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The Invertebrate Immune System Part 2 By Eric Borneman and Jonathan Lowrie Aquarium Net April 98

The Invertebrate Immune System By Eric Borneman and Jonathan Lowrie Part2 Aquarium Net has numerous articles written by the leading authors for the advanced aquarist

The Immune Response of Corals

Part Two: Models for "RTN"

by Eric Borneman and Jonathan Lowrie

In this article, we will discuss the disease which has become known within the reef aquarium hobby as rapid tissue necrosis (RTN). It should be noted that there is no disease in the wild designated as "RTN." There are a number of diseases which may share certain attributes or unknown factors as the aquarium malady. However, it is not yet known whether RTN is a single disease, multiple diseases sharing a similar pathology of tissue necrosis, a "new" disease, or if RTN is has a single or multi-component cause. There are simply too many variables and too many unknowns to assign modalities, causes, or treatments to RTN at this point.

In the last article, we described the basic coral immune system. This was done to not only describe a little known aspect of coral physiology to the hobby, but to lay groundwork for what we feel to be the implicit importance of the stress response of corals in the mechanism of RTN. Since encountering tissue slough in certain corals in captivity many years ago, it seemed that this was related to a "failure to thrive." Since first observing RTN, several discoveries and models have been proposed based on anecdote and experiment to explain the malady. It has been our experience that the models have been less than adequate in that they do not explain many aspects of RTN which are repeatedly demonstrated and experienced in the expression of RTN. Nonetheless, the aspects of these anecdotes and experiments could be quite accurate, and do not necessarily work against our hypothesis. In fact, they fit in well. We will hopefuly be able to extend this hypothesis for other coral diseases which have remained largely unexplained by studies to date.

Currently Proposed Models

1. Sam Gamble, former Aquarium Biologist for the John Pennekamp Marine State Park, proposed an interesting model for RTN. There have been numerous observations of how significant stirring of established "live sand" beds and intrusions into plenum areas can induce RTN in an established aquarium previously free of the disease. One hypothesis offered was that the significant numbers of Vibrio spp. that inhabit such communities were released pelagically to trigger an outbreak (Sprung 1997). Mr. Gamble proposed that the release of nutrients to the water column created complex shifts in autotrophs and heterotrophs that, in combination with prevailing static conditions, created imbalances that resulted in the sloughing of tissue (Gamble, pers. com.). While certainly an interesting hypothesis, and perhaps a factor in some instances, there are too many observations of RTN that remain unaccounted for by this model, including outbreaks of RTN in systems that do not employ sand.

2. Dr. Craig Bingman has collaborated to propose a model of RTN with Vibrio vulnificus as a causative bacterial pathogen (Bingman 1997). This model was based on findings of unusually large numbers of the virulent bacteria on RTN affected colonies that were not present on healthy colonies. Furthermore, the use of the antibiotic, chlormaphenicol, has an ability to significantly stop the progress of RTN in many affected corals. However, there are flaws with this model, despite the significance of the finding. The most notable being that RTN has been noted in corals many times now without the presence of V. vulnificus (Lowrie 1992, 1997, Borneman and Lowrie, 1998, Greco, 1998, etc.). Furthermore, rapidly necrotic tissue is a sign of disease in many corals with either no established pathogen or a different pathogen ( Peters, 1997, et. al.).

3. Diseases characterized by tissue sloughing are not new. They have been documented for at least twenty years. Antonius, Glynn, and others have failed as yet to isolate any causative pathogen in the tissue of many corals exhibiting necrosis. Some of the diseases which have been (recently) shown to have a pathogen involved also happen to display divergent appearances and signs from those which have not yet had any pathogen discovered. Some of the diseases with no determinate “cause” tend to show patterns of disease very similar to what is occurring in corals with RTN. The descriptions of Shut Down Reaction, by Antonius, describes a very clear parallel to RTN in the mode of coral death. In his limited descriptions, a rapid loss of tissue occurs in response to continued or extreme stress. The rate of tissue loss is very similar to that of RTN, and it is contagious to adjacent colonies. The time required for adjacent colonies to exhibit SDR is also very similar to what is seen with RTN. Finally, no causative pathogen was associated with SDR (Antonius 1985, et. al.). Peters (pers. com. 1998) states that no histology work was done in Antonius' work, despite Anonius' inferrence that isolation attempts were made. Until such time as we can discover more about what Antonius found in his studies, further comparisons can not be accurately made, though the parallels with RTN are interesting.

