY 6 Plot Studies 6 Calculation of Adjusted Mortalities 7 Relation of Gapers to Oyster Death Rate 8 Year Class Analyses 8 RESULTS AND CONCLUSIONS 8 PART I: STUDIES OF LABYRINTHOMYXA DISTRIBUTION 8 COMPARISON OF ANTIBIOTIC TREATMENTS 8 LABYRINTHOMYXA DISTRIBUTION IN FLORIDA 11 Relationship of Labyrinthomyxa to Season 11 Relationship of Labyrinthomyxa to Temperature 11 Distribution of Labyrinthomyxa Intensities 11 Relationship of Labyrinthomyxa to Salinity 13 Relationship of Labyrinthomyxa to Oyster Size 20 Relationship of Labyrinthomyxa to Oyster Depth 24 Relationship of Labyrinthomyxa to Oyster Condition 24 Annual Labyrinthomyxa Parasitism at Selected Sites 24 PART II: STUDIES OF OYSTER MORTALITIES 27 YEAR CLASS ANALYSIS AND ANNUAL MORTALITY 27 Determination of Oyster Age by Shell Contours 27 Aging Technique 27 General Results of Year Class Analysis 28 ETIOLOGY OF NATURAL OYSTER MORTALITIES 40 First Year of Plot Studies 40 Second Year of Plot Studies 40 DETERMINATION OF MORTALITY RATES OVER SHORT PERIODS 44 ANALYSIS OF INFECTION IN GAPERS 45
DESCRIPTION OF A LABYRINTHOMYXA Epizootic 46 Additional Epizootics 46 Damage to Oysters from Labyrinthomyxa 46 THE REDUCTION OF LABYRINTHOMYXA DAMAGE TO
COMMERCIAL OYSTERS 48 SUMMARY 49 ACKNOWLEDGMENTS 53 LITERATURE CITED 53

vii

### TABLES

1. Oyster condition rating key 5
2. Evaluation of Labyrinthomyxa infection intensities in F.T.M. assays 7
3. Results of paired Labyrinthomyxa tests in 1918 oysters 9
4. Comparison of detection of Labyrinthomyxa at each infection intensity 10
5. Pesticide residues in oysters at selected Labyrinthomyxa monitoring stations 20
6. Average trace metal concentrations in oysters from selected stations 21

FIGURES

1. Oyster sampling stations 2, 3
2. Shell measurements and features in 18-month oyster 6
3. L. marina infection by season 12
4. 

### L. marina infection relative to temperature 13
5. L. marina infection intensity frequency distribution 14
6. 

### L. marina infection relative to salinity 16
7. Parasitism by L. marina in Suwanne Sound 17
8. Salinity and temperature in Suwannee Sound 19
9. Parasitism by L. marina in Crystal River 22
10. Salinity and temperature in Crystal River 23
11. L. marina infection relative to oyster height 25
12. L. marina infection relative to depth 26
13. Oyster condition and associated infection by L. marina 29
14. Parasitism by L. marina in Tampa Bay 30
15. Salinity and temperature in Tampa Bay 31
16. Parasitism by L. marina in Sarasota Bay 32
17. Salinity and temperature in Sarasota Bay 33
18. Spatfall at two stations during 1969 34
19. Oyster shell configurations 35, 36
20. Oyster age calculator 37 21. L. marina infection during Station 23 plot test 38 22. Salinity and temperature during Station 23 plot test 38 23. Unadjusted oyster numbers and mortality during Station 23 plot test . 39 24. L. marina infection during Station 43 plot test 41
25. Salinity and temperature during Station 43 plot test 41
26. Unadjusted oyster numbers and mortalities during Station 43 plot test . 42
27. Incidence of L. marina infection intensity detected by P-FS F.T.M. assay among gapers 43 28. Labyrinthomyxa infection during an epizootic in Sarasota Bay 47 29. Oysters grown by the mortality minimizing technique 50, 51

viii

### OYSTER PARASITISM BY
LABYRINTHOMYXA MARINA

IN FLORIDA

ABSTRACT

The fungal oyster parasite, Labyrinthomyxa marina, was found to occur along the Gulf and Atlantic coasts of Florida. It was found concomitantly with the host throughout the year and at salinities from 6 100 to 36 °/... High temperature was implicated as the most important factor contributing to high rates of parasitism. Field studies indicated that L. marina kills an average of over 50% of Florida's adult oysters yearly. Sampling and observation of the intermittent oyster epizootics caused by this fungus showed that mortalities could be very rapid and intense. Oyster mortality estimation methods were evaluated and one technique was developed for measuring annual death rates and another for estimating high, short-term oyster kills. An oyster-growing program is proposed for use in central Florida to obtain the most oyster meats of highest quality in the shortest time with the smallest effective mortality.

INTRODUCTION
In 1950, Mackin, Owen, and Collier described a new parasite of the American oyster, Crassostrea virginica Gmelin. This pathogen of uncertain relationship was named Dermo cystidium marinum Mackin, Owen and Collier, but after a thorough study of the life history and other aspects, it was placed under the genus Labyrinthorny xa, becoming Labyrinthomyxa .marina (Mackin, Owen, and Collier) Mackin and Ray (1966).Research increased after the development of a simple quantitative assay for L. marina by Ray (1952b). Workers using this fluid thioglycollate medium culture (F.T.M.) technique found oysters infected with the fungal parasite from New Jersey (Anonymous, 1958) to Mexico (Mackin, 1962). Oyster deaths from L. marina were found to be particularly heavy in the states bordering the Gulf of Mexico (Mackin, 1951, 1962; Ray, 1954a, 1966a; Mackin and Wray, 1952; Ray, Mackin, and Boswell, 1953). The parasite was found in Florida in Pensacola Bay (Ray,1954b), Apalachicola Bay (Ingle and Dawson, 1953b), Cedar Keys (Ingle and Dawson, 1953a), and Tampa Bay (Stewart, 1966; Ray, 1966a). This laboratory began monitoring L. marina in Florida oysters in 1965, initially to determine the parasite's range beybnd these areas. The study was soon expanded to investigate the environmental limits, the seasonal occurrence, and the resultant oyster mortalities of this fungus parasite.

METHODS AND MATERIALS

STATION DESCRIPTIONS A total of 120 stations were sampledduring our study and one or more standardized Labyrinthomyxa samplings were made at 91 of them. All station locations are shown in Figure 1; eight of these, however, were extremely significant to this study and require further definition.

Station 15 (29. 17'30"N, 83-06'55"W): Located in the open Gulf about one mile off a large salt marsh near the mouth of the Suwannee River, these large oysterbars (reefs) are almost constantly submerged. The bottom near the bars is extremely soft, and currents readily create high turbidity.

Station 20 (28° 55'00"N, 82° 41'40"W): Located in the mouth of Crystal River near the Gulf, this station is characterized by several small intertidal oyster bars. The Contribution No. 164, Florida Department of Natural Resources Marine Research Laboratory2 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Figure 1. Oyster sampling stations for studies of Labyrinthomyxa marina. One or more twelve-oyster

samples were collected from stations 1 through 91. Stations with a letter suffix were alternates or were never collected in this manner. Exact station locations and coordinates may be obtained from the Marine Research Laboratory.



## 6 7 8D

8E 

## 8 ■ 9

8A 

## F 10A 

## 1 0

0

## F 

# 4

82 ° 81 ° 800

75B

78A- --\ 

### 0 78 ,--

----

78B AA 78c

78D OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 34 FLORIDA DEPARTMENT OF NATURAL RESOURCES

bottom near the bars is composed of gravel, shell, and firm clay.

Station 23 (27° 50'05"N, 82°46'15"W):Located in a rather narrow, mangrovelined bayou in upper Boca Ciega Bay nearLake Seminole, this station was characterized by numerous intertidal oyster bars interspersed with small mangrove islands. A ,soft black mud bottom surrounded the oyster bars. Because of construction inthe area this station is no longer productive.

Station 43 (27°54'32"N, 82°38'00"W): Located in a wide, shallow lagoon innorth Tampa Bay about three miles southwest of the Howard Frankland bridge, this station is characterized by many small, scattered intertidal oyster bars around andbetween mangrove islands. The surrounding bottom is a soft, muddy sand sparsely covered with Diplanthera.
The above stations 23 and 43 were most important to the study because they were the sites of the plot tests and weresampled more frequently than other stations. Station 75 (27°30'00"N, 82°34'05"W): Located in the Manatee River about four miles from the mouth, this station is a long intertidal oyster bar paralleling the railroad bridge. It is probably an old spoil bank and the bottom is primarily black mud. Station 78 (27°15'56"N, 82°33'15"W):This station is located in a small embayment on the west side of Little Sarasota Bay. It is a series of small intertidal oyster bars near the channel leading to a private boat basin. The bottom is primarily sand with some shell. Station 80 (26°42'00"N, 82°09'15"W):This station is located in a cove surrounded by mangrove swamp on the northwest end of Pine Island Sound off Charlotte Harbor. The oysters are subtidal and are scattered loosely over the sand bottom. Station 81 (26°30'48"N, 81°58'45"W):This station is in the mouth of the Caloosahatchee River in a small mangrove embayment on the southeast bank. The oysters are intertidal and are scattered loosely over the sand and mud bottom, attached to mangrove roots, or form small bars.

LABYRINTHOMYXA DISTRIBUTION STUDY Oysters from 91 stations in Florida(Figure 1) were monitored for the presence of Labyrinthomyxa from 1965 through 1969. Six of these (Nos. 15, 20,75, 78, 80, and 81) were sampled monthly for data on seasonal trends of parasiteincidence while the remainder were sampled one or more times for data on geographic range and environmental limits of the fungus. Oysters were taken at each station with oyster tongs, scallop dredge, or by hand and transported to the laboratory under moist, cool conditions for processing within ten hours of collection. Standardphysical and hydrological data were obtained at each sampling and biological observations were recorded.Twelve oysters were removed at random from each sample lot for parasite analysis. The height of the upper (right) valve of each oyster was measured parallel to a tangent of the major axis of growth(Figure 2) with linear calipers. Each oyster was opened and the animal's conditionrated according to an arbitrary scale (Table 1). Four tissue explants, two pieces each of mantle and gill, were removed just ventral to the labial palps for inoculation. Stock F.T.M. with dextrose (Difco No. ETM-0256-01) was rehydrated with a 2.0% aqueous sodium chloride solution and dispensed in 10 ml aliquots into 17 x 125 mm screw-capped culture tubes. Oneduplicate pair of these tubes was inoculated from each sample oyster. Every culture tube received one specimen of gill tissue and one of mantle tissue (Ray, 1952b, 1966b). One member of each duplicate pair of inoculated culture tubes was fortified by addition of 5,000 U.S.P. units each of Nystatin (E. R. Squibb and Sons, New York, List 5915) and chloramphenicol (Parke, Davis, and Co., Detroit, SY65 stock 35-65-1). The second tube of eachOYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 5

TABLE 1. OYSTER CONDITION RATING KEY

Condition Index Code No.

