Diseases of Wild and Cultured Shellfish in Alaska

Diseases of Wild and cultured shellfish In Alaska

Theodore Meyers and Tamara Burton

February 2009 Alaska Department of Fish and Game Fish Pathology Laboratories The Alaska Department of Fish and Game printed this publication at a cost of $12.47 in Anchorage, Alaska, USA. About This Booklet

The overwhelming success of the 2007 publication “Common Diseases of Wild and Cultured Fishes in Alaska” encouraged our publication of a companion book describing the common and not so common diseases of shellfish in Alaska. This book is a brief illustrated guide that describes many of the parasitic, infectious, and noninfectious diseases of bivalve molluscs and crustaceans encountered in Alaska. The content is directed towards lay users, as well as aquaculturists and biologists and is not a comprehensive treatise nor should it be considered a scientific document. Interested users of this guide are directed to the listed shellfish disease references for additional information.

Information contained within this booklet is published from the laboratory records of the Alaska Department of Fish and Game, Fish Pathology Section that has regulatory oversight of shellfish health in the State of Alaska. Alaska Department of Fish and Game, Fish Pathology Laboratories, 333 Raspberry Road, Anchorage, Alaska 99518; 3333 Glacier Highway, Juneau, Alaska 99802.

Manuscript Photographs: Alaska Department of Fish and Game, Fish Pathology Laboratory photo archives except where indicated by photo credits

Cover Photographs: Cultured juvenile geoduck clams at Alutiiq Pride Shellfish Hatchery, Seward, Alaska and (inset) negative stain of Aquareovirus isolated from wild adult geoduck clams

The Alaska Department of Fish and Game (ADF&G) administers all programs and activities free from discrimination based on race, color, national origin, age, sex, religion, marital status, pregnancy, parenthood, or disability. The department administers all programs and activities in compliance with Title VI of the Civil Rights Act of 1964, Section 504 of the Rehabilitation Act of 1973, Title II of the Americans with Disabilities Act of 1990, the Age Discrimination Act of 1975, and Title IX of the Education Amendments of 1972. If you believe you have been discriminated against in any program, activity, or facility please write ADF&G ADA Coordinator, P.O. Box 115526, Juneau, AK 99811-5526; U.S. Fish and Wildlife Service, 4401 N. Fairfax Drive, MS 2042, Arlington, VA 22203; or Office of Equal Opportunity, U.S. Department of the Interior, 1849 C Street NW MS 5230, Washington DC 20240.

For information on alternative fomats and questions on this publication, please contact the department’s ADA Coordinator at (VOICE) 907-465-6077, (Statewide Telecommunication Device for the Deaf) 1-800-478-3648, (Juneau TDD) 907-465-3646, or (FAX) 907-465-6078. Table of Contents

Bivalve Molluscs Crustaceans Viruses Viruses Aquabirnavirus...... 2 Aquabirna and Herpes Viruses...... 66 Aquareovirus...... 4 Bacteria Gametogenic Hypertrophy...... 6 Prokaryotic Intracytoplasmic Inclusions...... 7. 0 Herpesvirus...... 8 Shell Disease...... 74. Bacteria Fungi Bacterial Septicemia - Vibrio and Others...... 10 Black Mat Syndrome...... 76 Nuclear Inclusion X...... 12 Fungal Infection of Captive Red King Crabs...... 78 Pacific Oyster Nocardiosis...... 14. Protozoa Prokaryotic Intracytoplasmic Inclusions...... 16. Haplosporidian-like Parasite of Shrimp...... 80 Fungi Hematodinium - Bitter Crab Disease...... 84 Shell Fungus...... 1. 8 Hematodinium-like Disease Dungeness Crabs...90 Systemic Mycosis...... 2. 0 Mesanophrys sp. Ciliate Disease...... 92. Protozoa Thelohania and Other Microsporidia...... 94 Gill and Gut Ciliates...... 22 Helminths Haplosporidium sp...... 24 Carcinonemertes Egg Predator...... 98. Hexamita...... 2. 8 Crab Leech...... 102. Ichthyobodo-like Protozoan...... 3. 0 Encysted Trematode Metacercariae...... 104 Nematopsis and Other Gregarines...... 3. 2 Trypanorhynch Cestode Plerocercoids...... 106 Steinhausia and Other Microsporidia...... 3. 4 Arthropods Trichodinid Ciliates...... 3. 6 Briarosaccus callosus - Parasitic Barnacle ...... 108 Unidentified Kidney Coccidia and Others...... 3 8 Miscellaneous Helminths Digestive Tubular Degeneration and Bio-Foul..112 Gill and Gut Turbellaria...... 4. 4 Idiopathic Granulomatosis –Dungeness Crab. .11 4 Shell-boring Polychaetes...... 4. 6 Proliferative Lesions and Neoplasia...... 116 Trematode Metacercariae and Sporocysts...... 48 Arthropods Reference Gill and Gut Parasitic Copepods...... 52. Glossary of Terms...... 12 0 Miscellaneous Shellfish Disease References...... 12. 7 Foreign Body Granulomas and Pearls...... 56 Miscellaneous Invertebrate Pests and Pedators.58 Neoplasia...... 62 BIVALVE mOLLUSc VIRUSES Aquabirnavirus

I. Causative Agent and Disease III. Clinical Signs Aquabirnavirus is a recent new Bivalve molluscs are generally genus in the virus family Birnaviridae. asymptomatic carriers and/or vectors of These unenveloped icosahedral (~60 these viruses. nm) viruses (over 200 isolates) contain a bi-segmented double stranded RNA IV. Transmission genome that encodes 5 proteins and Transmission is horizontal animal to have been isolated in cell culture from a animal via water. Isolates from bivalve variety of marine and freshwater fish and molluscs may also represent bioaccumu- shellfish species worldwide. There are lation from filter feeding after the virus three and possibly four serogroups (A, B, is shed into the water column from a C & D) comprising at least 16 serotypes nearby fish host. of aquabirnaviruses. Molecular testing has determined there are currently 7 V. Diagnosis genogroups. Several of these viruses oc- Detection of Aquabirnavirus is ac- curring in finfish cause disease (such as complished either by direct examination IPNV in salmonids) while those infect- of shellfish tissues with transmission ing molluscs are mostly apathogenic, al- electron microscopy (TEM) or by isolat- though some isolates have been reported ing the virus in cultures of susceptible to cause cell pathology or mortality in fish cell lines that have been inoculated stressed bivalves. Some of these viruses with contaminated or infected shellfish that are shed into the water column by tissue. Cytopathic effect (CPE) is gener- a fish host may be bioaccumulated by ally a nondescript diffuse thinning and nearby bivalve molluscs through the necrosis of infected cells. Identification filter feeding mechanism. These viral is based on serology and TEM or by agents in shellfish are most often isolated polymerase chain reaction (PCR). There from asymptomatic adult animals during are no established bivalve mollusc or routine virus screening examinations. crustacean cell lines for isolating viruses that may have a strict host specificity for II. Host Species shellfish, although there has been some Currently, aquabirnaviruses have success in culturing primary cell mono- been isolated from many different layers from certain shellfish species. fish and shellfish species including 32 families of finfish, 11 species of bivalve VI. Prognosis for Host molluscs and at least 4 species of crus- Bivalve molluscs are asymptomatic taceans. In Alaska, Aquabirnavirus carriers and/or vectors of these viruses. has been isolated in fish cell lines from asymptomatic adult littleneck clams VII. Human Health Significance and an aquabirnavirus-like agent has There are no zoonotic human health been observed by transmission electron concerns associated with aquabirnavi- microscopy in a male blue king crab ruses in shellfish. associated with an adenocarcinoma-like proliferative lesion (discussed in the crustacean section).

2 3 BIVALVE MOLLUSc VIRUSES

 TEM of cultured bluegill fry cell with cytoplasmic aggregate of hexagonal- shaped aquabirna-like virus particles (arrow) isolated from littleneck clams



CPE in CHSE-214 cells showing areas of cell rounding and necrosis (arrow) typical of Aquabirnavirus

2 3 BIVALVE mOLLUSc VIRUSES Aquareovirus

I. Causative Agent and Disease III. Clinical Signs Aquareovirus is a recent new genus Fish and bivalve molluscs are gener- in the virus family Reoviridae. These ally asymptomatic carriers and vectors, icosahedral (60-80 nm) double-stranded respectively, of the virus. RNA viruses (over 50 isolated) with 11 genome segments have been isolated in IV. Transmission cell culture from a variety of marine and Transmission is horizontal via water. freshwater aquatic animals worldwide Isolates from bivalve molluscs likely including finfish and bivalve molluscs. represent virus bioaccumulated by filter There are several reports of reo-like vi- feeding after the virus is shed into the ruses observed in crustaceans but these water column from a nearby fish host. viruses have fewer or more genome segments and most have not been cultured. V. Diagnosis Genetic analyses have identified 7 (A, Detection of aquareoviruses is B, C, D, E, F, G) different genotypes of accomplished by isolating the virus in aquareoviruses. Except for isolates re- cultures of susceptible fish cell lines ported from 7 fish species, most of these that have been inoculated with contami- viruses produce self-limiting infections nated shellfish tissue. Nearly all of these of low pathogenicity in fish that are not viruses cause a unique cytopathic effect associated with extensive disease or mor- (CPE) characterized by focal areas of tality. In bivalve molluscs these viruses cellular fusion (syncytia) and cytoplas- are most likely bioaccumulated by the mic destruction creating a vacuolated or filter feeding mechanism. These viral foamy appearance. An exception is the agents in shellfish are most often isolated grass carp Aquareovirus that produces from asymptomatic adult animals during a diffuse CPE. Presumptive identifica- routine virus screening examinations. tions are based on the typical CPE and confirmed serologically, by electron II. Host Species microscopy or by polymerase chain Currently, aquareoviruses appear to reaction (PCR). There are no established be mostly of finfish origin with a wide bivalve mollusc or crustacean cell lines host range of marine and freshwater spe- for isolating viruses, although there has cies. In Alaska, Aquareovirus has been been some success in culturing primary isolated from ovarian fluids of returning cell monolayers from certain shellfish chinook salmon (genotype F) and adult species. geoduck clams (genotype A). Because aquareoviruses appear to be widespread VI. Prognosis for Host in many fish host species, it is likely Bivalve molluscs are asymptomatic other bivalve mollusc species in Alaska vectors of the viruses which are tran- may bioaccumulate these viruses from siently present in the water column. marine fish. Other isolates of aquareovirus (13p2) from bivalve molluscs have VII. Human Health Significance been reported from eastern oysters in There are no zoonotic human health waters off Long Island, New York. concerns associated with aquareoviruses.

4 5 BIVALVE MOLLUSc VIRUSES



Stained focal area of syncytial CPE (arrow) of geoduck Aquareovirus in bluegill fry

cells.

 Adult geoduck clam, a vector species for Aquareo-

virus in Alaska.

 

TEM showing cytoplasmic arrays of Negative stain of virus particles (arrow) geoduck Aquareovirus particles (arrow) in showing double capsid morphology cultured bluegill fry cell

4 5 BIVALVE mOLLUSc VIRUSES Gametogenic Hypertrophy

I. Causative Agent and Disease V. Diagnosis Gametogenic hypertrophy is caused Occasional extreme nuclear hyper- by an unclassified papilloma-like or trophy of male or female gametocytes polyoma-like virus previously referred is observed by histological examina- to as papovavirus. The virus is icosa- tion. Presence of virus particles having hedral, non-enveloped, about 40-50 nm the typical size and morphology within in diameter and is likely composed of the enlarged nuclei can be confirmed double-stranded DNA. The virus infects by transmission electron microscopy the nuclei of developing gametogenic (TEM). There are no bivalve mollusc cell cells in both male and female oysters and lines, therefore the virus has not been clams causing extreme cell enlargement cultured. (hypertrophy) first observed in female ova, hence referred to as “ovacystis”. VI. Prognosis for Host The number of hypertrophied gameto- The condition caused by the virus cytes per animal is generally low and results in no mortality or apparent harm the condition appears to have very little to the host animal. pathological significance to the host animal. VII. Human Health Significance There are no zoonotic human health II. Host Species concerns regarding gametogenic hyper- Ovacystis has been reported from trophy in bivalve molluscs. the east, west and gulf coasts of North America, France, Germany, Korea, Australia and Spain in at least seven oyster species and two clams, the Manila and soft shell. In Alaska and the Pacific Northwest, ovacystis has been observed at very low prevalences in both male and female adult Pacific oysters.

III. Clinical Signs There are no outward clinical signs of disease. The condition is found incidentally during routine histological examination of apparently healthy Pacific oysters.

IV. Transmission Transmission has not been established but presumably it is horizontal from animal to animal via ambient seawater. However, vertical transmission to progeny from infected parents may also be possible.

6 7 BIVALVE MOLLUSc VIRUSES



Histological section of enlarged gametocyte nucleus (arrow) in male Pacific oyster



Ultrastructural section of enlarged nucleus Higher magnification of virus particles containing arrays of virus-like particles (arrow)

6 7 BIVALVE mOLLUSc VIRUSES Herpesvirus

I. Causative Agent and Disease III. Clinical Signs Herpesviruses are icosahedral, Juvenile animals may exhibit slow or quasi-spherical viruses that have a lipid no growth with high mortality approach- envelope. They are members of the fam- ing 100% when seawater temperatures ily Herpesviridae and contain a genome are 25-26°C. Infection is usually associ- of linear double stranded DNA with a ated with intranuclear inclusion bodies particle size of 102-200 nm in diameter. and necrosis of host cells including There are approximately 120 herpesvi- connective tissues of the gills, inter- ruses infecting a wide range of verte- stitial cells around the digestive gland, brates and invertebrates including marine epithelium of the mantle and the velar bivalve molluscs. Herpesviruses can be epithelium and hemocyte precursor cells serious pathogens of hatchery reared ju- in larvae. Occasional secondary bacterial venile oysters and clams while significant infection of larvae and seed may further mortality in nature may require the pres- contribute to the severity of the mortality ence of environmental stressors. and confound the true etiology. However, no mortality of wild or cultured bivalve II. Host Species molluscs in Alaska has yet been associ- Herpes and herpes-like viruses have ated with a herpesvirus infection. been reported worldwide in several bivalve mollusc species in North America, IV. Transmission Mexico, Europe, South America, Aus- Transmission is horizontal from tralia, New Zealand and Asia. Infected animal to animal via ambient seawater bivalve hosts include 7 species of cupped or vertical transmission to progeny from and flat oysters, Manila clam, carpet infected parents. shell clam and French scallop. In Alaska, herpesvirus-like particles associated V. Diagnosis with Cowdry Type A intranuclear inclu- Diagnosis is based on histological sion bodies have been observed by observation of marginated chromatin and transmission electron microscopy (TEM) eosinophilic Cowdry Type A inclusion in mantle epithelium of rock scallops bodies within hypertrophied nuclei of and in the digestive gland epithelium of infected cells, although this cytopathol- native littleneck clams from several sites ogy is not always present. Association throughout the state. Typical inclusion of typical virus particles in infected bodies have also been observed in the cells is confirmed with TEM and PCR mantle epithelium of juvenile Pacific primers for certain strains of the virus oysters from the southeast panhandle. are available for definitive identification. Because herpesviruses in vertebrates are There are no bivalve cell lines available typically very host specific, bivalve her- to culture these viruses which will not pesviruses may be comprised of several replicate in the existing cell lines that are different genotypes. However, at least available. one virus, OsHV1, is capable of infecting several bivalve species. VI. Prognosis for Host There is no treatment for systemic

8 9 BIVALVE MOLLUSc VIRUSES

virus infections except avoidance and or spat deployment to infected culture prevention of virus introduction with areas has been avoided during periods of infected shellfish stocks. Herpesvirus high seawater temperatures. In Alaska, infection may result in high mortality optimum seawater temperatures for virus of juvenile bivalve molluscs. Juvenile replication and high mortality may only survivors and exposed adult animals occur in rare circumstances. become carriers of latent virus which may cause additional mortality during VII. Human Health Significance periods of stress and seawater tempera- There are no zoonotic human health ture extremes. Seawater temperature has concerns regarding herpesvirus infec-

been lowered to reduce juvenile mortal- tions of bivalve molluscs.

ity in certain cultured bivalve species

 

Histological section of inclusion body (arrow) in the mantle epithelium of purple-hinged rock scallop

Histological section of Cowdry Type A intranuclear inclusion bodies (arrow) in

the digestive epithelium of littleneck

clam 



 Histological section of intranuclear inclusion Ultrastructural section of a Cowdry Type bodies in the mantle epithelium of Pacific oyster A inclusion body in a digestive tubule epithelial cell from littleneck clam showing nucleocapsids (arrow) and empty capsids (arrowhead) of herpes-like virus particles

8 9 bivalve mollusc Bacteria Gram-Negative Bacterial Septicemia Vibrio and Others

I. Causative Agent and Disease III. Clinical Signs Gram-negative bacterial septicemia, Clinical signs of bacterial septice- also called bacillary necrosis (when mias include slow growth, failure to set, motile bacteria are involved), is the most high morbidity with reduced movement common disease in larval and juvenile and mortality of larval and juvenile shellfish worldwide, especially for bi- bivalves associated with attached or valve molluscs. Bacterial septicemia can swarming bacteria. Tissues become occur wherever nurseries and hatcheries necrotic and may detach into the water are operating and is indicative of poor column. sanitary procedures. The most common etiological agents are vibrios including IV. Transmission Vibrio sp., V. anguillarum, V. ordalii, V. These bacteria are normal marine tubiashii, V. alginolyticus, V. splendidus flora. Transmission is horizontal via and occasionally Pseudomonas sp. and incoming seawater, contaminated algal marine Flavobacteria. The motile vibri- food stocks and from brood stocks or os and pseudomonads attach and invade otherwise infected animals. soft tissues causing extensive necrosis and also produce exotoxins that can V. Diagnosis damage mantle and velar tissues without Numerous Gram-negative, motile direct bacterial invasion. Filamentous bacteria in wet mounts of dead or dying non-motile Flavobacteria can invade the larvae and tissues of larger juvenile hinge ligament (hinge ligament disease) bivalves provide a presumptive diagno- causing liquefaction, necrosis and loss of sis. Histological sections demonstrate hinge function allowing secondary infec- invasive bacteria in soft tissues and fla- tions by other bacteria. vobacteria in hinge ligaments. Definitive diagnosis of specific bacteria is based on II. Host Species isolation and biochemical characteriza- All species of cultured juvenile tion, fluorescent antibody tests or PCR. bivalve molluscs worldwide are likely susceptible to bacterial septicemia VI. Prognosis for Host which may also occur in adult animals Bacterial septicemia in larvae and ju- when poor environmental conditions are venile bivalve molluscs generally results present or when animals are otherwise in high mortality. Management of the stressed. Occurrence in nature has been disease is based on enhanced hatchery reported but the extent is unknown. In and nursery sanitation procedures as Alaska, occasional bacterial infections preventative measures. Lowering of sea- of juvenile bivalves has occurred at the water temperatures below the optimum shellfish hatchery in Seward. Also, a growth range of the bacteria (less than mortality of adult pink neck surf clams 25°C) can markedly inhibit the disease. near Juneau was associated with a marine flavobacterial septicemia when VII. Human Health Significance clams were stressed by very warm sunny Certain vibrios and pseudomonads weather during minus tides. infecting shellfish are known to be of

10 11 bivalve mollusc Bacteria

human health concern. However, these to be of zoonotic human health signifi- bacteria in juvenile life stages that are cance. too small for consumption are not likely





Mortality of adult pink-necked surf clams Histological section of bacterial septicemia

due to flavobacterial infection during warm and necrosis of the intestinal mucosa (ar-

weather minus tides rowhead) from Pacific oyster caused by Gram- negative bacteria (arrow) 

Wet mount of Gram-negative bacteria (arrow) swarming around the mantle periphery of larval Pacific oysters (Photo: R. Elston, AquaTechnics, WA)

10 11 bivalve mollusc Bacteria Nuclear Inclusion X

I. Causative Agent and Disease IV. Transmission Nuclear inclusion X (NIX) is a The mode of transmission is not unique rickettsia-like organism infecting known but is likely horizontal via ambi- the nuclei of non-ciliated branchial epi- ent seawater contaminated by nuclei thelium in razor clam populations from from ruptured host cells each containing Washington and Oregon. This patho- the parasite. The cycle of the disease gen has caused massive mortality in appears to begin by infection occurring Washington razor clams and has possibly in late fall or early winter. The para- caused declines in clam populations in site is detected in the spring and early Oregon as well. The organism completes summer followed by enlargement and its replication cycle by rupturing infected rupture of branchial host cells by fall gill epithelium causing respiratory and winter. This cell damage leads to failure and secondary infection by other secondary bacterial infection and death opportunistic bacteria. of the clams. There may also be other life stages that are infectious that have not II. Host Species been described and alternate reservoir This organism is exclusive to razor hosts or vectors. clams in Oregon, Washington and lower British Columbia, Canada. One sample V. Diagnosis of razor clams from Alaska, among two Diagnosis is made by wet mounts and others that were negative, was found to histological examination demonstrating be positive for NIX. However, the posi- enlarged branchial cell nuclei with typi- tive clams were held for about 2 months cal intranuclear inclusions containing the in infected state waters of Washington parasite (average 21 X 13 µm in diam- before they were examined. Consequent- eter). Transmission electron microscopy ly, it is very unlikely that NIX occurs in confirms the morphology of the parasite Alaska as it has not been found in other as rickettsia-like which is unique regard- samples above Vancouver Island, BC. ing its exclusive intranuclear occurrence Nonetheless, the pathogen is included during all observed life stages and its here for future reference. size that is larger than any known bacterium. III. Clinical Signs Infected clams present with extensive VI. Prognosis for Host swelling of branchial epithelium and The organism has been found where enlarged nuclei within intact cells or the populations of razor clams in Washing- nuclei are floating free nearby lysed cells ton have undergone extreme declines of when examined in wet mounts. Second- as much as 95% during 1983-1984 and ary infection by other opportunistic 1985-1986. This may have been peak bacteria is common. Histological ex- proliferative periods in the parasite life amination shows massively enlarged cell cycle, possibly influenced by unknown nuclei enclosing the basophilic rickettsial marine environmental conditions. Al- organism within the non-ciliated respira- though some populations of razor clams tory epithelium of the gills. appear to survive low intensity infec-

12 13 bivalve mollusc Bacteria

tions, the parasite has a proven potential VII. Human Health Significance to cause very significant population There are no known zoonotic human declines for reasons that are not well health concerns regarding NIX infec- understood. tions in razor clams.