What We Know and What We Don't Know About RTN

There are two defintions which should be considered before continuing :

"Disease: Any deviation from or interruption of the normal structure or function of any part, organ, or system (or combination thereof) of an organism that is manifested by a characteristic set of symptoms and signs and whose etiology, pathology, and prognosis may be known or unknown," (Friel 1985)

"Contagion: Literally, transmission of infection by direct contact; liberalized to include short-range airborne (waterborne) spread and transmission by freshly contaminated fomites (objects closely associated with the organism)."(Fox, 1970)

RTN fits the definition of both terms. It is a disease, whether it is a single or multiple disease(s), and whether or not it is described and understood. The problem with our interpretation of these definitions stems from a societally ingrained need to view things as traditional Western Medicine views them. We think in terms of disease = pathogen. If we find and kill the pathogen, we stop the disease. While this is true in some instances, it is decidedly untrue in others. For many diseases there may be a primary pathogen, a secondary pathogen, or no pathogen at all. An allergy is an excellent example of a disease where the cause is a normally non-pathogenic agent that results in disease. The "pathogenicity" involves the immune system reacting in such an abnormal fashion that normal system functions are impaired. RTN is also contagious to other corals, with some significant degree of inter-specific variability. However, the mode by which RTN is contagious does not necessarily require the presence of a pathogenic organism. Tissue or organ transplant rejection, and incompatibilities in blood type or sickle cell anemia during blood transfusions, are but two examples of disease that are contagious but do not require a pathogen as their agent. It is therefore possible that RTN is a contagious disease with or without an associated pathogen.

Rapid tissue necrosis, as it applies strictly to the loss of certain corals (notably Acropora sp.) in captivity is not so new as the aquarium literature would suggest. We first observed this disease in captivity over five years ago in wild collected Acropora colonies that showed a progression very similar to Antonius's described “white- band-turned-SDR." The corals, which had a (several month long) slow progressive white banded tissue recession from the base, suddenly turned necrotic. Their tissue peeled off overnight and affected other healthy Acropora within hours. The widespread hobby "scare" of RTN became more apparent as more colonies of RTN susceptible corals became available, and as more hobbyists began to try and keep these difficult animals in captivity. This may be considered to be an epidimiological false alarm, sometimes called a Type 1 error (Fox 1970). The increased number of such corals being imported, and the profit margins involved, may also have led to a decrease in the relative care with which specimens were being collected, transported and held. Such a decline in transient care would increase animal stress levels, decrease fitness, and increase the likelihood of disease.

Bacteria in the mucus rich coral surface microlayer are extremely active and are at levels comparable to eutrophic estuaries. In fact, bacterial populations on coral mucus are nearly 10,000 times more abundant than in the surrounding sea water (Paul 1986). Populations are so dense that they can affect coral respirometry experiments and must be separated in studies to determine coral from bacterial nutrient uptake from the water (Trench 1974)! They also grow at the expense of the coral mucus, which is critical to tissue integrity. A study of bacterial populations as an important coral food source by Linley and Koop (1986) discussed the consumption of coral mucus by bacteria. The composition of coral mucus can vary inter-specifically, and may vary in response to exposure to various stressors. The variance in mucosal composition may result in different metabolic substrates required for certain bacteria (Peters, pers. com.). Nonetheless, coral mucus, at least in terms of its composition and ability to be used as a medium for bacterial growth is consistent enough to question why same species corals would suddenly begin to harbor a new and pathogenic bacteria that was not previously expressed. New specimens harboring such bacteria could conceivably introduce pathogenicity to species with similar mucus compositions. Despite the increased reports of RTN outbreaks after introducing new specimens, there is no evidence to support the relative pathogenicity of a bacteria to one species, and not another, in causing RTN. Nor is there currently evidence supporting inter-specific mucus compositions as being either hospitable or inhospitable to these pathogens. Even more curious are the reports of RTN following sudden stress events of long term systems where a pathogenic bacteria, if one existed and were causative, must have been present at non-pathogenic levels. Supposing that RTN affected corals are exposed to a given pathogen, why would the exposure of healthy established colonies to a new specimen (hypothetically athogen) result in disease, while low-level exposure had not until a stressor was introduced. What is it about a new specimen addition that results in perceived stress that allows for pathogenicity to exist, if it must exist at all?