Very good 1

Good 2

Good minus 3

Fair plus 4

Fair 5

Fair minus 6

Poor plus 7

Poor 8

Very poor 9 Oyster Appearance

Animal firm and filling shell cavity; coloration creamy white and evenly textured; usually ready to spawn.

Not quite as firm or large as above; usually ready to spawn.

Coloration less opaque, often slightly yellow or gray.

Animal distinctly not filling shell cavity; coloration often mottled, with blood vessels and muscle fibers showing through the more translucent epithelium.

Oyster well-developed but not opaque or tending toward white; grayish and translucent; flesh flaccid.

Translucency more pronounced.

Oyster not well-developed, darker gray, often greenish; pericardial cavity clear; small portion of shell cavity filled.

Negative qualities more accentuated.

Animal distinctly atrophied; coloration dark and uneven, very translucent; seldom more than third of shell cavity occupied; adductor muscle often discolored and transparent even in the normally white sector.

pair received 5,000 U.S.P. units of penicillin G (E. R. Squibb and Sons, list 6735) plus 5.00 mg of streptomycin (E. R..Squibb and Sons, list 8377). The total volume of both additions to each tube was 0.2 ml. Inoculated, antibiotic fortified culture tubes were incubated in the dark at 25 to 30°C for seven days.
After incubation, each piece of tissue was removed from the culture medium, blotted to remove excess liquid, teased onto a microscope slide, and stained three to ten minutes by addition of Lugol'saqueous iodine potassium iodide. A coverglass was pressed onto the preparation and examination was made at 50X and 100X magnifications. Infections, exhibited in the cultured tissue by the presence of darkly stained hypnospores, were classed quantitatively (Table 2), similar to Ray's (1953, 1954b) method. A representativecode number was assigned to each infection on the basis of its intensity or theapproximate number of cells per unit volume of host tissue. Tissue explant size and slide preparation thickness were kept as constant as possible to increase precision in the infection intensity measurements. Averaging all the infection intensity code numbers for any selected group of assayed oysters produced a number whichreflected both the proportion of the population infected and the intensity of infection. This value is known as the weighted incidence (W.I.) of infection (Ray, 1953)and was very useful in comparing infections in various oyster populations. Theusual calculations of incidence and average intensity of infection were also used. The data from this parasite distribution study was analyzed with the aid of an IBM computer. A data input card was punched for each oyster assayed duiing the study. All applicable hydrologicaldata, as well as individual oyster test results and determinations, were includedon each card. The primary computer techniques used were card sorting with respectto selected parameters and statistical significance verifications.6 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Major Axis of Growth

Figure 2. Shell measurements and features in an 18-month oyster. FS - first summer's growth; SS - second summer's growth.

INVESTIGATIONS OF ETIOLOGY AND MORTALITY Plot Studies: Relationships between oyster mortalities and Labyrinthomy xaparasitism were evaluated, using an adaptation of the tray technique (Mackin, 1961; Hewatt and Andrews, 1954) similar to that of Owen (1955) and Ingle and Dawson (1952, 1953a, b). Locations were selected in oyster growing areas so that two very similar, adjacent plots of four square meters each could be delineated. Each square plot was cleared of oysters and a plastic pipe (3/4 inch PVC) was sunk vertically at each corner. One plot of each pair was then surrounded by heavy gauge galvanized welded fencing (1.8 x 3.8 cm mesh) with the lower edge buried 10 to 20 cm. At one location, a wire topwas secured over an enclosed plot to prevent the entry of predators during high tides which submerged the enclosure. Single adult oysters were then collected from the surrounding area, counted, and replaced in approximately the original oyster population density.Appearance Microscopic /Macroscopic

No hypnospores/tissue orange-brown

Hypnospores very scattered, usually larger than average/ tissue still orange-brown

Hypnospores in most fields but sometimes concentrated in specific areas/tissue still orange-brown

Hypnospores common, beginning to make up a significant portion of each field/tissue still orange-brown

Hypnospores numerous everywhere, seem to be occupying a significant volume of the tissue/darkened spots may show in tissue

All fields darkened by the presence of hypnospores, seem to occupy about half of tissue volume/tissue often bluish, takes up more than usual stain

All fields almost black with hypnospores occupying more than half of tissue volume; tissue often difficult to see, almost impossible to get thorough staining/tissue distinctly blue or black OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 7

TABLE 2. EVALUATION OF LABYRINTHOMYXA INFECTION INTENSITIES

IN F.T.M. ASSAYS

Intensity Description Code Number

Code Hypnospore

Concentration

Negative N 0 None in sample

Very light VL 1 1-10/sample

Light L 2 11-100/sample

Light medium LM 3 99-1,000/sample (1-30/5 mm field)*

Medium M 4 31-300/5 mm field

Medium heavy MH 5 301.1,000/5 mm field

Heavy H 6 1,001 and up/5 mm field 200 and up/2 mm field** (commonly to 3,000/5 mm field)

*B and L Dinoptic w/15X wide field oculars and 3.5X obj. gives 5 mm field **B and L Dinoptic w/15X wide field oculars and 10X obj. gives approx. 2 mm field

The plot experiments were sampled atselected intervals, usually monthly. All living oysters in each plot were counted and returned to their respective locations to determine how many oysters had died since the sampling. Separate counts weremade of gapers (oysters too weak to maintain shell closure), boxes (empty hinged shells), and all single left valves. Shells ofthese dead and dying oysters were examined for evidence of predation and removed from the plot. Hydrological measurements were taken and the sampling concluded by removing for analysis all gapers plus 12 living oysters at random from each plot. These sampled oysters were transported to the laboratory, measured (height), rated as to condition, and tested for Labyrinthomyxa. Calculation of Adjusted Mortalities: Month-to-month oyster mortalities weredetermined simply by subtracting the oysters counted at one sampling from those counted in the previous sampling. For8 FLORIDA DEPARTMENT OF NATURAL RESOURCES

longer periods, however, consideration had to be given to those living oysters removed for testing, since some of theseoysters may have died and thus contributed to the mortality in the plot. To avoid this possible error in mortalitycalculations, all oysters removed for testing were considered to have died at thesame rate as remaining oysters. The number of "surviving" test oysters was then added to the total living oysters to arrive at a more accurate figure. The resultingmortality rates, which include these projected deaths, are called "adjusted" mortalities. Relation of Gapers to Oyster Death Rate. Obviously, gapers should be present in any oyster area with a sufficiently high mortality rate. It was reasoned that if the period of time that some meat remained in each gaper was fairly constant, then there should be a quantitative relationship between the rate of mortality and the number of gapers in an area. The numberof gapers was compared with simultaneous mortalities derived from direct counts of oysters to determine any such quantitative relationships between gapers and mortality. This gaper count was later modified to a ratio between the gapers and the living oysters in the study area.Direct determination of this gaper to living oyster ratio was made thereafter by randomly placing a meter square wooden frame on the oyster bottom and counting the enclosed gapers and living oysters. All oysters were counted except very young ones, or spat, whose growing lips had not yet angled upward from the substrate and whose shells had not yet begun significant thickening. At first, the meter squareframe was placed inside the plots for oyster counts, but, later, these counts weremade in the area of oyster bar surrounding the plot to better simulate routine field use of the technique and to provide better accuracy due to the increased number of oysters counted. Sampling in the plots was increased to weekly for short periods to obtain more accuratedata. This method was also used at Station 78 where mortality rates were simply correlated with decreases in the average number of oysters per square meter. Year Class Analyses. Mackin (1961) used year class analysis to determine annual oyster mortalities in Louisiana.Large numbers of oysters from a designated area were measured and the resulting heights were graphed as height versusthe frequency of occurrence. The resulting graph was then read to establish year classes and the number of oysters in each annual group determined. The annual mortality was computed as the differencein the number of oysters in each successive year class, assuming that spatfallremained uniform. We attempted to duplicate these earlier Louisiana successes, but inconsistency in our attempts to establish year classes by height measurements caused us to examine other methods of establishing annual groupings.
The possible use of shell rings to establish oyster age and thus year classes was examined. The basic information on the occurrence and significance of shell rings and contours came from several years of field observations. Verification, however, required oysters of known age. During approximately two and a half years of sampling, several such verifications were possible. For example, Station 6 in north Florida produced a unique situation in which changes in water quality first killed all the oysters and then allowed only a single spatfall to survive in the fall of 1966. Additional shell observations were made at Station 43 where plot studiesprovided known mortalities for comparison. Observations of spatfall at this station also aided in the evaluation of shellcontours as a method of determining oyster age and thus, mortality.

RESULTS AND CONCLUSIONS

Part I: Studies of Labyrinthomyxa Distribution

COMPARISON OF ANTIBIOTIC TREATMENTS

Ray (1966a,b) demonstrated that infected oyster tissues cultured in F.T.M. assay higher Labyrinthomyxa intensities when fortified with either mycostatin or a mycostatin-dihydrostreptomycin-penicil-OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 9

lin mixture than duplicate tissues cultured

in ' F.T.M. containing a penicillin

- streptomycin mixture. Also, the number of enlarged parasite cells and the size of these
enlarged hypnospores was greater in
media with mycostatin.