Histological section of NIX-infected gill epithelium (arrow) of razor clam 

Histological section of inclusion bodies within enlarged nuclei (arrow) of NIX infected cells

12 13 bivalve mollusc Bacteria Pacific Oyster Nocardiosis

I. Causative Agent and Disease IV. Transmission Pacific oyster nocardiosis (PON), The bacterium is considered to be an previously known as “focal necrosis” opportunistic pathogen that is widespread and “fatal inflammatory bacteremia” in the marine environment. Transmis- (FIB) is caused by an actinomycete sion is likely horizontal and facilitated by bacterium Nocardia crassostreae that other physical environmental stressors. is Gram-positive or Gram variable, acid fast, PAS positive and catalase positive. V. Diagnosis The bacterium contributes to “summer Histological examination of typical mortality” of 2 year and older oysters in lesions in infected oyster tissues indicate shallow, warm eutrophic embayments Gram-positive or Gram-variable, acid fast during August through November when foci of bacteria within surface vesicles seawater temperature is 20°C or greater. and in granulomas eliciting a marked Infection produces small round yellow, host inflammatory response. Confirma- green or brown vesicles of bacteria and tion is by fluorescent antibody staining granulmoas 2 mm to 1 cm in diameter and by transmission electron microscopy throughout oyster tissues resulting in showing typical actinomycete morpholo- significant mortality. gy. Culture of the organism is possible on several medias for fastidious organisms II. Host Species but is not a routine procedure because the This disease is reported to be appearance of colony growth may take restricted to Pacific oysters, although a from 3 weeks to 3 months. similar disease has been observed in Eu- ropean flat oysters cultivated near areas VI. Prognosis for Host of infected Pacific oysters. PON has been Mortality of infected oysters may be reported in Pacific oysters from Califor- significant but has not been accurately nia, Washington, British Columbia, Japan determined in the field. However, ex- and most recently from the Netherlands. perimental injection of the organism has In Alaska, infection by this organism was produced 47-50% cumulative mortality detected in only two juvenile animals of oysters within 30 days. Management imported from the state of Washington techniques have not been investigated but but had been reared in Alaskan waters. culturing oysters in off-bottom gear and Therefore, it is unclear whether the avoidance of shallow warm embayments disease can spontaneously occur in the in the summer may reduce the prevalence colder seawater temperatures typical of and severity of the disease. Alaskan summers. VII. Human Health Significance III. Clinical Signs Aside from aesthetically displeas- Clinical signs of PON include moder- ing, there are no known zoonotic human ate to high mortality of young adult health concerns with Nocardia infection oysters that are undergoing physiological of Pacific oysters. stress from rapid gonadal development with presenting lesions of small, round, yellow, green or brown vesicles of bacteria and granulomas in the tissues. 14 15 bivalve mollusc Bacteria

 

Yellow vesicles (arrow) of nocardiosis Histological section of vesicles containing bac-

in the mantle tissues of a Pacific oyster teria (arrow) in infected mantle tissue (Photo: R. Elston, AquaTechnics, WA) 

Typical subsurface radiating granuloma (arrow) containing central Nocardia organisms sur- ounded by host inflammatory cells, histological section

14 15 bivalve mollusc Bacteria Prokaryotic Intracytoplasmic Inclusions

I. Causative Agent and Disease the cells of various tissues (gill, mantle, Prokaryotic intracytoplasmic inclu- digestive gland and kidney epithelium, sion bodies in marine molluscs are connective tissue, muscle and gameto- ubiquitous having been reported globally cytes) depending on the host species. in the epithelial cells and connective tissues of many different species. These IV. Transmission inclusion bodies contain organisms The mode of transmission has not that are mostly rickettsia-like with less been determined but could be horizontal reports of chlamydia-like organisms and via ambient seawater but an alternate fewer still of mollicute-like organisms reservoir host or vector as part of the life or mycoplasma. In most cases these cycle cannot be ruled out. organisms are incidental infections that do not cause significant disease except V. Diagnosis most notably the rickettsial agents caus- Diagnosis is by histological examina- ing mortality of sea scallops (Pectin tion to detect the typical intracytoplas- maximus and Placopecten magellani- mic inclusion bodies within the cells of cus), California black and red abalones host tissues. Specific identification of the with Withering Syndrome (caused by type of prokaryote within the inclusion Candidatus Xenohaliotis californiensis) bodies is based on the morphological fea- and an intranuclear rickettsia-like organ- tures specific for rickettsia, chlamydia or ism (NIX) causing mass mortality of mycoplasma as observed by transmission razor clams in Washington and possibly electron microscopy (TEM). Fluorescent Oregon (discussed in other section). antibody tests are available for some of these agents, although none have been II. Host Species isolated in culture using conventional Numerous species of marine and methods. Only rickettsia-like organisms some freshwater bivalve molluscs in all have been observed by TEM in the inclu- parts of the world have been reported sion bodies found in Alaskan bivalve with intracytoplasmic inclusion bodies molluscs. caused by these intracellular bacteria. Likewise in Alaska, similar cytoplas- VI. Prognosis for Host mic inclusion bodies of rickettsia-like These organisms appear to be well organisms (RLO) have been detected in: tolerated with no harm to the host, de- Pacific oysters; littleneck clams; razor spite sometimes heavy infections. clams; geoduck clams; butter clams; blue mussels; basket cockles; pink, rock and VII. Human Health Significance weathervane scallops. There are no known zoonotic human health concerns regarding infection of III. Clinical Signs marine bivalve molluscs by these poiki- There are no gross clinical signs of lothermic organisms which are different infection nor is there apparent disease. from similar organisms causing diseases During routine histological examination in higher animals. round, basophilic to purple intracytoplasmic inclusion bodies are detected in

16 17 bivalve mollusc Bacteria

  Histological section of RLO cytoplasmic inclusion (arrow) in a cell of the vesicular connective tissue of Pacific oyster

Histological section of RLO cytoplasmic inclusions (arrow) in digestive tubule

epithelium of Pacific oyster 



Histological sections of RLO cytoplasmic inclusions Histological section of RLO cytoplas-

(arrow) in digestive tubule epithelium of basket mic inclusion (arrow) in gill epithe- cockle  lium of basket cockle

TEM of similar littleneck clam muscle inclusion showing rickettsia-like organisms (arrow) RLO cytoplasmic inclusion in mantle muscle of littleneck clam 16 17 bivalve mollusc Fungi Shell Fungus

I. Causative Agent known but could lead to weakened shells A fungal agent resembling Penicil- and inability to close the valves properly lium sp. was associated with abnormal if conchiolin deposits become too thick. brown/black conchiolin deposits on the The abnormal conchiolin deposition is inner surface of butter clams collected a host response to wall-off or isolate an from Kodiak Island, Alaska. A differ- irritant. ent shell disease fungus, Ostracoblabe implexa, causes adductor muscle detach- VII. Human Health Significance ment and mortality characterized by There are no known zoonotic human inner shell conchiolin warts and severe health concerns caused by this shell thickening of the shell margin in oysters fungus. reported from Europe, India and on both Atlantic and Pacific coasts of Canada.

II. Host Species The Alaskan shell fungus isolate has been detected in only one sample of butter clams from Kodiak Island collected in late 2005.

III. Clinical Signs Clinical signs include brown to black semi-hard, flaky conchiolin deposits on the inside shell and around the mantle edges with no apparent mortality or unusual clam behavior.

IV. Transmission The mode of transmission is unknown but likely horizontal via seawater.

V. Diagnosis Abnormal brown to black conchiolin deposits are visible on the inside shell with associated septate fungal hyphae observed microscopically in wet mounts of the material. This fungus can be cultivated by inoculating the conchiolin material onto potato agar supplemented with 2% sodium chloride.

VI. Prognosis for Host The prognosis for the host is un-

18 19

bivalve mollusc Fungi 

Black conchiolin deposits (arrow) containing fungal hyphae on the inside shell surface of butter clams

Wet mount of conidiophore and typical conidia of Penicillium-like shell fungus from butter clams

18 19 bivalve mollusc Fungi Systemic Mycosis

I. Causative Agent and Disease Grocott’s method of methenamine-silver An unidentified marine fungus nitrate (GMS) for fungi. An opportunity causes a systemic mycosis in basket has not occurred for further character- cockles characterized by an intense ization of the fungus by attempted isola- inflammatory response with multiple tion on artificial media. granulomas containing necrotic cells and fungal hyphae. Infections have been VI. Prognosis for Host discovered incidentally as well as as- The fungal infection is highly sociated with high mortality. There are invasive and likely responsible for the no other known reports in the literature mortality of infected animals. The preva- of a similar systemic fungus infection in lence of this fungus in examined basket adult bivalve molluscs. cockle populations has been low but its overall importance for causing enzootic II. Host Species disease is unknown. This fungus has been observed only in basket cockle populations from three VII. Human Health Significance locations in southeast Alaska during 1987 It is unlikely that this fungal infec- to 2005. The geographic distribution of the tion in bivalve molluscs has any zoonotic fungus in Alaska and whether other species significance for human health but defini- of bivalves are susceptible are unknown. tive evidence is not available at this time.

III. Clinical Signs The fungal infection may be grossly visible as external focal tissue discolorations with or without observed cockle mortality. Histological examination shows variable infiltration of host tissues by fungal hyphae that are contained within large granulomas.

IV. Transmission The mode of transmission is unknown but is presumably horizontal via ambient seawater and/or the substrate where the cockles occur.

V. Diagnosis Histological examination of hematoxylin and eosin stained material show large granulomas containing necrotic tissues and branching brown septate hyphae with basophilic conidia-like reproductive structures. Tissue sections of hyphae stain black (positive) by

20 21

bivalve mollusc Fungi

 

Histological section of granuloma in connec- Higher magnification of same necrotic focus tive tissue with two necrotic foci containing with fungal hyphae (arrow) fungal hyphae (arrow)

GMS stain showing septate fungal hyphae (black)

20 21 bivalve mollusc Protozoa Gill and Gut Ciliates

I. Causative Agent and Disease V. Diagnosis The large thigmotrichid group Small (4.5 X 7.0 µm to 6.5 X 10.0 of relatively non-pathogenic ciliated µm) eosinophilic thigmotrich ciliates are protozoa found in several species of commonly found on or slightly embed- bivalve molluscs occupy an existence ded within the gill or palps tissues by between commensalism and parasitism. routine histological examination. Also These ciliates include small unidentified occurring on the gill and palps tissues, thigmotrichs and Sphenophrya sp. found Sphenophrya sp. is a larger basophilic exclusively on the gills and palps and crescent-shaped ciliate (maximum -15.0 others found on the gills, palps and in the X 23.0 µm) having a large elliptical GI tract that include Ancistrocoma sp., macronucleus. The unidentified cili- Stegotricha enterikos and large unidenti- ates (possibly more than one species) fied ciliates. are of variable size and found on the mantle, gills and within the GI tract. II. Host Species Ancistrocoma sp. and Stegotricha are These protozoa are ubiquitous and spindle-shaped ciliates (50-70 µm in commonly occur in Pacific, eastern and length) with large, granular, polymorphic European flat oysters on the Atlantic, nuclei. They are found mostly within the Pacific and Gulf coasts of North America digestive tubule lumens (sometimes the but are also reported from several other intestine) by histological examination or bivalve species from Europe. In Alaska, by tissue squashes mounted in seawater these ciliates have been observed on the where they can be observed as motile gills, palps and in the digestive tubules and attached forms. The morphology of and intestines of Pacific oysters, blue these ciliates is more clearly observed by mussels, basket cockles, rock and weath- staining tissue smears with silver protein ervane scallops and cultured juvenile or iron hematoxylin. For the ciliates in littleneck clams. the GI tract, the numbers and spacing of the somatic kineties (bands of cilia) are III. Clinical Signs more clearly observed with these stains There are no reported clinical signs which are useful for determining species of disease caused by these protozoa, identification. although high intensities may cause localized tissues damage on histologi- VI. Prognosis for Host cal examination and could be associated Often found at high prevalences but with physiological stress of the host. generally low intensities, these ciliates Occasionally, Sphenophrya sp. can are relatively non-pathogenic. At high produce xenomas (hypertrophied host intensities they are capable of producing cells containing one or many organisms) localized cell damage and excess mucus which do not appear to cause physical production. In gill infestations the lami- harm to bivalve hosts. nar water flow essential for food particle transport across the epithelial surface IV. Transmission may be disrupted. Because Ancistro- The mode of transmission is coma sp. is occasionally found on the gill horizontal from host to host via ambient tissues, the literature has suggested that seawater. those reported internally in both Pacific

22 23 bivalve mollusc Protozoa

and eastern oysters may be normal gill VII. Human Health Significance parasites which invade the GI tract during There are no zoonotic human health abnormal conditions of physiological concerns regarding the presence of these stress. This may also be true for some of ciliated protozoa on or within bivalve the unidentified ciliates. mollusc tissues.



Histological section of Ancistrocoma-like Xenoma of gill epithelial cell in blue mussel

ciliate in the digestive tubule of Pacific formed by Sphenophrya sp. (arrow)

oyster 

Histological section of small thigmotrich Histological section of large unidentified ciliate (arrow) on gill epithelium of Pacific ciliate on gill tissues of native littleneck clam oyster containing consumed host cells

22 23 bivalve mollusc Protozoa Haplosporidium sp.

I. Causative Agent and Disease in France, Britany and the Netherlands. Protozoa of the genus Haplospo- Other Haplosporidium species have also ridium sp. belong to the phylum Hap- been reported in California sea mus- losporidia, class Haplosporea within sels (H. tumefacientis) from California, the order Haplosporida in the family blue mussels (Haplosporidium sp.) from Haplosporidiidae. These are obligate Maine, gaper clams and native oysters parasites, some of which are extremely (Haplosporidium sp.) from Oregon, important pathogens of oysters. One carpet-shell and Manila calms ( H. such species, H. nelsoni, was respon- tapetis) from France, Spain and Portu- sible for MSX (multinucleate sphere gal. Unidentified Haplosporidium sp. unknown) later known as Delaware Bay were also found in cockles from France, Disease that caused the demise of the black-footed abalone in New Zealand eastern oyster in higher salinity (>15 and in toredo ship worms from New but < 25 ppt) areas of Delaware and Jersey, U.S.A. In Alaska, only one case Chesapeake Bays from 1957 through of a bivalve haplosporidian is on record 1960. Systemic plasmodial life stages regarding an unidentified Haplospo- of the parasite can cause extensive ridium sp. producing plasmodia in the necrosis of all tissues resulting in mass gill connective tissues of a single razor mortality. Occurring at about the same clam collected from the southcentral area time another species, H. costalis or SSO in 1991. No disease or mortality were (Seaside Organism), caused serious associated with this finding. mortality in eastern oysters rearing in higher salinity waters (> 25 ppt) on the III. Clinical Signs seaside coasts of Virginia and Mary- Non-specific clinical signs of land. haplosporidiosis parasitism are more severe in oysters and can include mantle II. Host Species recession, gaping valves, watery emaci- Haplosporidium nelsoni has been ated tissues, pale digestive gland and reported in eastern oysters on the rarely yellow-brown conchiolin deposits Atlantic coast of North America as far forming on the inner shell. Dissemina- north as Bras d’Or Lakes, Nova Scotia, tion of plasmodial stages of the parasite Canada south to Florida. A morpho- throughout all tissues with associated logically similar Haplosporidium sp. host cell inflammatory infiltration and in Pacific (Japanese) oysters in Korea cell necrosis are evident by histological and Japan and on the Pacific coast of examination. North America was determined by DNA sequence analysis to be H. nelsoni IV. Transmission apparently introduced to the Pacific Haplosporidium sp. produce opercu- Northwest from Asia. The parasite was late spores which are not directly infec- then introduced to the Atlantic coast with tious but likely require, a yet unknown, west coast Pacific oysters sometime be- intermediate host. The complete life cy- fore the epizootics occurred in Delaware cle and infective stage are unknown but Bay. Haplosporidium armoricana has once in the bivalve host a modified type been reported in the European flat oyster of shizogony gives rise to multinucleated

24 25 bivalve mollusc Protozoa

plasmodia. These plasmodia develop into VI. Prognosis for Host sporonts that become sporocysts enclos- Various species of Haplosporidium ing several spores that are released into sp. have caused significant bivalve the environment from moribund or dead mortality and are serious pathogens that bivalve hosts. should not be introduced into new areas via shellfish transports. Salinity and sea- V. Diagnosis water temperature are important limiting Preliminary diagnosis for all factors for parasite development and dis- Haplosporidium sp. is based on histo- ease that have been used to reduce oyster logical examination showing the typical losses due to H. nelsoni and H. costalis. plasmodia varying in size from 5 to 100 This strategy may also be useful to µm found in various host tissues. The control other haplosporidia. Haplospo- larger spores of H. nelsoni (5.4 X 7.5 µm) ridiosis in Pacific oysters has not been are rarely found in adult oysters but are associated with mortality of that species. frequent in juveniles occurring only in On the east coast selectively bred eastern the digestive gland epithelium. Smaller oysters that are genetically resistant to spores (3.3 X 4.3 µm) of H. costalis H. nelsoni have been deployed commer- occur throughout the connective tissues cially on a limited basis. but not in the digestive gland epithelium. Spores of both species (probably other VII. Human Health Significance haplosporidia as well) are red (acid-fast) There are no zoonotic human health using the Ziehl-Neelsen stain. Confirma- concerns associated with haplosporidian tory tests using PCR and DNA probes parasitism of bivalve mollusc tissues.

are available for H. nelsoni and possibly

other species of haplosporidia. 

Haplosporidia-like sporonts (arrow) in the gill connective tissues of Alaskan razor clam

24 25 bivalve mollusc Protozoa

Haplosporidium sp. 

Histological section showing various stages of haplosporidia-like sporonts (arrow) and spore development in the gill connective tissues of Alaskan razor clam



Histological section of eastern oyster with Histological section of eastern oyster with MSX plasmodia (arrow) in connective tissues MSX acid-fast spores (red) within the digestive tubule epithelium

26 27 bivalve mollusc Protozoa



 Histological section of eastern oyster with SSO plasmodia (arrow) and spores (arrowhead) in connective tissues

Histological section of eastern oyster exhibiting SSO acid-fast spores (red) in connective tissues

26 27 bivalve mollusc Protozoa Hexamita

I. Causative Agent and Disease V. Diagnosis Hexamita (nelsoni and inflata) Hexamita, as observed in hemo- is a zooflagellate protozoan that is a lymph smears or tissue squashes, is very free-living saprophyte occurring in the motile and pyriform in shape, about vicinity of oyster beds. It can become a 14-17 µm long by 7-10 µm wide and has facultative parasite when environmental 6 anterior and 2 posterior flagella. Mixed conditions are unfavorable or may be- populations of bacterial rods may also be come a secondary pathogen of oysters present in the hemolymph of moribund dying from other diseases. Hexamita is or dead oysters. Histological examina- commonly found in low numbers within tion demonstrates the protozoan in gut the intestinal tract of oysters with no lumens of healthy animals. In diseased associated pathology. animals the organism is present on mantle and gill surfaces and throughout II. Host Species all tissues as are various bacteria and tis- Hexamita can be found associated sue necrosis. Hexamita can be cultured with several different species of oysters in artificial media and in filtered seawa- worldwide. It has been associated with ter containing antibiotics. Identification mortality of the native oyster in winter of the organism to species requires silver during cold seawater temperatures in impregnation stains to reveal distin- Puget Sound, Washington and mortality guishing morphological characteristics. of the European flat oyster in recirculating seawater pits (Pit Disease) in the VI. Prognosis for Host Netherlands. Hexamita sp. has been Hexamita may be found at low detected once in southeast Alaska during intensities and generally does not invade 1987 as a secondary pathogen associated the tissues of healthy oysters except with opportunistic bacteria contributing when poor or extreme environmental to summer mortality in 18 month-old conditions (high temperatures, low Pacific oysters stressed by high seawater temperatures < 6°C, poor water quality) temperatures and rapid gonadal develop- cause physiological stress. In such cases, ment. overwhelming systemic disease can occur with reported mortality greater than III. Clinical Signs 50% in Alaskan Pacific oysters and up to Diseased oysters have no specific 75% in native oysters from Puget Sound, gross clinical signs but hemolymph Washington. smears may contain large numbers of the flagellate while systemic infestation by VII. Human Health Significance the parasite is evident in all tissues by There are no zoonotic human health histological examination. concerns regarding the presence of Hexamita in oyster tissues. IV. Transmission The mode of transmission is horizontal since Hexamita sp. occurs as a saprophyte in ambient seawater and as part of the normal gut flora of healthy oysters.