The acidic mucosal pH of cnidarians would certainly seem to be a stress for coral tissue to be in contact. Yet it is the low pH of mucus which has a bacteriostatic effect in the prevention of pathogens from reaching the coral tissue (Bigger, Hildemann 1982). Bacterial accumulations in coral mucus could attack the protein and polysaccharides of mucus and create by-products that are acidic, but bacteria on mucus flocs have been shown mainly to enhance the free sugar and nitrogen levels in their immediate environment (Ducklow, Mitchell 1979). Neither would explain such an acidic pH. Lowrie found a pH of coral mucus in the range of 6.0-6.5 to be most efffective in antimicrobial effects (Lowrie 1992). Corals must, therfore, spend energy to create a specialized acidic protective environment. Increased mucus production results in considerable energy loss to corals, notably ones such as Acropora acuminata (Crossland, et. al. 1980) and an increase in the bacterial population of the mucus, requiring enhanced immunological function (Ducklow,Mitchell 1979). Under stress, the mucus of corals becomes less acidic, and therefore less effectual in preventing bacterial accumulations (Lowrie 1997).

There is little doubt that marine Vibrios can be extremely pathogenic. But, Vibrio spp. are among the most common bacteria in marine environments. Many of them are normally found growing on and in coral mucus, and they are even cultivated in the interstitial branch space (Schiller 1989). It is interesting that the areas of highest bacterial concentrations are in the interstitial space, perhaps not coincidentally where RTN is most often found to appear. Some bacteria growing in coral mucus can produce exotoxins which can degrade both the mucus and coral tissue, and bacteria can also promote anoxia in the mucus medium (Peters pers. com.) Such populations, caused by overgrowth in response to various conditions, could cause normally non- pathogenic bacteria to become pathogenic, or to simply overwhelm corals through sheer numbers and anoxia. Segel and Ducklow (1982) formulated a mathematical model to predict the potentially explosive increase in coral mucus bacterial numbers when mucus secretion increases in response to sub lethal stresses. Such increases could bring about coral death by oxygen depletion, hydrogen sulfide production of bacterial predation. However, unstirred conditions exacerbate the situation twenty fold (Mitchell, Chet 1975). To be sure, the predatory bacteria, Desulfovibrio and Beggiatoa have been found to destroy the tissue of Platygyra exposed to chemical pollutants (Mitchell, Chett 1975), and theV. vulnificus found by Bingman and Dixon (1997) is hemolytic and capable of causing tissue necrosis. However, other potential pathogenic Vibrio species, such as V. alginolyticus and V. parahaemolyticus, are swarmers that are capable of rapid colonization and form often dominant bacterial aggregates in coral mucus, even in the most inhospitable high flow conditions (Ducklow, Mitchell 1979). Thus, the presence of potentially pathogenic Vibrios in the coral mucus may be viewed as a normal occurrence that does not ordinarily cause disease. The finding of V. vulnificus in RTN affected colonies d it may be significant in both etiology and pathology of certain cases of RTN. However, its presence is not unexpected, nor is it demostratably causative for all cases. Lowrie has failed to isolate any such causative pathogen in numerous samples of "RTN" affected corals (Lowrie 1992, 1997). It is thought that fewer than 1% of mucusal bacterial colonies are motile, and only a small percentage of those to be deemed pathogenic (Segel, Ducklow 1982). Most are simple normal microbial colonizers of mucus to be used by the coral in phagocytotic ingestion. However, Ducklow and Mitchell (1979) found Vibrios to comprise up to 30% of coral mucus populations. The incidence of RTN with so many variables of diverse conditions and specimens would make the probability of a novel, comparatively uncommon, and highly pathogenic bacteria being responsible for so many widespread and variant reports of RTN quite unlikely.