During 1 965 P *Id 1966, we assayed
1,918 oysters using paired F.T.M. tubes
containing duplicate tissues but having


### one fortified with mycostatin and chloramphenicol (M+C) and the other with
penicillin and streptomycin (P+S). Cultures containing M+C yielded an overall

W.I. of 1.41 while the P+S culture tubes
assayed a lower 0.99 W.I. (Table 3). The

incidences of infection were 62% and 56%. In addition, M+C cultures detected
233 infections not found by the paired
P+S tubes while the P+S cultures assayed
only 115 not found by the simultaneous

matched M+C fortified cultures.
Hypnospores treated with M+C were
larger (21% greater diameter) and appeared darker than those treated with
P+S. In the 348 paired tests where one
treatment was negative and the other positive (primarily low intensity infections),
M+C treated cultures produced 34% more
positive results than P+S. However, as the
infections increased, the difference between P+S and M+C intensity readings
became smaller (Table 4). It was possible 

### that intensity judgments (because of the

TABLE 3. COMPARISON OF PENICILLIN PLUS STREPTOMYCIN (P+S)

TREATMENT WITH MYCOSTATIN PLUS CHLORAMPHENICOL

(MTC) TREATMENT ON 1,918 OYSTERS

M+C Added to F.T.M. Difference in Results

62.04% 9.6%

1.41 29.8%

2.26 21.7%

229 49.8%

91.19% 9.6% P+S Added to F.T.M.

Infection incidence 56.10%

Weighted incidence 0.99

Average intensity of infected oysters 1.77

Oysters positive for infection in given treatment but negative in the other 115

Total oysters positive in one or both treatments 1305

Infection incidence derived from both treatments 68.04%

Relative accuracy (% of total positive oysters found by given treatment) 82.45%10 FLORIDA DEPARTMENT OF NATURAL RESOURCES

TABLE 4. COMPARISON OF DETECTION OF LABYRINTHOMYXA AT

EACH INFECTION INTENSITY USING THIOGLYCOLLATE CULTURE (F.T.M.) WITH PENICILLIN PLUS STREPTOMYCIN (P+S) AND WITH MYCOSTATIN PLUS CHLORAMPHENICOL (M+C) IN 2,062 OYSTERS A — Infections of Each Intensity Found by the Two Culture Variations:

Infection Intensity 0 1 2 3 4

F.T.M. with No. of oyster 899 584 336 156 56

P+S % of all oysters 43.6 28.3 16.3 7.57 2.72

F.T.M. with No. of oysters 778 436 328 323 129

M+C % of all oysters 37.7 21.2 15.9 15.7 6.24

B — Infection Intensities Detected in Assay Cultures with P+S and with M+C in Selected Groups of Oysters:

Oyster Groups A

No. of oyster

Infection intensity of all oysters in 899 584 336 156 56

each group 0 1 2 3 4

Infection intensities 0 0 1 2 3 found 1 1 2 3 4 2 4 4 5

5
Weighted incidence 0.4 1.4 2.6 3.3 4.1 5 6

18 13

0.88 0.63

42 26

2.02 1.24

F G

18 13

5 6

3 6 5 6

5.4 6.0 Groups selected & defined by P+S assay

Results of M+C assay on all oysters of each group

larger size and darker appearance of hypnospores) might have been a factor inthis difference, but our data did not support this. It therefore seems that M+C cultures caused more hypnospores to enlargeat low infection levels than did P+S cultures, but this advantage almost disappeared at heavier infection levels (Table 4).

The P+S combination was used to fortify F.T.M. in all other assays for Labyrinthomyxa made by this laboratory. This was originally done in accordance with Ray's (1952h) recommendation and later continued for the sake of uniformity. Ray (1966b) later concluded that the M+C antibiotic treatment was superior. We agree, particularly considering the greater accuracy of this method as shown above, but our experience indicated that the superiority of M+C may be somewhat compromised in practice. The insolubilityof mycostatin and chloramphenicol inconvenienced dispensing and sometimes interfered with microscopic observations of cultured tissues. The M+C seemed to produce some control of mycelial fungi but did not inhibit bacteria as well as P+S. Bacterial contaminants were damagingbecause sample softening and decomposition were often produced, thus complicating removal of tissues from the culture tubes and seriously compromising theOYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 11

quantitative aspect of the assay. This tissue softening or destruction was particularly serious in assays of gapers. Mycelialmolds were usually restricted to the surface and did not infiltrate the sunken tissues. When fungal mycelia did invade cultured oyster tissu ?•, however, they often improved firmness while not interfering appreciably with microscopic observations since hyphae remained transparent. The anaerobic condition' of properly preparedF.T.M. also inhibited growth of submerged fungi but had little effect on theoccurrence of bacteria. No significant differences could be found in the development of Labyrinthomyxa hypnospores between aerobic and anaerobic F.T.M. cultures in paired tubes from 33 infectedoysters. The presence or absence of contaminants, likewise, seemed to have no effect on hypnospore enlargement per se, although Mackin (1962) found that Labyrinthomyxa marina grew better in culture with the mold Fusarium sp. when compared with controls grown in sterile seawater. We often encountered this Ascomycete as a culture contaminant.

LAYBRINTHOMYXA DISTRIBUTION IN FLORIDA
Results of the Labyrinthomyxa distribution study showed the parasite's rangeto be continuous among oyster populations through the north Florida areas sampled by Ray (1954b, 1966b), Ingle and Dawson (1953a,b), and Stewart (1966). We also found the parasite's range to be continuous down the Gulf coast into the Florida Keys and northward up the Atlantic coast to Georgia. A total of 2,063 oysters were tested aspart of our study of the geographic, seasonal, and environmental distribution of Labyrinthomyxa. Infected oysters were taken from every county sampled and from 89 of the 91 stations (Figure 1). The overall W. I. was 1.02 with 1,158 oysters positive for the parasite to give a 56.1% incidence of infection. Relationship of Labyrinthomyxa to Season: The Labyrinthomyxa distribution study demonstrated this parasite to be prevalent throughout the year in Florida and to have an annual infection cycle of winter minimum and late summer maximum (Figure 3). Similar annual cycles have been observed by numerous authors(summarized by Mackin, 1962). The irregularities in our data (a February peak and a July decline)' coincide with the end of awinter drought and an unusually wet summer during sampling. Comparison of our data with the results of surveys for Labyrinthomyxa in more northerly mid-Atlantic coastal areas (Andrews and Hewatt, 1957; Dunnington, 1956; Anonymous, 1958) shows that theparasite was much more prevalent in Florida during the winter months and maintained high infections over a longer summer period. However, a comparison of our findings with data on parasitism in the northern Gulf of Mexico (Mackin, 1962; Mackin and Sparks, 1962; Ray, 1966a) reveals surprisingly small differences in both period and intensity of summer andwinter infestations; incidence and intensity seemed to drop slightly more during Florida winters. Relationship of Labyrinthomyxa to Temperature: Our distribution data reveals a strong positive temperature-infection relationship (Figure 4) similar to those observed by numerous other workers (summarized by Mackin, 1962). Incidence of infections and W.I. increased concurrently and almost linearly with temperature throughout the thermal range sampled. Critical temperatures, such as those affecting the parasite's well-being or the oyster's defense mechanisms, were not apparent. Distribution of Labyrinthomyxa Intensities: The number of distribution study oysters assigned each infection intensityrating is greatest in the "negative" intensity and decreases as intensity increases through "very light," "light," etc. (Figure5). Thus, there are about 50% fewer oysters in each successively higher infection Intensity level even though the range between the minimum and maximum hypnospore concentration increases byabout ten in each successively higher infection intensity level (Table 2). This progression is similar for infections rated in P+S and in M+C fortifications of the F.T.M. cultures. The most obvious break0 }.• Cr

• =C

CC CO 1.61LaINCIDENCE

NTENSITY 

### 100

90

80

70
ag

60Infection

50

40

c.) 30 C

-a • c.) 

### c 20

10 7 -

1.2

0.8 2 C

0.6 

## g

C)

0.4 C

0.2 12 FLORIDA DEPARTMENT OF NATURAL RESOURCES

in the pattern of decreasing numbers withincreasing infection is seen in the "medium heavy" and "heavy" groups (code 5 and 6) where the numbers are almost equal. This anomaly is probably validbecause these ratings are easily distinguished, and because the trend was apparent in both P+S and M+C treatments. Since oysters with "heavy" infections should experience a higher death rate than those with "medium heavy" infections, it would seem that numbers of heavily infected oysters would be substantially reduced, but this was not true. Possibly, the hosts were so debilitated by disease ofthis magnitude that the infections adFigure 3. Labyrinthomyxa marina infection by season.OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 

### 13

vanced very rapidly through "medium heavy" into "heavy." Oysters seemed to be removed from these infection groups only by death (see Analysis of Infection in Gapers), and it is the balance betweendeath rate and the rate of infection increase that ultimately determines the numbers of "medium heavy" and "heavy" oysters. R elationship of Labyrinthomyxa to Salinity: Many studies have shown Labyrinthomyxa to be inhibited by low salinFigure 4. Labyrinlhomyxa marina infection relative to temperature.

100 — 2.0

90



# F O 80

cD



### a , • 70



### 0
60UOIZ391U I

50

40 

# z

30

20

10

0 1.8

z t6

_ ▪

•

1.4 3

3

1.2 0 z _•

1.0

co tn

0.8 . c

0.6 fl

0.4

0.2

0. 0- 11- 16- 21- 26- 31- 36- 10 15 20 25 30 35 40

TEMPERATURE in ° 

# C

## Antibiotics in F.T.M.

1000

900

800

700 P+S

A M+C

U) 

