term pattern of codiversification of hosts and endosymbionts. The association between .... per 50 million years is roughly twice as fast as pre- vious approximate ...
39
Antonie van Leeuwenhoek 72: 39–48, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Non-cultivable microorganisms from symbiotic associations of insects and other hosts Paul Baumann1 & Nancy A. Moran2 1
Microbiology Section University of California Davis, CA 95616-8665, USA; 2 Department of Ecology and Evolutionary Biology University of Arizona Tucson, AZ 85721, USA
Received 18 September 1996, accepted 25 September 1996
Key words: Buchnera, cospeciation, endosymbionts, rRNA phylogeny, symbiosis, tryptophan biosynthesis, leucine biosynthesis
Abstract Many symbiotic associations involve microorganisms which cannot be cultivated on laboratory media. These organisms remained little known until the recent advent of methods of recombinant DNA analysis and molecular phylogenetics. Applications of these methods to endosymbionts have resulted in substantial new insights concerning the genetics and evolution of these organisms. This communication provides a listing of recently studied associations involving non-cultivable symbionts. The associations involve a diverse set of host taxa and a wide range of effects, both favorable and deleterious, on host biology. Among beneficial endosymbionts, a variety of nutritional interactions have been documented. One type of association has been demonstrated for a number of animal hosts, namely endosymbioses that result from a single infection of an ancestral host by a prokaryote. In these associations, endosymbionts are transmitted maternally and are not exchanged between host lineages, resulting in a longterm pattern of codiversification of hosts and endosymbionts. The association between aphids and non-cultivable prokaryotic endosymbionts is a well studied example of such a symbiosis. Abbreviations: kb – kilobase; rDNA – DNA coding for ribosomal RNA; rRNA – ribosomal RNA; S-endosymbiont – secondary-endosymbiont. Introduction Many symbioses involve microorganisms (symbionts) which are associated with a unicellular or multicellular eukaryotic host (Buchner 1965). The symbionts can be prokaryotes (Bacteria & Archaea) or eukaryotes and frequently they cannot be cultured on common laboratory media. During the past decade, the application of recombinant DNA methods to the characterization of non-cultivable pathogenic organisms and the development of methods for establishing phylogenetic relationships through analyses of rRNA sequences has led to a resurgence of interest in non-cultivable microbial symbionts (Amann et al. 1995; Baumann et al. 1995). As expected, microbial symbioses are very diverse with as yet few unifying principles. In this communication, we tabulate the non-cultivable micro-
bial symbionts whose phylogeny has been established or which are of interest due to physiological or genetic studies. In addition, we consider in greater detail some aspects of symbiotic associations of insects with emphasis on the lessons emerging from the studies of aphid endosymbionts. The original definition of symbiosis included longterm associations of two different partners which are in close proximity (Smith & Douglas 1987). Such associations may be intracellular in that the endosymbiont resides within the host cell or they may be extracellular in that the symbiont and the host remain separate but in close proximity. This definition of symbiosis encompasses mutualistic associations in which the members of the new composite organism experience favorable gain, frequently at the expense of their separate autonomy. In addition, it includes parasitic associations in
40 Table 1. General properties and taxonomic affiliations of resently studied, mostly non-cultivable, insect symbiontsa Host category
Principal host Symbiont food designationb
Aphids (Aphidoidea)
Plant sap
Whiteflies (Aleyrodoidea)
Mealybugs (Pseudococcidae) Planthoppers (Cicadelidae) Tsetse flies (Glossinidae)
Carpenter ants (Camponotus) Cockroaches (Blattaria) Termite (Isoptera)
Weevils (Curculionidae)
Many insect species and other arthropods Beetle (Coleoptera)
Number of Primery location host species of symbionts examined Intra- Extracellular cellular (endosymbionts)
Buchnera aphidicola 17c S-endosymbiont 2d
+ +
Rickettsia 1e Yeast-like organisms 13f
