Final Report

Sea Scallop Research

NOAA Grant Number: NOAA/NA12NMF4540036

Award Date: 2/10/2012

Start Date: 5/01/2012

End Date: 4/30/2013

Extended End Date: 4/30/2014

Project Title: What Causes Gray Meat in the Atlantic Sea Scallop

Placopecten magellanicus in Georges Bank Closed Areas?

Principal Investigator: Kevin D. E. Stokesbury, Ph.D. and Susan D. Inglis

Address: School for Marine Science and Technology,

University of Massachusetts Dartmouth,

200 Mill Road Suite 325

Fairhaven, MA, 02719

Phone: (508) 910-6373

Fax: (508) 910-6374

Email: kstokesbury@umassd.edu

Amount: We were granted (40,323 lbs) from the Open Area ($379,843) for research and

compensation.

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Introduction: Scallop meats are normally firm and creamy white in color. However, large numbers of older

scallops with small, darkened and stringy meat were observed in the rotational management

areas of Closed Area I (CAI) in 2009 and 2011, Closed Area II(CA II) in 2011, and the

Nantucket Light Ship (NLSCA) between 2004 and 2005 (Figure 1). These rotational areas are

part of Marine Protected Areas (MPAs) in Georges Bank. The occurrence of gray or

“discolored” scallop meat in the closed areas of Georges Bank has become a concern for

industry. The scallops have a low meat yield and the discolored and poor quality meat has a low

market value. Historically, large populations of these scallops have been observed in fishing

areas re-opened following closures. However, the cause of this meat quality issue is not well

understood. Determining the conditions that favor the production of these large, poor quality

scallops addresses both industry and management concerns as they represent a large biomass of

low commercial value scallops. We tested the following hypotheses as causes for this meat

quality condition; old age, poor nutrition, and parasitic disease. This study documents the

occurrence of “gray” meat in Closed Area I and II and presents the causative agent for the

disease and identification of a new species of bivalve parasite.

Objectives (from proposal): The occurrence of gray or “discolored” scallop meat in the closed

areas of Georges Bank has become a concern for industry. The scallops have a low meat yield

and the discolored and poor quality meat has a low market value. Significant numbers of these

scallops have been caught following the opening of Closed Areas I and II in August 2011.

Historically, large populations of these scallops have been observed in fishing areas re-opened

following closures. However, the cause of this meat quality issue is not well understood. We

propose to document the spatial and temporal occurrence of these “gray” scallops and investigate

possible causes for the decline in meat quality. The primary goal of this project is to identify

environmental and physiological factors that accompany the development of these large scallops

of low meat yield and discolored meat quality.

Methodology (from proposal): We will address our objectives through SMAST video digital

still image analysis of Georges Bank Closed Areas and whole scallop physiological examination

collected from the rotational access areas.

Digital Still Image Analysis: Digital still images from the SMAST–Industry cooperative video

survey will be analyzed:

1. For the occurrence of scallops exhibiting the external characteristics associated with

gray meat. Scallops exhibiting epifaunal growth, thickened and/or “pitted” shells will be

measured for shell height (SH) and standardized to individuals’ m-2

. To determine the ratio of

potentially “gray” scallops, the absolute number of scallops in the survey area will be calculated

by multiplying scallop density by the total area surveyed (Stokesbury 2002). Sponges and

hydrozoa/bryozoa will be recorded as present or absent within each quadrat (Stokesbury and

Harris 2006). The data will then be analyzed with respect to area and fishing status.

2. To establish habitat characteristics from collected gray scallop samples. The latitude

and longitude of gray meat samples collected from Industry, and research surveys will be entered

into the SMAST video data base and video from our nearest sample locations will be analyzed

for habitat conditions. These sample location will also be investigated historically with our

video archive.

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Whole Scallop Examination: In cooperation with commercial scallop fishing vessels, and

research surveys, whole scallop samples, grossly representing gray meat scallops, will be

collected from Georges Bank rotational areas and donated to the project. The date, location,

depth and general observations on the condition of the catch and other organisms found in the

dredge will be recorded.