28 29 bivalve mollusc Protozoa

 Hexamita sp. in a Giemsa stained smear

Histological section of Hexamita sp. (arrow) within the intestine of Pacific oyster associated with a bacterial infection



Histological section of Hexamita sp. (arrow) within the connective tissues of Pacific oyster

28 29 bivalve mollusc Protozoa Ichthyobodo-Like Protozoan

I. Causative Agent and Disease two flagella, that parasitize the same Protozoa of the genus Ichthyobodo or a different host. Motile forms attach belong to the family Bodonidae, order by means of a flat disc with two small Kinetoplastida of the class Kineto- microtubules extending into the host cell, plastidea within the flagellate phylum but retain flagella. of Mastigophora. Ichthyobodo neca- tor (synonym pyriformis) is a serious V. Diagnosis obligate ectoparasite of fishes in both the Diagnosis is by histological exami- freshwater and marine environments but nation showing very small pyriform- similar protozoa have not been described shaped (5-10 µm) feeding forms of the occurring on the surface of bivalve mol- parasite attached to epithelial cells of the luscs. Therefore, the disease potential of mantle surface. this ectoparasite in bivalves is unknown. VI. Prognosis for Host II. Host Species The prognosis for the host is There are no reports of a similar ap- unknown but in this case the parasite pearing parasite occurring on the surface is presumed to be relatively harmless tissues of bivalve molluscs, therefore the based on the lack of significant tissue potential host species are unknown. In pathology. However, it is plausible that Alaska, this parasite has been observed high parasite intensities on juvenile attached to mantle epithelium from 8 of animals could result in significant 15 adult weathervane scallops collected pathological changes. in Yakutat, Alaska during December 1990. No mortality or clinical disease VII. Human Health Significance were associated with this finding. There are no zoonotic human health concerns regarding the occurrence III. Clinical Signs of this parasite on external tissues of No gross clinical signs are evident bivalve molluscs. but the parasite can be observed attached to mantle epithelium by histological examination.

IV. Transmission The route of transmission is unknown but presumed to be horizontal via seawater by a motile flagellated non- feeding form as described for this group of organisms. The parasite alternates between a free swimming and a non- motile feeding stage that attaches to a host epithelial cell. Replication of the protozoan in fish is by asexual longitu- dinal fission where one cell produces two motile daughter cells, each with

30 31 bivalve mollusc Protozoa



Histological section of Ichthyobodo-like protozoa (arrow) attached to the mantle epithelium of weathervane scallop

30 31 bivalve mollusc Protozoa Nematopsis and Other Gregarines

I. Causative Agent and Disease rock scallops and weathervane scallops. Oocysts of several different species Localized gregarine-like trophont stages of Nematopsis gregarines of the family in the gut and gills have been observed in Porosporidae, subclass Gregarinia, class Alaskan Pacific oysters, blue mussels and Sporozoa infest various tissues of many rock scallops while systemically distrib- species of bivalve molluscs causing no uted gregarine-like trophozoites have been significant harm to the hosts. Encysting found in Pacific oysters, blue mussels and trophont stages of other gregarine-like littleneck clams. organisms observed primarily in oysters infest the epithelial mucosa of the gut, III. Clinical Signs again causing little tissue damage. A These gregarine organisms are found third type of systemic gregarine-like incidentally in apparently healthy bivalve trophozoite stage has been reported in molluscs showing no outward clinical bivalves infesting all tissues causing signs of disease. Oocysts and vegetative localized hemocyte accumulations and stages within bivalve tissues are discov- phagocytosis, but otherwise is not patho- ered by routine histological examination. genic. IV. Transmission II. Host Species The life cycle of gregarines requires Gregarine parasites are cosmopolitan two hosts, a marine mollusc as the in- in the oceans of the world while individual termediate host and a marine arthropod, species may have more confined distribu- generally a crustacean, as the final host. tions. Oocysts of Nematopsis species Trophozoites in the gut of a crusta- commonly reported from eastern and cean host give rise to gametocysts and Pacific oysters belong to N. ostrearum and gamete formation resulting in sporula- N. prytherchi that also infest other scallops tion and the production of gymnospores. and clams and use at least 5 xanthid spe- Gymnospores, released from ruptured cies of crab as final hosts. Oocysts of Nem- gametocysts, are eliminated with feces atopsis duorari infest several marine mol- and eventually come into contact with a luscs and the parasite uses the pink shrimp suitable mollusc. Gymnospores cause a as a definitive host. Other unidentified host cellular response and are engulfed gregarine-like encysting trophont stages in by phagocytes that pass back into the the gut mucosa have been reported from mollusc host through the mantle epithe- the eastern oyster on the Atlantic coast of lium. The engulfed gymnospore gives the U.S. and in Pacific oysters and Manila rise to sporozoites which, in the genus clams in the Pacific Northwest while the Nematopsis, form resistant oocysts each systemic gregarine-like trophozoite has containing one sporozoite. Sporozoites been reported from Washington State and in oocysts are eaten when the bivalve British Columbia, Canada in Pacific oys- host is consumed by the crustacean final ters and Manila clams. In Alaska, oocysts host and give rise to trophozoites which of Nematopsis sp. have not been observed completes the life cycle. in Pacific oysters but have been found in the gills and connective tissues of little- V. Diagnosis neck clams, basket cockles, blue mussels, Wet mounts of infected tissues

32 33 bivalve mollusc Protozoa

reveal typical oocysts while histological which spores have not been found to al- examination is less sensitive but allows low definitive identification. detection of both oocysts and vegetative troph stages within the bivalve tissues. VI. Prognosis for Host Nematopsis oocysts are characterized Gregarine parasites do not appear by a thick hyaline capsule enclosing a to negatively impact their intermediate densely basophilic worm-like sporozoite bivalve mollusc hosts. coiled within. Oocysts in oysters range in size from 14-19 µm X 10-16 µm and VII. Human Health Significance are found most commonly in the gill There are no zoonotic human health connective tissues. The term “gregarine- concerns regarding the occurrence of like” is used for organisms resembling gregarine parasites in bivalve mollusc the vegetative stages of gregarines for tissues.  

Histological section of oocysts (arrow) of Histological section of gregarine-like

Nematopsis sp. in the connective tissues of trophont stages (arrow) in the intestinal

weathervane scallop epithelium of Pacific oyster 

Histological section of gregarine trophont Histological section of gregarine trophont stages in the connective tissue of Pacific stages (arrow) within the water tubules of oyster the gill in basket cockle

32 33 bivalve mollusc Protozoa Steinhausia sp. and Other Unidentified Microsporidia

I. Causative Agent and Disease sporonts have been observed in the ova Microsporidia is a protozoan order of native littleneck clams and spores of within the class Microsporea within the unidentified microsporidia have occurred phylum Microspora. However, there in the nervous tissue, mantle connective is controversial genetic evidence for a tissue and foot muscle of littleneck clams closer affinity to the kingdom of Fungi and basket cockles. rather than Protozoa. A small number of Microspora parasitize marine bi- III. Clinical Signs valves. They are intracellular parasites Parasitized bivalves appear healthy producing smaller microspores (3 to with no clinical signs of disease or mor- 6 µm) and complete their life cycles tality. The microsporidia in ova and vari- in a single host cell, generally with no ous other tissues are incidental findings alternate hosts. Identified microsporid- during routine histological examination. ian species in bivalve molluscs include Steinhausia ovicola and S. mytilovum IV. Transmission that are parasites of ova in female Transmission is horizontal when bivalves and cause no significant harm. spores released from ruptured host cells Several other unidentified microsporid- are ingested by a suitable host. In the ia have been described in ova, connec- intestine of the new host each spore tive tissues and the digestive gland but releases a hollow polar tube attaching also causing no significant disease. the spore to a mucosal epithelial cell through which the internal amoeboid II. Host Species sporoplasm passes into the host cell. Steinhausia has been described para- The sporoplasm may replicate in the sitizing wild and cultured bivalves in the intestinal cell or may be injected into a ova of the European flat oyster S.( ovico- host phagocyte where it travels to other la), oocytes of blue and gallo mussels (S. target tissues. In addition to transmis- mytilovum) and cockles (Steinhausia sp.) sion via the alimentary tract, reports in France, in Mytilus sp. (S. mytilovum) have hypothesized that Steinhausia sp. from Korea and Japan, the blue mussel may also be vertically transmitted to (S. mytilovum) from the Atlantic coast progeny when lightly parasitized ova and in the gallo mussel (S. mytilovum) on develop normally. Replication of Stein- the Pacific coast of the U.S., Italy, Spain hausia sp. has also been reported in and Greece. Other unidentified mi- oocyte nuclei of some bivalve species. crosporidia have been reported from: the Once in the target host cell the parasite ova of Australian blacklipped oysters; undergoes further replication and devel- connective tissues surrounding the gut of opment involving merogony producing New Zealand dredge oysters; connective plasmodia and meronts followed by tissues of the digestive gland in queen sporogony producing sporonts that con- scallops in the United Kingdom; the tain sporoblasts that mature into spores. digestive gland of cockles in France; and The entire process is complex and may in the U.S. from ova of Macoma clams in have other intermediate stages depend- Maryland and from ova of Pacific oysters ing on the species of microsporidia. in California. In Alaska, Steinhausia-like

34 35 bivalve mollusc Protozoa

V. Diagnosis VI. Prognosis for Host Histological examination may detect Microsporidian parasites do not the presence of microspores within vari- appear to cause significant harm to their ous bivalve tissues or within sporocysts bivalve mollusc hosts. in the cytoplasm of maturing or well developed ova. Multiple immature VII. Human Health Significance sporonts (sporocysts) may be present There are no zoonotic human health with no visible spores. Genus and species concerns regarding the presence of mi- identification by histology alone is gener- crosporidian parasites within the tissues ally not possible and would require closer of bivalve molluscs. morphological examination by transmission electron microscopy.



Histological section of microspores (arrow) Higher magnification of microspores at

of an unidentified microsporidian in the pal- left lial nerve of basket cockle 

Microspores of an unidentified microsporid- Sporonts of a Steinhausia-like microspo- ian in the foot muscle of basket cockle ridian in an oocyte (arrow) of native littleneck clam

34 35 bivalve mollusc Protozoa Trichodinid Ciliates

I. Causative Agent and Disease glands, eroded and deformed gills with Peritrichous ciliates of the genera large numbers of the ciliates present. Trichodina, Urceolaria and Leiotrocha are common parasites of several spe- IV. Transmission cies of marine molluscs. Under normal Transmission of trichodinid parasites circumstances these ectoparasites are is horizontal via ambient seawater. Older regarded as more commensalistic and less susceptible adult bivalve molluscs harmless but when present in large num- are reservoirs for the parasite which can- bers they may cause disease. Trichodina not survive long outside a host. sp. is the most common genus and has been associated with recurrent large scale V. Diagnosis mortality of cockles in Germany and Tissue wet mounts of trichodinid cili- possibly oysters in France (a virus may ates demonstrate a saucer or disc-shaped also have been present) during warmer motile organism of variable diameter (33- summer/fall seawater temperatures. 103 µm) that often rotates in place. Vis- The mortality described in parasitized ible are a ventral ring of denticles (teeth) bivalves was likely due to overwhelming with a ciliary girdle and a dorsal adoral erosion of gill tissues that disrupts normal ciliary ring. Histological examination respiratory function followed by possible of gills, palps and mantle demonstrates microbial secondary infection. an eosinophilic helmet-shaped ciliate with a large horseshoe-shaped nucleus, II. Host Species the ends of which appear as two “eyes” Trichodina sp. has been reported from in most sections. Specific identification various species of clams, cockles, scallops of genus and species is accomplished by and oysters from Europe, the Atlantic silver impregnation staining of smears coast of the United States and the Pacific to reveal the arrangement and number rim. In Alaska, unidentified trichodinid of denticles as well as other features of ciliates have been found on the gills, palps parasite morphology. and mantle tissues of Pacific oysters, basket cockles, rock and weathervane scal- VI. Prognosis for Host lops. Trichodinid parasites usually occur in low numbers as harmless commensals III. Clinical Signs feeding on bacteria, surface detritus Trichodinids observed in Alaska and surface tissue cells of the host. were found incidentally during routine Significant tissue damage is avoided by histological examination of apparently the regenerative capacity of a healthy normal bivalve molluscs with no associ- host and the natural equilibrium between ated mortality or other clinical signs of rates of parasite multiplication and acci- disease. Cockle and oyster mortalities in dental parasite expulsion with the normal Europe and France that were associated flow of seawater maintained by the host with Trichodina infestations occurred in the pallial cavity. Environmental or mostly in juvenile or otherwise younger physiological stress of the host reduces animals. Clinical signs included emaci- ciliary activity and the pumping action ated soft tissues, grey-colored digestive of the gills. This reduces the frequency

36 37 bivalve mollusc Protozoa

of parasite expulsion leading to increases VII. Human Health Significance in parasite abundance, tissue damage and There are no zoonotic human health excessive mucus production followed by concerns with the occurrence of tricho- possible death of the host. Older adult bi- dinid parasites in bivalve molluscs. valves are reservoirs of the parasite that can infest the more susceptible juvenile molluscs.

Wet mount of Trichodina protozoan showing cilia and denticles

Histological section of a trichodinid ciliate on the mantle surface of Pacific oyster

Histological section of trichodinid ciliates on the gill surface of Pacific oyster

36 37 bivalve mollusc Protozoa Unidentified Kidney Coccidia and Others

I. Causative Agent and Disease Mediterranean Sea, England and Scot- Coccidiasina is a subclass within land, Pectin scallops and the European the class Conoidasida in the phylum flat oyster in France, cockles in Spain, Apicomplexa. These protozoa are blacklipped oysters in Australia and in intracellular parasites of vertebrates as blue mussels, bay scallops and the eastern well as invertebrates. Some members of oyster on the Atlantic coast of North two suborders, Adeleorina and Eimeri- America and on the Pacific coast in the orina, parasitize bivalve molluscs in the native littleneck clam in Washington State kidneys (Pseudoklossia glomerata, P. and in blue mussels, native littleneck and pectinis, P. pelseneeri, Klossia telli- Japanese littleneck (Manila) clams in nae), germ cells of the ovarian tubules British Columbia, Canada. In Alaska, an (Merocystis tellinovum), developing unidentified kidney coccidian (possibly ova (unidentified coccidia), connective multiple species) has been found in 12-33% tissues (unidentified coccidian sporo- of examined blue mussels, basket cockles cysts) and sometimes visceral ganglia (P. and native littleneck clams. Also, varying glomerata). Coccidia of bivalve molluscs prevalences of littleneck clams from all are of two types: those that complete part Alaskan locations (total prevalence ~ 62%) of their life cycle in bivalves and presum- have been parasitized by a sporocyst stage ably require an alternate host because of another unidentified coccidian dissemi- certain developmental stages have not nated throughout all connective tissues. A been observed; and those organisms that light intensity of similar sporocysts have complete their entire life cycle within also been found in the connective tissues their bivalve hosts where all develop- surrounding the gut of a single butter clam. mental stages have been described. De- pending on the species of bivalve host, III. Clinical Signs tissue degeneration and necrosis from the The kidney coccidian in native intracellular development of these para- littleneck clams from Washington State sites ranges from minor in developing reportedly causes behavioral modifica- ova to severe kidney damage or parasitic tion such that live parasitized clams castration of females when infecting are found on the surface of the sand germ cells. Severe kidney tubule infesta- substrate (“kick-outs”) rather than buried tion of an unidentified coccidian in Ca- underneath. Otherwise there are no other nadian bay scallops reportedly resulted gross clinical signs of disease or mortal- in systemic dissemination of the parasite ity in the other reported infestations. into various other tissues. No mortality Routine histological examination detects directly related to coccidian parasitism these coccidia within their respective in bivalves has been documented in feral target host tissues with varying degrees populations but has been reported in of cell damage or host response ranging scallops during at least one situation of from none to significant in the case of recirculating hatchery culture. certain kidney coccidia.

II. Host Species IV. Transmission Coccidia have been described Transmission of these coccidia is infecting Tapes and Tellina clams in the likely horizontal via ambient seawater

38 39 bivalve mollusc Protozoa

but many may require at least one alter- connective tissues of Alaskan littleneck nate host to complete their life cycles. clams typically contain elongate sporo- The typical life cycle producing the zoites (28 µm by 4 µm). Generic and spe- developmental stages of coccidia can cies identification of coccidia are based be divided into three phases: asexual primarily on the structure of oocysts merogony (schizogony) where multi- and sporocysts as well as the number of plication occurs inside the specific host sporocysts contained by the oocyst. target cells producing trophozoites giv- ing rise to schizonts containing mero- VI. Prognosis for Host zoites that infect other cells to continue The effects of coccidia on host merogony or to begin gamogony; sexual bivalve molluscs is variable ranging from gamogony (anisogomy) where merozo- no effect to significant kidney necrosis ites, upon entering host cells, transform and parasitic castration in females. In into gamonts of macrogametes (female) Alaska, moderate host inflammatory and biflagellated microgametes (male) infiltration with minor kidney necrosis that join to form a zygote or oocyst; and caused by infestation has been observed asexual sporogony where oocyst cyto- in two basket cockles. However, gener- plasm divides to contain sporoblasts that ally these parasites have appeared harm- develop into sporocysts each containing less in wild as well as cultured bivalve sporozoites that, when liberated from species. the oocyst, infect cells to start the entire cycle anew. For most coccidia in bivalve VII. Human Health Significance molluscs gamogony and sporogony oc- There are no zoonotic human health cur in the bivalve host with merogony concerns regarding these coccidian para- presumably occurring in other hosts sites in bivalve mollusc tissues. that have not been identified. The few remaining reported coccidia apparently complete all developmental phases in the bivalve host.

V. Diagnosis Large mature macrogamonts may be observed in squash preparations of kidney tissues while all intracellular developmental stages may be observed by histological examination. The kidney coccidian of the Alaskan littleneck clam appears identical to one reported in the same clam species in Washing- ton State and is likely an undescribed species based on: the apparent comple- tion of its entire life cycle in the clam; the production of a thick-walled spore in addition to a thin-walled oocyst; and the presence of tetrazoic (4 sporozoites) sporocysts instead of dizoic sporocysts. Large sporocysts of another unidentified coccidian disseminated throughout the

38 39 bivalve mollusc Protozoa Unidentified Kidney Coccidia

and Others 

Histological section of gamonts (arrow) of a Pseudoklossia-like coccidian in the kidney of native littleneck clam 

Microgamont (arrow) of a Pseudoklossia-like coccidian in the kidney of native littleneck clam

40 41

bivalve mollusc Protozoa 

Resting spore (arrow) of Pseudoklossia-like coccidian in the kidney of native littleneck clam



Oocyst (arrow) of Pseudoklossia-like coccidian in the kidney of basket cockle

40 41 bivalve mollusc Protozoa Unidentified Kidney Coccidia

and Others 

Histological section of sporocyst containing elongate sporozoites (arrow) of an

unidentified coccidian in the connective tissue of native littleneck clam  

Ultrastructural detail of littleneck clam sporozoites (arrow) and sporocyst wall (arrowhead), TEM

42 43 bivalve mollusc Protozoa Typical Coccidia Life Cycle

Stages occur in bivalve host

Asexual Sporogony oocysts



sporoblasts



sporocysts Stages occur in other host

sporozoites 

Asexual Merogony  sporozoites



trophozoites

schizonts 

merozoites

Sexual Gamogony merozoites

 

male female microgametes macrogametes 

oocysts

42 43 bivalve mollusc Helminths Gill and Gut Turbellaria

I. Causative Agent and Disease colonize another or the same host. Turbellaria found associated with bivalve molluscs are flatworms of the V. Diagnosis phylum Platyhelminthes, class Turbel- Diagnosis is by observation of small, laria, order Rhabdocoela and family oval or pyriform worms up to 2 mm long Graffillidae. These flatworms exist as having a ciliated body surface in wet both endocommensals and parasites mounts of gill or mantle tissues. Special able to pass freely between the mantle stains of mounted worms are used for cavity and alimentary canal of the host species identification. Also, routine bivalve. They generally are found in histological examination may demon- low prevalences and intensities causing strate the presence of ciliated flatworms no apparent harm to the bivalve host. A in the intestinal tract, digestive tubules common species is Urastoma cyprinae or kidney. The presence of external cilia found on the gills of several bivalve spe- differentiate turbellarians from other cies or free-living in muddy sediments. classes of flatworms.

II. Host Species VI. Prognosis for Host Turbellarians have been reported Turbellarians cause no apparent harm from eastern oysters and giant scallops to bivalve hosts. in Atlantic Canada, in various clams, cockles, mussels and oyster species in VII. Human Health Significance Europe and in geoduck clams and Pacific There are no zoonotic human health oysters in British Columbia, Canada. In concerns with the presence of turbellar- Alaska, turbellaria have been observed ians on the surface or within shellfish on the gills, within alimentary tracts and tissues that may be consumed uncooked. kidneys of weathervane scallops, basket cockles, blue mussels and native littleneck clams. Turbellaria are likely global in distribution although individual species may be confined to certain ranges and bivalve hosts.

III. Clinical Signs The only gross clinical signs of infestation may be the appearance of white to pink colored “gill worms” up to 2 mm long on gill and mantle surfaces.