+
16S rRNA group References and/or other taxonimic affiliation
-Proteobacteria
-Proteobacteria,
Baumann et al. 1995 Unterman et al. 1989, Enterobacteriaceae Chen & Purcell 1996 -Proteobacteria Chen et al. 1996 Body cavity Pyrenomycetes Fukatsu et al. 1994, Fukatsu & Ishikawa 1996
-Proteobacteria Clark et al. 1992
P-endosymbiont
3g
+
S-endosymbiont
1h
+
Plant sap
P-endosymbiont
3i
+
Clark et al. 1992 Enterobacteriaceae -Proteobacteria Munson et al. 1992
Plant sap
Yeast
3i
+
Pyrenomycetes
Noda et al. 1995
Animal blood Wigglesworthia glossinidia S-endosymbiont
5k
+
-Proteobacteria
Aksoy 1994, 1995
Plant nectar, honeydew Universalists
P-endosymbiont
4m
+
Blattabacterium cuenoti Blattabacterium cuenoti Spirochetes
7n
+
1o
+
2o;p
Gut
Multiple types
1q
Gut
Metadevescovina extranea Pentatrichomonoides scroa P-endosymbiont Symbiotaphrina buchneria S. kochiia Wolbachia pipientisu Rickettsia
1o
Gut
Proteobacteria, Bacteroides, Spirochetes, Low G+C Grampositive bacteria, methanogens Trichomonad
1o
Gut
Trichomonad
Plant sap
Dead wood
Stored grain
-Proteobacteria
-Proteobacteria,
2l
2r
+
1s 1t Many
+ + +
1v
+
Beard et al. 1993 Enterobacteriaceae
-Proteobacteria Schr¨oder et al. 1996 FlavobacteriumBacteroides FlavobacteriumBacteroides Spirochetes
-Proteobacteria,
Bandi et al. 1994, 1995 Bandi et al. 1995 Berchtold et al. 1994, Paster et al. 1996 Ohkuma et al. 1995, Ohkuma & Kudo 1996
Berchtold & K¨onig 1995
Campbell et al. 1992 Enterobacteriaceae Related to Noda & Kodama 1996 Discomycetes (yeast) -Proteobacteria O’Neill et al. 1997, Werren et al. 1995 -Proteobacteria Werren et al. 1994
41 a Symbionts which have been cultivated outside the host are underlined. b P=primery, S=sectondary endosymbiont. c In addition to the host species given in Figure 1, B. aphidicola was detected in the following aphid species: Uroleucon ambrosiae, U. astronomus, U. caligatum, U. erigeronensis. Their assignment to B. aphidicola is based on trpB. d A. pisum, Macrosiphum rosae. e pisum. f For list see Fukatsu et al. 1994. g Bemisia tabaci, Siphoninus phillyreae, Trialeurodes vaporariorum. h B. tabaci. i Dysmicoccus neobrevipes, Pseudococcus longispinus, P. maritimus. j Laodelphax striatellus, Nilaparvata lugens, Sogatella furcifera. k Glossina austeni, G. brevipalpis, G. morsitans, G. palpalis, G. tachinoides. l Glossina pallidipes, G. morsitans. m Camponotus floridanus, C. herculeanus, C. ligniperdus, C. rufipes. n Blaberus craniifer, Blattella germaninca, Cryptocercus punctulatus, Nauphoeta cinerea, Periplaneta americana, P. australasiae, Pycnoscelus surinamensis. o Mastotermis darwiniensis. p Nasutitermes lujae. q Reticulitermes speratus. r Sitophilus oryzae, S. zeamais. s Stegobium paniceum. t Lasioderma serricorne. u Causative agent of reproductive disorders in insects and other vertebrates. v Adalia bipunctata (ladybird beetle).
which the symbiont may derive some advantage at the expense of the host. In practice, it is often difficult to fit a symbiotic association into these simple categories. In some cases, the principal function of the symbiont in terms of its contribution to the host has been well established. In other cases, the parasitic nature of the symbiont is reasonably clear. Unfortunately, for most non-cultivable symbionts, the beneficial, neutral or deleterious effect on the host is not known.
Insect symbiotic associations Table 1 presents a summary of the recent studies of non-cultivable symbionts of insects. The best understood symbiotic association involving insects is that between termites and gut-inhabiting microorganisms (protozoa and bacteria) (Breznak & Brune 1994). This is an extracellular association in that the symbionts are located in a specialized enlargement of the termite gut known as the paunch. Most of the bacteria and some of the protozoa found in the termite paunch have not been cultured. Two additional and contrasting endosymbioses have been the subject of considerable recent work. These are the mutualistic associations between aphids and members of the genus Buchnera and the parasitic associations between Wolbachia pipientis and a variety of insect species and other arthropods (Baumann et al. 1995; O’Neill et al. 1996; Werren et al. 1995). Neither of these organisms has been cultured. A book devoted to Wolbachia pipientis is currently in press (O’Neill et al. 1997).