Each scallop will be externally examined and photographed for evidence of shell boring

activity. The shell thickness and height will be recorded and the scallop dissected. Following the

methodology of Sarro and Stokesbury (2009), the shell height/meat weight and gonadal somatic

index will be calculated. The meat will be analyzed for proximate composition (lipid, protein,

water and ash content) using Official Methods of Analysis (1990) to determine the nutritional

status of the animal. Reproductive tissues will be prepared to determine the presence of

senescence. The meat will also be prepared for histological examination for myodegenerative

changes in the muscle indicating both nutritional stress and possible proykaryotic infection. The

top and ventral shells will be examined by radiography for extent and type of shell boring

activity and a sub sampled aged using δ 18

O carbonate analysis.

Time Line: Samples were collected from Industry during the spring and summer 2012 and from

the Yellowtail Bycatch survey of Georges Bank from May 2012 through March 2013 for

analysis.

Figure 1: Map of Georges Bank Closed Areas showing Closed Area I (CAI), Closed Area II

(CAII) and Nantucket Lightship (NLCA). The areas shaded in light blue represent the rotational

management access areas.

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Methods:

Whole Scallop Examination for Causative Agent:

Scallop samples, frozen whole for gray meat analysis were collected from Georges Bank

Closed Areas I and II, the Northern Edge. These samples were obtained opportunistically from

commercial scallop fishermen and research survey trips including the Scallop RSA survey of the

Northern Edge by the Virginia Institute of Marine Sciences. We also collected samples monthly

from the Scallop RSA Project-Yellowtail Bycatch Survey of Georges Bank (12-SCA-06) starting

May 2012.

These monthly samples included:

A random sample of thirty scallops with shell height (SH) > 145mm frozen whole from

stations in Closed Area 1 South (CA1 S), Closed Area 1 North(CA1 N) and Closed Area

II (CA2) (10 from each area). These samples were dissected while frozen, meat quality

(color) recorded and the adductor muscle shipped to the New Jersey Feed Laboratory for

proximate analysis to determine the water, lipid, ash, protein and carbohydrate content of

each tissue to test the nutrition hypothesis.

Twenty samples from confirmed gray meat scallops for pathological and histological

analysis (Figure 2). Each adductor muscle and gonad was preserved in formalin and the

corresponding shell labeled for subsequent analysis (Figure 8B). The shells from these

samples were treated in a fresh water bath to collect polychaetes present in the shell.

Tissue and polychaete samples were sent to the Kennebec River Biosciences Laboratory

in Maine and Dept. of Fish Diseases at the Institute of Pathology, University of Iceland

the University of Iceland Fish Pathology Laboratory for identification and analyses to test

the disease hypothesis and provide shell height data for the age hypothesis.

Data collected from stations selected for shell height/meat weight analysis for the

Yellowtail Bycatch Survey were also analyzed for meat quality in 2013 (Figure 3). A

bushel of randomly selected scallops from each tow in designated stations were collected

and processed for shell height (mm): meat weight (g) and meat quality. This data set

was used to map the occurrence of gray meat scallops in Closed Area I and II and

document the shell height frequency associated with the discolored meat to test the age

hypothesis.

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Figure 2. Stations in Georges Bank where gray meat samples were collected for

histopathology (circled stations).

Figure 3. The YT Bycatch Survey Stations (Coonamessett Farm Foundation) in the Georges

Bank Closed Area I and Closed Area II study area. These stations were sampled monthly for

shell height:meat weight (n=12 samples per assigned station). Meat quality was added to the

analysis in 2013.

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Digital Still Image Analysis:

The latitude and longitude of gray meat samples collected from Industry and research

surveys were entered into a “nearest neighbor” query for the SMAST video data base which

identified video survey results for the “gray meat” locations. Video footage was analyzed

following Stokesbury 2002, and Stokesbury and Harris 2006. The number of clappers and mega

fauna representing potential predators such as sea stars, crabs, sponges and tunicates were

identified.

Substrate sediments were visually identified following the Wentworth particle grade scale

from the video images, where the sediment particle size categories were based on a doubling or

halving of the fixed reference point of 1 mm; sand = 0.0625 to 2.0 mm, gravel = 2.0 to 256.0 mm

and boulders > 256.0 mm (Lincoln et al. 1992). Gravel was divided into two categories,

granule/pebble = 2.0 to 64.0 mm and cobble = 64.0 to 256.0 mm (Lincoln et al. 1992). Shell

debris and detritus were also identified.

