Biotechnology-Aquaculture Interface:

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Appendix
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Workshop Report
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-Final Report

MEMORANDUM March 20, 1998

No Animal is an Island: Understanding the Dynamics of

Persistent Animal-Microbial Interactions

Margaret J. McFall-Ngai

Pacific Biomedical Research Center

Kewalo Marine Laboratory

University of Hawaii

41 Ahui Street

Honolulu, HI 96813

Email: mcfallng@hawaii.edu

Abstract

All animals are believed to form persistent relationships with communities of microorganisms. Only in a few mammalian and insect species has the nature of the microbial community that associates with the host been defined to any extent. However, recently-developed methods promise to allow biologists to: 1) determine whether such multi-species alliances actually exist as a primitive character of the metazoa; and, if so, 2) define their biological structure and function. An understanding of the dynamics of animal-microbial communities will allow biologists to determine more precisely what goes awry when a pathogen becomes an interloper in the delicate balance between the host and its coevolved consortium. This contribution examines these new methods for the study of animal-microbial interactions and considers their possible application to aquaculture.

Key words: microbiota, consortium, metagenomics

Introduction

The study of the interactions between animals and their associated bacterial consortia has focused principally on several mammalian (e.g., humans, ungulates, rodents; Savage, 1977, 1986) and a few economically important insect (e.g., termites, cockroaches; Lysenko, 1985) species. These complex communities are numerically dominated by the prokaryotic components. For example, in the best-defined system, that of the human body, 90% of the cells are prokaryotic, i.e., the average person has 1013 body cells, 1014 bacteria in the intestinal consortium, and 1011 bacteria on the skin. Analyses of these prokaryotic consortia suggest that they are highly coevolved, stable and essential components of the animal host’s biology.

Outside of these few better-defined systems, little is known about the microbial communities that associate with animals. Several very basic questions remain unresolved. For example, is it a primitive character of animals to coevolve with a complex microbial community, or is this character restricted to certain taxa? Aquaculture species, which can be experimentally manipulated, offer unique opportunities to address such very basic questions. In addition, the results of such studies should lead to more comprehensive and successful approaches for the development of measures that would both combat microbial disease and promote health.

Technical Impediments

Even in these better-studied systems, technical difficulties (e.g., the non-culturability of some of the bacterial partners) have prevented a thorough understanding of their dynamics. For example, in the human intestine, although approximately 400 prokaryotic species have been unequivocally defined as constituents of the consortia, it has continued to be difficult to determine whether some bacteria are ‘native’, coevolved species or casual ‘tourists’, for which the principal niche is not the human intestine. In addition to the difficulties in defining such complex systems, the multi-species nature of these associations has rendered experimental approaches very difficult. Isolating individual species and studying their biology in culture have provided limited information about their function as members of a complex community. To make an analogy, the behavior of a person in solitary confinement will differ significantly from his/her behavior as an active member of society. Similarly, rendering the host "germ free" or gnotobiotic (colonized by a small, defined subset of the community) presents difficulty both in the design of the experimental approach and in the interpretation of the data, i.e., extrapolation of these data to a model that seeks to describe the workings of the whole community.

With all of the difficulties in studying normal animal-bacterial consortia, the response on the part of biologists has been, most often, to ignore this "complicating" feature in considerations of the host’s biology. However, studies of these alliances, although challenged by technical difficulties, have resulted in some very basic and important findings. Most notably, recent research in this field has shown that the interactions of the host with its microbiotia are essential for the host’s health and development, particularly the maturation of the immune system and the morphogenesis of the tissues interacting directly with the microbial population (for review see McFall-Ngai, 1998). These types of findings, along with the development of new methods, have encouraged pioneering efforts to increase the resolution of our understanding of beneficial animal-bacterial interactions.

An Urgent Need

The development of new methods is timely, not only so that biologists might further our understanding of these basic biological processes, but so that we might better define the differences between cooperative animal-bacterial interactions and pathogenic ones. The globalization of society has led to the realization that one of our principal challenges in the coming decades is the control of traditional and emerging bacterial pathogens (McCormick, 1998). Whether they are natural or man-made (e.g., in the payload of bioweapons), such pathogens present a threat, not only to the animal host, but also to the entire, complex community of beneficial bacteria that it harbors. Therefore, a more sophisticated grasp of nature of the normal, benign, animal-bacterial associations is essential to the development of a better-integrated understanding of what goes wrong when pathogenic bacterial ‘interlopers’ disrupt the dynamics of this community. Specifically, by gaining insight into the molecular strategies for co-existence that have arisen between hosts and their benign bacteria, we can hope to discover when and how some of these strategies have been subsequently expropriated by pathogens.

Principal Questions and Experimental Approach

In this context, these basic questions present themselves. In a given animal species:

    1. Is there evidence that the host has coevolved with a microbial community?
    2. Who are the constituent species in this community, and what are their relative proportions?
    3. What is the community doing as a whole to support the biology of the host, and what

is the contribution of individual microbial species to the activities of the community?