## r, 600 (;)

500

.0 

## 41400

E



# z 300

200

100

0 6

1 2 3 4 Infection Intensity 14 FLORIDA DEPARTMENT OF NATURAL RESOURCES



### ities. Infection rates and mortalities due to this parasite are depressed in the lower salinity areas of Louisiana (Ray, Mackinand Boswell, 1953; Mackin, 1956). Similarly, the parasite is totally absent upstream of certain isohalines of Chesapeake Bay (Andrews and Hewatt, 1957). The inhibitory effect of low salinity has also been demonstrated in the laboratory (Ray, Mackin and Boswell, 1953).
In most respects, our data is not superFigure 5. Labyrinthomyxa marina infection intensity frequency distribution.OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 15

ficially in agreement with the salinityparasite relationship mentioned above, but there is no disagreement between our findings and those of previous workers ( concerning a positive salinity-parasitecorrelation) if the rationale we have devised is valid. It is also worth noting that the habitats we allude to as being "lowsalinity" do not meet the criterion required by one of us (Mackin) to be so styled. By this standard, in order for a habitat to qualify for low salinity status, the range must lie between ()V.'. s and 10./ • •. This is a rigorous definition which none of our sampling stations could meet. The listing of all oysters by salinity taken at the time of collection (Figure 6)reveals an unusual trend in which infections decrease to either side of a peak in the 6-15•/•salinity grouping. Such atrend might be apparent if some circumstance had caused the measured salinities to be abnormally low, but several factors tend to discount this possibility. The large number of samples and the regularity oftheir collection should have caused measured hydrographic values to average out very near the true mean values. Although data from one-time samples or someminimal samplings may have been abnormal, this should have been offset by data from the many other samples so that the net effect would have been to widen modal peaks rather than to change theirvalues. If any data prejudice was produced, it should have been in favor ofhigher, rather than lower, salinities because our hydrographic values were measured just above the bottom, the layer into which higher salinity waters moveearliest and leave latest under most conditions. In addition, most samples were taken by boat so that favoritism wasprobably given to time of high tide, usually the period of highest salinity in an estuary. Consequently, our salinity data appears to be representative of the areas under study and some other reason must be found for this apparent lack of support to past findings.Several circumstances relating to Florida oysters may explain this apparent contradiction: 1) our seasonal data showed that Labyrinthomyxa infection drops, but does not disappear, during the winter and rises very early in the spring, showing a great increase prior to May; 2) southern oysters are much more restricted to low salinities (Ingle and Dawson, 1953b; Menzel and Hopkins, 1954; Galtsoff, 1964) than their northern counterparts with commercial populations seldom found above 25°/ — mean salinity even in north Florida (Pearse and Wharton, 19 3 8) — Florida oysters are, therefore, more restricted to the propinquity offresh water sources; 3) Florida characteristically experiences a summer rainy season from June to September, and one exceptionally wet period occurred during our sampling; and 4) Mackin (1955, 1962) has shown that previously established Labyrinthomyxa infections fall only slowly, if at all, in the presence of reduced salinities. Consequently, the following cycle of events probably accounts for the relationship observed between salinity and infection. Young oysters become infected during or before the fall of their first year but fail to develop higher intensities due to winter. Infections in older oysters also drop during the winter, and most oysters start each new year with low infection levels. These established infections begin to increase and new infections start as water temperatures rise after the short Florida cold season. Substantial infections develop before the summer rainy season produces increased freshwater runoff and a resultant drop in estuarine salinities.During this lower salinity period, established infections tend to remain the same or even to increase slightly due to the high Florida water temperatures (Figures 8, 10, 15, and 17). As a result, low salinities may exist throughout the months when heavy infections are recorded. Data may thus be misleading, since summer low salinity infections actually developed at higher salinities. Data from stations sampled continuously (TampaBay composite, Figures 13 and 14; Sarasota Bay, Figures 15 and 16) supported this hypothesis. Dawson (1955) also found little effect of salinity on Labyrinthomyxa in Apalachicola Bay, Florida. Mackin (1962) noted incompleteness in Dawson's data and used information of

### 16 FLORIDA DEPARTMENT OF NATURAL RESOURCESuoiloajui



### 50 Figure 6. Labyrinthomyxa marina infection relative to salinity.



### 100

90

■ 

### z 0 80 

# a al

= n a) 

### 70

0 -I, 60

40 0.8 2.0

1.8

1.6

1.4uolloalui



### 0- 6- 16- 26- 31- 5 15 25 30 40

SALINITY in %o 

### at 30

20

10 0.6

0.4

0.2

0.0OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 17

Figure 7. Parasitism by Labyrinthomyxa marina in Suwannee Sound (lower salinity station 15).



### Intensity as W.I. A-- — 0 0 0 0

C) 

### 0

. 1 0

I I I I I I I I I I I 

### 0 0 0 0 0 0 0 0 0 0 0 

### 0 O) CO l'- CD 0 "4'M C•1

r - 

### % Incidence 111--111 

### 2 Cr)

u

## z

0

Ct 

### oa

a



### u_

z 0 U)

-,

2
u_



## z

0 (1) 

### tD 

## a 6018 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Ingle and Dawson (1953b) to argue that salinity had been effective. Since north Florida also experiences a summer wet season, it would seem reasonable that thesame events indicated by our central Florida stations also occurred in Apalachicola Bay. We also observed high infections and mortalities in two areas of continual low salinities. Station 15, near the mouth of the Suwannee River, had salinities ranging from 2.5 to 247.. with an average of 12.67 — (Figures 7 and 8). Although noepizootics were observed in monthly sampling from 1965 to 1969, gapers andboxes were present on numerous occasions and assays showed potentially lethal infections. The usual Labyrinthomyxa seasonal cycle was also demonstrated at this location. A similar situation occurred at the mouth of the Crystal River (Station20) which receives a continuous freshwater supply from deep limestone springs(Figures 9 and 10). This station had a salinity range of 0 to 30°/ 0. with an average of 12.7 0 /0. although salinity varied rather widely with each tidal change, due to the proximity of the Gulf. The incidences of infection in the Crystal River ranged near those at the Suwannee River but infection intensities ran significantly higher.

Factors known to promote Labyrinthomyxa infection were checked as possible causes of the high infections observed in low salinities at Suwannee and Crystal Rivers. Mackin (1961, 1962) and others have demonstrated that rapid and heavy infestations are promoted by reduced water circulation. Inadequate circulation,however, was not the case since both stations experience almost continuous, andoften rapid, water flow. It does not appear that chemical agents were responsible since both sampling stations were devoid of detectable pollution, domestic or industrial, with the exception of very low pesticide residues (Table 5; Butler and Springer, 1963; Butler 1966a, b). Other factors such as turbidity (averages of k 0.3 at Station 15, fc-- 9.3 at Station 20; Mackin and Hopkins, 1962), trace metal content (Table 6; Hammerstrom et al., 1967; Kopfler and Mayer, 1969; Pringle, et al., 1968), and freshwater sources (swamp fed Suwannee River and deep limestone spring fed Crystal River) varied widely and thus probably had no effect on infection.

Although it would appear that temperature must again be the controlling factor, Mackin classified these two stations as being high or medium salinity locations by his definitions. He classes locations as high, medium, or low, according to the highest salinity reached, rather than the mean, because it is this highest salinity at which infections will develop, and he has found that succeeding lower values willcause very little reduction in these established infections as mentioned previously. Infections, therefore, are enhanced by exposure to the highest salinities reached,even though the length of exposure necessary to increase infection is unknown, probably being less than three weeks. Noknown oyster areas in Florida would qualify as permanent low salinity locationsunder this definition because all are subject to occasional periods of higher salinity.

The high temperatures at Stations 15 and 20, as elsewhere in Florida, certainly contributed to maintaining or increasing established high infections. The maximum temperatures in most previous field and laboratory work have been 30 0 C or lessand the highest field temperature recorded in association with work on Labyrinthomyxa was 32"C (Ray, 1954). Summer water temperatures at our stations commonly reached 32°C and remained above 30 0 C for four months or more each year. Twenty-four regular samples fromcentral and south Florida came from waters of 35 and 39 0 C. Thus, it may be that temperatures in excess of 30°C not onlymaintain infection but induce a slow increase, even in the presence of lowered salinity. A tendency of temperature to dominate was also demonstrated in the converse situation where infections failed to proliferate at low temperatures even in the presence of highly saline waters, in both our study and others (Mackin, 1961; Andrews and Hewatt, 1957).OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 

### 19

Figure 8. Salinity and temperature in Suwannee Sound (Station 15).



### r I



### 0

------ <1---

_ I I I I I I 1 

### Lo 0 IA 0 in 0 Lc) 0 C.) cl cv of ,-- Temperature in ° C and Salinity in Voo 

### 0-0 41----4

### 20 FLORIDA DEPARTMENT OF NATURAL RESOURCES



### Relationship of Labyrinthomyxa to Oyster Size: Analysis of the distribution data with respect to oyster height (Figure11) revealed that both small and large oysters escaped the heavier Labyrinthomyxa infestations. By relating these oyster height groupings to oyster age (Figure 20, scale C') it can be seen that uniformly high infection ratings were present inthose height groupings containing substantial numbers of oysters in their second year. Mackin (1951) and Ray (1953) also found young oysters to experience low infections, and Ray even suggested thatsome form of immunity may exist. Laboratory studies by Andrews and Hewatt ( 1957), however, did not verify this hypothesis and they felt, with Mackin's ( 1962) concurrence, that smaller and younger oysters probably ingested fewer infective cells. Earlier work had indicated that the intensity of infection and its rateof development in adult oysters were diTABLE 5. PESTICIDE RESIDUES IN OYSTERS AT SELECTED

LABYRINTHOMYXA MONITORING STATIONS*

Pesticides (ppm)

DDT DDD DDE Dieldrin

Suwannee River (Station 15) High 0.011 0.0 0.012 Low 0.0 0.0 0.0 Mean 0.007 0.0 0.004 Crystal River (Station 20) High 0.016 0.0 0.012 Low 0.0 0.0 0.0 Mean 0.008 0.0 0.007

Manatee River (Station 75) High 0.040 0.088 0.042 Low 0.010 0.014 0.013 Mean 0.017 0.037 0.025 Sarasota Bay (Station 78) High 0.049 0.043 0.049 Low 0.0 0.010 0.010 Mean 0.018 0.026 0.025
Pirate Harbor (Station 

### 80) High 3.700 0.760 1.200 0.039 

### Low 0.0 0.0 0.0 0.011 Mean 0.028 0.029 0.011 0.020 Caloosahatchee River (Station 81) High 2.80 1.40 1.50 0.011 Low 0.0 0.016 0.0 0.011 Mean 0.58 0.26 0.37 0.007 *Analyses provided by Bureau of Commercial Fisheries Field Laboratory, Gulf Breeze, Florida.OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 21

rectly proportional to the number of pathogen cells received by the host (Ray, 1953; Mackin, 1962). However, it would seem that smaller animals would require proportionately fewer pathogen cells for infection. Consequently, we now feel thatother reasons for this apparent low infection rate of small oysters must be found. The presence of relatively low parasite intensities, even though incidences are high, among small oysters (Figure 11)indicates that environmental circumstances may have protected them. These young Florida oysters generally showed rapid growth and many apparently grew to 4 cm or more before intense infections had time to develop. Our measurements (267 oysters from Stations 23 and 43) showed oysters to average 6.9 cm at theirfirst spawning. Growth rates on commercial beds have usually been even greater with average lengths of 12 to 15 cm being measured in 19 month old oysters (Ingle and Dawson, 1953b) and edge growth of 1.9 cm occurring in 42 days (Ingle 1967). In addition, several investigations have shown a period of near two months to be required for lethal infections to develop in the laboratory (Mackin, 1962; Ray, 1954a, b; Ray and Chandler, 1955; Ray