The habitat characterization of the gray meat areas was determined by the percent

absence: presence of each of these sediment and mega fauna categories.

Results:

Digital Still Image Analysis:

Results from the video analysis of “nearest neighbors” stations to gray meat locations in

Closed Area I and II for 2011 and 2012 are presented in Figures 4 and 5. This analysis is being

expanded and continued the 2014 RSA project entitled “Tracking the Occurrence of Gray

Meat in Atlantic Sea Scallops, Placopecten magellanicus”. Subsequent results that include

physical characteristics of gray meat habitat including scallop density and bottom water

temperature will be presented in the new RSA project reports.

Gray meat stations were characterized by a predominantly sand substrate with shell

debris. There were very few clappers observed that would indicate natural mortality events. Sea

stars were the most abundant predator in gray meat areas.

Figure 4. Percent occurrence of substrate in locations where gray meat were found in Closed

Area 1 (CAI) and Closed Area II (CAII) in 2011 and 2012 (n=13 stations and 52 quadrats).

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Figure 5. Percent occurrence of different habitat characteristics in locations where gray meat

were found in Closed Area 1 (CAI) and Closed Area II (CAII) in 2011 and 2012 (n= 8 Stations

and 32 quadrats).

Distribution

Result for the distribution of gray meat scallops in Georges Bank Closed Area I and

Closed Area II are presented in Figures 6 and 7. Gray meat scallops are found throughout

Closed Area I with the greatest occurrence in the southeast portion where over 50% of the

scallops sampled had discolored meat. This is consistent with reports from fishermen. Although

not as concentrated as in Closed Area I, gray meat scallops are also found in high concentrations

(25-49%) throughout Closed Area II. From this study the condition does not seem to have

moved into open area south of Closed Area II yet.

Gray meat scallops (n=71) were also found in “pockets” in the Nantucket Lightship

access area during a fall and spring survey conducted for the RSA Project - Combined high-

resolution video survey and biological sampling using a modified sled dredge of the sea scallop

resource in Nantucket Lightship Access area (Figure 8A). .

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Figure 6. The percent of the sample (bushel) that contained gray meat per station for Closed Area

I Sept 2013-March 2014 surveys > 50%, 25-49%, 1-24%, 0%.

Figure 7. The percent of the sample (bushel) that contained gray meat per station for Closed Area

II and Open Areas Sept 2013-March 2014 surveys > 50%, 25-49%, 1-24%, 0%.

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Figure 8. Examples of gray meat scallops from the Nantucket Lightship access area (A) and

Closed Area 1 (B).

Age:

Results from shell height analysis of scallop samples collected specifically for pathology

and histology (n=218) did not show a significant difference in shell height (mm) with gray meat

occurrence (Figure 9). These findings were confirmed in the random samples collected for shell

height meat weight analysis (Figure 10; n=395). A Pearson’s chi-squared test found no

significant difference between the shell height (mm) of white and gray meat scallops in Closed

Area 1, Closed Area II and Open Area of Georges Bank. Running the test with a simulated p

value did not change the significance result. Scallop age is associated with shell height. Thus,

these finding suggest that the gray meat condition is not directly caused by old age.

Pearson's Chi-squared test:

CA1: X-squared = 209.6667, df = 195, p-value = 0.2241

CA11: X-squared = 152.5278, df = 130, p-value = 0.08621

Open Area: X-squared = 85, df = 70, p-value = 0.107

We were unable to use stable isotope δ18

O analysis to age the gray meat scallops we

collected due to the poor condition of the shell. Stable isotope analysis of the shell carbonate is

compromised by contamination by boring organism tissue and debris. The gray meat scallops

often exhibited extensive polychaete infestation of the shell. The University of Michigan Stable

Isotope Laboratory is working on a process to extract the contamination from the shell carbonate

samples but these results are not available at this time.

A

B

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2012 2013 2014

Shell

heig

ht

(mm

)

90

100

110

120

130

140

150

160

170

Figure 9. The mean shell height for gray meat scallops collected in 2012, 2013, 2014 for

histopathology analysis (n=218).