Aphids and their endosymbionts General properties Aphids can be thought of as living syringes that insert their needle-like stylets into the plant phloem tissue and suck up plant sap (Baumann et al. 1995). Due to this mode of feeding, aphids vector plant virus-
es which cause major losses to agricultural crops in addition to the direct feeding damage. Inside the body cavity of most aphids is a bilobed structure called the bacteriome consisting of 60-90 polyploid cells called bacteriocytes. These cells contain host-derived vesicles which house gram-negative spherical or oval bacteria assigned to the genus Buchnera (Table 1). Each aphid acquires Buchnera from its mother. A typical fully mature aphid weighs about 500 g and contains 5.6 million cells of Buchnera. Treatment of aphids with antibiotics results in the elimination of the endosymbiont with a concomitant reduction in the rate of aphid growth and eventual sterility of the aphid. Surrounding the bacteriome is a sheath consisting of a thin layer of flattened cells. In some aphids, these cells may harbor a gram-negative, rod-shaped bacterium called the S-endosymbiont (Table 1). The S-endosymbionts, which are also maternally transmitted, are generally fewer in number than Buchnera. Many species of aphids lack S-endosymbionts, and some species show variation among strains in whether or not S-endosymbionts are present, suggesting that they are not essential for host survival (Chen & Purcell 1996). It is not known whether S-endosymbionts have any beneficial effects on their hosts. Coevolution of Buchnera and the aphid hosts Phylogenetic analyses based on small subunit (16S or 18S) rDNA are routinely used to elucidate evolutionary relationships within both prokaryotes and eukaryotes. Analyses of bacterial 16S rDNA show that Buchnera from a diverse assemblage of aphids form a single monophyletic group (Figure 1). Furthermore, the relationships obtained within Buchnera agree with the established host classification and with reconstructions of aphid phylogeny based on morphology and on 18S rDNA (Baumann et al. 1995; Moran & Baumann 1994; Moran et al. 1995; von Dohlen & Moran 1995). This congruence of phylogenetic trees between aphids and corresponding Buchnera strongly supports the view that a single ancient infection of a common ancestor of
42
Figure 1. Evolutionary relationships of Buchnera and the aphid hosts. The endosymbiont tree is based on 16S rDNA; the aphid tree is based on 18S rDNA and the fossil record. Full generic and specific names are used for free-living bacteria. Aphid generic abbreviations and species names are used in the Buchnera lineage. A, Acyrthosiphon; C, Chaitophorus; D, Diuraphis; M, Myzus; Me, Melaphis; Mi, Mindarus; P, Pemphigus; R, Rhopalosiphum; S, Schizaphis; Sl, Schlechtendalia; U, Uroleucon; MY, million years.
all aphids has been vertically transmitted through the various lineages of aphids as they diversified. Because the common ancestor of all aphids is estimated, from fossil evidence, to be 150-250 million years old, the original infection must date back at least this far. Following this initial infection, endosymbionts and aphid hosts appear to have diversified in parallel, resulting in the present strains of Buchnera which are associated with the present species of aphids. Using the fossil record to estimate times of divergence for aphid hosts and extending the same dates to the corresponding endosymbionts, it has been possible to calculate the rate of nucleotide base substitution within the 16S rDNA gene of Buchnera. The rate of 1-2% per site per 50 million years is roughly twice as fast as previous approximate estimates of evolutionary rates in 16S rDNA of free-living prokaryotes (Moran & Baumann 1994). An accelerated rate of evolution appears to be a characteristic of endosymbiotic bacteria in general (Moran 1996). One hypothesis for the cause of this acceleration is that endosymbionts are subject to bottlenecks in population size that result in more frequent fixation of slightly deleterious mutations through genetic drift (i.e., by chance events). This interpretation is consistent with the observation that the acceleration in evolutionary rate is concentrated at sites in the DNA sequences that are more subject to conservative natural selection (Moran 1996). Possibly natural selection is
less effective at slowing rates of substitution at such sites, due to the population structure of these bacteria. Mealybugs and whiteflies are groups of insects that are related to aphids. Like aphids, these insects feed on plant sap as their sole diet and harbor endosymbionts. Phylogenetic studies based on 16S rDNA have indicated that the endosymbionts of each of these groups descend from infections separate from the one giving rise to Buchnera (Table 1). Similar investigations on the endosymbionts of cockroaches, carpenter ants, and tsetse flies have shown that these also result from additional, independent infections by free-living bacteria (Table 1). Together, these molecular phylogenetic studies of endosymbionts of diverse insect groups suggest that infections that lead to endosymbiotic associations have occurred repeatedly in different groups of hosts and have arisen from a variety of free-living bacterial groups. These results also suggest that endosymbiotic associations within insects can be evolutionarily stable for very long timespans and through periods of codiversification of hosts and bacteria. In contrast to Buchnera, which forms a clade rather distant from any known bacteria in the same division, the S-endosymbionts of Acyrthosiphon pisum (Figure 1) and Macrosiphum rosae fall within the monophyletic bacterial group Enterobacteriaceae, which includes such well studied organisms as Escherichia coli and Proteus vulgaris (Baumann et al. 1995; Chen & Purcell 1996).
