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2) Nonspecific immune defenses are often prominent at portals of entry (skin, gills, gut) and thus can prevent invasion before tissue damage occurs

3) Many components of nonspecific immunity can be upregulated very rapidly (within hours or a few days) to respond to a pathogen. This is in contrast to specific immune responses, which require much longer periods to respond, especially under suboptimal conditions (e.g., low temperature)

4) Nonspecific defenses can protect against multiple pathogens, and in some cases are extremely broad-spectrum

However, nonspecific immunity is less effective than specific immune responses in being relatively short-lived and having no memory (i.e., the response does not "improve" after exposure to a pathogen). Thus, it's application must be designed with these limitations in mind.

Components of Nonspecific Immunity

There are many components of nonspecific immunity. The cellular arm of nonspecific immunity in fish includes natural cytotoxic cells, granulocytes (neutrophils, eosinophilic granule cells) and cells of the monocyte/macrophage lineage. Analagous cells occur in invertebrates (granulocytes, amoebocytes, etc.). Among the most well-recognized humoral components are complement, tranferrin, lectins, interferons, C-reactive protein, prophenoloxidase, clotting factors, and various defensive enzymes (Yano 1996, Noga 2000).

One of the most interesting and potentially important components of nonspecific immunity which has recently been been discovered in aquatic animals is the presence of polypeptide antibiotics. Polypeptide antibiotics are host-produced antimicrobials having broad-spectrum activity (i.e., inhibitory/lethal to multiple pathogens)(Scott and Hancock 2000, Tossi et al 2000). For example, histone-like proteins (HLPs) are active against bacteria, water molds and parasites. HLPs have been isolated from numerous fish, including channel catfish, rainbow trout and hybrid striped bass (Robinette et al 1998; Noga et al, Accepted) and are probably present in most, if not all, fish. An increasing number of other polypeptide antibiotics have also been recently isolated from various commercially important aquatic species, including flatfish, shrimp, crabs, and bivalve molluscs (see Douglas et al 2001, Mitta et al 2000, Khoo et al 1999 and Robinette et al 1998 for recent reviews).

Practical Applications of Nonspecific Immunity

Knowledge of nonspecific immunity can be applied to aquaculture in developing biomarkers of health/stress, upregulating it to protect against infections, and genetically enhancing resistance to disease:

Health Biomarkers

Development of field-friendly methods for accurately assessing the health of aquatic animal populations would be a highly valuable tool in managing aquaculture populations. However, while a large amount of work has been done in evaluating possible indicators of health in aquatic animal populations, no assay has gained widespread commercial use. This is due to failure to satisfy at least one of the following criteria (Noga 2000):

• Ease of collection - a tissue sample to be tested must be easily, simply, and rapidly collectable under

real-world field conditions by untrained personnel (e.g., farmers). This includes being unaffected by the stress of capture.

- Stability - a tissue sample must remain unchanged between collection and testing

• Measureability in the field - ideally, the biomarker should be measureable by simple and economical

means on site

- Correlation with animal health - the biomarker should be a sensitive bioindicator of the aquatic animal's health

Improved Methods for Managing Epidemics

Stimulation of nonspecific immunity has the potential to enhance disease resistance in several scenarios:

- Preventing epidemics during stressful events - the rapid response of nonspecific immunity (often within hours) suggests that it is possible to protect fish against an anticipated acute stress (e.g., hauling, grading, etc.). This is not feasible with specific vaccines.

- Treating active epidemics - the ability of nonspecific immunity to be rapidly induced suggests that one could literally "vaccinate" with this method even during an active epidemic, which is not possible with classical vaccines based upon stimulation of the specific immune response. This could be useful as both a stand-alone treatment, where stimulation of nonspecific immunity could be used as an alternative to drugs (e.g., classical antibiotics, etc.) in treating epidemic diseases, and as an adjunct, where stimulation of nonspecific immunity could be used in conjunction with drugs, reducing the amount of drug needed for successful therapy.

- Preventing epidemics via vaccination - incorporation of nonspecific immunostimulants into traditional specific vaccines might enhance the efficacy of these vaccines

Genetic Markers of Resistance

Using components of nonspecific immunity as markers to select for disease resistance may be superior to selecting for resistance to a single disease. The latter approach has not met with strong success in most instances (Fjalestad et al 1993). This could be approached by selecting for biomarkers of genetic resistance to disease. It would also be promising to look for biomarkers of genetic resistance to stress. For example, different defenses might be involved in resistance to an acute stress compared to a biomarker which maintained aquatic animal health in "unstressful" environments. Alternatively, genetic engineering could be used to either overexpress the genes coding for these factors or they could be cloned into species which are lacking such genes.

Promising Candidates for Practical Applications of Nonspecific Immunity

Nonspecific immunity is being used extensively in commercial aquaculture (Sakai 1999) and promises to be as useful as the specific arm of the immune response in controlling diseases of cultured aquatic animals. While immunostimulation of nonspecific defenses is practiced widely in aquaculture, there is a need to better define the specific conditions for optimizing these protective responses, including understanding how environmental and microbial cues up- or down-regulate their activities. Many of the in vitro tests currently considered to be most useful in assessing these responses (e.g., phagocyte activity) are expensive to perform, limiting the ability to rapidly screen potential immunostimulant regimens for efficacy. Nor do we often understand what changes in some components mean to the health of the host. This is partly due to the fact that nonspecific immunity consists of many diverse defenses which play different roles in protection. In many cases, these protective roles are not directly toxic to pathogens but rather enhance other mechanisms (e.g., C-reactive protein enhancing complement activity).