Figure 10. Shell height analysis of scallops collected from Closed Area I (CAI), Closed Area II

(CAII) and the open areas for gray and white meat scallops (n=395). No significant difference in

shell height (mm) between gray and white meat scallops was found using a Pearson's Chi-

squared test. Further analysis using a simulated p-value did not change the significance.

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Nutrition:

Samples were tested at the New Jersey Feed Laboratory for percent fat, carbohydrate,

protein, moisture and ash in the muscle tissue using standard analytical methods AOAC (1990).

The samples were categorized as white, brown, or gray adductor meat color. There was a

significant decrease the protein content of scallops with discolored meat, and the darker the meat

color (brown to gray) the greater the decrease in protein content. There was also a reduction in

carbohydrate and lipid levels in gray meat scallops (Figure 11). Lipid levels were low in all meat

samples which is consistent with scallops that preferentially use carbohydrate for energy

(Shumway and Parsons, 2012).

In 1992, the USA Food and Drug Administration (FDA) determined that scallops with a

total moisture content of 80% or less, if not subjected to processing conditions utilizing

excessive water and/or phosphate treatment, could be labeled simply as “scallops”.

Moisture:protein ratios are used as indicators of conditions representing “water-added” when

marketing scallop meats and are carefully monitored by the FDA and US Department of

Agriculture (USDA). In Atlantic sea scallops the moisture:protein ratio is considered to be 4.0

to 4.9 (Lampilla, 1993). Thus, a ratio about 5.1 would indicate a product with added water or

“altered”. Scallops with gray meat have a significantly higher moisture:protein ratio of 12.9

(±4.2), with brown meat scallops at 6.46 (±1.36) (Figure 12). These results generally indicate

nutritional stress in individuals due to either a reduction in energy intake or assimilation, or an

extra metabolic burden. However, both of these conditions can also be caused by a disease

process.

Figure 11. Proximate analysis of scallop adductor muscle (mean ± SD) found a significant

reduction in % protein and carbohydrate and increase in moisture content in gray meat scallops

n=68; (ANOVA: p>0.05).

Moisture Lipid Protein Ash Carbohydrate

% t

issu

e c

om

po

sitio

n

0

20

40

60

80

100

white meat

brown meat

gray meat

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Figure 12. The moisture:protein ratio found in white, brown and gray meat scallops. In Atlantic

sea scallops a ratio of 4.0-4.9 is considered “normal” by the FDA and USDA.

A controlled laboratory experiment conducted to determine the effects of food

availability on growth and shell isotopic signatures in Atlantic sea scallops resulted in a finding

of gray meat scallops in the reduced ration group. This study maintained scallops from Georges

Bank Closed Area 1 of approximately 100 mm shell height in a controlled laboratory setting on

either a full or reduced ration of a commercial Shellfish diet (Reed ShellFish Diet). There was a

significant difference in meat quality between the experimental groups, with the reduced ration

groups presenting with small, soft, and in some instances discolored meat. We observed five

gray meat scallops out of twenty in this reduced ration group. These scallops did not show any

signs of boring parasite infection in the shells. The appearance of gray meat in this experiment

was an unexpected result, and suggested that poor nutrition may be a physiological factor or

stressor in scallops exhibiting gray meat (Figure 13).

Figure 13. Examples of meat quality observations in the diet studies; A. Gray meat in reduced

ration group, B. White meat from full ration group.

mois

ture

:pro

tein

ratio

0

2

4

6

8

10

12

14

16

18

20

white meat

brown meat

gray meat

A B

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Disease:

Results from the shell analysis show a high density of polychaete, Polydora species

activity (Figure 14). The level of infestation was moderate to high among the majority of the

shells from large scallops (shell height > 150 mm) with gray meat. Burrow holes were present on

both valves, but at a higher density on the left valves. In the more advanced stages of infestation,

polychaete burrows had penetrated the scallop shell and formed lesions on the interior surface.

These areas and adductor muscle attachment sites were marked by conchiolin deposition.

Conchiolin is secreted by the mantle in response to shell damage. Interior shell morphology

suggested possible partial deterioration of muscle attachment sites in some of the samples. Mud

blisters were also visible along the interior shell wall in more advanced infections, observed in

approximately 30% of samples.