43 Buchnera resembles free-living bacteria The nucleotide sequence of over 65 kb of DNA has been determined for Buchnera from the aphid Schizaphis graminum. Analyses of these data show that Buchnera has many of the genes present in free-living bacteria. The detected genes include ones coding for proteins that are involved in DNA replication, messenger RNA synthesis, protein synthesis, amino acid biosynthesis, glycolysis, ATP generation, protein secretion, and protein folding. The presence of these genes with diverse functions is consistent with previous studies indicating that isolated cells of Buchnera can synthesize over 210 proteins and can incorporate radioactive precursors into DNA and rRNA (Ishikawa 1989). During growth of the aphid, the increase in number of Buchnera parallels the increase in aphid weight (Baumann et al. 1995). These observations imply a strict regulation of endosymbiont number and also suggest that the orderly events characteristic of bacterial growth, involving the expression of many genes and the synthesis of a large number of proteins, occur during Buchnera growth. The doubling time of Buchnera in the aphid is about 1.5 to 2.0 days, a timespan much longer than doubling times attainable by many freeliving bacteria. Since bacteria growing at a slow rate have a reduced demand for ribosomes, many bacteria with low doubling times have only one or two copies of the rRNA operon (Baumann et al. 1995). This is also the case with Buchnera, which contains a single copy of the genes for rRNAs. In addition, these genes show an unusual organization in Buchnera. In most free-living bacteria, rRNA genes form a single transcription unit (16S-23S-5S rRNA), whereas in Buchnera they are arranged as two transcription units (16S rRNA and 23S-5S rRNA) (Baumann et al. 1995). Endosymbionts sometimes are postulated to represent a transitional stage between free-living bacteria and organelles, such as mitochondria or chloroplasts, that originated from prokaryotes. A characteristic of mitochondria and chloroplasts is a major reduction of the genome size, with a concomitant decrease in gene coding capacity, and the transfer of many essential genes to the host cell nucleus. Buchnera differs from organelles in its large genome size and the retention of many genes that are present in free-living bacteria but absent from organelles (Baumann et al. 1995). The basis for this difference may involve the characteristics of the host in which the endosymbiotic association first originated. The ancestors of mitochondria and chloroplasts infected a unicellular eukaryote; in contrast, the
ancestor of Buchnera infected a multicellular animal host in which it was sequestered within somatic cells separate from those of the germ line. This separation may have limited the opportunities for the permanent transfer of genes from the prokaryotic chromosome to the nuclear genome of the host, since such transfer would persist only if it occurred in the germ line. The role of Buchnera in the production of tryptophan for the aphid host Plant phloem sap, the diet of aphids, is rich in carbohydrates but deficient in certain nitrogenous compounds including some of the ten essential amino acids required by insects and other animals. The biosynthetic activities of endosymbionts have been proposed as the source of these amino acids for aphids (Baumann et al. 1995). Evidence for this interpretation has come from a variety of nutritional studies of aphid growth on synthetic media. Recent investigations have presented evidence for the synthesis by Buchnera of the amino acids tryptophan, cysteine and methionine (Baumann et al. 1995; Douglas 1990; Douglas & Prosser 1992). The tryptophan biosynthetic pathway, from chorismate to tryptophan, consists of seven enzymatic reactions. The genetics and biochemistry of this pathway have been elucidated in many different prokaryotes (Crawford 1989). In almost all cases, anthranilate synthase, the first enzyme of the pathway, is rate-limiting and is feedback inhibited by tryptophan. In free-living bacteria, this mode of regulation assures that tryptophan is not made when this amino acid is available, thereby conserving carbon and energy for other cellular processes. If tryptophan is to be overproduced by Buchnera in order to supply its aphid host, this regulatory mechanism would have to be modified so as to allow the synthesis of tryptophan even under conditions of its accumulation. All of the genes for the tryptophan biosynthetic pathway are present in Buchnera from the aphid S. graminum (Figure 2). These endosymbionts show an unusual modification that permits the increased synthesis of tryptophan for use by the aphid host. The genes for anthranilate synthase (trpEG ) are located on a plasmid. The trpEG plasmid in this species consists of four tandem repeats of a 3.6 kb unit that contains trpEG. The remaining genes [trpDC(F)BA] are on the Buchnera chromosome. About four such plasmids are present per Buchnera cell, resulting in an approximately 16-fold amplification of trpEG (Figure 2). Gene amplification would lead to an increase in
44
Figure 2. Genetics of the tryptophan biosynthetic pathway in Buchnera from the aphids S. graminum and Sl. chinensis. Thin line, DNA; thick line, protein coding regions. In Buchnera from S. graminum trpEG is amplified by being on a plasmid consisting of four tandem repeats of a 3.6 kb unit. In Sl. chinensis trpEG is located on the endosymbiont chromosome. Each enzymatic reaction is designated by an arrow. Lines link the enzymes (arrows) to their corresponding genes. Stippled bar on the plasmid designates a region having properties of a DNA origin of replication (ori?), arrow heads on plasmid indicate direction of transcription, chromosomal genes are transcribed left to right.