TABLE 6. AVER AGE TRACE METAL CONCENTRATIONS IN OYSTERS

FROM SELECTED STATIONS*

Cu Cd M e t a

Cr Pb 1 s (ppm)

Hg Zn Fe

Suwannee Sound (Station 15) High 4.84 0.75 1.85 0.00 285.0 75.70 High 4.84 0.75 0.69 1.85 0.00 285.0 75.70 High 4.84 0.75 0.69 1.85 0.00 285.0 75.70 Low 2.50 0.31 0.24 0.67 0.00 122.9 40.00 Mean 3.77 0.53 0.39 1.17 0.00 212.1 42.81 Crystal River (Station 20) High 28.00 1.65 0.67 1.63 0.00 105.0 57.00 Low 13.00 0.86 0.24 0.56 0.00 71.0 40.20 Mean 17.20 1.19 0.39 1.13 0.00 86.8 46.40
Manatee River (Station 75) High 68.00 2.00 0.64 2.10 0.20 1084.0 57.00 Low 21.70 0.53 0.00 0.82 0.00 574.3 40.82 Mean 37.14 1.23 0.36 1.46 0.02 718.86 48.82
Pirate Harbor (Station 80) High 4.92 0.72 0.61 1.59 0.00 213.5 78.10 Low 2.37 0.32 0.00 0.76 0.00 145.0 49.30 Mean 3.73 0.50 0.33 1.12 0.00 173.4 65.00 San Carlos Bay (Station 81) High 8.50 0.89 0.52 1.70 0.00 200.0 76.10 Low 2.06 0.26 0.51 1.16 0.00 151.0 69.00 Mean 5.28 0.58 0.52 1.43 0.00 176.0 72.55

*Analyses provided by Gulf Coast Marine Health Sciences Laboratory, USDHEW, Dauphin Island, Alabama.

### 22 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Figure 9. Parasitism by Labyrinthomyxa marina in Crystal River (lower salinity station 20). Intensity as W.I. 

### A— — 0 C 1 1 N T 

### 0



# ❑

z 0

U)



## co 

# rn

Uz 

# 0

tt)



### 0 0 0 0 0 0 0 0 0 0 0 0 0) c0 t0 LO CN1 

### r- % Incidence 

# 0---4

# o z 0 41-____ 

# - IA 

# -- --- "1. 

# - a

■ 41 _ n (

„, 41- _ n Cr) T'

0 '

...

. 

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• ■■ 

# 1

-

-------- .7;70.1 <?.;:". -
• ..X1 ------- -4 -.. • OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 23

Figure 10. Salinity and temperature in Crystal River (Station 20).



# c:=

:: -- -

-- •=::::::ft 

# __a'

- In

- 0

.. .%1 - ct r.... -------

, - - 

# ..„ 4.......

.....

- 1

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' 

## _ " In 0 to 0 LO 0 10 0 0101N01 Temperature in ° C and Salinity in 0 /00 Li 

## ----d 

# .....

•

•



## ,-24 FLORIDA DEPARTMENT OF NATURAL RESOURCES

and Mackin, 1954), and infections have been found to develop even more slowly in the field (Mackin, 1962), thus further reducing the parasite intensity apparent in small oysters even though incidence may be high.Low winter temperatures may be a necessary integrant to this juvenile disease phenomenon, serving to reduce infectionintensity in young oysters, since even oysters set during the first spring peak (May) must survive only four months before water temperatures begin to fall. Data from Station 43 showed an average age of 8.2 months at first spawning in April andMay. Spat set occurs throughout the summer months; therefore, most of the 8.2month prespawning juvenile period occurred during the winter. This allowed only a few summer months for both thecontraction and intensification of the disease before proliferation was attenuated or arrested during the colder months. Thus, small oysters probably contract Labyrinthomyxa, but a combination ofrapid growth rates and cold weather precludes the development of substantialinfection intensities. Observations indicated that, once established, infectionsincreased nearly as rapidly in young oysters as in older ones.
Lower infection rates and intensities were also observed in larger, generally older oysters (Figure 11). Further analysis of the 113 oysters in the 13-21 cm group revealed that only a single heavy infection (0.9%) was assayed in contrast to 42 heavy infections (2.8%) among the 1,486 oysters in the 4 through 12 cm groups. Only 7 heavy infections (1.8%) were found among 377 oysters in the 11 through 12 cm length grouping. It would appear that oysters have some form of acquired or innate resistance to Labyrinthomyxa infection after their first year as adults. Relationship of Labyrinthomyxa to Oyster Depth: Analysis of Labyrinthomyxa distribution data with respect to the depth relative to mean low water atwhich each sample oyster was living (Figure 12) revealed that disease incidencewas similar at all depths, being only slightly higher in the upper littoral zone. The average infection intensities, however, increased with decreasing depth. The harsh conditions of intertidal life, such astemperature extremes and shortened feeding periods, may have been responsible forthis increased parasitism. In a later experiment, temperatures of the tissues of exposed oysters were measured by placing minute thermistor probes inside the shells of living animals. These temperatures were found to vary from 10 ° C above theair temperature in oysters exposed to direct sunlight to about 2°C below air temperature in oysters exposed on a clear night. Diseased intertidal oysters may also become autoinfective during long periods of closure if the parasite cells contained in leucocytes voided by diapedesis are still viable and infective. Therefore, intertidal oysters experience severe environmental extremes which seem to be reflected not only in their small size but in their high incidence of Labyrinthomyxa infection. Relationship of Labyrinthomyxa to Oyster Condition: Distribution data indicated that disease incidence and intensity increased steadily as oyster condition worsened (Figure 13). However, since condition index is closely correlated with seasonal changes, this observation might be coincidental. In fact, oysters in very good condition (code 9) have occasionallybeen found harboring very high intensities. Nevertheless, most of these oysters with highest intensities were in poorer condition than the contemporary average. This lower quality may he due to Labyrinthomyxa.
Annual Labyrinthomyxa Parasitism at Selected Sites: Figures 14, 15, 16, and 17display systematic hydrological and parasite data taken at two different localesduring the distribution study. As in Figures 7, 8, 9, and 10, the typical summerinfection highs and winter low are obvious. These two graphs are significant because heavy mortalities were experiencedat both stations during epizootics in September 1968 and are discussed later. Thesalinities and other environmental parameters are more typical than, and provide good contrast to, the low salinity oyster areas illustrated in Figures 7, 8, 9, and 10.

### 2.0 Figure 11. Labyrinthomyxa marina infection relative to oyster height (length). —



### 1.8

1.1 

### 1.6 -,,

CD 

### 0 p4 

# a 1.4 3

0 rpi



### 0.6

0.4

0.2

0.0 0- 3 4-

5 6-

8 9- 10 11-

12 13-

21
--Juveniles One Year Old Adults—Two Years & Up—



### OYSTER LENGTH in cm. OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 25

### 100

90 —

80 —

70 —

60--

50—

40



## 5 30 at



# 111 20

10



## 0uolpajuiJoaouappui



### a) co

0.8 co co'

8

. 0.6 a

c.)

0.4 a o iii:
3 0 M 26 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Figure 12. Labyrinthomyxa marina infection relative to depth.



### Above MW- 0.4- 2.0- MW 0.3 m 1.9m 3.0m
DEPTH

### 2.0

1.8

0.6

0.4

0.2

0.0 20

10



## 0 100

90

80

70

60

50



# 3 40

30aouappul

0uolloalu OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 

### 27

Figure 13. Oyster condition and associated infection by L. marina



### 1 2-5 6-7 8-9
CONDITION INDEX

## g

28 FLORIDA DEPARTMENT OF NATURAL RESOURCES Figure 14. Parasitism by Labyrinthomyxa marina in Tampa Bay oysters (medium salinity stations

18-48). Note infection peaks associated with general epizootic of late summer, 1968.

Incidence of Infection in

0 81

. F) (.•3 A (Ti Cr) 0 0 0 0 0

DECEMBER _ I I I I I I I ok

... „....0 -' ...I CO t 0 0 0 0 I I I 

### I

AUGUST -
SEPTEMBER JANUARY -
FEBRUARY
MARCH -
APRIL -
MAY - —L. JUNE co C) JULY - a) AUGUST -
SEPTEMBER -
OCTOBER -
NOVEMBER -
DECEMBER -
JANUARY -
FEBRUARY
MARCH -
APRIL
MAY —L. CD JUNE Cr) JULY V AUGUST -
SEPTEMBER
OCTOBER
NOVEMBER -
DECEMBER -
JANUARY
FEBRUARY -
MARCH -

APRIL -
MAY - 1 CD JUNE -
O IULY - O AUGUST -
SEPTEMBER
OCTOBER
NOVEMBER
DECEMBER -
JANUARY -
FEBRUARY

- MARCH -
APRIL
MAY - _L. CD JUNE - O IULY - O



### I I I I 1 I l 2 o ..I. - na n.) P 50 3 A o (Ti 6 Ln

- 0 al o a, o Infection Intensify as W. I. Ar------I

=MI.. 

### co (0 CD 1—



# _.

M

c:C 

### M

Li

### N-- to C) 

### 1— OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA

Figure 15. Salinity and temperature in Tampa Bay (Stations 28-48). 29



### L I I I I I I 1 

## 1

o ul 

## o us) 

## o us) 

### o in 0 

### Tr el C) 01 Cd /-- r_ Temperature in ° C and Salinity in 0 /00 A- - - -A O

-- 

### -

o

### —L _A. K.) N W W 

### 42.

Infection c.71



### Intensity as W. I. 

### 30 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Figure 16. Parasitism by Labyrinthomyxa marina in Sarasota Bay (higher salinity station 78). A

severe epizootic occurred here in September 1968.



### Incidence of Infection in o io rs. .....04
_L IQ CA.) .0. 

### al a) ".1 CO 

### tO 0 0 0 0 0 0 0 0 0 O — L 

### O O

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### UOYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 31

Figure 17. Salinity and temperature in Sarasota Bay (Station 78).



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1 32 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Figure 18. Spatfall at two stations during 1969 expressed as macroscopic spat per adult oyster shell. 