IV. Transmission Bivalve turbellarians are hermaphroditic having a direct life cycle where eggs are produced that hatch in ambient seawater. The juvenile stage may then

44 45

bivalve mollusc Helminths 

Histological section of a ciliated turbellarian (arrow) in the intestinal lumen of weathervane scallop

44 45 bivalve mollusc Helminths Shell-Boring Polychaetes

I. Causative Agent and Disease IV. Transmission Segmented polychaete worms belong The abundance of shell boring poly- to the phylum Annelida, class Polychaeta chaetes is largely influenced by the pres- and family Spionidae. These worms ence of a mud bottom. Host infestation is utilize the shells of bivalve molluscs as a from a simple direct life cycle. Juvenile substrate by constructing burrows on the worms settle on the edge of the shell and shell surface or burrowing into the shell begin burrowing or, in the case of P. to form tunnels lined with compacted ligni, build their tubes on the surface of mud. Hence, the name of the condition the shell. Hermaphroditic adults produce is known as “mud blister”. The species egg capsules in the burrow that hatch Polydora websteri and P. ligni, among into larvae released into seawater which others, are commonly associated with disperse to a new host. mud blisters in bivalve molluscs causing unsightly internal shell surfaces and, V. Diagnosis in severe cases, causing formation of Diagnosis is based on gross observa- debilitating pustules or abscesses in tion of 2 mm diameter sinuous burrows soft tissues that can result in substantial or 1 cm diameter patches of mud and bivalve mortality and economic losses. debris in or on the shell matrix when held next to a bright light. “Muscle II. Host Species pearls” or nacreous excrescences in the Shell-boring polychaetes occur muscle scar of the shell is evidence of worldwide causing mud blisters in vari- healed abscesses in the muscle tissues. A ous species of bivalve molluscs including polychaete may be removed by break- oysters, mussels and scallops. In Alaska, ing the shell along the burrow path and mud blisters have only been observed submerging the shell fragments in sea- in weathervane scallops from Shelikof water to extract the worm with forceps Strait and may have been associated with and needle. Species identity is based on mortality in the population. the morphological characteristics of the worm, particularly the armature of the III. Clinical Signs setae. Gross clinical signs include mud blisters on the inner surface of the VI. Prognosis for Host shell and possibly yellow pustules in Mortality of infested bivalves is soft tissues contacted by the burrows. generally rare when prevalences and in- Severely infested bivalves may show tensities of mud blisters are low, but the gaping valves and an overall poor body half-shell marketability may be reduced. condition. Polydora ligni does not bore In the majority of cases oysters are able directly into shell but builds tubes on the to better tolerate infestation by walling bivalve shell surface and secretes thick off the burrows with newly secreted mucus that retains sediment, oyster feces shell nacre. Scallops are more seriously and rejected material. Decomposition of affected because they are less able to this material produces hydrogen sulfide produce nacre towards the interior of the easily detected by odor that can also shell where burrows may damage the at- cause massive bivalve mortality. tachment of the adductor muscle. Violent

46 47 bivalve mollusc Helminths

contractions of the valves can pull the VII. Human Health Significance damaged adductor muscle loose resulting There are no zoonotic human health in eventual scallop mortality. Preva- concerns regarding polychaete infes- lences and intensities of mud blister may tation of bivalve molluscs except for be reduced by off-bottom bivalve culture the reduced aesthetic quality of meats techniques. consumed in the half-shell market.

Eastern oyster shell with Polydora tunnels (arrow) on inner surface of valves (Photo: Dorothy Howard, NOAA Cooperative Oxford, MD Laboratory)

Another eastern oyster with Polydora tunnels (arrow) on inner surface of valves (Photo: Dorothy Howard, NOAA Cooperative Oxford, MD Laboratory)

46 47 bivalve mollusc Helminths Trematode Metacercariae and Sporocysts

I. Causative Agent and Disease mussels and razor clams while unidenti- Trematodes or flukes are members fied encysted metacercariae have been of the phylum Platyhelminthes and the observed in blue mussels, basket cockles, class Trematoda. Adult worms of the littleneck clams and weathervane scal- subclass Digenea are endoparasites oc- lops. curring in all classes of vertebrates and use invertebrates as the first and second III. Clinical Signs intermediate host and rarely as the final Clinical signs of trematode sporocyst host. A metacercaria is the encysted ju- infestation may include increased flesh venile trematode usually occurring in the yield regarding initial sporocyst devel- second intermediate host that requires opment that ultimately results in poor ingestion by the final host to become growth and debilitation in later stages, an adult worm. A sporocyst is formed weakness in valve closure, reduced as part of the trematode developmen- byssal thread production in mussels, tal cycle in the first intermediate host. infertility (parasitic castration), chalky Depending on the trematode species, shell deposits, induction of pearl forma- sporocysts and metacercariae are found tion reducing marketability and bivalve in the tissues of a variety of intermediate mortality. Encysted metacercariae hosts including bivalve molluscs. Low most often are benign depending on the numbers of encysted metacercariae in trematode species and intensity. How- most molluscs generally cause no overt ever, some investigations have shown disease but when present in significant metacercariae to cause shell gaping and numbers can cause general debilitation, deformities, production of pearls and behavioral changes favoring preda- chalky shell deposits, behavioral changes tion, tissue destruction and host death. that favor predation, reduced tolerance Sporocyst stages are overtly destructive to stress, and host mortality in heavy in- causing parasitic castration, weakness festations. Metacercariae and developing and gaping of valves with gradual de- sporocysts may be observed in various struction and replacement of molluscan bivalve tissues during routine histologi- tissues resulting in eventual mortality cal examination. of the host. There are several different trematode families utilizing marine mol- IV. Transmission luscs as intermediate hosts, most notably Trematode life cycles involving the family Bucephalidae and the genus marine molluscs generally include two Bucephalus. intermediate hosts and one definitive host (only two hosts for Sanguinicolidae) II. Host Species with several possible combinations of Various species of digenetic trema- pathways that include gastropods and tode sporocyst and metacercarial stages bivalves (or other invertebrates) for inter- occur in many marine bivalve mollusc mediate hosts and fish, birds or bivalves species worldwide including oysters, (or other invertebrates) as final hosts. clams, scallops, mussels and cockles. Trematodes of the family Bucephalidae In Alaska, sporocysts of unidentified are of great importance because their trematodes have been observed in blue larvae affect numerous commercially

48 49 bivalve mollusc Helminths

important mussels and oysters and food as small foci having varying degrees of fishes such as flatfishes and cods. The encapsulating host fibrous tissues and pathway for Bucephalus sp. typifies the inflammatory cells. family and begins in a bivalve followed by a fish then on to a second fish as the VI. Prognosis for Host final host. Eggs in the alimentary tract of Effects of encysted metacercariae on the final predatory fish host are depos- the bivalve host vary from none to being ited with feces in seawater and hatch responsible for killing young spat. In the into ciliated miracidia that parasitize the Pacific Northwest and Alaska, metacer- first intermediate bivalve host. Once in cariae are considered relatively harmless the bivalve a miracidium develops into to bivalve hosts except when encountered a mother sporocyst that produces more in significant numbers. Sporocysts may sporocysts that eventually produce cer- cause severe tissue damage and debilita- cariae released from the host. These cer- tion resulting in parasitic castration and cariae drift with the currents and parasit- bivalve mortality. ize the second intermediate fish host and encyst as metacercariae. The encysted VII. Human Health Significance metacercariae find their way to the gut of Trematodes in the family Echinos- the final host fish when the second inter- tomatidae parasitize birds as the final mediate fish host is eaten. Metacercariae host. Echinostome metacercariae, such become adult trematodes in the gut of the as Himasthla sp., found in bivalve tissues fish where eggs are produced to begin consumed raw have been implicated in the cycle again. Encysted metacercariae human gastrointestinal disturbances. found within bivalve host tissues indicate members of other trematode families that may follow different life cycle pathways of having a bivalve mollusc as a second intermediate host rather than a fish.

V. Diagnosis Wet mounts of fresh tissues may show the encysted metacercariae (Gym- nophallidae are unencysted) which can be extremely difficult to find if in low prevalences. Sporocysts of some trematodes may be grossly orange in color within the bivalve tissues and differentiated by species based on the morphology of the cercariae. Sporocysts and encysted metacercariae can also be observed in tissue sections during routine histological examination. Sporocysts may extensively infiltrate host tissues, exhibit dichotomous branching and are strongly basophilic containing germ balls and cercariae. Metacercariae may exhibit trematode features such as oral and ventral suckers and appear in various tissues

48 49 bivalve mollusc Helminths Trematode Metacercariae and Sporocysts



Histological section of encysted trematode metacercaria (arrow) encapsulated by host inflammatory cells and fibrous tissue within the connective tissues of blue mussel

 Histological section of a Bucephalus-like trematode sporocyst containing germ balls (arrow) in the connective tissues of blue mussel

50 51 bivalve mollusc Helminths

 Histological section of a different type of sporocyst containing cercariae (arrow) in razor clam

Bucephalus sp. Life Cycle

Predatory fish final host for adult trematode

Mature egg passes in feces

Intermediate fish host with encysted metacercaria Ciliated larva – miracidium

Free swimming cercaria Infected bivalve where sporocysts develop

50 51 bivalve mollusc Arthropods Gill and Gut Parasitic Copepods

I. Causative Agent and Disease Canada with seed oysters from Japan. It Parasitic copepods are arthropods occurs in the European flat, Pacific and belonging to the subphylum Crustacea, native oysters and several other spe- class Maxillopoda and subclass cies of marine bivalves and has recently Copepoda. Copepods parasitizing been introduced into France. Mytilicola bivalve molluscs are mostly within porrecta occurs in ribbed and hooked the order Cyclopoida and can be mussels and may also occur in other divided into two basic groups; obligate bivalves in the Gulf of Mexico. External endoparasites inhabiting the alimentary parasitic copepods have been reported in tract including the more common species the mantle and on the gills of various bi- of Mytilicola (intestinalis, orientalis, valve species from Europe and from the porrecta) and ectoparasites inhabiting Atlantic, Gulf and Pacific coasts of North the mantle and gill tissues including America, including British Columbia, several species within the genera Canada. In Alaska, unidentified parasitic Conchyliurus, Modiolicola, Myicola, copepods have occurred on the gills Myocheres, Ostrincola, Pseudomyicola and in the alimentary tracts of Pacific and Paranthessius. An exception to the oysters, basket cockles, blue mussels, latter group is Pseudomyicola spinosus rock scallops and native littleneck clams. that apparently is able to move back Many of these copepods have been and forth between the gill/mantle areas present both externally and internally ap- and the alimentary tract of bivalve pearing more like Pseudomyicola rather hosts. Mytilicola has been implicated in than Mytilicola. causing intestinal damage, poor growth and mortality in European mussels but III. Clinical Signs conclusive evidence is lacking regarding Copepod infestations are mostly its true pathogenicity. Most ectoparasitic found by routine histological examina- copepods appear to be commensals but tion. There are no clinical signs of their attachment to gill surfaces may external or internal copepod infestation cause localized tissue damage. When except in European reports attributing found within the GI tract, P. spinosus poor growth and mortality in mussels to may damage mucosal epithelial cells infestations by M. intestinalis. Infesta- followed by perforation, subsequent tions in the GI tract may cause meta- invasion into connective tissues and plasia (columnar epithelium reduced to encapsulation of the parasite by host low cuboidal or squamous) of mucosal granulomatous tissues. epithelium and in rare cases erosion and perforation from the appendages with or II. Host Species without host encapsulation by hemo- Mytilicola intestinalis is apparently cytes. limited to Europe occurring in mussels, clams, cockles and the flat oyster in IV. Transmission waters from Denmark to Italy includ- Parasitic copepods of bivalve mol- ing the British Isles and Ireland but not luscs have a direct life cycle involving the Baltic. Mytilicola orientalis was copulation of two adult sexes and the introduced to the Pacific Northwest and production of eggs that hatch directly in

52 53 bivalve mollusc Arthropods

ambient seawater or, as with endopara- the parasites are present in great num- sitic species, the egg cases are released bers. The pathogenicity of endoparasitic with feces to seawater. The hatched copepods in bivalve molluscs has been nauplii may undergo molts into other enigmatic with many reports indicating naupliar stages or into metanauplii and no pathogenicity. Other investigators in- finally to one or more copepodid stages. dicate poor growth and bivalve mortality One of the copepodid stages is usually caused by erosion of the mucosal epithe- the initial parasitic stage that either at- lium with perforation and encapsulation taches to the external target host tissue of the parasites in surrounding connec- or, in the case of endoparasitic forms, is tive tissues. Internal copepods may also drawn into the GI tract by filter feed- cause apparent occlusion of the large ing of the mollusc host. Other parasitic ducts connecting the stomach and diges- instars may occur until the copepod be- tive diverticulae. Mytilicola sp. occurs comes an adult. However, the number of more commonly in larger host animals molts and specific stages vary depending inhabiting the bottoms of sheltered areas on the species of parasitic copepod and and rarely occurs in bivalves less than 10 remain unknown for many. mm in length and in Pacific oysters less than or equal to 20 mm. In Alaska, there V. Diagnosis has been no bivalve mortality or poor Specific diagnosis is based on body condition associated with the pres- observing external copepods attached to ence of parasitic copepods. gills and mantle tissues or by dissect- ing reddish colored elongate worm-like VII. Human Health Significance copepods from the stomach and intestine There are no zoonotic human health of internally infested bivalves. Enzy- concerns with the occurrence of parasitic matic digestion also may be used for copepods in the tissues of bivalve mol- quantification. The body of Mytilicola luscs. sp. is about 5 to 12 mm long (depending on species), relatively dedifferentiated with reduced limbs and body segmenta- tion and overall is less recognizable as compared to other ectoparasitic genera that resemble free living copepods. Spe- cies is determined by various external morphological features (body length or size, mouth parts and antennae) of female copepods since the occurrence of males is generally transient. Histological examination of a parasitic copepod demonstrates general features of a complex body structure with carapace, segments, appendages, GI tract, gonads and striated musculature.

VI. Prognosis for Host The effects of ectoparasitic copepods on their hosts have been minimal and generally considered harmless, unless

52 53 bivalve mollusc Arthropods

Gill and Gut Parasitic Copepods 

Histological section of Pacific oyster with a Histological section of Pacific oyster parasitic copepod (arrow) attached to the with a parasitic copepod in the intestinal gill tissues causing localized inflammation lumen



Mytilicola sp. in gut (arrow and inset) of the clam Macoma balthica (Photo: Dorothy Howard, NOAA Cooperative Oxford, MD Laboratory)

54 55

bivalve mollusc Arthropods 



Histological section of dead parasitic Similar dead parasitic copepod (arrow) copepod (arrow) surrounded by host surrounded by host inflammatory cells inflammatory cells after penetrating into in connective tissue of digestive gland of the connective tissues from digestive gland blue mussel

tubules in Pacific oyster 

Histological section of a parasitic copepod (arrow) on the mantle tissues of weathervane scallop

54 55 bivalve mollusc miscellaneous Foreign Body Granulomas and Pearls

I. Causative Agent and Disease small to be observed except when tissues Foreign body granulomas are inflam- are examined histologically. matory cell foci within host tissues in response to a non-self object or irritant. IV. Transmission These granulomas are usually benign and Additional details regarding trans- are found incidentally when examining mission of metazoan parasites and Sporo- histologic sections of bivalve molluscs. zoa that may cause either unidentified The foreign body stimulating the forma- foreign body granulomas and/or pearls tion of a granuloma often cannot be may be found in other sections of this identified by examining the section due booklet. Granulomas and pearls caused to degradation by the host response or by non-infectious foreign bodies (sand, because it is outside the plane of section. shell, debris) occur spontaneously and Foreign bodies that cause granulomas are not transmissible. An exception is most commonly include trematode meta- found in the Japanese pearl industry cercariae, cestode and nematode larvae where artificial transmission of a non- and parasitic copepods (see other chap- infectious foreign body is accomplished ters). Bacterial, fungal and protozoan by manual insertion of nacreous shell agents may also be responsible for granu- material between the mantle layers of lomas as well as inert material that may pearl oysters. These shell chips cause become embedded in shellfish tissues. tissue irritation that is walled off by cal- Pearls are calcium carbonate concretions careous secretions which form cultured precipitated in the soft tissues by bivalve pearls. molluscs in response to the same irritants or foreign bodies that cause granulomas. V. Diagnosis Diagnosis of unidentified foreign II. Host Species body granulomas and pearls is generally Unidentified foreign body granulo- made by routine histological examina- mas and pearls may be found in a variety tion. Granulomas are composed of vari- of marine and freshwater bivalve mollusc ably sized foci of host inflammatory cells species worldwide including clams, sometimes surrounding a necrotic center. oysters, cockles, scallops and mussels. In Pearls appear as eosinophilic layered Alaska, foreign body granulomas have concretions within soft host tissues, most been observed in Pacific oysters, blue often the mantle. mussels, basket cockles, littleneck clams and weathervane scallops while pearls VI. Prognosis for Host have been found in weathervane scallops, Unidentified foreign body granulo- Pacific oysters and most commonly in mas and pearls in bivalve molluscs are the blue mussel. considered benign unless present in large numbers that may impair normal tissue III. Clinical Signs functions. High intensities of metacer- Generally, there are no obvious clini- carial infestation that may cause either or cal signs of either foreign body granu- both conditions can result in host mortal- lomas or pearls, both of which are too ity and debilitation. However, in the

56 57 bivalve mollusc Miscellaneous

Pacific Northwest and Alaska, trematode may be unfit for human consumption for metacercariae are usually encountered at obvious reasons. Some concern may be low prevalences and intensities causing warranted in cases where metacercarial no significant harm to bivalve hosts. infestation is implicated as causing granulomas or pearls. Echinostome VII. Human Health Significance metacercariae, such as Himasthla sp., Unidentified foreign body granu- have been implicated in human gastro- lomas and pearls in bivalve molluscs intestinal disturbances when present in are of no direct zoonotic human health bivalve tissues consumed raw. concern. Bivalve tissues containing high numbers of calcareous pearl concretions



Unidentified foreign body granulomas Unidentified foreign body granulomas (ar- (arrow) encompassing digestive tubules row) in the mantle connective tissue of blue

in weathervane scallop, histological mussel, histological section

section 

Histological section of a pearl (arrow) with concentric layers in the mantle tissue of blue mussel 56 57 bivalve mollusc miscellaneous Miscellaneous Invertebrate Pests and Predators

I. Causative Agent and Disease III. Clinical Signs Marine invertebrates that are consid- Physical presence of the pest or ered pests and predators do not necessar- predator is obvious causal evidence but ily cause disease. They may kill bivalve in their absence there are occasional molluscs by directly feeding on them or clinical signs to indicate they have been use them as a substrate which inadver- there. Signs of pea crab infestation can tently causes debilitating damage that include minor damage or atrophy of may affect the health of the bivalve or its mantle and gill tissues. Boring sponges marketability. Besides the numerous bio- burrow into calcium carbonate by local- fouling organisms other known pests of ized secretion of enzymes that etch bivalves in Alaska include three species bivalve shells causing many small holes of pea crabs found inhabiting the mantle and a porous appearance. The stomach of cavities of several bivalves and the in- a starfish is everted between the valves troduced non-indigenous boring sponge, of a mollusc to digest the soft tissues Cliona thoosina, that uses the external leaving the empty cleaned valves. Crab shell of several bivalve molluscs as a sub- predation generally leaves a jagged to strate. Common invertebrate predators rounded hole in one of the empty valves of bivalves in Alaska include starfish and of the eaten bivalve. Although drills have various species of crabs, most notably not been reported in Alaska, these snails of the genus Cancer. Although two snail bore through the shell leaving a small species of oyster drill are present in the perfectly round hole with an indented Pacific Northwest, there have been no margin from the scouring action of their confirmed reports that either has become rasping mouthpart, known as a radula. established in Alaska through importation of Pacific oyster spat. IV. Transmission Invertebrate pests and predators are II. Host Species normal fauna of the marine environ- Upper ranges of pea crabs in Alaska, ment that reproduce in the water column. depending on species, include Akutan, Adult crabs and starfish are mobile and Prince William Sound and Prince of able to seek out hosts or prey. Boring Wales Island and are most commonly sponges are disseminated horizontally found in the mantle cavities of mus- in ambient water by sexual reproduction sels, horse clams and occasionally other producing planktonic larvae or asexually bivalve species. Boring sponge was intro- by formation of spore-like gemmules or duced into Prince William Sound and fragmentation and/or budding from the can occur on various substrates including main sponge body that drift and settle the shells of several bivalve molluscs, elsewhere. most notably Pacific oysters. Various starfish species and numerous species of V. Diagnosis crabs are common marine fauna through- Diagnosis is based on observation of out Alaska and opportunistically feed on clinical signs or physical presence of the any available species of bivalve mollusc. pest or predator.