The Immune Response Hypothesis

From the information provided herein regarding the many contradictory and unexplained aspects of RTN, and from the information provided in the previous installment of this work, it is now possible to construct a model that is based primarily on the fact that stress seems to be the overwhelming common denominator in the occurrence of this disease. Stress has been implicated and found to be causative in a myriad of diseases crossing countless phyla. Stress has been shown to elicit disease in plants and animals, from laboratory conditions to humans in their respective environments. Often, stress has been found to act through a repression or maladaptive response of the immune system. Through our investigations and studies, we feel our immune response hypothesis can be made to explain, at least in part, the observations and reaction of corals with RTN.

Based on what is known and has been observed, the tentative hypothesis for RTN is as follows:

At least some corals, when subjected to stressful conditions, potential pathogens, or quantitatively/qualitatively significant antigens, are reacting in an undetermined autoimmune manner, responsible not only for the loss of their own tissues through possible autolysis or loss of host cell adhesion, but for the potential of triggering a similar response in similar corals. Inappropriate or decreased immune function may also play an undetermined role in the expression of pathogenic, physical, or chemical stress related events that result, ultimately, in tissue necrosis. The contagia of RTN is not necessarily due to any pathogen, but to histocompatability factors, likely to be present in the mucus of corals."

Acropora and similar corals have high metabolic rates that would predispose them to be the first to react to such disturbances and through the aforementioned "drastic" means of autoimmune reactions. All of the normally occurring, as well as the likely-to-be-present, enzyme and chemical releasing immunodefensive cell types have been previously shown to exist,and are capable of releasing substances that would easily cause the massive and rapid tissue necrosis that characterizes the disease. Immune mediated loss of host cell adhesion (Gates 1992, et. al.) could also be responsible. Calcium and other divalent cations are readily available in calcareous invertebrates to catalyze hemolytic type events in both the colony and the surrounding water medium. The likelihood of the presence of a2-macroglobulin (or a similar molecule) is already required under normal conditions to prevent protease mediated self digestion. Both of these aspects were discussed in our last article.

Certain species of corals seem to be more susceptible to RTN than other species (Sheimer 1997). As a genus, Acropora seems to be the most susceptible. Other species appear to be completely unaffected by RTN or immune. Acroporids are fairly recent corals in an evolutionary sense (Veron 1995). They are highly specialized and possess numerous features that have allowed them to become dominant species in coral reefs (ibid.). Such specializations could include specialized immune responses, or novel reactions that are directly or indirectly a result of their specialized physiology. The following are but a few cases where RTN susceptible corals showed increased sensitivity to environmental stressors: Unusual low temperature conditions in Saudi Arabia created a hypothermic stress that resulted in coral bleaching. However, all Acropora were dead after having sloughed their tissues. Platygyra, another recent coral in evolutionary terms, was also sloughing tissue, though less so than Acropora. Other scleractinians, such as those rarely observed with RTN (Favia, Porites, Psammocora, Pavona, Leptastrea, Cyphastrea, Favites, and Turbinaria), showed no tissue sloughing or mucus extrusion (Coles and Fadlallah, 1991). Temperature extreme causes host cell detachment in Pocillopora. (Gates et. al. 1992). Offshore oil drilling caused preferential loss of Acroporid and Pocilloporid corals (Hudson, 1982). Chronic oil pollution caused reduction of Pocilloporid and Acroporid corals (Fishelson, 1973). Pocillopora least resistant to thermal pollution (Acropora not present) (Jokiel and Coles, 1974). Acroporids were most susceptible to recreational activities (Tilmant and Schmahl, 1983). Acropora and Pocillopora most susceptible to thermal stress (Neudecker, 1981). Tissue disintegration of A. cervicornis after exposure to drilling mud (Thompson, 1980). Acropora formosa most susceptible to elevated temperature showing increased mucus production within hours (Neudecker, 1983). A. palmata least tolerant of exposure to calcare ents, causing death of underlying coral tissue (Rogers, 1984)(note: previous 8 studies referenced in Brown, Howard, 1985).