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1969

MAMJ J ASOND

1969OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 33

Part II: Studies of Oyster Mortalities

YEAR CLASS ANALYSIS AND ANNUAL

MORTALITY
Mackin (1961) successfully used shell heights to establish year classes of oysters and, from these (assuming a constant annual spatfall), calculated past annual mortalities. No such year class patterncould be determined in the Florida oysters tested. This lack of distinct year class height differential is probably due to acombination of rapid growth rates discussed previously and an extended setting period of over seven months each year with peaks in spring and fall. Ingle and Dawson (1953b), in extensive studies inApalachicola Bay, found setting commonly from April or June through October or November each year. Ingle and Dawson (1953a) observed oyster larvae in theplankton during every month except January, February and March in Cedar Keys and cited year-round spatfall during some years in this more southerly location. We made limited spatfall observations at the Tampa Bay area stations 23 and 43, still further south, by simply counting the macroscopic spat on each oyster in the regular monthly sample. Since larger spat counted in any sample may have actuallyset more than one month prior, and because microscopic spat were not counted, the data obtained is not comparable to the more precise methods of Ingle andDawson (1953a,b). Nevertheless, our studies did show significant setting for ninemonths of the year and some set throughout the year (Figure 18), thus effectively masking the size differential between year classes. This, coupled with very rapid growth rates, undoubtedly prevents any year class determinations based on length (height) frequencies.

Determination of Oyster Age by Shell Contours: Since year classes could not be separated by length frequency, another method had to be developed. Analyses of shell growth rings indicated that they could be utilized with some degree of accuracy to separate year classes. As oysters grow, minute edges are formed approximately parallel to the generating curve and perpendicular to the direction of growth.These "micro-annuli" are the distal margins of prismatic layers of shell (Figure 19a, b, c) and are approximately 0.1 mm thick. Counts of micro-annuli in oysters of known age, and even oysters of the same age growing adjacently, indicated thatthere was no set rate or regularity of formation. In addition, the counts variedalong different radii of each oyster because individual micro-annuli were notcontinuous around the shell circumference. Nevertheless,. the average distancebetween micro-annuli increased with increasing growth rate. Conversely, the distance between micro-annuli decreased during periods of slow growth and individual annuli were sometimes even repressed under the edge of overlying shell layers during periods in which no growth occurred. The thickness of individual micro-annuli became greater during warm seasons, especially during periods of spawning. During rapid growth, the increased width plus the decreased micro-annulus thickness results in wide, relatively smooth expanses of shell. Slower summer shell growth is characterized by rough areas composed of numerous, closely packed, irregular, thickened micro-annuli. Thickening of the prismatic shell layers in spawning oysters accompanied by slowing of growth usually produces a "spawning shelf" along the generating curve (Figures 2 and 19e,f,). This distinctive shell feature was found to be a reliable indicator of spawning in central Florida oysters.

Aging Technique: It was found that oyster shells could be aged with 80% accuracy to within four months by using observations of micro-annuli and shell layer trends along with general information on growth rates and spat set intensities. A slide rule-like device was constructed as an aid to age estimation (Figure 20). Its stationary arms carry five scales as follows: A, a scale of time calibrated in months and years from left to right which serves both for calculations and as the horizontal time axis for all34 FLORIDA DEPARTMENT OF NATURAL RESOURCES

graphic scales; B, a graphic representation of typical micro-annuli configurations as they would have been laid down in adult oysters over each year represented; C, a graphic representation of generalized growth rate for reference; D, a graph of the average oyster death rate for reference; and E, a graph of the average spat set rate for reference purposes. The movable slide carries two scales: F, a scale of time identical in spacing to Scale A, but calibrated in consecutively numbered months, years, and fractions of years from right to left; and C,' a scale of measurements giving the average oyster size at various ages. In use, the slide is placed so that 0 on the F scale is directly opposite the date on the A scale on which the test oyster was killed or on the current date if the oyster is alive. The appropriate date on the A scale closest to the right end is selected. The hairline is then moved from 0 on the F scale over all micro-annuli configurations present on the test oyster as illustrated on the B scale. Mirco-annulus configurations are matched in order from the growing posteroventral margin toward the beak of the test oyster. When the hairline reaches the point on the B scale comparable to the growth the test oyster produced as a postlarval spat, the age is read from the F scale under the hairline in months and years. This instrument and system were designed to use with the right (upper) valve since this one is usually flatter and its more linear growth allows more detail to be seen. The left valve was sometimes used but curvature, oblique growth, and substrate influences had to be considered. Experience and familiarity with oysters are of great assistance in using this system. Scales B and C, for example, are repetitions of 12-month cycles based on first year adults but, as seen on scale C', juveniles grow much faster and do not produce a spawning shelf, while older oysters grow more slowly. In addition to changing with age, individual oysters vary widely and the investigator must recognize each oyster as an individual problem and usecare and discretion in matching microannulus configurations and growth rates with the generalized scales. Even when oysters are carefully aged, the results must be considered accurate only to the values stipulated previously (80% of the oysters to within four months). This method of year class separationfor the calculation of annual oyster mortality was initially field tested in conjunction with experiments which includedmortality determination by counts. Average annual mortalities obtained by comparison of year classes varied only 4.3% from those calculated from periodic counts of the same oysters over three one-year periods at Stations 23 and 43. These results also agreed well with thoseof another method of mortality estimation based on gapers. General Results of Year Class Analysis: The results of mortality calculations from year classes established by shell contours demonstrated that the annual mortality of Florida oysters varied greatly betweenyears and between different locations during the same year. The mortality variation at Station 43 is typical, with annual deaths near 52% in 1965, 87% in 1966, 70% in 1967, 98% in 1968, and only 49% in 1969. The rates at Station 23, however, were 61% in 1967, 91% in 1968, and 100% in 1969. These rates were extremely high, averaging 71% at Station 43 whichseemed to be typical of the central Florida area tested. The fact that medium tolarge oysters were always present in abundance at Station 43 in spite of these substantial death rates is further evidence of the rapid and continuing recruitment and growth in Florida.The validity of this type of annual mortality calculation in Florida is hampered by the extreme variability in abundance of spatfall and the high rate of mortality causing few oysters to survive past two tothree years. These difficulties were minimized by sampling each area annually,preferably between October and February. This allowed field determination of annual spatfall and mortality from only one sample and was much simpler than the tray and plot methods of mortality determinations. Shell contour analysis also providedFigure 19. Oyster shell configurations. A - photomicrograph of a prismatic shell layer forming by growth of calcium carbonate crystals on a conchiolin membrane; B - a completed prismatic layer at the growing edge of an oyster shell (phase contrast); C - the lip of an oyster showing the intact prismatic layer edges or micro-annuli stranded on the surface of the shell by progression of the growing edge; D - micro-annuli as they appear after weathering has destroyed the delicate edges; E - two oysters collected in October at ages of 14 and 12 months, upper and lower, showing spawning shelves (scale in cm); F - an 18-month old oyster showing a particularly prominent overhanging spawning shelf on the right (upper) valve. 

## 100 p 100 p

A36 FLORIDA DEPARTMENT OF NATURAL RESOURCES



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FOYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 

### 37

Figure 20. The oyster age calculator. A - calculator; B - working scales of calculator set for a 26-month

oyster which set during May and was aged or died in July.

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preliminary information on first spawning times and sizes, number of seasons in which spawning occurred, survival, and growth data. Although the spring setting peak resulted in more spat than the fallpeak, the majority of yearling oysters surviving until the following summer were members of the fall set. About 50% of the Florida oysters which set in April or May spawn in September or October of the same year (4 to 5 cm height). About 5% of the oysters setting in summer or fall do not spawn the following year even though they are of adult size. Oysters whichspawn generally grow less than nonspawning oysters of the same size.

ETIOLOGY OF NATURAL OYSTER MORTALITIES

Plot tests were used to determine the oyster mortality caused by Labyrinthomyxa and the effect of other parasites,predators, and physical or chemical conditions on oysters. First Year of Plot Studies: Data wastaken from paired plots maintained at Station 23 from early March to early September 1967 (Figures 21, 22, and 23). Twolater plot tests at this location were tampered with and had to be terminated. Basically these oysters showed the usual warm weather increase in Labyrinthomyxa parasitism, even during loweredsalinities caused by an unusually wet central Florida summer. The greatest mortalities closely paralleled rises in the weighted incidence of the fungus and amounted to 57% in the open plot and 44% in the closed plot during the six-month period.Data from other stations cited above indicated that these high death rates were representative of or lower than usual summer mortalities among adult oysters in Florida.Mortality due to predation would normally be represented as the difference between the percentages of death in the open and closed plots, but certain factorsprevent this from being completely accurate. For example, small predators such as Thais were occasionally found in the enclosure. Other organisms, such as Stylochus, usually classified as scavengers, are possibly significant as predators of weak oysters and could also have entered the closed plot. In longer plot tests, predatorsmay also have entered as larvae or juveniles and grown to destructive size. Independent observations in Louisiana by Mackin indicated that the zinc in new galvanized oyster trays (and thus possibly the plot fencing) caused slight oyster kills; however, the majority of our fencing was not new. Observations indicated that predation of adult oysters in the enclosed plot was negligible, although one adult blue crab, Callinectes sapidus, and seven small Thaiswere found in the plot. Both plots harbored the usual smaller fauna associated with oysters. ExaMination of empty shells provided direct evidence that substantial predation did occur in the open plot. 'Total adjusted mortality was 13% greater in the open plot during the entire test, but the month-to-month difference between the plots varied widely (Figure 23) and mortality was slightly greater in the enclosed plot during June and July. Stone crabs were a significant predator in this study, accounting for 4.1% of the verified mortalities in the open plot. This crab probably also caused a portion of the unclassified loss (shells not located) by crushing shells beyond recognition or by carrying them off, as reported by Menzel and Hopkins (1954), since local currents were not sufficient to remove them. The area appeared to be relatively free of other predators of adult oysters because a 0.7% loss to conchs was the only other evidence of attack. A slight mortality occurred in June, but no marks were left on shells and infection intensities were very light; no cause could be found. Second Year of Plot Studies: A fourth set of plots was established at Station 43 the following year (1968). The summer of 1968 was unusually sunny, hot, and dry and estuarine temperatures and salinities climbed to high levels. These conditions apparently contributed to an epizootic in this test plot (Figures 24, 25, 26) which killed so many oysters that work had tobe discontinued for lack of survivors. Although the mortality was high (80%), it4 . 0

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### 1968 OYSTER PARASITISM BY LABY RINTHOMYXA MARINA IN FLORIDA 41

Figure 24. Labyrinlhomyxa marina infection during Station 43 plot test.I 

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### 42 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Figure 26. Unadjusted oyster numbers and mortalities during Station 43 plot test.

