Nor'Easter Spring/Summer 1996
"Our quahogs are dying and I think we need some help," Stephan Nofield said over the phone. "Could you pull together a group of people to meet with my clam farmers and see if you can determine what is going wrong here?"
It was August 1995 and, though they had no way of knowing it at the time, Nofield, an economic advisor for Provincetown, Mass., and several of the town's clam farmers had just started the wheels turning to lead eventually to the discovery of a parasite that causes severe mortality in farmed quahogs, or hard clams, Mercenaria mercenaria.
As a scientist, I have worked with bivalves for the past 15 years, including studies of the prevalence of hematopoietic neoplasia, a leukemia-like illness, in the soft-shell clam, Mya arenaria (see Nor'easter Vol. 5, No. 1). I first heard about the Provincetown quahog farmers' problems in 1994, when I started as the Woods Hole Oceanographic Institution (WHOI) Sea Grant program's fisheries and aquaculture specialist in the Marine Advisory Service.
Intrigued by the Provincetown quahog mortality mystery, I met with over 20 clam farmers, along with Roxanna Smolowitz, a veterinarian from the University of Pennsylvania's Laboratory for Aquatic Animal Medicine and Pathology (LAAMP), located at the Marine Biological Laboratory (MBL) in Woods Hole. The farmers told of mortality levels that increased each year. Common characteristics and behaviors of the dying clams included a post-harvest shelf life of less than 24 hours, slightly gaping valves, a tendency to rise to the sediment surface just prior to death, and the appearance of very small "nibble" marks along the outer edge of the empty shells of the dead clams. Generally, only clams nearing the legal size limit for harvest (1-inch width at the hinge) were dying; younger clams planted on the same farms were growing and surviving as expected for healthy farmed quahogs.
Molly Benjamin, a Provincetown grower, describes the cycle: "The clam beds are usually thick with clams up through year two. You think, 'This is it, this is the year I make some real money.' Then when year three rolls in, they start to die - especially as they reach the legal size."
As in any good mystery novel, there was one clue that sent several investigators down a dead-end street: the nibbled valve (shell) margin. That single symptom led many scientists, experienced growers, and shellfish regulators to attribute the mortality problem to crab predation, common in clam farming. This seemed like an open-and-shut case.
Provincetown clam growers had been complaining about mortality losses since starting their farming operations in 1990. In 1991, quahog mortality from the Provincetown flats was approximately 20 percent, climbing to an alarming 80 percent in 1995. As the numbers worked against the farmers, it became apparent that predation alone could not account for the drastic numbers of empty shells.
In August 1995, farmers descended upon Provincetown's town hall with bucketsful of dead and dying quahogs. We had a chance to perform visual inspections on several of these clams and found signs of arrested growth, nibbled valve margins, and gaping valves. Furthermore, the mantle of the clam - the thin layer of tissue along the edge of the shell - was retracted from the shell margin and appeared thicker than normal.
Our curiosity piqued, we decided to conduct diagnostic tests on the clams as part of a standard investigation for possible causes of mortality. Smolowitz selected 10 clams that looked sick, based on the descriptions above, and five clams with no obvious symptoms. She returned them to the histopathology laboratory at the LAAMP, where they were processed for routine microscopic histological observation of the soft tissues. What Smolowitz discovered was anything but routine for quahogs.
Quahogs Typically Hardy Species
In general, the quahog is considered a hardy bivalve species with very few illnesses. The few reported cases of sickness in quahogs were mostly confined to clam hatcheries where bacterial infestations infected larval clams. Because quahogs don't typically get sick, the possibility of disease as the cause of high mortality in Provincetown's clams had not been pursued. Yet when Smolowitz looked through the microscope, she observed a severe bacterial infection in several of the animals. What she didn't expect to find, however, was the single-cell organism growing within the soft tissue of the quahog, predominantly in the gill and mantle. At that time, Smolowitz could not determine whether the single-cell organism was a primary invader responsible for the mortality, or a secondary invader affecting an already weakened clam.
To find out, Smolowitz and I went to Provincetown in October to obtain quahog samples from two stricken clam farms. Emergency funding support was provided by WHOI Sea Grant and WHOI's Coastal Research Center. We employed a combination of field measurement techniques to determine growth and mortality and conducted a 'directed collection' to select some clams that appeared sick and some that appeared healthy. We returned to the laboratory with 80 clams for analysis. This time the single-cell parasite was observed in 90 percent of the clams grossly identified as sick and in only 10 percent of the clams identified as healthy. The deciding factor in this second look at the sick clams was that, in all of the infected clams, the sole problem observed by Smolowitz was the presence of the single-cell parasite. There was no confounding secondary bacterial infection.