Some Acropora displaying RTN, and even immediately before active RTN takes place, show significantly increased production of mucus that may even have a strong aromatic nature implicating the excess production of biochemicals as a response to unknown factors. Prior to contracting RTN, we observed a noticeable change in the mucus of a large colony of wild caught A. gemmifera a week after introduction to a tank. The mucus became extremely aromatic, thick, and darkened in color. The following day, tissue necrosis began on that colony. This finding was corroborated by Carl delFavero with several Acropora colonies of different species from the same source over a period of several weeks (Borneman, delFavero pers. obs.). Hypersecretion of mucus occurred colony wide, and not near a localized area of stress or proposed pathogen action. Acropora are normally prolific mucus shedders and depend, to a larger degree than many other corals, on mucus production for their defense and feeding (Sorokin 1973, et. al.). Jonathan Lowrie has found evidence of undifferentiated granular cells within the coral mucus of Acropora, suggesting that the mucus may even play a more significant role in immunodefense in these corals. This may be due, in part, to the thin tissue of Acroporids which have less mesogleal space with which to harbor tissue-indigenous immunodefensive cells. Other corals (Pachyseris, Pocilloporids) that are commonly affected by RTN share these characteristics.

Corals exposed to strong water motion have been reported and observed to have a reduced incidence of contracting RTN. Reduced water flow has already been shown to have bacterial and immunological implications. It would make sense that corals in the wild, which are exposed to virtually infinite water volume and significantly higher water movement would, if such a model existed, be far less likely to contract RTN. Strong water movement would tend to wash away mucus with pathogenic bacteria into the vast ocean and prevent mucus densities that would allow for greater microbial populations. However, strong water movement in a closed system would seem to merely increase the chance that mucus containing a pathogen would contact or even be consumed by other corals in an aquarium. The effects of strong water movement in reducing incidence of RTN, coupled with comparatively less epidemic incidence in the wild, could be explained through the dilutatory and washing effects that any histocompatabilty factors would have, even between reasonably proximate allogeneic or xenogeneic species. Reports have indeed been made that adjacent colonies in the aquarium are most susceptible to RTN contagion. Unfortunately, analogies of wild and captive exposures and incidents of disease in adjacent colonies are not terribly valid. The description of increased prevalence of disease in adjacent colonies in the wild occurs over an area that is vastly larger than captive systems and exposed to huge water dilution effects. For all practical purposes, if such a pathogenic bacteria is responsible for disease, all corals in captivity are efffectively "adjacent." Furthermore, since many Vibrios are swarmers and other bacteria are likely already present and capable of actively growing in the mucus, this model makes less sense than one in which there was another mediator or carrier of the RTN response. However, strong water movement increases colony fitness and would allow for a more normal immune response to stress or pathogens. Sea sta owing seawater do not show bacterial invaders in their coelomic fluid, but those in aerated but stagnant water are overwhelmed by bacteria (Bang 1973). Such findings relate to reported reductions of RTN when corals are placed in high flow conditions. Although bacteria are the relevant agent in this case, the immune reaction of corals to bacteria is what is applicable to this paper.

In terms of its occurrence and progression, RTN does not always follow the widely described "from the base up" pattern of tissue loss. It may also occur from the tips down or affect whole brances. This is concurrent with Esther Peters (1997) description of WBD initiated in the middle of A. cervicornis branches. Until this description, it had been assumed through Antonius' work that WBD began at the center or base of a coral and proceeded outward toward the margins. The most commonly observed "base-up" loss of tissue in RTN could further indicate a relatively consistent point of weakness for a hypothetical pathogenic mechanism to occur. One possible explanation is that both branch tips and bases are marginal areas of the coral and are inherently weaker (Scheimer, pers com.). However, the margin of a corallite might just as easily be construed as such an area when discussing organisms the size of bacteria. The current lack of an attributable pathogen in both WBD and in some cases of RTN, coupled with inconsistent and often contradictory modes of progression, creates difficulties for such causative models. These other patterns of RTN are somewhat more explainable using histocompatability models, as they are primarily mucosal in nature and do not depend on sites of infection. The effects of strong water movement work the same for an immune hypothesis as they do for a bacterial one, but may be slightly better because chemical factors are more predictable in hydronamic and simple diffusion models than pathogens which actively attach to and colonize various mucosal substrates. Allogeneic corals, xenogeneic corals, or even fragments of the same parent colony would likely display increased incidence of RTN within closed systems where any histocompatability factors would be easily considered within required contact distance. Even within aquaria, similar corals nearby to RTN-affected corals are usually affected first. The concentrations and likelihood of histocompatability factors would still be more likely t mate corals.