--corollaries: a. How is the consortium maintained between generations?

b. Does the composition of the community change through the ontogeny of the host?

Methods: The methods associated with addressing the first two questions, while they continue to be refined, have been extensively used by microbial ecologists to define microbial communities in environments such as soil and seawater. More recently, these techniques have been applied to the study of microbial communities that associate with animal hosts (Paster et al., 1996; Suau et al., 1999; Thimm et al., 1998). The third question represents a pioneering area of research, where methods are rapidly developing.

Question 1—Goal: To define whether a given animal species has coevolved with a microbial consortium. The methods available to address this type of question include denaturing or temperature gradient gel electrophoresis (DGGE or TGGE, respectively; Muyzer and Smalla, 1998; Simpson et al., 1999) or terminal restriction fragment length polymorphism (TRFLP; Kerkof et al., 2000; Marsh et al., 2000). Such approaches provide a ‘fingerprint’ of the community, defining its complexity within a given sample, and allowing direct comparison of the community composition between individual samples.

Question 2—Goal: To determine the precise species composition of the community, and the relative abundance of the constituents. The numerous advances in sequencing technology in the past several years make possible the defining of the constituents of a complex community. While DGGE and TGGE cannot be used reliably to define the relative abundance of species in a consortium, TRFLP has been successfully used for this purpose. Thus, TRFLP can be used to identify abundant species. If a community appears to be highly complex, the sequencing regime can be prioritized with data provided by the TRFLP analyses.

Question 3—Goal: To characterize the activity of the consortial community and its members. In addition to defining the composition of the community, several techniques are being developed that promise to provide insight into the physiology of the community. For example, the commercially available Biolog microtiter plates (Biolog, Hayward, CA) can be used to determine the metabolic activity of a microbial community, as well as the assess the impact of environmentally induced changes on this activity (Choi and Dobbs, 1999; Kerkof et al., 2000; O’Connell et al., 2000). Other methods seek to link the genotype and phenotype of the bacterial species comprising a consortium. In one promising method, the DNA of an entire, defined community, or the ‘metagenome’, is introduced as large fragments into E. coli using a bacterial artificial chromosome (BAC) vector (Osburne et al., 2000; Rondon et al., 2000). The bacterial species is identified by the sequencing of a gene, such as the rDNA or rpoB (RNA polymerase beta subunit) gene, and then long stretches of DNA flanking these genes are sequenced to define genes that suggest aspects of the metabolic potential of this bacterium, such as antibiotic production. This approach is particularly valuable in defining the activity of communities that are composed largely of nonculturable microorganisms.

Recommendations

Overall goal: To generate a prototype set of methods for efficiently and accurately defining the composition and activity of microbial consortia that associate with aquaculture species.

  1. Short-term—(1-3 years)

    2. Define appropriate aquaculture species for a protracted study of their associated consortia. This effort would include preliminary analyses to determine whether a coevolved community of microorganisms exists with a given host species and, if so, to determine its complexity and reproducibility between individuals.

  2. Mid-term—(4-7 years)

    3. Characterize the activity of the community through approaches such as metagenomics. Define indicator species that are characteristic of a healthy community.

  3. Long-term—(4-7)

Develop protocols for the application of methods to a wide variety of aquaculture species.

References

Choi, K.H., Dobbs, F.C., 1999. Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities. J. Microbiol. Methods 36, 203-213.

Kerkhof, L., Santoro, M., Garland, J., 2000. Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis. FEMS Microbiol. Lett. 184, 95-101.

Lysenko, O., 1985. Non-sporeforming bacteria pathogenic to insects: incidence and mechanisms. Annu. Rev. Microbiol. 39, 673-695.

Marsh, T.L., Saxman, P., Cole, J., Tiedje, J., 2000. Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. Appl. Environ. Microbiol. 66, 3616-3620.

McCormick, J.B., 1998. Epidemiology of emerging/re-emerging antimicrobial-resistant bacterial pathogens. Curr. Opin. Microbiol. 1, 125-129.

McFall-Ngai, M.J., 1998. The development of cooperative associations between animals and

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Muyzer, G., Smalla, K., 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73, 127-141.

O’Connell, S., Lawson, R.D., Watwood, M.E., Lehman, R.M., 2000. BASIC program for reduction of data from community-level physiological profiling using biolog microplates: rationale and critical interpretation of data. J. Microbiol. Methods 40, 213-220.

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Paster, B.J., Dewhirst, F.E., Cooke, S.M., Fussing, V., Poulsen, L.K., and Breznak, J.A., 1996. Phylogeny of not-yet-cultured spirochetes from termite guts. Appl. Environ. Microbiol. 62, 347-352.

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Savage, D., 1977. Microbial ecology of the gastrointestinal tract. Annu. Rev. Microbiol. 31, 107-133.

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Simpson, J.M., McCracken, V.J., White, B.A., Gaskins, H.R., Mackie, R.I., 1999. Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota. J. Microbiol. Methods 36, 167-179.

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