Culprit Revealed
As this story was unfolding in Provincetown, a similar situation was playing out just across Massachusetts Bay in Duxbury. A quahog farmer in that town experienced a sudden onslaught of clam mortality in an isolated bed of clams on his farm. Clams collected from this site ended up not only in Smolowitz's pathology lab, but also in the laboratory of Frank Perkins, a well-known clam pathologist from North Carolina State University. Perkins - like Smolowitz, but independently - identified a single-cell parasite in the tissues of the clams from Duxbury. Although the gross signs of sickness were not similar - that is, the onset of mortality in the Duxbury case was sudden, all of the clams had exhibited good seasonal growth, there was no chipping of the shells or gaping of the valves, and the afflicted clams came from an isolated spot within a larger bed of clams - both pathologists confirmed that they were looking at what appeared to be the same animal. So what was this mystery parasite?
QPX, short for quahog parasite unknown, earned its moniker in 1989, when a quahog nursery in Prince Edward Island, Canada, reported high mortality due to a protozoan. This was actually the second occurrence of this parasite: It had been reported in a dense population of wild quahogs in the St. Lawrence River during the late 1950s and early 1960s. QPX has been tentatively identified as either Thraustochytriales or Labyrinthulales, both of which are non-chlorophyll-containing, single-celled protists that have been placed in a separate phylum (Labyrinthomorpha) in the subkingdom protozoa. The QPX protozoa have also been described as fungus-like. Members of this phylum appear to be abundant in the marine environment, although disease-causing organisms from this phylum appear to be rare.
Word within the scientific community that this organism had been observed in quahogs from Cape Cod jogged the memories of other research pathologists who worked with clams. For example, in 1976, an episodic mortality associated with a QPX-like protist was noted in wild clams from Barnegat Bay, N.J. And, in 1992, Smolowitz observed QPX-like parasites in a small sample of moribund clams from Mitchell River, Chatham, Mass. Also, the QPX-like parasites observed in both Provincetown and Duxbury were similar to those identified in QPX disease of Canadian quahogs. Their life cycle within the quahog was readily observable in the clams sampled.
QPX-like parasites and accompanying intense inflammation were found primarily in the sinusoidal spaces and surrounding connective tissues of the infected organs or tissues in the quahog. The most commonly infected organs or tissues in the clams were the mantle and the gill - a good indicator that the parasite is present in the water column, because these are the two organs constantly exposed to the water column.
Method of Infection: Many Unsolved Questions Remain
The method of infection of the clams exhibiting QPX-like parasites is unknown. One possibility is direct infection. Direct infection of adjacent animals - via a flagellated zoospore - has been shown to occur in a similar disease in abalone, a Gastropoda snail that superficially resembles a clam. It is likely that direct infection of adjacent clams occurs in QPX and QPX-like disease also.
Obviously, direct infection would have major implications for hard clam aquaculture operations, nurseries, and wild populations. It is our feeling that the clams in Provincetown and Duxbury are going into the field as clean, uninfected clams. With any infection or disease, it takes a certain amount of time and a certain dose - called "loading dose" - for a parasite to take hold. Once the parasite takes hold, the spores can form flagellated zoospores that can leave a dying animal, swim through the water column, and infect another animal. "I think that if the clams (seed) came to Provincetown or Duxbury with the disease, they'd be dying sooner," said Smolowitz. "Instead, we're seeing the highest mortality just prior to harvest size."
The need for routine screening of seed (and suspect adult populations) before transplantation in a new area is obvious. However, if our hunch is correct - that the animals are getting the disease once planted - the risk to aquaculture operations using imported seed would not be increased.
While the life history of the organism is unknown, it is possible that QPX or the QPX-like parasite may be a 'facultative parasite.' This means that the organism may be able to lead an independent existence, say, as a free-living spore in the sediment. It is not possible to rule out intermediate hosts, in which the parasite goes through a two-host life cycle (in other words, the parasite cannot survive with only one host). This is the case with MSX (multinucleated sphere with unknown affinity, caused by the protozoan parasite Haplosporidium nelsoni) disease in oysters. Or the clam might be an aberrant host, meaning that the parasite has not evolved sufficiently to exist in harmony with the host. (For example, the parasite causing equine encephalitis can exist in mosquitos and some birds with little or no harmful effects, whereas it can be fatal to horses or humans.)
Much of the mystery about QPX and QPX-like disease remains. The single-cell organism provided the major clue in solving this puzzle, but we still have to piece together critical evidence to arrest this quahog disease event. We have to better identify the disease organism - conducting electron microscope examinations and culture studies to better define its position within the Labyrinthomorpha phylum - and we have to answer critical questions, such as how the parasite infects clams, how the infection spreads, and how long it takes the parasite to kill its host. We need to learn whether clams younger than the 1.5- to 2-year olds examined in our study are infected. And we need to determine if the environment plays a role.
We are working as fast as grant money comes in to understand more about QPX and QPX-like disease so we can close the file on this mystery killer and end the biological and economic devastation on the outer Cape clam flats. One of the Provincetown growers called recently. "I've lost 95 percent of my clams," she said. A stressful environment for the just-undersized quahogs - created by a combination of QPX and bad weather - has taken its toll on more than the clams. "I'm not planting seed this year," she said flatly. "I just can't."
Dale F. Leavitt is Fisheries and Aquaculture Specialist for WHOI Sea Grant Marine Advisory Service
Tracey I. Crago is Communicator for WHOI Sea Grant.