58 59 bivalve mollusc Miscellaneous

VI. Prognosis for Host tors and pathogens alike into new areas Predators generally kill their prey. by the importation of shellfish such as Pests may debilitate or weaken their Pacific oysters. hosts. Pea crabs are generally benign pests. Boring sponge can weaken the VII. Human Health Significance host shell that may increase vulnerability There is no zoonotic human health to predation or in the worse case dissolve concern with the presence of inverte- the shell killing the host. Oysters with brate predators or pests associated with shells scarred by boring sponge gener- bivalve molluscs except for the potential ally are not marketable. There is always loss of aesthetic quality to the consumer. risk of introducing bivalve pests, preda-

Irregular to round holes in the shells of juvenile Pacific oysters produced by crab predation

Japanese oyster drill eggs on the shell of an adult Pacific oyster (Photo: R. Elston, AquaTechnics, WA)

58 59 bivalve mollusc miscellaneous

Miscellaneous Invertebrate

Pests and Predators 

Boring sponge etching (arrow) on the external shell of a Pacific oyster (Photo: R. Elston, AquaTechnics, WA) 

Boring sponge etching (arrow) that has penetrated to the internal shell surface of the same oyster (Photo: R. Elston, AquaTechnics, WA)

Starfish predation on bay scallop (Photo: Dorothy Howard, NOAA Cooperative Oxford, MD Laboratory)

60 61 bivalve mollusc Miscellaneous

Oyster drill Urosalpinx cinerea (Photo: Dorothy Drill holes (arrows) in shells of the eastern Howard, NOAA, Cooperative Oxford, MD oyster (Photo: Dorothy Howard, NOAA Laboratory) Cooperative Oxford, MD Laboratory)

Pea crab (Pinnotheres) in the gill mantle cavity of an eastern oyster (Photo: Dorothy How- ard, NOAA Cooperative Oxford, MD Laboratory)

60 61 bivalve mollusc miscellaneous Neoplasia (Tumors)

I. Causative Agent and Disease mesenchymal (connective tissue origin) Cancers or neoplasms are growths of tumor in a blue mussel, disseminated abnormal cells that proliferate uncontrol- neoplasia (hemocyte origin) in a blue lably. In bivalve molluscs, neoplasms mussel and littleneck clam and a secre- are rare in comparison to vertebrates but tory cell adenoma of the gill arch in a more common than in other invertebrate geoduck clam. phyla. Neoplastic lesions have been reported most commonly in oysters but III. Clinical Signs also in other marine clams and mussels Clinical signs of neoplasia may including mesenchymal tumors, epithe- include a grossly visible abnormal tissue lioma, myofibroma, germinoma, neu- mass but more commonly neoplasia in rofibroma, sarcomas and disseminated bivalve molluscs is an incidental finding neoplasia resembling leukemia. Bivalve during routine histological examination. molluscs develop cancer in much the High prevalences of disseminated or same way as do higher animals. Known hemic neoplasia in blue mussels and soft and suspected factors contributing to shell clams have been associated with neoplasia in molluscs include viruses, significant mortality of 30-80%. environmental chemicals (carcinogens), repeated physical trauma, hormones, IV. Transmission age, sex and genetic predisposition. Most neoplasms described in bivalve molluscs are considered spontaneous II. Host Species resulting from environmental contami- Neoplasia has been reported in: nation, congenital malformation, age or Pacific and Olympic (native) oysters on genetic predisposition and are not trans- the Pacific coast of North America as missible in nature. A major exception is well as the eastern oyster on the Gulf disseminated neoplasia in clams, mussels and Atlantic coasts; ocean quahog, hard and cockles which has been transmit- and soft-shell clams and bay scallop ted in mussels by cohabitation and in on the Atlantic coast; Pacific oyster mussels and cockles by injection of and mussel in Japan; pearl oyster in cancerous cell-free tissue homogenates Australia; Chilean oyster in Chile and suggesting an infectious agent. In soft- New Zealand; the European flat oyster shell clams mixed results using cell-free in Spain and Yugoslavia; the common homogenates are reported but a retro- cockle in Ireland and Spain; the Mediter- virus is suspected to cause the disease ranean mussel in Spain; the blue mussel in all three bivalve species. The highest in Great Britain, Scandinavia, Tasmania prevalences of disseminated neoplasia and North America including British Co- occur during autumn and winter months lumbia, Canada. Disseminated neoplasia with highest bivalve mortality occurring is reported to occur in at least 15 marine in late winter and early spring. bivalve species from four continents and every ocean except the Antarctic. In V. Diagnosis Alaska, neoplasms observed in bivalve Neoplasms are diagnosed and clas- molluscs have included germinoma (go- sified using histological methods to nadal origin) in an adult Pacific oyster, determine the cell or tissue of origin and

62 63 bivalve mollusc Miscellaneous

are further grouped based on benign or VI. Prognosis for Host malignant characteristics. Benign tumors Neoplasia usually results in death of are often well-differentiated, grow the affected bivalve mollusc, although slowly, are well circumscribed without remission has been known to occur with invading surrounding normal tissue disseminated neoplasia. High preva- and do not metastasize. Most benign lences of disseminated neoplasia are neoplasms usually end in the suffix associated with high mortality of bivalve “oma”. Exceptions are benign neoplasms mollusc populations, especially those of the brain and some endocrine organs that have been previously unexposed. in higher vertebrates that can be life Due to the transmissible nature of this threatening due to their location and cancer, known infected stocks of bivalve deleterious physiological effects on the molluscs should not be introduced to new host. Malignant tumors are often not areas. well differentiated, may grow rapidly, infiltrate normal tissues and tend to me- VII. Human Health Significance tastasize. The names of these neoplasms Although aesthetically disturbing, are often preceded by the word “malig- there are no zoonotic human health nant” or with the suffixes “sarcoma” or concerns associated with neoplasia in “carcinoma”. Disseminated neoplasia bivalve molluscs. Except for dissemi- in bivalve molluscs is characterized by nated neoplasia that has a suspected viral intense infiltration of tissues by ab- etiology, cancer is generally a rare event normal hemocytes that have very little in bivalve molluscs affecting one animal cytoplasm and enlarged pleomorphic in several thousand. Should these other nuclei with frequent mitotic figures. The tumors occur more frequently in a popu- neoplastic cells can be examined from lation, an indirect human health concern wet and stained preparations of collected would be whether the cause is linked to

hemolymph and during histological environmental contamination. examination of cut tissue sections. 

Histological section of mesenchymal neo- Higher magnification of fibroblastic plasm (arrow) in blue mussel type cells composing the mesenchymal neoplasm 62 63 bivalve mollusc miscellaneous

Neoplasia (Tumors) 

Histological section of disseminated neoplasia (arrow) in blue mussel

Higher magnification of typical leukemic-like cells with large nuclei and scant cytoplasm comprising disseminated neoplasia

64 65

bivalve mollusc Miscellaneous 

Two white nodular foci (arrow) of a secretory cell adenoma on the gill arch of a

geoduck clam 





Histological section of geoduck adenoma Higher magnification of collagen (arrowhead) composed of concentric whorled layers and secretory-type cells (arrow) containing of basophilic mucinous material (arrow) eosinophilic droplets also found free in the containing secretory type cells and areas of tumor mass collagen

64 65 crustacean VIRUSES Aquabirna and Herpes Viruses

I. Causative Agent and Disease let, Bristol Bay, Pribilof Islands and the Many different viruses occurring western Aleutian Islands. The aquabirna- in wild shrimp and crabs have been like virus was found in a single male described globally but few have been blue king crab from Glacier Bay in documented in Alaska. In part this may southeast Alaska while the intranuclear be due to the paucity of studies con- inclusions were observed in 4% of the ducted on wild crustacean populations, Dungeness crabs collected from Excur- the geographic isolation of Alaska and sion Inlet, Freshwater Bay and Bridget the absence of a commercial mariculture Cove near Juneau. industry for shrimp that has prevented importation of exotic crustacean vi- III. Clinical Signs ruses from other parts of the world. Two No outward or internal clinical viruses have been reported from king signs are reported for either virus. The crabs in Alaska, a herpes-like virus and herpes-like virus is associated with mas- an aquabirna-like virus, while suspicious sive destruction of the bladder, antennal intranuclear inclusion bodies have been gland and sometimes hindgut epithelium, observed in Dungeness crabs. strongly suggestive of a lethal disease. A herpes-like virus reportedly infects The aquabirna-like virus was contained the bladder and antennal gland epithe- within cytoplasmic inclusion bodies in lium in red, blue and golden king crabs. the labyrinth epithelium of the antennal The virus nucleocapsid is 140 X 165 gland. In the same tissue an adenocar- nm in diameter with two electron-dense cinoma (discussed in other section) of layers surrounding a central electron- probable tegmental gland epithelial ori- dense cylinder of approximately 55-60 gin was also present. However, there is X 90-105 nm. Enveloped particles were no evidence that the virus was the cause not observed by transmission electron of or otherwise was specifically associ- microscopy and no other biochemical ated with the neoplasm. The intranuclear features are known. An aquabirna-like inclusions in Dungeness crabs were virus was reported infecting labyrinth incidental findings on routine histologi- epithelium of the antennal gland from a cal examination. single asymptomatic blue king crab. The virus particles were icosahedral, about IV. Transmission 65 nm in diameter and present within The mode of transmission is un- cytoplasmic inclusion bodies containing known for either virus but suspected to microtubular structures. Intranuclear be horizontal from animal to animal via inclusion bodies of possible virus origin ambient seawater. have been observed in the cells of the urinary bladder, hepatopancreas and V. Diagnosis female seminal receptacle of Dungeness Neither virus has been isolated crabs. in cell culture. The herpes-like virus infection is recognized by cytopathol- II. Host Species ogy in histological sections comprised of The herpes-like virus infects blue, hypertrophied epithelial cell nuclei of the red and golden king crabs from Cook In- bladder and antennal gland. Examina-

66 67 crustacean VIRUSES

tion by TEM confirms that the enlarged These inclusions were basophilic caus- nuclei contain marginated chromatin, a ing nuclear enlargement, compression rarified eosinophilic stroma with one or and eccentricity of the nucleoplasm and more pleomorphic eosinophilic inclusion nucleolus with margination of chromatin. bodies associated with typical herpes- like virus particles scattered throughout VI. Prognosis for Host the nuclear stroma. TEM confirmation The host prognosis regarding infec- of virus particles has been done for only tion by either virus is unknown but the one such diseased blue king crab. The herpes-like virus is suspected to cause aquabirna-like virus may have been king crab mortality. an incidental subclinical finding that was only recognized by serendipitous VII. Human Health Significance observation with TEM unless the grossly There are no known zoonotic human visible neoplasm was somehow virus- health concerns associated with infection associated. The intranuclear inclusion of king or Dungeness crabs by poikilo- bodies in the Dungeness crabs were thermic viruses.

detected by histological examination. 

TEM of a labyrinth epithelial cell of the antennal gland in blue king crab with cytoplasmic inclusion (arrow)

 

Higher magnification of inclusion showing icosahedral aquabirna-like virus particles (arrow) with associated microtubular structures (arrowhead)

66 67 crustacean VIRUSES

Crustacean Viruses 

Higher magnification of blue king crab aquabirna-like virus particles with associated microtubular structure (arrow)



Histological section of intranuclear inclusion body (arrow) in a bladder epithelial cell of Dungeness crab

68 69 crustacean VIRUSES



Histological section of blue king crab antennal gland epithelium with hypertrophied nuclei (arrow) containing one or more eosinophilic intranuclear inclusion bodies associated with a herpes-like virus (Photo: J. Frank Morado, National Marine Fisheries Service, Seattle)



TEM of herpes-like virus particles (arrow) associated with intranuclear inclusions in antennal gland epithelium of blue king crab (Photo: J. Frank Morado, National Marine Fisheries Service, Seattle)

68 69 crustacean Bacteria Prokaryotic Intracytoplasmic Inclusions

I. Causative Agent and Disease Alaska reported a similar rickettsial Unlike bivalve molluscs, there are agent infecting one blue and one golden fewer reports of prokaryotic intracy- king crab held captive after collection toplasmic inclusion bodies in crusta- from Glacier Bay and Lynn Canal near ceans of which 13 cases were caused Haines, Alaska. by rickettsia-like organisms and one other by chlamydia-like bacteria. All III. Clinical Signs infections produced intracytoplasmic Clinical signs vary from normal in inclusions or microcolonies infecting a appearance to lethargy with mortality wide range of tissues in both freshwater and tissue discolorations. King crabs and marine crustaceans. The organisms infected by rickettsial organisms may are either confined to the hepatopan- have arrested ovarian development, creatic epithelium or are systemic and become lethargic and die when held vary in their significance from mildly for prolonged periods of time. Infected to seriously pathogenic causing cell en- tissues in king crabs have included the largement with organ and tissue necrosis epithelial cells of the antennal gland and including demonstrated or suspected hepatopancreas that become indurated mortality. However, in several of these and friable. Histological examination of cases other serious pathogens were also infected tissues demonstrates enlarged present which may have contributed to cells with intracytoplasmic inclusions the mortality and the observed disease composed of microcolonies of rod- syndrome. shaped bacteria causing disseminated granulomatous foci and caseous necrosis II. Host Species of the tissues. The single documented chlamydia- like agent was reported from a large IV. Transmission Dungeness crab mortality in Puget Certain rickettsia-like organisms Sound, Washington. Rickettsia-like in cultured and wild shrimp have been agents have been reported from transmitted horizontally by cannibalism crustaceans in the United States from and via seawater exposure. Similar stud- Florida, Hawaii and Washington, and ies with other rickettsias either have not from British Columbia, Canada, France, been successful or the mode of transmis- Sweden, Mexico, Ecuador, Madagascar, sion is unknown. China, Malaysia and Australia. Host species have included wild freshwater V. Diagnosis amphipods and cultured crayfish, at Histological examination of infected least five wild and cultured species of tissues reveal the typical enlarged cells penaeid shrimp, one species of wild containing microcolonies of organisms. pandalid shrimp, and one cultured and Confirmation of typical rickettsial or three wild crab species including a 1984 chlamydial morphology is by transmis- report of one blue king crab from the sion electron microscopy. PCR may be eastern Bering Sea near St. Lawrence available for certain agents described in Island. Later studies (1990) in southeast the literature. None of these agents have

70 71 crustacean Bacteria

been isolated in culture using conven- VII. Human Health Significance tional methods. There are no known zoonotic human health concerns regarding infection of VI. Prognosis for Host crustaceans by these poikilothermic or- Infections can be light and insignifi- ganisms which are different from similar cant but low to high mortality has also organisms causing diseases in higher been reported for both wild and cultured animals. shrimp and crabs.

Caseous necrosis of hepatopancreas from golden king crab infected with a rickettsia-like organism that has destroyed hepatopancreatic tubules

70 71 crustacean Bacteria

Prokaryotic Intracytoplasmic

Inclusions 

Histological section of tubular necrosis (arrow) and intense host inflammatory infiltration caused by infection with rickettsia-like organisms



Histological section of infected hepatopancreas epithelium showing microcolonies (arrow) of rickettsia-like organisms

72 73

crustacean Bacteria 

Higher magnification of rickettsia-like organisms (arrow) 

TEM of an infected hepatopancreatic cell showing a colony of rickettsia-like organisms (arrow)

72 73 crustacean Bacteria Shell Disease

I. Causative Agent and Disease III. Clinical Signs Shell disease is a progressive Crustaceans with shell disease degradation of the crustacean cuticle present with brown to black focal shell characterized externally by melanized erosions of varying size and severity that brown to black focal lesions of varying may penetrate into the soft tissues. size and severity regarding penetration of the surface exoskeleton. The disease can IV. Transmission occur in nearly all freshwater and marine Transmission is horizontal via crustaceans, usually at low prevalences water containing the common flora of except when host animals are stressed by chitinoclastic bacteria. Initiation of the poor environmental conditions caused by external lesions generally requires previ- intensive aquaculture, animal impound- ous shell injury or degradation as a portal ment or waters polluted by chemicals, of bacterial invasion. In Alaska, shell sewage or heavy metals. The shell ero- disease, loss of limbs, lower fecundity sion is largely attributed to colonization and mortality in Dungeness crabs have by chitinoclastic bacteria in areas of shell been associated with benthic deposits of injury initially caused by poor environ- decomposing bark and sulfide at logging mental conditions, predation or cannibal- transfer sites. ism. Studies of shell disease in some crab species have shown that resulting shell V. Diagnosis ulcerations lead to limited septicemia by Diagnosis is determined by typical these and other opportunistic bacteria shell surface lesions associated with resulting in damage to internal organs Gram-negative bacterial rods that can be and tissues. Many different bacteria have cultured on conventional media and iden- been associated with shell disease but tified by biochemical tests. Histological common isolates include species from examination indicates varying degrees several Gram-negative genera such as of melanization, erosion and ulceration Achromobacter, Acinetobacter, Aero- of the shell layers with bacteria and oc- monas, Plesiomonas, Pseudomonas and casional protists colonizing the surface Vibrio, all of which are commonly found accompanied by inflammatory exudate on the shell surface of healthy animals. in the underlying dermis and epider- Fungal organisms are rarely encountered mis. Hemocytes may form a protective in the larger more severe lesions. pseudomembrane overlying some lesions.

II. Host Species VI. Prognosis for Host Shell disease has been reported to oc- In the initial stages, shell disease is cur in many different species of crusta- likely not fatal but mortality is known ceans worldwide. In Alaska, shell disease to occur from adhesion of molted shells has been observed in feral populations of at the lesion sites leading to incomplete red, blue and golden king crabs, Tanner withdrawal from the old exoskeleton. crabs and Dungeness crabs. However, all Mortality can also occur from second- freshwater and marine crustaceans are ary bacterial septicemia if shell erosion considered susceptible to the disease. progresses to the soft underlying tissues.

74 75 crustacean Bacteria

VII. Human Health Significance have been no reports on which to base Shell disease in crustaceans is any zoonotic human health concerns unsightly and some of the associated associated with shell disease in crusta- vibrios and pseudomonad bacteria are ceans. known human pathogens. However, there



Erosion of carapace (arrow) due to shell disease in red king crab 

Erosion of carapace (arrow) due to shell disease in golden king crab

Dungeness crab from a logging transfer site with shell disease and loss of limbs (Photo: C. E. O’Clair, National Marine Fisheries Service, Juneau)

74 75 crustacean Fungi Black Mat Syndrome

I. Causative Agent and Disease by direct contact with infected crabs. Trichomaris invadens, the causative agent of Black Mat Syndrome, is V. Diagnosis a chitinoclastic, obligate Ascomycete Varying degrees of a black encrust- fungal parasite of Tanner crabs. The ing fungal mat on the exoskeleton of fungus proliferates on the surface of the Tanner crabs is confirmed as Tricho- exoskeleton invading the shell and inter- maris invadens by wet mounts or nal soft tissues causing destruction with histological examination showing the little or no host response. Infected crabs external thick-walled, black pigmented, have varying degrees of a dense black infrequently septated hyphae with tarry encrustation on the surfaces of the fruiting bodies or perithecia contain- carapace and appendages. ing many asci each of which contains eight ascospores. Each ascospore has a II. Host Species long thin filament tightly wound around In the 1970’s and early 1980’s, the each end that unwinds when the spore is fungus was prevalent in the Alaskan discharged. Internal tissues are invaded Tanner crab fishery around Kodiak by dense proliferations of non-pigmented Island and in the northwestern Gulf hyphae also having infrequent septa. In- of Alaska, primarily infecting Chion- ternal fungal hyphae are positive (black) oecetes bairdi at prevalences as high as when stained by Grocott’s methenamine- 75%. The fungus rarely has been found silver method for fungi. The fungus infecting C. opilio and C. tanneri but has not been cultured successfully on has also been reported in C. bairdi from artificial media. deep fiords in northern British Columbia, Canada. VI. Prognosis for Host Moderate fungal infection likely III. Clinical Signs prevents molting and causes blindness if Infected crabs have varying degrees eyestalks are invaded. Severe infections of encrusting nodules or pustules form- likely result in crab mortality, however, ing a dense, hard, black, tar-like covering the pathogenesis of the disease has not over parts of the exoskeleton. Histologi- been studied in the laboratory. Black cal examination of internal soft tissues mat appears to occur more frequently in shows dense proliferation of non-pig- old shell or skip molted crabs. Infected mented fungal hyphae and tissue necro- sublegal-sized animals that fail to molt sis. There is a significant increase in the will not reach legal size and immature percentage of eosinophilic granulocytes females will not reach sexual maturity. in circulating hemolymph of infected crabs while infected female crabs may be VII. Human Health Significance barren. There are no known zoonotic human health concerns regarding consump- IV. Transmission tion of Tanner crab meat infected with The mode of transmission is un- Trichomaris invadens. known but is likely horizontal via infectious ascospores in ambient seawater or

76 77 Crustacean Fungi



Black mat fungus (arrow) infecting the underside of the carapace of a Tanner crab

Severe black mat fungus infection of the dorsal carapace in each of two Tan- ner crabs (Photo: J. Frank Morado, National Marine Fisheries Service, Seattle)

GMS stain of black mat fungal hyphae and fruiting body penetrating the shell surface of an eyestalk in Tanner crab (Photo: J. Frank Morado, National Marine Fisheries Service, Seattle)

76 77 crustacean Fungi Fungal Infection of Captive Red King Crabs

I. Causative Agent and Disease mation of granuloma formation around An unidentified marine fungus branching aseptate fungal hyphae that caused mortality of four captive adult red are positive (black) when stained with king crabs producing a disease charac- Grocott’s method of methenamine-silver terized by carapace discoloration of the nitrate (GMS) for fungi. An opportunity walking legs and multiple granulomas for further characterization of the fungus and necrosis in the underlying epidermal by attempted isolation on artificial media and sub-epidermal tissues. There are no has not occurred. other known reports in the literature of a similar fungus infection in adult red VI. Prognosis for Host king crabs. Whether the fungus infection This fungal infection of the carapace occurs in wild crabs or is related to the results in deep localized invasion of stress and environment of captivity is not underlying soft tissues, secondary bacte- known. rial infection, leg loss and crab mortality. Even minor shell damage may result in II. Host Species adhesion of the old carapace to the site The fungus occurred in captive adult of the wound during molting causing red king crabs collected from Cook Inlet, incomplete ecdysis and mortality of the Alaska, but other crab species may be crab. Whether this fungus occurs in wild susceptible. populations of king crabs or is an artifact of captivity is not known. III. Clinical Signs Focal yellow-brown discoloration VII. Human Health Significance appears on the carapace of walking legs Although unsightly, this fungal infec- followed by deep necrotic ulceration tion of captive red king crabs is unlikely of the underlying soft tissues, loss of to have any zoonotic significance for affected legs and crab mortality. The human health. lesions are localized and not systemic characterized histologically by severe hemocyte infiltration and granulomas forming around fungal hyphae.