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II ° OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 43



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### Infection Intensities44 FLORIDA DEPARTMENT OF NATURAL RESOURCES

occurred over a period of almost two months. These new plot tests substantiated some of the previous results. The stone crab was again shown to be the major predator, but there was more evidence of predation by gastropods with Busycon contrarium and Melongena corona often observed in the open plot. Blue crabs, Callinectes sapidus, and other smaller crabs and fishes seem to attack only young oysters under 3 cm in height; thismay be beneficial in many areas of Florida where the heavy spatfall often produces serious overcrowding. The oyster "leech," Stylochus frontalis (Pearse and Wharton, 1938; Galtsoff,1964) was observed in the plots and elsewhere preying on very young oysters and barnacles. This polyclad worm was also found feeding upon many adult oysters, but in all Labyrinthomyxa assays of the host oyster, a potentially lethal infection was found. Thus, this leech appears to be a predator of young oysters but only a scavenger of adults. A slight mortality of unknown origin occurred in both plots in early June, as observed the previous year at Station 23.It is possible that these light June mortalities may have been caused by an undescribed parasite, currently being investigated by Mackin. Another unexplained series of events occurred in the plot tests at Stations 23 and 43. Around September 1967 and 1968 (at the different sites) mortalities in the closed plots exceeded those in the open plots. This unusual occurrence was coincidental with highest Labyrinthorny xa mortalities (reaching epizootic proportions at Station 43). Other workershave observed similar occurrences in mortality studies using oysters maintained in closed trays (Owen, 1955; Andrews andHewatt, 1957). Possible contributing factors are: 1) reduced current, such as may have resulted from summer fouling of the enclosures, has been shown to increase infection (Mackin, 1962); and 2) dying oysters which cannot be scavenged may liberate greater numbers of infective spores as the tissues decay (Valiulis and Mackin, 1969). Plot tests indicated that predation is a relatively minor cause of adult oyster mortalities in central Florida. Labyrinthomyxa, however, causes considerable mortality.

DETERMINATION OF MORTALITY RATES OVER SHORT PERIODS

Observations indicated that gapers were present in larger numbers during periods of higher mortality and attempts were made to quantitate the relationship to provide another method of determining oyster death rates. Gaper counts were made at the Station 23 plot test as a pilot study and results were encouraging. Box counts made for comparison varied widelyas noted by Mackin (1961). Since the preliminary tests indicated that this method had potential, further and more detailed tests were conducted at Station 43. The average numbers of gapers (animals unable to maintain closure of shells but with meat present) per square meter in 10 meter square areas were compared with the average number of living oysters per square meter in the same area. The ratio of gapers to living oysters was then compared with the known death rates from plot tests and a conversion factor was calculated. The following relationship resulted:

CG>K=

where G = gapers/m 2 , L = living oysters/m 2 , K = a conversion constant, and M is the -tokareowt oyster mortality/week. The value of K was calculated to be 6.2 in this
test. Further applications of this techniquewere made at several locations where mortalities were high. Back calculations ofmortalities using estimates of original populations and the counts of oysters and gapers per square meter indicated that gaper counts provided accurate evaluationof current mortality rates if these exceeded 10% per month. The value of theOYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 45

conversion factor, K, seemed to be somewhat temperature dependent, decreasing with temperature, but the average value at 20 to 31 °C was 5.5. The value of K is related to the length of time a gaper may exist. Since colder temperatures slow bacterial decay and usually reduce the activities of scavengers,gapers (which by definition must still contain oyster tissue) can persist longer under cooler conditions. Calculations of gaper "life" indicate an average of about 17 hours from the time the oyster ceases tomaintain shell closure until no more tissues remain in the shell. This figure is based on a daytime temperature of about 30°C. However, boxes (shells still hinged but with no tissues remaining) were found to persist from one hour to over 60 days and maybe much longer.

ANALYSIS INFECTION IN GAPERS Data of infected gapers (Figure 27) shows that the lethal level of Labyrinthomyxa varies among individuals but was usually "medium heavy" or above. Dataalso indicated that lower infection intensities may have resulted in mortality inthe early fall, apparently because of oyster debilitation by spawning. In addition, many gapers assayed for Labyrinthomyxa showed little or no infection. These oysters were undoubtedly dying from other causes as yet undetermined.

DESCRIPTION OF A LABYRINTHOMYXA EPIZOOTIC
A very short but severe oyster epizooticoccurred in late September 1968 at Station 78. A sharp rise in infection rate accompanied by the appearance of a fewgapers in July 1968 prompted us to increase the sampling frequency, first to biweekly and then weekly, continuing into the following winter. Determinations of gapers per square meter were also begun,
The incidence of Labyrinthomyxa rose rapidly from 18% in May to 100% in July (Figure 28) and continued near that level until after the mortality. The infection intensity lagged behind the incidence, increasing slowly from 0.8 in June to 2.0 in September, and then advanced rapidly to over 4.0 in two weeks and remained near that extremely high level into the epizootic (about four weeks). Death rates calculated from gaper counts varied but showed a distinct rise during the month just prior to the massive oyster kill. During the oyster epizootic, incidence of infection dropped rapidly and thencontinued to fall slowly until the last sampling of adult oysters in November. The new young oysters on the bed then tookup the normal winter cycle of low incidence starting at 20%. The intensity of infection in living oysters also dropped rapidly during the kill but appeared to be rising again by the November sampling. These declines are undoubtedly caused by the death and removal of oysters from the living population. Hypnospore enlargement in F.T.M. just prior to and during the oyster epizootic was very meager, particularly in tissues from gapers. Small 5-15 ht hypnospores, which stained slowly and seldom got darker than a translucent blue, resulted. Small hypnospores were often mixed withlarge in varying proportions with intermediate sizes and colors also being observed occasionally. Although small, these hypnospores were extremely numerous and produced infections many times as intense as any previously rated.
One possible cause of these unusual minute hypnospores was the infection by an organism other than Labyrinthomyxa marina or by another organism concurrently. Culture studies supported this hypothesis indicating the presence of a Labyrinthomyxa very similar to L. marina but with a distinct sporulation pattern. It is possible that a substantial number of the oysters killed at the height of the epizootic succumbed to this undescribed organism. As mentioned earlier, Mackin has isolated several other closely related Labyrinthula oyster parasites. One ormore of these forms may have been responsible for disease and death during this epizootic. Several of these do not respond46 FLORIDA DEPARTMENT OF NATURAL RESOURCES

to the F.T.M. assay we used but can be found only by histological preparation or culture. The peak of this massive epizooticoccurred just prior to sampling on September 11, 1968. At that sampling the odor of rotting flesh was strong at the beds and strings of bacterial scum weredraped over the exposed oysters. The oyster bar was littered with fresh shells, boxes, and gapers in various stages ofdeath and decay. Small mud snails, particularly Nassarius, were numerous, especially in and on the many fresh boxes. Small crabs, primarily young blue crabs,were unusually numerous and were feeding on gaping oysters. Schools of small fish were prevalent in the water around the oyster bar. The following week (week 10), the bar was apparently beginning to return to normal but several large and small blue crabs were still feeding on dying oysters. The mortality rate, however, had dropped only slightly from the previous week'ssampling and fresh boxes far outnumbered living oysters. The small remnant population continued to suffer significant mortality and from the second week of October on, several hours were required to find enough oysters for a sample. Two persons searched the bar four hours and were able to find only ten adult oysters for the November sample. The mortality was essentially complete. However, a heavy spatfall had begun just prior to the epizootic and continued into October; the spat grew rapidly and provided material for subsequent samples. By the followingspring, the oyster population had essentially fully recovered. Additional Oyster Epizootics: Several other oyster epizootics were visited andsampled during late August and September of 1968, and observations indicated that from 80 to 97% of the oysters in stricken areas were killed. The death ratesvaried widely, however, even between different locations along a single oyster reef. We would estimate that over 60% of the Gulf coast from Apalachee Bay southwardto the Florida Keys was ravaged by epizootics. Mortalities were also reported from the Atlantic coast but were not confirmed. Of all the oyster kills observed, that atStation 78 was the most rapid, the majority of deaths occurring in less than two weeks, and most complete with essentially the entire adult oyster population beingkilled over a wide area. The observed characteristics of other kills were similar, with oysters of less than about three months old or 3 cm long seemingly unaffected. This combined with a heavy fall spat set to prepare the beds for rapid rejuvenation Damage to Oysters from Labyrinthomyxa: The data gathered in the severalstudies described has repeatedly incriminated Labyrinthomyxa as the cause of major oyster mortality in Florida. The parasite alone is generally responsible for a 50 to 60% annual mortality among the adult population and is, therefore, a majorfactor in the limitation of oyster populations in Florida growing areas. In additionto this normal yearly enzootic destructiveness, this pathogen is capable of causing massive epizootics. Also, the synergistic effect of this fungus probably causes many nonlethal situations to result indeath and may accelerate other lethal conditions. For example, most of the earliest oysters which died at Station 23 after a salinity drop to 07 o o (1969) showed L. marina of "medium" to "heavy." Unusually high parsitism was also encountered in the severely polluted Pensacola Bay area (Hopkins, 1969; Butler, 1969) during January and February of 1968 and 1969. The parasite greatly affects the oyster industry in Florida, as well as in other states (Mackin, 1962; Ray, 1954b; Hewatt and Andrews, 1954; and others). Labyrinthornyxa not only selectively and severelyreduces the numbers of marketable oysters but prevents mass production oflarger, older oysters. In addition, the destruction of oysters after their first spawning hinders natural development of resistance because deaths before the second spawning are so numerous that disease - selected breeding stock have little or no chance of competing reproductively with the first year unselected breeding stock.OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 

### 47

Figure 28. Labyrinthomyxa infection and oyster mortality during a severe epizootic in Sarasota Bay

(Station 78).



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Fortunately, the effect of this parasite on the oyster industry is restricted to the oyster itself. Labyrinthomyxa marina isno direct threat to man from eating infected oysters (Hoese, 1964). Various project personnel consumed the raw oysters remaining after sampling on numerous occasions with no ill effects, although analysis often showed both high incidence and intensity of L. marina.