Histological analysis of the adductor muscle (n=85) exhibited degeneration of muscle

fibers (myodegeneration ) in all gray meat samples at ‘moderate to extensive’ level (per Gulka et

al. 1983) and the presence of a previously unknown apicomplexan parasite. This parasite is

similar to an apicomplexan parasite identified in the Iceland Scallop Chlamys islandica and was

the cause of a mass mortality event in the year 2000 (Figure 15; Eiriksson et al 2010,

Kristmundsson et al 2011). Research conducted by Dr. Arni Kristmundsson with the

Department of Fish Diseases at the Institute of Pathology, University of Iceland and Dr. Mark

Frement with the Institute of Biological Science at the University of Malaya in Malaysia

identified this new apicomplexan species (parasite), which caused visible gross pathology and

extensive muscular necrosis or myodegeneraion in the Iceland scallop. In the case of the Iceland

scallop, high summer sea bottom temperatures were thought to have made the scallops

susceptible to this parasitic infection (Eiriksson et al 2013). This parasite is non zoonotic and

only affects bivalves. It has also been found in the queen scallop, Aequpecten opercularis, L and

king scallop, Pecten maximus.

In the Atlantic sea scallop increased muscle discoloration, from white to brown to gray,

was associated with increased muscle degeneration and parasite intensity. The parasite was

found in high intensity in all gray meat samples, in moderate intensity in brown meat scallops

and rarely in white meat scallops (Table 1).

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Figure 14. Interior shell from a scallop with gray meat and exhibiting myodegeneration.

Figure 15. Examples of the “gray” meat found in the Iceland Scallop infected with the

apicomplexan parasite. Reproduced from Eiriksson et al 2000.

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Table 1: Summary of pathological findings in white, brown and gray scallop adductor muscle.

Adductor

Muscle Color

Muscle Degeneration Hemocyte

Neoplasia

Apicomplexon

White none rare rare

Brown slight-stringy elevated, low-

moderate

moderate sporozoites

Gray moderate to severe with

deterioration of muscle

attachment sites

high free sporozoites extensive in

moderate, less in severe; secondary

infection in some samples

Apicomplexan parasites have a complex life cycle and can reproduce sexually and

asexually. Direct transmission from infected tissues and animals is possible (Figure 16). All

apicomplexan stages (both sexual and asexual stages): free sporozoites, trophozoites, meronts,

merozoites were found in Placopecten adductor muscle samples.

Figure 16. Generalized schematic of Apicomplexan parasite life history.

The pathology results suggest the following progression of the disease in the Atlantic sea

scallop.

Hemocyte infiltration with no detectable changes in the muscle fiber structre, hemocytes

morphologically normal but at 2-3X normal density, some presence of acid-fast postive

cells indicating secondary infection.

Reduction in the diameter of muscle fibers (thinning), appearance of apicomplexan zoite

stages in pockets amoung fibers and structures which appear consistent with neoplastic

hemocytes in other bivalve shellfish species. High level of neoplastic hemocyte presence

in other tissues (gills, giestive organ, gonads).

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Further thinning of muscle fibers, increased numbers of neoplastic hemocytes,

concentrated in pockets and in conjucntion with zoites.

Extensive thinning of muscle fibers, gaps among fibers with debris, greater observation

of other structures such as acid-fast cells and other potential apicomplaxan stages.

Fused muscle fibers, initally observed nearer to shell attachment sites, then deeper into

muslc tissue, decreased hemocyte number and near absence in fused areas.

A histological comparison of the parasite infecting Placopecten magellanicus and

Chlamys islandica found the morphology of sporozoites and effect on muscle tissue to be

identical (Figures 18, 19, 20). DNA results on the apicomplexan parasite found in the Atlantic

sea scallop indicate it is conspecific with the parasite found in the Iceland, Queen, and King

scallop. This has now been recognized as a new species of bivalve parasite.

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Figure 18. Histological comparison of parasite infecting Placopecten magellanicus and

Chlamys islandica. The morphology of sporozoites appears identical.

18

Figure 19. Histological comparison of muscle necrosis from parasite infection in Placopecten

magellanicus and Chlamys islandica.

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Figure 20. Zoite stages of parasite within adductor muscle fibers.