For these reasons, polypeptide antibiotics have especially strong potential application to aquaculture because they have potent, usually lethal activity against many disease-causing agents (viruses, bacteria, fungi, parasites, tumor cells) and thus probably play a vital role in protecting against many diseases. They can be directly and easily measured using relatively simple assays. They can also be upregulated with various types of stimulants (Scott and Hancock 2000) and there is increasing evidence that they are significantly downregulated with stress (Noga et al 1994, Simmacco et al 1997, Robinette and Noga, Accepted). The molecular mechanisms controlling these regulatory events are also being elucidated (Scott and Hancock 2000).

Economic Benefits of Optimizing Nonspecific Defenses

The optimization of nonspecific defenses, in conjunction with the use of other immune enhancement techniques, should be a major priority in aquaculture because it will result in numerous major benefits to producers, including:

1) Decreased costs in time, labor and materials for treating aquatic animals with drugs

2) Reduced need/costs to develop new therapeutants for aquaculture

3) Decreased costs in time, labor and materials in treating fish with conventional (specific) vaccines

4) Higher yields due to decreased losses from morbidity and mortality

5) Higher yields due to increased feed conversion efficiency

6) Higher prices due to enhanced public perception of quality (both specific markets and aquaculture in general)

7) Value-added products for penetration of growing niche markets (e.g., "organically" grown)

8) Reduced trade barriers and enhanced ability to export products

9) Reduced regulatory pressure due to lessened environmental impacts and concerns

While there will certainly be economic costs for using this technology (increased costs in time, labor and materials for monitoring aquatic animal health and treating aquatic animals with nonspecific immune enhancers), there is considerable potential for a large, positive, cost/benefit ratio. These benefits should become more clear as uncertainties about this techology are resolved.

CONCLUSIONS AND RECOMMENDATIONS

Application of nonspecific immunity to aquaculture management has great potential, but in order to capitalize on this potential, we need a much better understanding of the mechanisms by which one can most efficiently regulate these responses. While a significant amount of this information will probably be provided by studies in other animals (especially insects and mammals) where considerable research is ongoing, there will certainly be areas relating to aquatic animals which will need elucidation. The timeline for specific aquaculture sectors will vary because our knowledge of nonspecific defenses varies with species. However, a general timeline for implementation would be as follows:

Short-term (1-3 years)

1. Identify candidate nonspecific immune (NSI) defenses (e.g., the major polypeptide antibiotics) in

target species which are constituitively expressed. Identify the pathogens which might be controllable by these defenses. While genomics will play an increasingly important role in identifying these targets as our knowledge of the chemical structure of various defenses is determined, protein purification technology will probably play the key role in initially identifying defenses such as polypeptide antibiotics. This is because of the great diversity of structures which define this defense. For example, to date, none of the antibiotics described from fish have been isolated from all major fish species (histone-like proteins might be an exception).

2) Identify candidate NSI defenses in target species which are expressed after immnostimulation. For example, while at least some polypeptide antibiotics may be the same as those identified in #1 above, it is likely that novel antibiotics may also be discovered as well.

Mid-term (4-7 years)

3) Clone the genes for candidate NSI defenses in important aquaculture species. This is needed to eventually understand the genetic mechanisms which up- and down-regulate these factors.

5) Optimize immunostimulation regimes for defending against specific, important diseases.

6) Begin technology transfer of relevant technologies to commercial interests and end-users.

Long-term (8-10 years)

7) Further validate the importance of candidate NSI defenses in maintaining health in an aquatic animal population.

8) Select for populations which express high levels of candidate NSI defenses, either constituitively or during stressful events.

ACKNOWLEDGEMENTS

Research of my laboratory described in this review has been supported by Saltonstall-Kennedy Project #NA67FD00500, Grants #NA86-RG-0036 and #NA46-RG-0087 from the National Sea Grant College Program, National Oceanic and Atmospheric Administration, to the North Carolina Sea Grant College Program, Binational U.S.-Israel Agricultural Research and Development Projects #US-2206-92 and #US-3030-98, the USDA NRI Competitive Grant Program (Project #97-35204-7722) and the NCSU College of Veterinary Medicine.

REFERENCES

Douglas SE, JW Gallant, Z Gong and C Hew. 2001. Cloning and developmental expression of a family of pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum). Developmental and Comparative Immunology 25:137-147.

Khoo L, D Robinette and EJ Noga. 1999. Isolation of callinectin, an antibacterial peptide from blue crab, Callinectes sapidus, hemocytes. Marine Biotechnology 1:41-51.

Noga EJ, Z Fan and U Silphaduang. (Accepted). Histone-like proteins from fish are lethal to the parasitic dinoflagellate Amyloodinium ocellatum. Parasitology

coincides with increased prevalence of shell disease in blue crabs, Callinectes sapidus. Diseases of Aquatic Organisms 19:121-128.

Robinette DR and EJ Noga. (Accepted). Histone-Like Protein (HLP): A novel method for assessing chronic stress in fish. Diseases of Aquatic Organisms

Sakai M. 1999. Current research status of fish immunostimulants. Aquaculture 172:63-92.

Simmacco M, A Boman, ML Mangoni, G Mignogna, R Miele, D Barra and HG Boman. 1997. Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin. FEBS Letters 416:273-275.

Tossi A, L Sandri and A Giangaspero. 2000. Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 55:4-30.