IV. Transmission The mode of transmission is unknown but assumed to be horizontal within the water column, probably requiring an injury to the carapace as a portal of entry.

V. Diagnosis Diagnosis is by observation of typical focal discolorations of the carapace (see similar lesions in shell disease section) followed with histological confir-

78 79

Crustacean Fungi 



Histological section of fungal hyphae within Higher magnification of granuloma and localized granuloma (arrow) in soft tissues fungal hyphae (arrow) beneath the carapace of infected red king crab

GMS stain of aseptate fungal hyphae (black) from figures above

78 79 crustacean Protozoa Haplosporidian-like Parasite of Shrimp

I. Causative Agent and Disease of about 14-20 µm in diameter with a Haplosporidian protozoa are within single central nucleus having a distinct the phylum Haplosporidia, class Hap- outline and no dinokaryotic figures. This losporea within the order Haplosporida nuclear profile distinguishes the parasite in the family Haplosporidiidae. Reports from the Hematodinium dinoflagellate of haplosporidians in crustaceans are in Tanner crabs (discussed elsewhere). few and, although they have potential Histological examination demonstrates as serious pathogens, their prevalences the uninucleate form observed sys- have been low and distributions rare. temically within the hemal sinuses and An unidentified haplosporidian-like a larger multinucleated plasmodial form protozoan, previously described as within the tissues. Transmission electron dinoflagellate-like, occurs in spot and microscopy of nuclear detail further pink shrimp where unconfirmed preva- demonstrates less condensed nuclear lences of 10-50% have been reported. chromatin that does not cause irregular However, documented prevalences have bulging of the well-defined nuclear en- been less than 1% but parasitized prawns velope. These features produce a distinct did not survive in captivity. Uninucleate nuclear profile. Various other ultrastruc- stages of the parasite replace hemocytes tural features distinguish this parasite in the hemal sinuses and multinucleated from the Tanner crab Hematodinium sp. plasmodial forms occur in tissues causing systemic disease. VI. Prognosis for Host This haplosporidian-like protozoan II. Host Species causes mortality in parasitized shrimp by This haplosporidian-like parasite has displacement of hemocytes and normal been reported in spot and pink shrimp organ tissues. However, the actual popu- from the Strait of Georgia in British lation prevalence may be quite low with Columbia, Canada and from Yakutat and insignificant mortality. Prince William Sound in Alaska. VII. Human Health Significance III. Clinical Signs There are no apparent zoonotic hu- Parasitized shrimp are lethargic, have man health concerns with the presence an unusual red color with chalky white of this haplosporidian-like parasite in musculature and characteristic cloudy shrimp. However, the meats of parasit- or milky hemolymph containing myriad ized shrimp may be unsuitable in texture numbers of the parasite. for human consumption.

IV. Transmission The mode of transmission is unknown.

V. Diagnosis Wet mounts and stained smears of tissue exudate and hemolymph demonstrate a circular non-motile protozoan

80 81 crustacean Protozoa

Red “cooked” appearance of spot shrimp parasitized by a haplosporidian-like protozoan

Smear of mucinous milky tissue exudate containing myriad numbers of organisms

80 81 crustacean Protozoa

Haplosporidian-like Parasite

of Shrimp 

Histological section of a haplosporidian-like parasite (arrow) in the hepatopancreas of spot shrimp

A higher magnification of multinucleated plasmodial forms in histological section

82 83 crustacean Protozoa

A stained smear of fixed uninucleate forms of the parasite

Ultrastructural detail of the parasite showing less condensed nuclear chromatin and a well defined nuclear profile

82 83 crustacean Protozoa Hematodinium sp. - Bitter Crab Disease of Tanner Crabs

I. Causative Agent and Disease IV. Transmission Bitter crab disease is caused by a Although the natural route of trans- parasitic dinoflagellate,Hematodinium mission has not been established, there sp. belonging to the protozoan phylum are likely to be several modes in which Alveolata, subphylum Dinoflagellida, the parasite is transmitted horizontally order Syndinida and family Syndiniceae. via: stages of the parasite in seawater A major feature of classification for di- that enter breaks in the cuticle during noflagellates is the biflagellated grooved mating and molting; cannibalism or dinospore stage. Obligate Hematodinium feeding on detritis containing resting parasites have been described from spores; sexual transmission via semi- several species of crustaceans, certain nal fluids of parasitized male crabs and fishes and cephalopods. In crustaceans possibly through a reservoir host such as the type species is H. perezi parasitizing bottom dwelling amphipods. The para- the European shore crab. All Hema- site and disease have been transmitted in todinium described from crustaceans the laboratory by injection of hemo- parasitize the hemolymph causing lymph containing vegetative trophonts systemic disease and mortality affecting and dinospore stages. Life history stages at least 26 species of crustacean hosts in include: trophonts which are single cells Europe, Australia and North America and have slow division; larger plasmo- including many commercially important dia with multiple nuclei; pre-spores crab species and 13 species of benthic with multiple nuclei and rapid division amphipods. followed by sporulation of biflagellated spores with a single nucleus. Spores II. Host Species are of two types, a large macrospore The bitter crab Hematodinium is and a small microspore, generally with reported from Tanner and snow crabs (C. only one spore type occurring per host. bairdi, C. opilio) in southeast Alaska, the Spores may possibly have a dissemina- Gulf of Alaska and the eastern and west- tory or resting stage function rather than ern Bering Sea; from deep water Tanner transmission. Laboratory studies suggest crabs (C. tanneri) in British Columbia, that the life cycle of the parasite occurs Canada and from snow crabs in Atlantic over a 15 to 18 month period. Infestation Canada. likely occurs from the trophont stage during the spring molting period from III. Clinical Signs mid-March to May. Crabs dying in the Parasitized crabs are lethargic and die summer and fall following sporulation when handled, have an exaggerated red were likely parasitized in the spring color of the carapace, flaccid chalky tex- of the previous year. After sporulation tured meat and white opaque hemolymph in the fall the prevalence of parasitism caused by the myriad numbers of dinofla- becomes almost undetectable (eclipse) gellates. The cooked meats of parasitized until the new infestations of the previous crabs have an astringent aspirin aftertaste spring build to detectable levels that can and are unmarketable, hence the “bitter be observed the following late winter and crab” name of the disease. early spring.

84 85 crustacean Protozoa

V. Diagnosis lowed by a reduction or eclipse until late Diagnosis is based on typical clinical winter and early spring. Peak mortality signs with the occurrence of myriad occurs in August through September due numbers of dinoflagellate stages (usually to sporulation. Chronic mortality occurs non-motile trophonts) in wet mounts or in weakened hosts from secondary infec- stained smears of hemolymph. Trophonts tions by other pathogens. Options for are 15.4 X 20.7 µm, have an indistinct control of bitter crab disease in southeast nuclear profile and foamy cytoplasm Alaska include: harvest Tanner crabs with droplets exuding from the surface from October through November during pellicle in a stained hemolymph smear. the eclipse period of the disease when A key diagnostic feature visible in many crab meats may be less full but of accept- trophonts is the dinokaryon nuclear able quality (requires no culling of para- division producing condensed V-shaped sitized crabs); grossly parasitized crabs pairs of chromosomes. Nonmotile should be properly disposed of (landfill; plasmodia are similar in appearance but incinerated; ground and cooked or chlo- larger and multinucleated. Prespores are rinated) rather than released; discourage similar to plasmodia but each nucleus culling through education programs to has a distinct lobed nuclear outline. Mac- recognize the disease and follow proper rospores are oval 15.2 X 11.4 µm having disposal; provide an economic incentive slow motility and a beaked protrusion by developing alternative use products on one end. The nucleus is longer than for harvested parasitized crabs (surimi, it is wide with a distinct outline. The chitin products, etc.). microspore is elliptical and smaller at 12.0 X 4.4 µm and has rapid motility VII. Human Health Significance with a refractile body at the posterior There are no zoonotic human health end and 2 flagella more often visible on concerns regarding bitter crab disease in stained smears. The microspore develops Tanner crabs. However, parasitized crabs an obvious bent corkscrew shape within have an unpalatable flavor and undesir- 6 days after sporulation. Histologi- able meat texture. cal examination demonstrates parasite stages within hemal sinuses throughout the tissues of the crab host. PCR primers are available for certain Hematodinium sp. parasitizing commercial crab species.

VI. Prognosis for Host Virtually 100% mortality occurs in Tanner crabs naturally parasitized by Hematodinium sp. when maintained in the laboratory. A similar mortality of parasitized crabs is assumed to occur in wild Tanner crab populations. Signifi- cantly higher prevalences of bitter crab disease occur in new shell crabs and crabs less than 60 mm carapace width. There is a seasonal prevalence/intensity peaking in July through October fol-

84 85 crustacean Protozoa Hematodinium sp. - Bitter Crab Disease of Tanner Crabs

Exaggerated reddening of the crab carapace (bottom) typical of bitter crab

disease 

White viscera with milky hemolymph (arrow)

86 87 crustacean Protozoa



Vegetative stages in a hemolymph smear SEM showing droplet exuding from pel- with irregular nuclear profile and droplets licle of vegetative stage (arrow) exuding from pellicle

Twisted microspores with flagella visible in SEM showing the smooth surface of a phase contrast microscopy microspore and two flagella

86 87 crustacean Protozoa Hematodinium sp. - Bitter Crab Disease of Tanner Crabs

Wet mount of beaked macrospores

Beaked macrospore (SEM) showing

warty surface and one of the two

flagella

 

Histological section showing vegetative Histological section showing typical stages (arrow) inside spermatophore dinokaryon nuclear division (arrow)

88 89 crustacean Protozoa Possible Hematodinium Life Cycle

Possible Transmission By: Damaged cuticle during molt

Insemination from parasitized male

Cannibalism

Trophont ------Slow Reproduction 15 - 18 months

' Vegetative Plasmodia Stages '

Pre-Spore ------Rapid Reproduction Sporulation 10 - 14 days

' Dinospore ------Crab Mortality 24 - 48 hours

 

Microspore  Tanner Crabs

Macrospore Disseminatory resting Amphipods - reservoir hosts? stages transmitted to detritus feeders?

88 89 crustacean Protozoa Hematodinium-like Disease of Dungeness Crabs

I. Causative Agent and Disease developmental stages if confirmed to be Obligate parasitic dinoflagellates Hematodinium sp. that belong to the genus Hematodinium sp. are in the protozoan phylum Alveo- V. Diagnosis lata, subphylum Dinoflagellida, order Stained blood smears contain myriad Syndinida and family Syndiniceae. numbers of apparent prespore stages, A major feature of classification for some of which have dinokaryon type all dinoflagellates is the biflagellated condensed chromosomes in V-shaped grooved dinospore stage. Hematodimium configuration. Tissues were not available parasites have been described from for further diagnostic analysis. several species of crustaceans, certain fishes and cephalopods. In crustaceans VI. Prognosis for Host the type species is H. perezi parasitizing The single parasitized crab was one the European shore crab. All Hema- of several being held in crowded 10 todinium described from crustaceans foot circular tanks for a size at maturity parasitize the hemolymph causing study. Minor mortality, beginning in systemic disease and mortality affecting early July, occurred in the captive crab at least 26 species of crustacean hosts in groups, some of which was due to stress- Europe, Australia and North America caused bacterial septicemia while other including many commercially important mortality was reportedly caused by this crab species and 13 species of benthic Hematodinium-like parasite. amphipods. VII. Human Health Significance II. Host Species There are no zoonotic human health A Hematodinium-like parasite was concerns regarding dinoflagellate para- observed in a single captive subadult sitism in crabs. However, parasitized Dungeness crab collected from the crabs often have an unpalatable flavor waters of Kodiak Island, Alaska in and undesirable meat texture. mid-May of 2003. It is the only known case of dinoflagellate parasitism in the Dungeness crab on record in the Pacific Northwest.

III. Clinical Signs Clinical signs include lethargy followed by death associated with milky white hemolymph and grossly abnormal viscera characterized by pallid color and a white viscous exudate.

IV. Transmission The mode of transmission is unknown but likely complex (see bitter crab disease section) involving several

90 91 crustacean Protozoa

Milky-white viscera and exudate of a Dungeness crab parasitized by a Hematodinium- like organism



Stained hemolymph smear of parasitized Dungeness crab showing Hematodinium-like prespores with dinokaryon type condensed chromosomes in V-shaped configuration (arrow)

90 91 crustacean Protozoa Mesanophrys sp. Ciliate Disease

I. Causative Agent and Disease form of host debilitation or external Mesanophrys sp. (pugettensis) (syn injury as a portal of entry. The life cycle Anophrys, Paranophrys) is a scuticocili- of this ciliate is simple and direct with ate protozoan with holotrichous ciliation reproduction by binary fission allowing and a long trailing caudal cilium. These parasite numbers to increase exponen- ciliates are primarily facultative patho- tially. gens of injured or captive crustaceans but have also been found in wild popula- V. Diagnosis tions, most often in recently molted Diagnosis includes the typical gross animals. Infestation is generally fatal clinical signs of injury, lethargy and causing a systemic disease with high mortality in conjunction with myriad mortality. numbers of elongate holotrichous ciliates each having a long trailing cilium in wet II. Host Species mounts of hemolymph and tissue smears. Mesanophrys has a broad range with White focal areas of necrosis and coagu- most reports occurring from several lated hemolymph containing ciliates may marine crab species in Europe and the also be observed on the surfaces of soft Pacific Northwest. Closely related tissues (see bottom figure of blue king species have been reported in lobsters crab on page 111). from Maine and Atlantic Canada and in cultured penaeid shrimp from China. In VI. Prognosis for Host Alaska, Mesanophrys has been reported Mesanophrys generally causes high in wild isopods collected from Afognak mortality in injured or captive crus- Island and in captive blue and golden taceans. Some measure of control for king crabs, Dungeness crabs and Tanner impounded animals may be achieved crabs from southeast. through improvement of environmental conditions and reducing crustacean III. Clinical Signs densities to further reduce mechanical Parasitized crustaceans are generally damage. injured or otherwise stressed by captivity and exhibit lethargy, anorexia and ataxia VII. Human Health Significance followed by death. The hemolymph is There are no zoonotic human health cloudy or opaque from the presence of concerns regarding ciliate infestation of massive numbers of motile ciliates. Tis- crustacean tissues. sue pathology observed by histological examination is characterized by nearly complete destruction of peripheral and tissue hemocytes accompanied by massive tissue infiltration of the ciliates with severe systemic necrosis of major organs.

IV. Transmission Transmission is horizontal from ambient seawater, usually requiring some

92 93 crustacean Protozoa

Mesanophrys-like ciliate in hemolymph Higher magnification of Mesanophrys-like smear from parasitized Tanner crab ciliates in histological section from blue king crab



Histological section of Mesanophrys-like ciliates (arrow) beneath the epidermis of a parasitized blue king crab

92 93 crustacean Protozoa Thelohania and Other Microsporidia

I. Causative Agent and Disease have been reported parasitizing many Microsporidia is a protozoan order different species of crabs, shrimps and within the class Microsporea within the other crustaceans in Europe, Thailand, phylum Microspora. However, there Australia and on the Atlantic, Gulf and is controversial genetic evidence for a Pacific coasts of North America. In closer affinity to the kingdom of Fungi Alaska, microsporidia have been found rather than Protozoa. All microsporid- in red, blue and golden king crabs from ians are intracellular parasites that Bristol Bay and the Bering Sea and blue produce microspores (3 to 10 µm) as the king crabs and coonstriped shrimp from infectious stage and complete their life southeast Alaska. cycles in a single host cell, generally with no alternate hosts. Microsporidia III. Clinical Signs are one of the more common groups of Clinical signs may include lethargy, parasites in crustaceans with over 140 mortality and tissues that are white or species reported from all orders. Some curd-like in appearance caused by tissue microsporidians cause significant pathol- damage and replacement with the spores ogy, mostly affecting skeletal muscle. of the parasite. Shrimp often exhibit Thelohania duorara parasitizing pink, opaque white abdomens with a milky white and brown shrimps causes an fluid exudate. opaque white abdomen typifying “cot- ton” or “milky” disease that destroys IV. Transmission muscle and connective tissues. Ag- Transmission is horizontal when masoma penaei also parasitizes shrimp spores released from ruptured host cells causing destruction of many different are ingested by a suitable host. In the tissues as well as parasitic castration. intestine of the new host each spore Two different undescribed species of releases a hollow polar tube attaching the Thelohania cause “cottage cheese” dis- spore to a mucosal epithelial cell through ease in both red and blue king crabs from which the internal amoeboid sporoplasm Bristol Bay and the Bering Sea charac- passes into the host cell cytoplasm. In terized by massive numbers of spores some cases the parasite replicates in the in all major visceral organs causing the host cell nucleus rather than in the cyto- tissues to appear white and curd-like. plasm. The sporoplasm may replicate in Another unidentified microsporidian the intestinal cell or may be injected into in the family Nosematidae parasitizes a host phagocyte where it travels to other mostly the musculature, including the target tissues. In addition to transmission heart, of golden king crabs while other via the alimentary tract some species in unidentified microsporidians have been amphipods may also be vertically trans- observed in the antennal gland of blue mitted to progeny from parasitized ova. king crabs and in the body musculature Once in the target host cell the parasite of a single coonstriped shrimp from undergoes further replication and devel- southeast Alaska. opment involving merogony producing plasmodia and meronts followed by II. Host Species sporogony producing sporonts that con- Microsporidia are ubiquitous and tain sporoblasts that mature into spores.

94 95 crustacean Protozoa

The entire process is complex and may VI. Prognosis for Host have other intermediate stages depend- Microsporidians in several crusta- ing on the species of microsporidian. cean hosts are serious pathogens causing Infected host cells and their nuclei also significant mortality and/or debilita- may respond by marked hypertrophy in tion resulting in predation or secondary which the enlarged cells become cysts or pathogen infections. The prevalences xenomas containing myriad numbers of of parasitism are generally low in most the parasite. wild populations of crustaceans but can be very high in cultured shrimp. The V. Diagnosis king crab microsporidians are considered Stained and unstained hemolymph to be lethal parasites as well but have and/or tissue smears from parasitized occurred at low prevalences (2-10%) that animals generally demonstrate the may not significantly impact king crab microspores that can also be observed by populations. histological examination. Major characteristics for classification of microspo- VII. Human Health Significance ridia are the size, shape and number of Affected shrimp are generally un- spores produced within sporonts. Thelo- marketable due to poor aesthetic quality. hania has 8 spores each approximately However, there are no zoonotic human 3 X 5 µm. DNA probes are available to health concerns regarding the parasitism

identify some genera (Agmasoma sp.) of of crustacean tissues by microsporidia.

microsporidia. 

Histological section of Thelohania microspores (arrow) within digestive gland of red king crab (Photo: J. Frank Morado, National Marine Fisheries Service, Seattle)

94 95 crustacean Protozoa Thelohania and Other Microsporidia

Stained tissue smear showing packets of 8 spores typical of Thelohania sp. 