Wild caught corals have been observed to have a higher incidence of initially contracting RTN than captive raised corals. It may be hypothesized that wild communties are subjected to a larger group of potential pathogens they may harbor in their mucus, tissue, or skeleton. However, the stress corals must endure after collection from natural reef communties can be extreme. Some facilities further stress corals by subjecting them to oxidative antibacterial or antiseptic dips to "sterilize" the coral of potential pathogens. The net effect, and possibly some of the increased incidence of RTN, may be because such "dips" are serving to decrease the ability of the coral to utilize its own mucus-based defenses to further stresses (which are already being taxed after collection). Wild caught corals are also transported in bags of water that are functionally stagnant. Such conditions create a number of conditions that would be easily explained by an immune model. Low oxygen levels have been shown to increase phagocytosis. Stagnant bag water would increase the production of mucus and would also be unable to wash excess mucus away. Such increased mucus production would increase the available food source for resident bacterial populations, possibly to the point of pathogenicity. It would also have the effect of suffocating the coral, lowering tissue oxygen levels, producing H2S (Segel, Ducklow 1982), and possibly increasing phagocytic or autolytic events at the tissue level. Mucosal fueled growth of bacteria would increase the production of lysozymes to cope with the larger populations. Such enzymes would not be able to be washed away in the mucus and may begin to exert a stressful, if not digestive action on coral tissue and mucus, liberating further enzymes and antigens. The result of dumping such highly immune-responsive corals, coral mucus, and even bag water after shipping/holding may have hyper-heightened effects and acute responses on any other corals within a system. Increases in other water parameters, i ature (already shown to have serious immunologic consequences), are also likely, if not certain, in the shipping and holding conditions. Starvation (common previous to acquisition by an aquarist) accompanied by sediments can change the compostion of the mucin and mucoid containing cells, with symbiotic corals showing an increased rate of atrophy, erosion and necrosis (Peters and Pilson, 1985). The same study showed starved corals (starvation = decreased immunocompetence?) with a cell architecture breakdown, decreased epidermal mucus production, increased intracellular basophilic material, changes in mucosal pH, decrease in epidermal mucosal cell function (with sediment application) and the increased mucoid material within certain cells to be indicative of pathologic dysfunction of skeleton- producing cells. In other words, when stressed, the corals defense mechanisms are likely weakened and many cellular functions go awry. Higher temperatures cause more rapid self-/non-self rejection in corals, characterizing a "sharply heightened immune reaction," while at temperatures below 21 degrees C, Montipora verrucosa may not even elicit a reaction (Johnston, et. al. 1981). Temperature stress can cause histopathologic abnormalities that include, "deterioration of epidermal mucous secretory cells, erosion of epidermal and gastrodermal cell layers, atrophy of longitudinal retractor muscles, loss of mesogleal pleat structure, appearance of necrotic nuclei in calcioblastic layer and epidermal and gastrodermal cells, disappearnace of spermaries and developed ova...The histopathological abnormalities observed in this study (were) a general atrophy and necrosis of coral tissues..." Furthermore, and perhaps most notably, "foreign organisms, such as bacteria, fungi, algae and protozoans, were found only occasionally in coral tissues in these studies."(Glynn 1990).

These same stressors are largely not present in captive raised corals, despite the certainty that commerical facilties (and any marine captive habitat) can contain most, if not all, of the normal antigens and potentially pathogenic microorganisms found in the wild. Once established, the overall vigor of captive bred corals seems to impart a slight advantage towards resistance when exposed to RTN affected colonies. However, once exposed to another coral which is releasing histocompatibility factors, the lack or presence of pathogens would not necessarily be a factor as even the captive bred colonies would have an immune response elicited. There may be less captive bred specimens affected because of their generally stronger immune system from already being adapted to captive conditions, and from their resultant lowered stress response. Tank raised corals or those already within established aquarium may also have conferred on each other some degree of already-present antigen immunity as shown in our previous article.

