Current Research Projects (click on the links for more details)

Full publication list.

I am interested in how environmental variables such as dissolved oxygen, salinity and temperature affect marine organisms.  Recently my research has focused on the effects of low dissolved oxygen (hypoxia) and elevated carbon dioxide (hypercapnia) on disease resistance in a variety of organisms including fishes, oysters, and crustaceans.  I am not confined to any particular taxon in my research and enjoy a comparative approach to investigating problems.

We have made excellent progress in understanding the influences of environmental hypoxia (low O₂) and hypercapnia (elevated CO₂) on the defensive responses of a variety of marine organisms to bacterial challenges.  We have used several approaches to document the responses of the innate immune systems of fishes, crabs, shrimp, and oysters to these environmental challenges.  At the biochemical and the cellular levels we have documented a large and significant reduction in the production of reactive oxygen species (ROS) to bacterial challenge.  The production of highly reactive oxygen species is one of the first lines of defense against an invading pathogen in most organisms.  Graduate student John Boyd has shown that ROS production in oyster hemocytes is reduced by two thirds in hypoxic conditions.  Graduate student Kim Boleza has found a similar phenomenon in the killifish Fundulus heteroclitus.  Sensitivity to oxygen is confirmed in the killifish using a completely different assay in which fish macrophages are allowed to “kill” a pathogen such as a Vibrio bacterium.   Graduate student Tina Mikulski demonstrated that the oxygen sensitivity of these mechanisms also manifests itself in a reduced fitness of the whole organism to bacterial challenge. 

We have begun looking more closely at the mechanisms involved in these responses.  In the Pacific white shrimp, Litopenaeus vannamei, graduate student Joe Burgents has shown that Vibrio campbellii injected into the abdominal muscle appear in a variety of different tissues within a few minutes.  The shrimp lymphoid organ, which is very small, appears to be very important in rendering the bacteria harmless.  The gills and the hepatopancreas also play an important role in removing or “clearing” bacteria from the shrimp especially when the water is hypoxic.  In these studies, Joe Burgents was able to use real time PCR to quantify the number of intact bacteria in specific tissues.  The number of culturable bacteria in the same tissues was also determined using the more traditional counting of colonies in a Petri dish. 

We have used the Atlantic blue crab, Callinectes sapidus, as another model to study the mechanisms of innate immunity.  Blue crabs are larger than shrimp and hemolymph his more easily sampled from a single individual.  Undergraduate student Jeremy Holman has shown that blue crabs eliminate Vibrio injected into the circulatory system within minutes.  Forty minutes after injection the concentration of Vibrio circulating in the hemolymph is near zero.  Furthermore, if a crab is held in hypoxic water prior to injection and during and after the injection, the rate of clearance of the Vibrio from the hemolymph is dramatically and significantly reduced.

We were also interested in testing the hypothesis that the mechanism of bacterial removal causes hemocytes to aggregate, lodge in the gills, and reduce the effectiveness of the oxygen delivery system.  This appears to be the case in both the blue crab and the Pacific white shrimp.  Oxygen uptake within 30 minutes to over an hour is significantly reduced in both C. sapidus¹ and L. vannamei².  In the blue crab, the arterial oxygen pressures are significantly reduced indicating respiratory obstruction.  In L. vannamei, oxygen uptake is significantly lowered and it stays this way for at least 24 hours.  The hydrostatic pressures driving the hemolymph through the gills increases¹, a result consistent with the hypothesis suggestion that the gills are obstructed.  We have preliminary evidence from microscopy suggesting that hemocyte aggregates do indeed block the gills.

Effects of hypoxia and air exposure on disease resistance in oysters.

Oysters encounter hypoxic water on a routine basis.  Furthermore, their intertidal habitat exposes them to air on a daily basis.  Air exposure causes Eastern oysters to close their shells tightly, nearly completely isolating themselves from the air.  Inside the shell, the tissues of the oyster become hypoxic and severely acidic, due largely to the buildup of CO₂.  Thus, air exposure resembles hypoxia in many ways.  Graduate student John Dwyer investigated the responses of oysters to air exposure and to the oyster pathogen Perkinsus marinus and found that both caused a significant acidosis in oysters tissues.  Graduate student Libby Willson extended these observations and found that oyster metabolism was sensitive to both hypoxia, elevated water CO₂, but not to infections of Perkinsus marinus.  Using the environmental conditions known to occur during hypoxia and air exposure, graduate student John Boyd found that the production of reactive oxygen intermediates by oyster hemocytes, thought to be one of the main defense mechanisms against pathogens, was severely inhibited by low oxygen and low pH.

This information was used by graduate student Steve Allen to investigate the ability of oyster hemocytes to kill bacteria under conditions that occur during air exposure and hypoxia.  Steve studied this process in the Pacific oyster Crassostrea gigas as a part of a larger project funded by the Oyster Disease Research Program.  Both low oxygen and air exposure are hypothesized to be associated with summer mortalities of C. gigas in the Pacific northwest.  Using an assay in which Vibrio were exposed to oyster hemocytes in vitro, Steve was unable to show an effect of low oxygen or low pH on the ability of hemocytes to kill Vibrio.

Effects of air exposure on Dermo infections in oysters.

Chris Milardo has just completed a study on the influences of environmental variables on the growth and metabolism of the oyster parasite Perkinsus marinus in culture.  Oysters exposed to the air and the hot summer sun close themselves completely from the ambient environment.  Within the shells oysters become hypoxic, carbon dioxide builds up and a large acidosis occurs, especially at the elevated temperatures experience by intertidal oysters in the summer months.  We have mimicked these conditions in cultures of Perkinsus and have found that elevated CO₂ at 35^oC greatly increases the aerobic metabolism of Perkinsus.  Under these conditions the growth rate of Perkinsus is dramatically reduced.  We suggest that these environmental variables, while they do not prevent the infection of oysters by Perkinsus, act to inhibit heavy infections.  These results may explain why heavy mortalities of oysters are not always observed in oysters in the southeastern United States.

We have also developed a bacterial “clearance” model using oysters.  Oysters are injected with Vibrio campbellii and at some specific time point, the oyster is shucked and all of the internal tissues are collected in a buffered saline and the number of intact bacteria are counted using real-time PCR and the number of viable bacteria are assess by counting the number of colony forming units.  The difference between these two numbers gives a measure of bacterial killing.

Other Related Projects

ENHANCED DISEASE RESISTANCE IN PACIFIC WHITE SHRIMP, LITOPENAEUS VANNAMEI, USING A YEAST CULTURE FEED SUPPLEMENT

Burgents, J. E., K. G. Burnett, L. E. Burnett. 2003. Disease resistance of Pacific white shrimp, Litopenaeus vannamei, following the dietary administration of a yeast culture food supplement. Aquaculture 231:1-8.

Please see Students and Recent Publications for examples of my other research interests.

Updated:  20 Aug 2007