TEM of spores (arrow) within sporont of Thelohania sp. (Photo: J. Frank Morado, National Marine Fisheries Service, Seattle)

96 97 crustacean Protozoa



Histological section of degenerating unidentified microsporidian spores (arrow) in the antennal gland of blue king crab

96 97 crustacean Helminths Carcinonemertes Egg Predator

I. Causative Agent and Disease III. Clinical Signs Worms belonging to the phylum Clinical signs of infestation include Nemertea include the genus Carcinone- dull pink to orange adult worms visible mertes sp. in the family Carcinonemerti- within the egg clutch, usually found on dae. These nemerteans are specialized the funicular strands (stalks binding ectosymbionts that feed on the eggs of fascicles of setae) attaching the eggs to decapod crustaceans. Feeding is accom- the pleopods of ovigerous female crabs. plished by puncturing the egg membrane Empty or partially eaten eggs may be ob- with a knife-like stylet on the end of a vious in the egg mass where worms can proboscis that is everted with the foregut be found inside when infestation is high. into the egg to ingest the contents. These Juveniles may be present ensheathed in egg predators can be major sources mucus on the uncalcified cuticle of the of egg mortality as demonstrated for ventral surface of the host abdomen. several economically important decapod crustacean species. Egg predation was IV. Transmission implicated in the collapse of the Dunge- Variations occur depending on worm ness crab fishery in southern California species but generally female Carcinon- in 1960 and localized brood failure in emertes are fertilized internally by en- the early 1980s for red king crabs in the trance of waterborne male sperm through Kodiak, Island and Cook Inlet areas of tegmental pores. The embryos are Alaska. At least six species of Carcinon- extruded through the gonoduct to hatch emertes have been described from crabs; externally where the haplonemertean C. carcinophila (with 2 subspecies), C. larvae undergo direct development into coei, C. epialti, C. errans, C. mitsukurii the juvenile stage during a planktonic and C. regicides. The effects of another existence. Juveniles settle out on crabs of nemertean egg predator, Pseudocarcino- any age or sex, produce a sheath of mu- nemertes homaris, on the American lob- cus and attach to various protected areas ster have been more difficult to quantify. on the host exoskeleton. Attached worms apparently subsist by absorbing dissolved II. Host Species amino acids through the skin that leak Nemertean egg predators have been from the crab arthrodial membranes. reported on many host species of crabs When female crabs oviposit the resident primarily in North America including worms migrate to the egg mass to feed commercially important species such as and reproduce. Infested male crabs can Dungeness crab, Tanner crab, red king transmit juvenile worms to female crabs crab, blue crab and the American lobster. during mating and transfer also occurs The nemertean worm described from from the old to the new cuticle when a Alaska is C. regicides found on Tanner host crab molts. and red king crabs where significant egg predation/mortality in the latter host was V. Diagnosis implicated as causing previous localized Diagnosis of Carcinonemertes infes- population declines. tation is determined by finding worms on the cuticle or in the egg mass of the host

98 99 Crustacean Helminths

crab and/or the presence of empty or par- poor recruitment into the population tially eaten crab eggs. Species identifica- if the prevalence of infestation is high. tion is based on morphological charac- Epibiont fouling of egg masses may also teristics which, for C. regicides, include result indirectly from worm feeding among others a smaller adult length of activity that produces a rich organic 1.0 mm, presence of an excretory system substrate from punctured eggs and worm and a large anterior proboscis chamber or feces. rhynchocoelum. VII. Human Health Significance VI. Prognosis for Host There are no zoonotic human health In ovigerous female crabs, heavy in- concerns regarding the presence of festations of Carcinonemertes may cause nemertean egg predators on decapod extensive loss of eggs and subsequent crustaceans.

Drawing of Carcinonemertes regicides (J. Shields, Virginia Institute of Marine Science)

98 99 crustacean Helminths Carcinonemertes Egg Predator

Egg mass of a female red king crab infested by Carcinonemertes (Photo: J. Shields, Virginia Institute of Marine Science)

Carcinonemertes worms removed from egg mass above (Photo: J. Shields, Virginia Institute of Marine Science)

100 101 Crustacean Helminths

Higher magnification of king crab eggs infested by Carcinonemertes regicides (Photo: J. Shields, Virginia Institute of Marine Science)

Higher magnification of single Carcinonemertes regicides worm (Photo: J. Shields, Virginia Institute of Marine Science)

100 101 crustacean Helminths Crab Leech

I. Causative Agent and Disease crabs and probably occur on the shells of Leeches belong to the phylum An- other crab species as well. nelida (segmented worms) within the class Hirudinea consisting of over 500 III. Clinical Signs species of freshwater, marine and ter- Gross clinical signs include obser- restrial worms. They possess anterior vation of attached adult leeches and/or and posterior suckers and a large number closely adherent brown to black convex of leeches are not ectoparasitic. Com- cocoons on the shell surfaces. mon leeches in the North Pacific area include Johanssonia arctica, a parasite IV. Transmission of several marine fish including cod and The leech eggs hatch on the shell sur- a vector for Trypanosoma murmanensis, face of the crustacean and the juveniles a blood parasite of fish. Another leech, find a fish host to parasitize and obtain a Notostomum cyclostoma, is parasitic blood meal. Leeches are hermaphroditic, on several fish species including cod, reproduce sexually and produce eggs. At pollock, flatfishes, dogfish and skates some point a leech leaves its fish host to and is a vector for the hemoflagellate seek out a hard substrate, often a crus- fish parasite, Cryptobia sp. A smaller tacean shell, on which to lay eggs. Each fish leech, Malmiana sp., is less com- egg is encased in a single cocoon. mon. Most notably, these leeches do not parasitize crabs but favor the carapace as V. Diagnosis a substrate for depositing eggs each in a Diagnosis is by visual observation of single cocoon. The crustaceans function typical leech cocoons on the shell sur- as transport hosts to disseminate the face of various crustaceans, especially leeches and are unharmed. king and Tanner crabs. Attached adult leeches may also be present and can be II. Host Species identified by various morphological char- Several crab species may serve as acteristics. Cocoons of J. arctica ( 1.5 transport hosts for the eggs of these mm X 1.0 mm) as well as adult worms leeches which are circumpolar in distri- (21.5 mm) are smaller than cocoons (7 bution. Notostomum cyclostoma occurs mm X 5.5 mm) and adults (70 mm to 110 in the Seas of Japan and Okhotsk, the mm) of N. cyclostoma. Bering Sea and along the Alaskan coast south to the Stikine River. In northern VI. Prognosis for Host British Columbia, Canada it has been There is no harm to the crustacean observed on golden and red king crabs transport host caused by adult leeches and Tanner crabs in the Portland Inlet or leech cocoons attached to the shell system. Johanssonia arctica has been surfaces. reported in the Bering Sea and was likely transported into the Barents Sea with VII. Human Health Significance the introduction of red king crabs. In There are no zoonotic human health Alaska, adult leeches and cocoons, most concerns with the presence of leeches likely Notostomum cyclostoma, have or their external cocoons on the shell been observed on the shells of red, blue surfaces of crustaceans. and golden king crabs, Tanner and snow

102 103 Crustacean Helminths

Crab leech 

Leech cocoons (arrow) deposited on the Crab leech attached to the carapace of a red undersurface of the carapace of a golden king crab king crab

Crab Leech Life Cycle

Leech attaches Hatched, leeches to fish host seek fish host

Engorged leech falls off host producing eggs

Eggs

Eggs encased in cocoons attached to crustacean shell

102 103 crustacean Helminths Encysted Trematode Metacercariae

I. Causative Agent and Disease parasitizing the Atlantic and Gulf coast Trematodes or flukes are members of blue crab that often itself is parasitized the phylum Platyhelminthes and the class by the haplosporidian, Urosporidium Trematoda. Adult worms of the subclass crescens. The hyperparasite causes the Digenea are endoparasites occurring in fluke to become enlarged and darkly pig- all classes of vertebrates and use inverte- mented resulting in black spots visible brates as the first and sometimes second within the flesh of the crab, a condition intermediate host. A metacercaria is the known as “buckshot” or “pepper” crab encysted juvenile trematode usually oc- that renders the meats unmarketable. curring in the second intermediate host that requires ingestion by the final host IV. Transmission to become an adult worm. Depending Microphallid life cycles generally on the trematode species, metacercariae include a snail host producing swim- are found in a variety of tissues and in- ming cercariae shed into seawater that termediate hosts including crustaceans. horizontally parasitize a crustacean Encysted metacercariae in most hosts host producing metacercariae that must generally cause no overt disease unless be eaten by a final host, usually a bird, present in numbers large enough to dam- mammal or rarely a cold-blooded verte- age major organs and tissues. Many of brate. The adult trematode matures in the these worms have been identified belong- intestine of the final host and produces ing to the family Microphallidae includ- eggs released into seawater with feces to ing the genera Microphallus, Spelotrema begin the cycle again (see metacercariae and Opecoeloides. in bivalve section).

II. Host Species V. Diagnosis Encysted digenetic trematode meta- Wet mounts of fresh tissues may cercarial stages have been reported in show the encysted metacercariae which several marine crustaceans including: are small and extremely difficult to penaeid shrimps from the southeastern find. Usually, encysted metacercariae U.S.; various Gulf coast crabs including are observed during routine histologi- the blue crab and blue crabs from Rhode cal examination in connective tissues, Island; crangonid shrimp and Dunge- musculature, nervous tissues, hepatopan- ness crabs from Washington State. In creas and gonads. Alaska, unidentified metacercariae have been observed in Dungeness crabs and VI. Prognosis for Host undoubtedly occur in other indigenous Encysted trematode metacercariae crab species as well. generally cause no harm to the host except in isolated cases where large III. Clinical Signs numbers may cause tissue necrosis and There generally are no clinical signs dysfunction. Ataxia resulting from ne- of parasitism because the encysted crosis or compression atrophy of nervous metacercariae cause no significant tissue tissues could result when metacercariae damage and are too small to observe occur in the nerves, brain or thoracic grossly. An exception is the microphallid ganglion as reported for Dungeness

104 105 Crustacean Helminths

crabs and crangonid shrimp. host there could be a zoonotic human health concern if encysted metacercariae VII. Human Health Significance in parasitized crab meats are consumed Because microphallid trematodes uncooked.

use warm-blooded animals as the final 

Encysted trematode metacercaria (arrow) in cranial nerve of Dungeness crab

104 105 crustacean Helminths Trypanorhynch Cestode Plerocercoids

I. Causative Agent and Disease ported in decapod crustaceans except for Trypanorhynch cestodes belong encapsulation and granuloma formation to the phylum Platyhelminthes, class in the hepatopancreas and possibly other Cestoda, order Trypanorhyncha and tissues. Conceivably, some host mortality those having decapod crustaceans as or debilitation could result from heavy intermediate hosts are found as adults infestations. in the alimentary tracts of elasmobranch final hosts. Reports of trypanorhynchid IV. Transmission plerocercoids in crustaceans have largely Trypanorhynchid life cycles involv- been restricted to shrimp where high ing decapod crustaceans can include two prevalences and intensities have been general pathways starting with eggs re- observed. In some shrimp species the leased with feces from the elasmobranch worms are destroyed by host reaction final host followed by: eggs or hatched causing granulomas in the hepatopancre- free-swimming ciliated coracidia are as but no significant related mortality has eaten by or infest a bivalve mollusc that been recognized. In a report of trypano- is eaten by a decapod crustacean that is rhynch plerocercoids (Trimacracanthus eaten by an elasmobranch; alternatively aetobatidis and Dollfusiella martini) eggs or coracidia are eaten by or infest in green crabs the worms cause epithe- small crustaceans (copepod, amphipod) lial metaplasia and loss of surrounding that are eaten by a larger decapod crus- secretory parenchyma in digestive gland tacean that is eaten by an elasmobranch. tubules and are also destroyed by a host Coracidia develop into procercoids in the mediated inflammation and encapsula- first intermediate host that later develop tion. In heavy infestations crab survival into plerocercoids in the last intermedi- may be reduced. ate host.

II. Host Species V. Diagnosis Penaeid shrimp species within Diagnosis may be made by gross or Gulf coast waters of North America histological observation of plerocercoids are the most common crustacean hosts within the tissues of infested crusta- reported to harbor trypanorhynch ceans. The scolex or head of a trypano- plerocercoids but the distribution of this rhynch plerocercoid has 2-4 bothria and order is worldwide and likely occurs in 4 spiny eversible proboscides (tentacles). many other crustacean species such as reported from green crabs in Australia. VI. Prognosis for Host In Alaska, a single unencysted specimen There are no reports of significant of a trypanorhynchid plerocercoid was decapod crustacean mortality or pathol- recovered from the hepatopancreas of ogy associated with larval trypano- a captive red king crab collected from rhynch infestations. An exception may southeast waters. be the trypanorhynchid plerocercoids infesting Australian green crabs where III. Clinical Signs the parasites may possibly contribute to There have been no significant clini- reduced crab survival. cal signs of plerocercoid infestation re-

106 107 Crustacean Helminths

VII. Human Health Significance rhynch cestode larvae in the tissues of

There are no zoonotic human health decapod crustaceans. concerns with the presence of trypano- 

Trypanorhynch plerocercoid from red king Trypanorhynch plerocercoid from red king crab showing posterior end crab showing scolex with bothria and 4 spiny proboscides (arrow) Trypanorhynch Cestode Life Cycle

Elasmobranch final host

Eggs

Ciliated larva – coracidium Decapod crustacean eats infested animal

Eggs or coricidia eaten by or infest bivalve mollusc or small crustacean

106 107 crustacean Arthropods Briarosaccus callosis - Parasitic Barnacle

I. Causative Agent and Disease crabs have an enlarged, raised abdominal Briarosaccus callosus is a para- flap and exaggerated growth of coxal sitic barnacle belonging to the phylum setae. Histological examination confirms Arthropoda, subphylum Crustacea, class the extensive infiltration of tissues by the Maxillopoda, subclass Thecostraca, basophilic branching tubules of the root- infraclass Cirripedia (barnacles) and lets generally causing no host inflamma- the superorder Rhizocephala (parasitic tory response. However, encapsulation barnacles). Rhizocephalan barnacles are of rootlets has been observed when the primarily parasites of decapod crus- inflammatory response of the crab host taceans but also occur in free-living is stimulated by a concurrent rickettsial barnacles and bivalve molluscs. They are infection. noted for their invasiveness but general non-lethality in the natural host and IV. Transmission endocrinological suppression of host The complete life history of B. cal- fecundity at the population level. losus is not known but a portion of larval biology has been established to allow II. Host Species analogy with other known rhizocepha- Briarosaccus callosus is a cos- lans. The externa is a double cylinder mopolitan rhizocephalan parasitizing with an internal chamber containing ova several species of lithodid crabs most that move into the outer chamber as the notably from the southeast coast of North embryos mature causing the externa col- America, Antarctica and sub Antarctic or to become pale orange. Stage I nauplii areas, southwest Indian Ocean, the Ber- released from the externa become plank- ing Sea, the Gulf of Alaska, southeast tonic during which successive molts Alaska and British Columbia, Canada. occur to the final stage IV before molting Known crab species parasitized by B. into a cyprid. As with other rhizocepha- callosus in Alaska include the red, blue lans, these cyprids are presumed to be and golden king crabs. the infectious stage and are sexually di- morphic in that one externa may produce III. Clinical Signs cyprids that are small and female while External clinical signs include a another produces larger male cyprids. large orange/red sausage-shaped sac The smaller female cyprids attach to the known as an externa that is attached by gill lamellae of a crab host and molt to a stalk underneath the abdominal flap. the kentrogon stage which injects a cell The externa contains ova and/or larvae mass through a hollow stylet. These cells of the parasite. Radiating into the crab proliferate in the crab host becoming the viscera from the externa is an emerald female interna and reproductive externa. green dendritic mass or root system Newly emerged virginal externa do not called the interna which largely replaces reach sexual maturity until fertilized by the hepatopancreas extending rootlets larger male cyprids to begin the cycle into all major organs and tissues of the again. visceral cavity including nerves and the bases of gills and muscle within the V. Diagnosis coxal joints. Gonads of both crab sexes Diagnosis is made by observation of are atrophied or absent. Parasitized male the typical orange/red externa attached 108 109 crustacean Arthropods

by a stalk underneath the abdominal flap and necrotic causing an intense inflam- in lithodid crabs and the emerald green matory foreign body response that may interna within the visceral mass. Parasit- kill the crab host. The parasite alters the ized crabs, including males, have femi- endocrinology of the host crab causing nized traits of an enlarged abdominal atrophy of the gonads (castration) in both flap and a thick ventral growth of coxal sexes and suppressed growth which ef- setae, atrophied gonads and a protective fectively limit recruitment. The negative behavior towards the attached externa. impact of this parasite on a population The externa utilizes hemoglobin as a scale may be significant in certain areas respiratory pigment and bleeds red when depending on prevalences which have damaged. Histological examination varied in southeast Alaska from less than demonstrates the dendritic branches of 1% in red king crabs, 20% in golden king the interna that retain the green pigment crabs and up to 76% in blue king crabs. granules in fixed material. VII. Human Health Significance VI. Prognosis for Host There are no zoonotic human Crabs are able to molt successfully health concerns regarding parasitism with attached externa but the likelihood of king crabs by Briarosaccus callosus. for long-term survival is not known once However, the rootlets may cause green the parasite externa is lost or becomes discoloration of knuckle meats despite senescent. Externa do not regenerate and cooking. the interna rootlets become atrophied



Dual parasitism of red king crab by Bria- Cyprid larva of Briarosaccus callosus from the rosaccus callosus showing viable externa externa of parasitized blue king crab (orange) and second senescent externa that has become necrotic (arrow)

108 109 crustacean Arthropods Briarosaccus callosis - Parasitic Barnacle



 Histological section of blue king crab Histological section of Briarosaccus callosus thoracic ganglion invaded by Briarosaccus in another blue king crab infected with a callosus rootlets (arrow) of the interna with rickettsia-like organism showing encapsula- no host inflammatory response tion of rootlets (arrow) by host cells

Briarosaccus callosus in blue king crab with typical green rootlet mass from healthy parasite externa

110 111

crustacean Arthropods 

Parasitized blue king crab with missing externa resulting in black, atrophied and necrotic internal rootlet mass (arrow)



Blue king crab parasitized by Briarosaccus callosus showing atrophied yellow ovary (arrow) and obvious green rootlets of the interna in the hepatopancreas; compare with normal ovary on next page; note coagulated hemolymph nodule (arrowhead) resulting from concurrent infestation by the ciliate , Mesanophrys sp.

110 111 crustacean miscellaneous Digestive Tubular Degeneration and Bio-Fouling

I. Causative Agent and Disease growth. Digestive tubular degeneration ap- IV. Transmission pears to be a non-infectious lesion of the No infectious agent has been found hepatopancreas observed in only two for digestive tubular degeneration which captive golden king crabs collected from may be related to the stress of captiv- southeast Alaska. Whether the condi- ity. Bio-fouling epibionts occur and tion occurs in wild crabs or is related to reproduce in the water column and are the stress of captivity is not known but passively transmitted horizontally. affected crabs appear clinically normal. Bio-fouling is the external growth of V. Diagnosis algae, bacteria, bryozoa, barnacles and Digestive tubular degeneration is other epibionts on the cuticle and shell diagnosed by gross observation during surface of crustaceans. Generally, the necropsy and confirmed by histologi- presence of these epibionts causes no cal examination. Bio-fouling may also harm to the crustacean host. However, be obvious but is confirmed by routine heavy growth on the surface of gills or histological methods. eyestalks could reduce respiratory capacity and sensory perception, respectively. VI. Prognosis for Host The clinical outcome of digestive II. Host Species tubule degeneration is unknown but af- Although noninfectious digestive fected crabs appear normal. Bio-fouling tubular degeneration has been observed is generally harmless to adult crusta- in only two captive Alaskan golden king ceans which shed the epibionts with the crabs, the condition may occur in other old shell during molting. Severe fouling captive crab species if related to stress. of gills may result in shellfish mortality Bio-fouling may occur on any marine or in areas having high organic pollution. freshwater crustacean species worldwide, Heavy epibiont growth by filamentous the severity of which depends on the bacteria is known to cause mass mortal- specific environmental conditions. ity of captive larval red and blue king crabs when maintained at high densities. III. Clinical Signs Digestive tubular degeneration causes VII. Human Health Significance no outward clinical signs but is visible There are no zoonotic human health on necropsy as black, brittle sections of concerns regarding digestive tubular de- hepatopancreatic tubules. Histological generation or bio-fouling in crustaceans. sections indicate areas of yellow-brown atrophied and necrotic digestive tubule acini surrounded by normal tubules. No causative agent is apparent using conventional stains. Bio-fouling may or may not be apparent by gross observation depending on the degree and the nature of the epibiont growth. Routine histological sections confirm the presence of epibiont

112 113 Crustacean miscellaneous



Female golden king crab with black sections of degenerated hepatopan-

creatic tubules (arrow); note normal yellow ovary underneath

 



Atrophy and necrosis of tubule acini (arrow) in

histological section of golden king crab with

hepatopancreatic tubular degeneration Histological section of eyestalk from  Dungeness crab covered with bryo- zoan bio-fouling (arrows)



Histological section of gills from red rock crab dem- Histological section of gills from blue onstrating varied microbial bio-fouling (arrow) king crab demonstrating bacterial bio-fouling (arrow)

112 113 crustacean miscellaneous Idiopathic Granulomatosis –Dungeness Crab

I. Causative Agent and Disease suggest a potential toxic etiology. Other Idiopathic (cause unknown) granu- environmental causes may be associated lomatosis is a condition in Dungeness with granulomatosis described in Dunge- crabs where multiple granulomas are ness crabs from different locations. present histologically within the connective tissues of the midgut wall and rarely V. Diagnosis other tissues including the heart and Diagnosis is by routine histological bladder. Extensive histological examina- examination of Dungeness crab tissues tion using special stains have shown no showing multiple granulomas in the evidence of an etiologic agent. It is pos- connective tissues of the midgut wall. sible that the cause may be environmen- These lesions are typically of two types. tal rather than a foreign body response Type one is smaller, more frequent, to a biological entity. The granulomas consists of a melanized center with few appear to have no affect on crab health. surrounding hemocytes and is located throughout the midgut wall. The second II. Host Species type is larger, less common and is Granulomatosis has been described located deeper within the connective tis- in 34% of Dungeness crabs from Puget sues of the midgut wall and has a central Sound, Washington and affecting 100% melanized core surrounded by a large of Dungeness crabs inhabiting bark collar of hemocytes. The differences in sediments at some logging transfer sites the size, morphology and location of the in southeast Alaska. Similar idiopathic two types of granulomas have caused granulomas have been described in pe- speculation that they are reactions to two naeid shrimp and probably occur in other different stimuli. These granulomas can crustacean species in various oceans of be found in other tissues including the the world. heart and bladder.

III. Clinical Signs VI. Prognosis for Host There are no obvious clinical signs There has been no obvious host of idiopathic granulomatosis in Dunge- morbidity or debilitation associated with ness crabs which can only be detected by idiopathic granulomatosis in Dungeness routine histological examination. crabs.