Perhaps of significant interest is that RTN is inducible under non-pathogenic conditions in healthy corals. In other words, in the absence of time or conditions to attain a bacterial population capable of causing disease, RTN can occur. RTN is inducible by various chemical, biological, and mechanical means that initiate acute or prolonged stressful conditions that do not necessarily allow for a statistically significant probability of vector induced disease. Lowrie (1997) has repeatedly induced RTN in healthy colonies and fresh sea water through the acute stress of blowing diatomaceous earth onto the branches. Prolonged exposure to low light, low dissolved oxygen, stagnant water, high temperatures, extremely low temperatures, overhandling, shipping stresses, and other unstable or detrimental water conditions have also resulted in the expression of RTN (Lowrie 1997, et. al.) . Environmental stressors, including pH, temperature, chemicals, sedimentation and other antigens can all elicit immune responses and impair the normal immune function of corals. Lasker, Peters and Coffroth (1984) showed swollen mesoglea and abnormally increased mucus production under stressful in situ bleaching-type conditions that was concurrent with atrophy and necrosis in some corals. Autolysis of coral cells was present in Agaricia, along with one colony found with a "white band type" granular basophilic body. Agaricia is in the same family as Pachyseris, a coral commonly afflicted with RTN.

RTN seems to be a surface mediated event carried within the mucus to tissue necrotic events that, in general, follow a more or less linear pattern of loss across a colony. Esther Peters (pers. com.) notes that differences in cells and mucus production in different areas can occur, and that Acropora have an internal system of gastovascular canals to exchange fluids throughout the colony. She offered this as a proposed mechanism for which a pathogenic microbe could be carried throughout the colony or selectively proliferate in an area. Fragmentation of branches or sections of coral, and their removal to separate conditions without any antimicrobial treatment, is effective in stopping RTN. It does not matter if a branch tip or entire section of unaffected coral is removed. The transmission of a vector through intavascular canals would seemingly limit the usefulness of this procedure. The fact that the source corals of histocompatability effectors are no longer present to elicit an autoimmune response would explain this finding. Such sections or fragments would, however, have microbial mucus populations of a very similar to nature to the those theorized to be causing RTN in the affected corals. In fact, the time required for RTN to be "contagiously" spread fits in very closely with the time usually required for immune responses to be elicited. Ten minutes to several hours has been given as the normal time for such immunodefensive action to begin. Theodor (1970), in a tremendously relevant work, theorized that a diffusable substance caused "induced suicide" in corals. He showed how visible necrosis was the late stages of cytolytic response in gorgonians where supposedly "killer tissue" induced the target tissue to kill itself. The time required to induce 100% lysis was 30 minutes; a very appropriate number and sequence of events relative to RTN. Cytotoxicity occurs within 1-2 minutes. Induced suicide may also explain cytotoxicity from non-immune mechanisms. In a later work, (Theodor, Senelar 1975), it was purported that the diffusable "toxic factor" is already present in the "killer tissue" and different species have fundamental differences in histocompatability. In other words, the mere presence of a "new" antigenic substance (mucus, etc.) can effect cnidaria through autolysis in an autoimmune response. Longer durations or RTN resistance in some species can be explained by genetic disparity, inhibitory factors, lack of receptors for the histocomaptability effector, or higher immune function resulting from longer establishment in its environment and the resulting lowered stress. Metabolic shock persists after removal of environmental stressors (Bak 1978). This implicates the sudden occurence of RTN even some time after introduction to comparatively "stress free" conditions. A valuable study by Buss, Moore and Green (1985) showed a defect in self- tolerance in Hydractinia echinata. Large scale migration of multipotent interstitial cells begins with a proliferation of nematocystic cells, which then produces an autoreactive system. The hydroids used this system in an "autoimmune phenomenon" which only occurred after colonies had reached sexual maturity. The lessened chance of RTN in fragmented colonies are usually new growth of similarly lesser maturity.