THE REDUCTION OF LABYRINTHOMYXA DAMAGE TO COMMERCIAL OYSTERS Many methods of protecting oysters from Labyrinthomyxa have been proposed, but most depend on the inhibitionof the parasite by continuously low salinities and would apparently not work in Florida. Juvenility and low temperature were the only factors we found to limit Labyrinthomyxa parasitism of oysters. Juvenility obviously provides only a shortreprieve from the fungus. Avoiding dangerously high temperatures for extended times is, unfortunately, also impossible in Florida. Even in Apalachicola Bay in the northern part of the state, temperaturesregularly remain above 25°C from midApril until mid-October and commonlyexceed 30°C from mid-July until September (Ingle, 1951). In Cedar Keys, temperatures averaged over 25 ° C from mid-March to late October and often exceeded 30°Cfrom late May until early September during 30 years of monitoring (Ingle andDawson, 1953a). Afternoon water temperatures in Tampa Bay in central Florida average above 25°C from late May until mid-November and average above 30°C from mid-April until mid-October, evenexceeding 34°C during June through September. Temperature further south couldbe expected to surpass these values. Additionally, minimum temperatures are insufficient to cause disappearance or even serious attenuation of established infections since the coldest month averaged 13.5°C in Apalachicola (Ingle and Dawson, 1953b), 15.1°C in Cedar Keys (Ingle and Dawson, 1953a) and 15.4°C at Tampa Bay Station 43. A plan for growing oysters to avoid extensive mortalities from Labyrinthomyxa marina and other causes wasformulated based on the following factors: 1) most Florida oyster areas receive spatfall throughout the summer; 2) since several months are required for young oysters to develop their first infection, these oysters are temporarily safe, even when exposed to summer temperatures;3) several months are required for infections to intensify to lethal levels in both old and young oysters after thewinter low; and 4) Florida oysters are capable of extremely rapid growth under favorable conditions. Consequently,oysters spawned in midsummer would experience little mortality before winter andlittle more until midsummer of the following year, thus providing a year of growth with low mortality. The onlyother problems were obtaining an adequate set at the proper time and inducinggrowth rates sufficient to produce Marketable oysters in the necessary 12-month period.
This method was utilized in the Indian River in 1968-69 and proved extremelysuccessful. Spat were collected in midsummer on subtidal cultch in an area with relatively high salinity and good current. After two to three months the oysters were 3 to 5 cm in height (length) andwere moved to a carefully selected growing area with a narrow range of salinity(Ingle and Dawson, 1950) near 257.0(private lease) where they were evenly spreadover the gravel and shell bottom. Harvesting was initiated in May, prior to heavy spawning, when the oysters had good shape, little fouling, excellent quality and averaged 12 cm in height (Figure 29). Additional harvesting was done in July when the oysters were of slightly reduced quality but averaged 15 cm in height. The apparent success of this method in one isolated incidence is indicative, butfurther testing is needed to verify its usefulness. In addition, the application of other knowledge of oyster biology could further improve the method. Current data indicates that successful application of these methods could yield a mortality from planting to harvest of only 20%.Excellent quality and substantial production are also indicated.OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 49

SUMMARY

A new parasite of the American oyster, Crassostrea virginica, was described in 1950 by Mackin, Owen, and Collier and named Dermocystidium marinum. As a result of extensive studies, this parasitic fungus was assigned to another genus and became Labyrinthomyxa marina (Mackin, Owen, and Collier) Mackin and Ray (1966). Several authors demonstrated extensive oyster mortalities caused by this parasite in many areas, notably Louisiana and Texas. A simple, quantitative assay for L. marina in the host was developed by Ray (1952b), and an adaptation of this fluid thioglycollate medium (F.T.M.)culture technique has been used extensively in testing Florida oysters since 1965.
Antibiotics were originally used in F.T.M. assays to control contaminationbut they also affected the essential enlargement of the parasite cells. Ray's (1966b) findings that mycostatin induced enlargement of the individual parasite cells and caused a greater proportion of the cells to enlarge were confirmed by our studies. Duplicate tissue cultures showed parasite cells (hypnospores) averaged 21% greater in diameter when cultured with mycostatin plus chloramphenical (M+C) than when cultured with penicillin plus streptomycin (P+S). We also found that about 30% more cells enlarged in M+Cthan in P+S cultures at low infection intensities but this percentage decreased as infection intensities increased. Although M+C improves the accuracy of the assay, it has some deficiencies which, under certain conditions, make P+S fortification preferable.
Extensive sampling of oysters from over 100 stations demonstrated that L. marina was present in oysters throughout both the Atlantic and Gulf coastal areas of Florida. The results of sampling over several years revealed that L. marina goes through annual cycles of low winter and high summer incidences and intensities similar to those observed in more northerly Gulf states. Infection rates and intensities in Florida, however, remained substantial throughout the winter and began a rapid rise toward summer peaks much earlier (February).
A strong positive correlation was demonstrated between temperature and infection in nature. The relationship applied toinfection rates and intensities and conformed with the findings of many workers (Mackin, 1962) for field and laboratory studies. Infection intensities generallyincreased gradually through the "medium" rating but increased rapidly through "medium-heavy" and into "heavy." The reasons for this occurrence are not known but it perhaps indicates a decline in theefficiency of the host's defense mechanisms.

It was demonstrated that after infections have developed, high temperatures can maintain or increase these intensities, even in the presence of low salinity. Inareas of relatively low salinity, short periods of higher salinity may combine topromote infection increases. These findings establish the overriding importance of temperature rather than salinity as an infection promoting mechanism.

Juvenile oysters were not found to have special immunity to L. marina. The failure of juvenile or yearling oysters to suffer loss from this parasite is probably because the time between setting and fall cooling is insufficient for lethal infections to initiate and develop. In addition, oyster growth rates are so rapid in many areas of Florida that spat can grow to adult-like sizes of 4-5 cm height during the several months required for lethal infection to develop.

The analysis of infection among oystersof various age groups indicated that individuals of two years or older, although very few in number, possess some form of special resistance to 

### L. marina. These oysters had fewer and lighter infections.

Infections were found to be greatest in

### 50 FLORIDA DEPARTMENT OF NATURAL RESOURCES

Figure 29. Oysters grown by the mortality minimizing technique. A, B - ten month old oysters harvested in May for best meat quality (scale in cm); C, D - oysters harvested at larger size but lower meat quality in July after 13 months of growth; E, F - survivors harvested at the end of the summer with severe fouling and boring damage after 18 months.OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 

### 51

Figure 29. (Cont.)52 FLORIDA DEPARTMENT OF NATURAL RESOURCES

both incidence and intensity in high intertidal oyster, diminishing with increasing depth. Although data indicated that moreheavily infected oysters had poorer condition, the correlation was probably false,being produced by the coincidental debilitation of oysters by summer spawning rather than by Labyrinthomyxa. Severe infections ("medium heavy" and above), however, do seem to cause sporadic, andsometimes extreme, lowering of host condition.
Mackin (1961) used size frequency analysis to establish year classes from which he determined annual mortalitiesfor Louisiana oysters. Attempts to determine year classes of Florida oysters by this method were unsuccessful because no dependable size modes were present. This lack of size modes seemed to result from the long Florida spawning season and rapid growth rates.
Further efforts to delimit year classesfor mortality calculations led to the development and testing of shell contours as a method of aging oysters. The system was simplified for routine use. Calculations demonstrated that oyster mortalities vary greatly from year to year. The analysis of shell configurations was also used to determine the season each oyster set and the number of times it spawned. This data indicated that the survival rate of oysters setting in fall was significantly greater than of those setting in spring.

Field tests indicated that the normal annual mortality from L. marina parasitism is about 50% of the adult population. Deaths from other causes averaged about 15% of the adult population. The stone crab. Menippe mercenaria, was the most active predator in the areas studied, accounting for a four to eight percent annual adult oyster kill. Significant spring mortalities of unknown origin occurred. All adult oysters found to contain the oyster "leech," Stylochus frontalis, were in advanced stages of L. marina infection.

The ratio of gapers to living oysters present at any time in an area was found to be quantitatively related to the short term oyster mortality rate. The method of determining the gaper/living oyster ratiowas standardized and a conversion formula to mortality rate is presented. Fieldtesting indicated that the conversion factor was temperature dependent because individual gapers were present for longer periods at lower temperatures.
Several epizootics caused by L. marina were observed and one kill was studied extensively. The oyster mortality from these infrequent occurrences was severe and rapid. Spat and very small oysters,however, were almost unaffected and oyster beds destroyed by disease displayed essentially complete recovery in nine months. The appearance of unusual minute hypnospores at the height of the oysterepizootics suggested the presence of a second lethal oyster parasite, and culture studies demonstrated a second species of Labyrinthomyxa with a distinct sporulation. In other investigations, Mackin has isolated and studied several other closely related oyster parasites, some of whichmay have been significant in the epizootics or other studies since they cannot be detected by the F.T.M. assay we used. Analysis of gaper infection intensities confirmed that infections of L. marina as low as "medium" may result in death of the host. However, most animals do not succumb until heavy or medium heavy parasite intensity is reached. Theexistence of other pathogenic or unfavorable conditions often combine to cause death at abnormally low levels of L. marina.
An oyster culture schedule was devised to minimize L. marina mortalities while also lessening other oyster losses and producing the best product in the shortest time. A field evaluation of this schedule under optimum conditions showed that the time from set to harvest was 10 to 13 months, average harvest oyster height was 12 to 15 cm, mortality from L. marina was about 10%, and the overall post spat loss was approximately 20%. Oysters were moved once and planted at medium to high density.OYSTER PARASITISM BY LABYRINTHOMYXA MARINA IN FLORIDA 53

ACKNOWLEDGMENTS

We wish to thank Mrs. Violet Stewart and Messrs. Dale McCall, Robert Cissel, Don Lockard, and Andrew Kaloski, who worked directly on the project, and many other members of the laboratory staff who have assisted in numerous ways. Our thanks also to Mr. R. M. Ingle, Chief ofthe Bureau of Marine Science and Technology, for assistance and direction throughout the study and to Mr. E. A. Joyce, Jr., Supervisor of the MarineResearch Laboratory, who helped in preparation of the manuscript. BUTLER, P. A. 1969. Monitoring pesticide pollution. BioScience, 19(10): 889-891.

BUTLER, P. A. and P. F. SPRINGER 1963. Pesticides a new factor in coastal environments. Trans. 28th N. Amer. Wildl. Manage. Res. Conf.: 378-390.

DAWSON, C. E. 1955. Observations on the incidence of Dermocystidium marinuminfection in oysters of Apalachicola Bay, Florida. Tex. J. Sci., 7(1): 47-56.

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