IV. Transmission VII. Human Health Significance There is no current evidence to indi- There is no zoonotic human health cate that Dungeness crab granulomatosis concern associated with this condition in is caused by a transmissible infectious Dungeness crabs. agent. The high prevalence of granulomatosis accompanied by shell disease, loss of limbs, lower fecundity and mortality in Dungeness crabs associated with benthic deposits of decomposing bark and sulfide at logging transfer sites (see shell disease section) in southeast Alaska

114 115 Crustacean miscellaneous

Typical Dungeness crab  

Histological section of smaller type 1 granu- Histological section of larger type 2 granulo- lomas (arrow) in the midgut connective mas (arrow) in the midgut connective tissues tissues of Dungeness crab of Dungeness crab

114 115 crustacean miscellaneous Proliferative Lesions and Neoplasia

I. Causative Agent and Disease position and are not transmissible in Reports of abnormal proliferation nature. Exceptions are cancers caused of tissues in marine decapod crusta- by infectious viruses of which none have ceans have been extremely rare. There been documented as causing neoplasia in have been less than a dozen cases in the decapod crustaceans. There have been literature of which only four have shown three cases of probable tegmental gland convincing evidence of cancer. Cancers adenocarcinoma in two blue and one or neoplasms are growths of abnormal golden king crab in which an aquabirna- cells that proliferate uncontrollably and like virus (discussed elsewhere) was ob- have no useful function as opposed to served in one affected crab but the true cells that are normal that may proliferate etiology of that neoplasm is not known. excessively (hyperplasia) due to some functional demand or stimulus. V. Diagnosis Neoplasms are diagnosed and clas- II. Host Species sified using histological methods to Reports of neoplasia have included determine the cell or tissue of origin and lymphosarcoma of hematopoietic tissues are further grouped based on benign or in two cultured penaeid white shrimp malignant characteristics. Benign tumors from Hawaii, a carcinoma of the hindgut are often well-differentiated, grow and an adenocarcinoma of the anten- slowly, are well circumscribed without nal gland in Alaskan red and blue king invading surrounding normal tissue crabs, respectively, and embryonal and do not metastasize. Most benign carcinoma in developing embryos of cul- neoplasms usually end in the suffix tured grass shrimp in Taiwan. Additional “oma”. Exceptions are benign neoplasms studies in Alaska have discovered two of the brain and some endocrine organs other cases of probable tegmental gland in higher vertebrates that can be life adenocarcinoma in a blue and golden threatening due to their location and king crab. deleterious physiological effects on the host. Malignant tumors are often not III. Clinical Signs well differentiated, may grow rapidly, Clinical signs of proliferative dis- infiltrate normal tissues and tend to me- eases can include obvious external tissue tastasize. The names of these neoplasms growths but more frequently the abnor- are often preceded by the word “malig- mal tissues are found during routine nant” or with the suffixes “sarcoma” or necropsy and histological examination. “carcinoma”. Three of the four neoplasms observed in Alaskan king crabs IV. Transmission are considered to be tegmental gland Certain proliferative lesions may be adenocarcinomas affecting the anten- simple hyperplasia and inflammation in nal gland, midgut, hepatopancreas and response to an infectious agent or foreign muscle tissues and appear to metastasize. body. Most neoplasms or cancers are These neoplasms range in histological considered spontaneous resulting from appearance from: mostly inflammatory environmental contamination, congenital with hemocyte infiltration, fibroplasia malformation, age or genetic predis- and cell necrosis to a highly cellular

116 117 Crustacean miscellaneous

mass of streaming and nested fibroblastic depending upon the tissue location and if cells surrounding glandular basophilic the irritating cause can be removed. True acinar-like structures or a highly cellular neoplasia usually results in death of the solid mass of large basophilic cells with affected animal. bizarre nuclei that also form occasional glandular acinar-like structures. The VII. Human Health Significance underlying neoplastic cell present in Although aesthetically disturbing, varying frequencies in all presentations there are no zoonotic human health con- is an anaplastic large basophilic cell with cerns associated with proliferative lesions a large clefted bizarre shaped nucleus or neoplasia in decapod crustaceans. having one to three eosinophilic nucleoli. Proliferative inflammatory lesions may These cells sometimes form multinucle- suggest an infectious agent that could ated giant cells and occasionally are negatively affect the desirability of the observed inside phagocytic host cells. meats for human consumption. Cancer is Mitotic figures in acinar-like structures an extremely rare event in decapod crus- are present. taceans and, therefore, should neoplasia be found to occur more frequently in a VI. Prognosis for Host population, an indirect human health con- Proliferative lesions of an inflamma- cern would be whether the cause is linked tory or hyperplastic nature are gener- to environmental contamination. ally reversible and not life threatening

Note: all figures are of suspected tegmental gland adenocarcinomas in blue and

golden king crabs

 

White neoplastic nodular masses on hepatopancreas (arrow) and midgut (arrowhead) of a blue king crab

 

Histological section of midgut neoplasm above showing streaming fibroblasts (arrow) and acinar-like structures (arrowhead)

116 117 crustacean miscellaneous

Proliferative Lesions and Neoplasia 

A second histological section of nodule on previous page

showing occasional large anaplastic cell (arrow) with

large nucleus 

Golden king crab with a white nodular neoplasm in dorsal connec-

tive tissue and musculature (arrow)



 

Histological section of Inflammatory cells, Same neoplasm showing multinucleated necrotic debris (arrow) and large cells with giant cell (arrow) bizarre-shaped nuclei (arrowhead) from golden king crab nodular mass 118 119

Crustacean miscellaneous 

Histological section of a second blue king crab with basophilic

cellular neoplasm (arrow) in antennal gland (published report) 

Large cells with bizarre-shaped clefted nuclei and occasional acinar-like structures (arrow) from blue king crab neoplasm above

Higher magnification of same neoplastic cells with bizarre nuclei

118 119 reference Glossary of Terms

Acid-fast - a physical property of the cell clams, scallops, or mussels, of the class walls in some bacteria and protozoa that Bivalvia having two shells hinged to- resists de-colorization by acids during the gether, a soft body, and lamellate gills. staining procedure. Bothria- shallow sucking grooves on the Amphipod - a small crustacean of the scolex of a tapeworm. order Amphipoda, having a laterally compressed body with no carapace. Carapace - The shell or exoskeleton covering the head, thorax, and legs of a Anaplastic - relating to cells that have crustacean. become less differentiated and more embryonic. Carrier - an individual animal with an asymptomatic infection which is capable Antennal gland - one of the pair of of being transmitted to other susceptible excretory organs (functional kidney) in individuals. each side of the head region of decapod crustaceans, emptying at the base of the Catalase - enzyme found in most plant antennae. and animal cells that functions as an oxidative catalyst; decomposes hydrogen Arthropod - belonging to the phylum peroxide into oxygen and water. Arthropoda, an insect or crustacean that has a cuticle made of chitin forming an Cephalopod - any mollusc of the class exoskeleton with segments and jointed Cephalopoda, having tentacles attached appendages. to the head, including the cuttlefish, squid, and octopus. Ataxia - loss of the ability to coordinate muscular movement. Cercaria - free swimming larva of flukes of the class Trematoda, having a tail-like Bacteria - any of a large group of unicel- appendage; stage usually released from lular prokaryotic organisms that lack a the mollusc first intermediate host. cell nucleus, reproduce by fission or by forming spores, and in some cases cause Cestode - a tapeworm possessing a modi- disease. fied end segment called a scolex that is used for attachment. Tapeworms gener- Basophilic - tissue components having an ally require three hosts for development. affinity for dye under basic pH conditions (as in histology) that generally stain blue. Chlamydia - genus including coccoid to spherical Gram-negative intracellular Bioaccumulation - the accumulation of parasitic bacteria that have three develop- particles such as viruses or bacteria in mental forms. various tissues of bivalve molluscs by the filter feeding mechanism. Chromatin - the readily stainable substance of a cell nucleus consisting Bivalve - any mollusc, such as oysters, of DNA and RNA and various proteins;

120 121 reference

during mitotic division it condenses into Decapod - crustaceans characteristically chromosomes. having five pairs of locomotor appendages each joined to a segment of the thorax. Commensalism - a symbiotic relationship between two organisms of different Denticles -small tooth or tooth-like species in which one derives some benefit projections. while the other is unaffected. Dinoflagellate - any of numerous min- Conchiolin - a fibrous protein substance ute, chiefly marine protozoans of the or- that is the organic basis of mollusc shells. der Dinoflagellata that have a trilaminar alveolate pellicle covering and produce Conidia - an asexually produced fungal spores having two flagella. spore, formed on a conidiophore. Diplobacilli – paired rod shaped bacterial Copepod - small planktonic crustaceans cells. which are an important part of the aquatic food chain. DNA - deoxyribonucleic acid that contains genetic information for the re- Copepodid - juvenile stages that follow production, development and function of the naupliar stages in copepods, often living organisms including some viruses. similar in body form to the adult. Ecdysis - shedding of the old shell or the Coracidium - a ciliated larval stage of a process of molting. tapeworm hatching from the egg which infests the first intermediate mollusc or Ectoparasite - a parasite that lives on the crustacean host. surface or exterior of the host organism.

CPE (Cytopathic Effect) - damage to cul- Elasmobranch - any of numerous fishes tured cells caused by virus infection. of the class Chondrichthyes, characterized by a cartilaginous skeleton and Crustacean - an arthropod having a placoid scales that includes the sharks, segmented body and jointed appendages, rays, and skates. with two pairs of antennae at some stage in their life cycle. Electron microscopy - use of an electron microscope that generates an electron Cuticle - the outer, non-cellular layer of beam focused through a series of objec- the arthropod integument, composed of a tives and lenses to create an image for mixture of chitin and protein. observing ultrastructural details at a much higher magnification than a tradi- Cyst - a capsule of connective tissue tional light microscope. formed by the host around a foreign body, such as a parasite, that acts as an Encyst – to enclose in a cyst. irritant. Endoparasite - a parasite that lives Cytoplasm - the fluid-like substance that within the body of another organism fills the cell, consisting of cytosol and rather than on the surface. organelles excluding the nucleus. Eosinophilic - a red color of cells or

120 121 reference

tissues in histological sections or stained sitic relationship to obtain their nutrients. smears that have been stained with eosin or other acid dyes. Gametocytes - a cell from which gam- etes (spermatocyte or oocyte) develop by Epibiont - an organism that uses the body meiotic division. surface of another as a substrate but takes no nourishment or other benefit. Gamogony - sexual reproduction in protozoa (anisogomy). Epithelium - one or more layers of specialized cells forming the covering Gastropod - any of various molluscs of of most internal and external surfaces of the class Gastropoda characteristically the body and its organs and comprising having a single, usually coiled shell or no glandular elements of certain organs. shell at all, a ventral muscular foot for lo- comotion, and eyes and tentacles located Epizootic – an outbreak of a disease in an on a distinct head. animal population or an unusually large increase in prevalence and/or intensity of Genome - the total genetic content con- a parasite. tained in a haploid set of chromosomes in eukaryotes, in a single chromosome Exoskeleton - a hard outer structure, such in bacteria, or in the DNA or RNA of as the shell of an insect or crustacean, viruses. that provides protection or support for an organism. Germ ball - a group of undifferented cells in digenetic trematode miracidial larvae Exotoxin - a poisonous substance se- and sporocysts that form either sporo- creted by a microorganism and released cysts or rediae and finally cercariae. into the medium in which it grows. Gram-negative rod - a rod shaped bacte- Extracellular - outside the cell. rium that does not retain the crystal violet in the Gram stain process, but retains the Facultative parasite - an organism that counter stain and is pink in color. may either lead an independent existence or live as a parasite. Gram-positive rod - a rod-shaped bacterium that retains the crystal violet from Final host - (definitive host) the host in the Gram stain process and is dark purple which a parasite develops to an adult in color. form and reproduces. Granuloma - a chronic focal inflamma- Fluorescent antibody test (FAT) - a test tory lesion that walls off a foreign body using antibody against a specific patho- or necrotic tissues and may consist of gen that is conjugated with a fluorescein several elements including different types dye. The conjugated antibody sticks to of host inflammatory cells and fibroblas- the target organism causing fluorescence tic connective tissue. when viewed with a fluorescent microscope. Gregarine - a sporozoan parasite utilizing molluscs as intermediate hosts and Fungi - heterotrophic organisms that may crustaceans as final hosts. exist in a symbiotic, saprophytic or para-

122 123 reference

Hemocyte - general term for several Latent - remaining in an inactive or hid- types of cellular components of the blood den phase; dormant. in an invertebrate. Lethargy - a state of sluggishness or Hemolymph - the circulatory fluid of in- inactivity. vertebrates, including all arthropods and most molluscs, that have an open circula- Macronucleus - the larger of two nuclei tory system. Hemolymph is analogous to present in ciliate protozoans which con- blood and lymph in vertebrate animals trols cell metabolism and growth. and consists of water, amino acids, inorganic salts, lipids, sugars and usually Mantle - an outgrowth of the body wall hemocyanin as a respiratory pigment. that lines the inner surface of the valves of the shell in molluscs and brachiopods. Hepatopancreas - a large digestive gland of shrimps, lobsters, and crabs that Melanin - a tyrosine derived polymeric combines the functions of a liver and brown to black pigment produced by pancreas. melanocytes within the tissues of vertebrates and invertebrates. Hermaphrodite - an individual having reproductive organs of both sexes. Merogony - also called schizogony, the asexual reproduction of various proto- Histological (histology) – the micro- zoans involving multiple fission of a scopic anatomy of cells and tissues as trophozoite or schizont into merozoites. viewed in thin stained tissue sections on glass slides. Meront - a stage in the life cycle of various protozoans in which multiple asexual Hyperplasia - an increase in the growth fission (schizogony) occurs, resulting in of cell numbers of a tissue or organ that the production of merozoites. may or may not increase in overall size; usually stimulated by an irritant. Metacercaria - an encysted cercaria of a digenetic trematode that develops into Hypertrophy - a non-cancerous enlarge- a juvenile trematode, usually within the ment of a cell, organ or tissue as a result second intermediate host. of an increase in the size rather than the number of constituent cells. Metaplasia - the transformation of one type of tissue or cell into another. Hyphae - long branching vegetative fila- ments of a fungus. Metastasize - dissemination of neoplastic cells from a primary tumor to other parts Inflammation - a host cellular response of the body producing secondary tumors. to tissue damage or irritation in invertebrates sometimes causing dysfunction of Micronucleus - the smaller of two nuclei the tissues and organs involved. in ciliate protozoans that contains genetic material that controls reproduction. Intermediate host - a host in which there is development of the asexual or imma- Miracidium - the ciliated larval stage of ture stage of a parasite. a digenetic trematode hatching from the egg which infests the first intermediate

122 123 reference

mollusc host. nucleolus and chromatin.

Mollicutes - an unusual class of very Obligate parasite - an organism that small bacteria that live a parasitic ex- cannot lead an independent nonparasitic istence within host cells and are distin- existence. guished by the absence of a cell wall; commonly called mycoplasmas. Oocyst - the encysted zygotic stage in the life cycle of some sporozoans. Mollusc - any invertebrate of the phylum Mollusca, typically having a calcare- Palps - in bivalve molluscs - folds of gill- ous shell of one, two, or more pieces like tissues surrounding the mouth which that wholly or partly enclose the soft, receive food collected by the gills. unsegmented body, including the chitons, snails, bivalves, squids, and octopuses. Parasite - an organism that lives on or in another organism at whose expense it Mycoplasma - any of numerous parasitic obtains some advantage. microorganisms of the class Mollicutes comprising the smallest self-reproducing PAS - periodic acid-Schiff stain which prokaryotes, lacking a true cell wall and is primarily used to identify glycogen able to survive without oxygen. in histological sections that is useful in detecting fungi and some protozoa. Mycosis - fungal infection. Pathogen - an infectious agent that can Nauplius - the free-swimming first larval cause disease. stage of various crustaceans having an unsegmented body with three pairs of ap- PCR – polymerase chain reaction is a test pendages and a single median eye. that amplifies targeted DNA or RNA that has been converted to DNA through a Necropsy - a postmortem examination of reverse transcription process. an animal. Phagocytosis - process in which host Necrosis - the death of cells or tissues hemocytes engulf and digest microorgan- from traumatic injury or disease, gener- isms and cellular debris; an important de- ally in a localized area of the body. fense against infection for invertebrates.

Neoplasms/neoplasia - cancer caused by Plaque Forming Unit (PFU) - number of uncontrolled abnormal growth of tissue infectious virus particles per unit volume cells. based on the number of holes or plaques in the monolayer of the infected cell Nucleolus - one or more small, typically culture. round granular body composed of protein and RNA in the nucleus of a cell, usually Plasmodia - multinucleated reproductive associated with a specific chromosomal cells in various protozoa that develop site and involved in ribosomal RNA syn- into sporonts and sporocysts containing thesis and the formation of ribosomes. or producing spores.

Nucleoplasm - the jelly-like material Plerocercoid - the third larval stage of within a cell nucleus containing the cestodes that has an obvious scolex.

124 125 reference

Generally found in the second intermedi- haplosporidians. ate host. Pycnidia - an asexual structure contain- Poikilothermic - animals or agents that ing conidia, found in certain fungi. occur in animals with internal body temperatures that cannot be self regu- Rickettsia - any member of the family lated, often determined by the ambient Rickettsiaceae, comprising rod-shaped temperature of the environment; cold to coccoid microorganisms that resemble blooded. bacteria and reproduce only inside a living cell. Polar tube - a hollow tube in microsporidians that anchors the spore to the intes- RNA - ribonucleic acid; the nucleic acid tinal wall of the prospective host through that is used in key metabolic processes which the organism is injected. for all steps of protein synthesis in all living cells and carries the genetic informa- Polychaete - any of various often tion for many viruses. brightly colored annelid worms of the class Polychaeta. Each segment of a Saprophyte - an organism, such as a fun- polychaete has a pair of fleshy append- gus, bacterium or protozoan that grows ages that are tipped with bristles (setae), on and derives its nourishment from dead used for swimming or burrowing. or decaying organic matter.

Proboscis - any of various elongate Scolex - the head segment of a cestode feeding, defensive, attachment or sensory that attaches to its host. organs of the oral region, found in certain leeches and worms. SEM - scanning electron microscopy.

Procercoid - the first stage in the aquatic Septate - describing fungal hyphae that life cycle of certain tapeworms following are divided by crosswalls or septa. ingestion of the newly hatched coricidi- um larva by a mollusc or copepod. Septicemia - presence of bacteria or other organisms in the blood. Prognosis - a prediction of how a disease will progress, and the chance for recov- Serotype - a unique antigenic property of ery. a bacterial cell, virus or other organism identified by serological methods. Prokaryote - an organism of the kingdom Monera (or Prokaryotae), comprising Setae - stiff hairs, bristles, or bristle-like the bacteria and cyanobacteria, char- processes that are part of the body of an acterized by the absence of a distinct, organism. membrane-bound nucleus or membrane- bound organelles, and by DNA that is not Spat - a juvenile oyster or similar bivalve organized into chromosomes. mollusc that has settled out of the water column and has developed a shell. Protozoa - any of a large group of single- celled, usually microscopic, eukaryotic Sporangia - a single or many celled organisms, such as amoebas, ciliates, structure from which spores or zoospores flagellates, sporozoans, microspora and are produced in a fungus.

124 125 reference

Spore - a reproductive structure that is TEM - transmission electron micros- adapted for dispersion and survival for copy. extended periods of time in unfavorable environmental conditions. Trematode - a flatworm having a complex (digenetic) life cycle involv- Sporoblast - in some sporozoa, an ing two or three hosts. Also referred to early stage in the development of a as a fluke. sporocyst, prior to differentiation of the sporozoites. In microspora a sporoblast Trophozoite - active, feeding stage of a matures into a spore. protozoan.

Sporocyst - in sporozoa, a walled body Turbellarian - belonging to the Tur- resulting from multiple division which bellaria, a class of platyhelminths or produces one or more sporozoites; in flatworms, mostly aquatic and having microspora, haplosporidia and Hemato- cilia on the body surface. dinium sp. a walled body that contains or produces spores; in trematoda, an Vector - any agent (person, animal or elongated stage in the first intermedi- microorganism) that carries and trans- ate host that produces more sporocysts mits a disease. or rediae depending on the species of fluke. The end product of both is Virulence - the pathogenicity or abil- cercariae. ity of an infectious agent to produce disease. Sporogony - asexual reproduction by multiple fission of a spore or zygote. Viscera - internal organs of an animal. Sporogony in sporozoans results in the production of sporozoites, or sporonts Virus - a very small infectious agent in microspora, haplosporidia and He- composed of a nucleic acid core (RNA matodinium sp. or DNA) surrounded by a protein coat that replicates only within living host Sporont - in the sexual reproduction of cells. certain sporozoans, an encysted spore developed from a zygote, which under- Zooflagellate - any flagellated proto- goes sporogony to form sporozoites. zoan that lacks photosynthetic pigment In certain microspora, sporogony pro- and feeds on organic matter; often duces sporonts that produce sporoblasts parasitic. that mature into spores. In haplosporidia and Hematodinium sp. sporonts Zoonotic - a disease of an animal that become sporocysts that produce spores. can be transmitted to humans.

Sporozoite - minute undeveloped infectious stages of sporozoans produced by multiple fission of a zygote or spore.

Syncytia - the fusing of separate cells to form a larger multinucleated cell.

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129 Notes

130