Results from this study indicate that gray meat in the Atlantic sea scallop is caused by an

apicomplexan parasite infection. Age, nutritional stress, as well as secondary infections and

shell parasites such as Polydora sp. may be covariates of this condition by reducing the fitness of

the scallop and thus the scallop’s ability to control the parasite infection and subsequent disease

process. Therefore, a combination of physiological and site specific environmental conditions

that supports the proliferation and transmission of this parasite is likely responsible for the

outbreaks of “gray meat” in Georges Bank scallop stocks. Unfortunately, we currently do not

understand the interactions between these variables well enough to be able to predict these

events. We hope to continue and expand this research to address this question.

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Outreach and Publications:

Susan Inglis presented the results of this project to the Scallop Plan Development Team (PDT)

on April 8 2014, Woods Hole, MA. These data have also been presented at the SMAST

Fishermen’s Steering Committee meetings and to the public at the 2012 High School Marine

Science Symposium in New Bedford, MA.

Publications:

A paper entitled “ The apicomplexan parasite, parasarcocystis pectinis, and gray meat in

the Atlantic sea scallop Placopecten magellanicus” is in preparation for the Journal: Diseases of

Aquatic Organisms.

Conferences/Seminars:

1. Inglis, S.D., Kristmundsson, A and K.D.E. Stokesbury. What Causes Gray Meat in the

Atlantic Sea Scallop Placopecten magellanicus in Georges Bank Closed Areas? Northeast

Fisheries Science Center-Invertebrate Subcommittee. Sea Scallop Stock Assessment

Meeting (SARC-59). Woods Hole, MA. March 17-21.

2. Inglis, S.D. What Causes Gray Meat in the Atlantic Sea Scallop Placopecten magellanicus in

Georges Bank Closed Areas? Department of Fisheries and Ocean Sciences Seminar Series.

University of Massachusetts, Dartmouth. Fairhaven, MA. April 30 2014.

3. Inglis, S.D. and K.D.E. Stokesbury. What Causes Gray Meat in the Atlantic Sea Scallop

Placopecten magellanicus in Georges Bank Closed Areas? 44th

American Fisheries Society

Annual Meeting, Quebec City, Quebec. Poster Presentation. August 18-22. 2014.

4. Kristmundsson, A, Inglis, S.D., Stokesbury, K.D.E. and M.A. Freeman. Apicomplexan

infection of the Atlantic sea scallop Placopecten magellanicus. Seventh International

Symposium on Aquatic Animal Health, Portland , Oregon , August 31-September 4th

2014.

Literature Cited

Association of Official Analytical Chemists. 1990. Official methods of analysis. 15th

edition, Vol. 2, Arlington, AOAC. 1298 pp.

Eiriksson,H., Thorarinsdottir,G.G., Jonasson,J.P.,and A. Kristmundsson. 2010. Increase in

natural mortality of the Iceland Scallop (Chlamys Islandica) in West Iceland and collapse

of the fishery in the early 2000s. ICES CM: 20.

Gulka, G., Change, P.W., and K.A. Marti. 1983. Prokaryotic infection associated with a mass

mortality of the sea scallop, Placopecten magellanicus. J Fish Dis 6:355-364.

Kristmundsson, A., Helgason, S., Bambir, S.H., Eydal, M., and M.A. Freeman. 2011. Previously

unknown apicomplexan species infecting the Iceland scallop Chlamys Islandica (Muller,

1776), a queen scallop, Aequpecten opercularis, L and king scallop, Pecten maximus,L. J.

Invertebr. Pathol., 108. 147-155.

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Lincoln, R.J., G.A. Boxshall, and P.F. Clark. 1992. A dictionary of ecology, evolution and

systematics, Vol. Cambridge University Press, Cambridge.

Naidu KS. 1970. Reproduction and breeding cycle of the giant scallop Placopecten magellanicus

(Gmelin) in Port au Port Bay, Newfoundland. Canadian Journal of Zoology, 48(5): 1003-

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Sarro, C. and K.D.E. Stokesbury. 2009. Spatial and temporal variation in the shell height/meat

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Shumway, S.E and J. Parsons. 2012. Scallops: Biology, Ecology and Aquaculture:

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Stokesbury, K. D. E. 2002. Estimation of sea scallop abundance in closed areas of Georges

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