Research Interests—Louis E. BurnettI am interested in how environmental variables such as dissolved oxygen, carbon dioxide, 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.
Hypercapnia. Hypercapnia, or elevated environmental CO2, is prevalent in coastal marine and estuarine environments (Burnett, 1997; Burnett & Stickle, 2001). Estuarine organisms appear to have CO2-specific mechanisms that mitigate the acidic effects of hypercapnia (Mangum & Burnett, 1986) and compensate for the acidosis associated with hypercapnia (review – Burnett, 1997). Work on oysters has shown that they experience extremely large fluctuations of CO2, due largely to their intertidal habitat and exposure to temperature extremes (Dwyer & Burnett, 1996; Willson & Burnett, 2000; Allen & Burnett, 2008). The presence of the oyster disease called dermo (Perkinsus marinus) produces an acidosis within the tissues of oysters (Dwyer & Burnett, 1996). We have subsequently shown that the elevated CO2 that occurs during air exposure at high temperatures offers some protection against this disease (Milardo & Burnett, in prep), however, the benefits of hypercapnia in this case may be offset by the overall decrease in immune function brought about by elevated CO2 (discussed below).
Immune Responses. 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) in response 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. In oysters, ROS production is greatly reduced in hypoxia and mild hypercapnia (Boyd & Burnett, 1999). Similar findings were documented in the killifish Fundulus heteroclitus using a completely different kind of assay in which macrophages were allowed to “kill” Vibrio, a bacterial pathogen of fish (Boleza et al., 2001). Interestingly, it was not the low oxygen alone, but low oxygen in combination with mild hypercapia that inhibited immune function. We also demonstrated at the whole animal level, measuring survival in shrimp, that the immune system is sensitive to hypercapnic hypoxia (Mikulski et al., 2000). As with the fish, the immune response of the shrimp was highly sensitive to CO2 in combination with hypoxia.
Organisms integrate their responses to bacterial challenges. We have begun to understand how the parts work to make up the whole response. Shrimp use a lymphoid organ to render bacteria harmless and they also use gills and hepatopancreas as immune organs (Burgents et al., 2005 a, b). Gills play a particularly important role when the water is hypoxic. In these studies, we were able to use real time PCR to quantify the number of intact bacteria in specific tissues and track the ability of the individual tissues to render the intact bacteria harmless.
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 is more easily sampled from a single individual. Crabs can eliminate bacteria from their blood within minutes if the water is well-aerated, but the rate of elimination is significantly reduced when the crab is exposed to hypercapnic hypoxia (Holman et al., 2004). This study was also important because it showed us that there was a significant decline in aerobic metabolism during the time that the bacteria were being eliminated.
Hemocytes (macrophages in fishes) are the cells circulating in blood that are known to bind to bacteria and ultimately assist in eliminating them. In this process, hemocytes bind to bacteria and other hemocytes forming aggregates. These aggregates can get quite large and previous researchers had theorized that these aggregates can clog the thin blood channels of the gills and reduce gill function in important things like respiration and ion regulation. We tested this hypothesis directly, documenting that significant aggregations of hemocytes occur in the gills when we inject crabs with bacteria (Ikerd, Burnett & Burnett, in prep). These aggregates dramatically disrupt respiratory function by blocking the channels of the gills, causing a rise in blood pressure and decreasing oxygen uptake (Burnett et al., 2006). Large (30 to 40%) decreases in oxygen uptake occur in crabs (Burnett et al., 2006) and shrimp (Scholnick et al., 2006).
Metabolic Depression. This decline in aerobic metabolism is profound and represents a true reduction in overall metabolism that is rapid and that persists for 24 hours (Burnett et al., 2006; Scholnick et al., 2006; Thibodeaux et al., 2009). Such a metabolic depression has not been documented under these conditions as far as we know. We were interested in determining if this metabolic depression had a negative influence on the capability of the blue crabs to sustain a high level of activity, a behavior that is ecologically relevant to this organism. We have developed treadmill systems that allow us to elevate the activity of both crabs and shrimp and this allows us to understand the consequences of metabolic depression on their behavior. One of the first major results of this effort reveals that blue crabs injected with a sublethal dose of the pathogenic Vibrio sustain 30 min of treadmill activity (at 8 m/minute) with no problem (Thibodeaux et al., 2009). It now remains to determine how moderate to severe hypoxia and hypercapnia influence this performance.
A large part of the energy consumed by animals goes toward protein synthesis. Our reading of the literature leads us to hypothesize that the metabolic depression we have observed can be explained by a reduction in protein synthesis. Such metabolic depression is commonly observed in hypoxic conditions as animals reduce their aerobic metabolism. With this in mind, we embarked on a large genomic study of our shrimp (Litopenaeus vannemei) under hypoxic and hypercapnic hypoxic conditions. The results of this study are in and are being prepared for publication (Rathburn, Burnett & Burnett). They indicate that a suite of genes that are important in protein synthesis are downregulated and this is consistent with a downregulation of protein synthesis. We are now interested in seeing if we can measure protein synthesis directly to further test this hypothesis. We are also interested in seeing if our observations of a significant metabolic depression resulting from bacterial challenge can also be explained by a decline in protein synthesis.
This thread of research experiences shows that organisms face numerous challenges in their aquatic environments and that their response to any one variable is linked and integrated into their responses to other variables including oxygen, carbon dioxide, temperature, and disease challenges.