If a Vibrio species, or any bacterial pathogen, were responsible for RTN, then antibiotic treatments should prove beneficial. Chloramphenicol is a broad spectrum antibiotic of large molecular size, and is effective in treating many cases of "RTN." According Dr. Bingman, and substantiated by our own experiences and the reports of others, chloramphenicol is not only effective in treating RTN, but it also seems to impart a reduced if not total resistance toward the same coral acquiring another case of RTN. This occurs despite the fact that other antibiotics known to be as effective or more effective on gram-negative bacteria (including Vibrios) do not have the same effect. Neither is chloramphenicol is always effective in treating RTN in corals. We have treated numerous Acroporids and Pocilloporids with this drug that failed to respond to the protocol. Such findings would imply a multi-causative model of RTN, resistant strains of bacteria, or a difference in the uptake or pharmacokinetics of chlormaphenicol in different coral species. Based on our observations, this would have to be extended to include different colonies of corals from the same species and even same RTN event; an unlikely possibility. There is also no clear reason to support the view that this antibiotic has any method of action that would impart long term resistance, especially in a filtered marine environment. Furthermore, the mechanism of chloramphenicol is primarily bacteriostatic, and the pharmacokinetics of the drug are unlike most antibiotics. Chloramphenicol works by inhibiting protein synthesis of the 50S ribosomal subunit, preventing further reproduction of the bacterium. It would not necessarily prevent already present bacteria from continuing their necrosis of coral tissue. Chloramphenicol does, however, present a rare human contraindiction for a type of acute anemia (aplastic) that surpresses bone marrow white blood cells (possibly due to the nitrobenzene moiety). It is, in effect, a type of severe immunosuppression. It

chloramphenicol is effective on some corals through actions not akin to antibiotic action, it is conceivable that certain species have their immune systems affected in such a severely repressive manner. If such action could be demostrated, it would certainly support the theory that RTN is not caused by any particular microbe . The fact that chlormaphenicol is used only as a 24 to 48 hour bath for affected corals may mean that the proposed immunosuppressive action in isolation is substantial enough to stop autolytic events. The coral can is replaced into a low-stress tank condition, relieved of histocompatability effectors and an hypersensitive immune response, and is then perceived as "miraculously" (though deceptively) free of the disease. Even if no such immunosuppression exists, a short duration bath in chloramphenicol could slow bacterial growth, allowing the immune system of the corals to recover in quarantine conditions, and allow for histocompatability factors in the aquarium to disperse or be lost.

Our experimental initial use of certain steroidal and non-steroidal anti- inflammatory drugs and antihistamines has also slowed or stopped the progression of RTN in various affected tanks and corals. Some of the drugs attempted have unfortunately caused coral death through acute cytotoxicity, although they did halt the progression of RTN. However, the work in discovering an invertebrate compatable, non-toxic, immunosuppressive substance has barely begun.


Our hypothesis of an immune mediated response as being of primary importance in the affliction known as RTN is based on its overwhelming occurrence in stressful or recently stressful conditions. The pattern of tissue loss, with tissue often being lost as intact sheets with no gross evidence of bleaching or degradation, suggests that the host tissues have lost adhesion. The ability of corals to lose cell adhesion in response to stress is well documented in the Pocilloporids' poly bail-out response (Sammarco 1982, et. al.) and to thermal stress, etc. (Gates 1982). The lack of consistent findings of associated pathogens in RTN affected corals lends further credence to the theory that stress can cause such a reaction. The presence of potential pathogens in coral mucus would only exacerbate the stress response, with digestive action of autolysis and already present bacteria accounting for further tissue degradation. The physiologic and biochemical attributes required for such a reaction have been shown to be present. Furthermore, many descriptions of coral disease in the wild, especially those studied in conditions of stress, have been often reported to have tissue necrosis at varying rates. The relative levels of stress in the wild are, in most instances, of an order many magnitudes lower than those which are present in the collection, shipping and placement of corals into a new and captive environment. The RTN reaction in both numbers and severity would be fit logically within the confines of an stress related immune reaction hypothesis. Antonius's work, the role of stress in studies and observations of wild disease and necrotic events (Peters, et. al.), as well as the presence of the highly pathogenic V. vulnificus found by Bingman, would be synergistic with the impaired immune function suggested in this paper. We are currently compiling the sum total of experimental data we have to date, and will be working on more studies in the future. These will be published at a later date.


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