the amount of anthranilate synthase protein, thereby resulting in overproduction of tryptophan by Buchnera for the aphid host. In both prokaryotes and eukaryotes, gene amplification is widely documented as a common mechanism for increasing the amount of an enzyme, whenever growth is limited by the level of an enzyme activity (Anderson & Roth 1977). Gene amplification of trpEG has been detected in Buchnera from the other members of the Aphididae that have been examined, including the aphids Rhopalosiphum padi, R. maidis, A. pisum, Diuraphis noxia, Uroleucon rubale and U. caligatum (Lai et al. 1996; Rouhbakhsh et al. 1996). All of these aphids have a rapid development time reaching maturity less than two weeks after birth. In contrast to these aphids, Schlechtendalia chinensis (Figure 2), has a development time of over 6 weeks. In Buchnera from Sl. chinensis, trpEG is not amplified and is present as one copy on the endosymbiont chromosome (Baumann et al. 1995). The difference between Sl. chinensis and the other aphids may be linked to a difference in the demand for tryptophan. Aphids with a short development time would require a higher rate of tryptophan provision, hence amplification of trpEG. In contrast,
Figure 3. Plasmid containing genes of the leucine biosynthetic pathway from Buchnera of the aphid R. padi. Each enzymatic reaction is designated by an arrow. Lines link the enzymes (arrows) to their corresponding genes. Arrow heads on plasmids indicte direction of transcription. Redrawn from the nucleotide sequence of Bracho et al. 1995.
in aphids with a long development time, the demand for tryptophan is reduced and gene amplification may be unnecessary. Phylogenetic analyses based on trpEG sequences result in trees with the same order of branching as trees based on chromosomal trpB, 16S rDNA, and mitochondrial rRNA genes and cytochrome oxidase (Rouhbakhsh et al. 1996). These results add support for the long-term vertical transmission of these genes and indicate a lack of genetic transfer between plasmids and endosymbionts of different aphid lineages. In addition, the congruence of trees based on trpEG and on other genes indicates that the amplified trpEG originated from a chromosomal trpEG and not from an exogenous source. Plasmid location of the genes underlying leucine biosynthesis Recently it has been found that Buchnera from the aphid R. padi harbors a 7.8 kb plasmid containing the genes of the leucine biosynthetic pathway (Figure 3) (Bracho et al. 1995). Leucine is an essential amino acid and is a member of the pyruvate family which includes valine and isoleucine. The leucine portion of the pathway is initiated from -ketoisovalerate which is also a branch point for the synthesis of valine. Ketoisovalerate is converted to leucine by means of four enzymatic reactions. The Buchnera plasmid contains the genes coding for three enzymes of the pathway (Figure 3). The fourth reaction, which is a transamina-
45 Tabel 2. General properties and taxonomic affiliation of some recently studied, non-cultivable, symbionts from a variety of animals Host category
Location Intracellular (endosymbionts)
Vestimentiferans
+
Vestimentiferans
+
Bivalve molluscs
+
Comments
+ +
Marine annelids
+ Animal surface
Marine nematode
Animal surface
Luminous fish
Specialized organs
Aerobic protozoa
+
Anaerobic protozoa
+ +
16S rRNA group
References
-Proteobacteria
Cavanaugh 1994
-Proteobacteria
Cavanaugh 1994
Extracellular
Cell surface
Sulfur-oxidizing chemoautotrophs Sulfur-oxidizing chemoautotrophs Sulfur-oxidizing chemoautotrophs Methanotrophs Cells contain both sulfer-oxidizing chemoautotrophs and methanotrophs Sulfur-oxidizing chemoautotrophs Sulfur-oxidizing chemoautotrophs Sulfur-oxidizing chemoautotroph Emit light
-Proteobacteria
-Proteobacteria
-Proteobacteria
Distel et al. 1994, Kim et al. 1995 Distel & Cavanaugh 1994 Distel et al. 1995
-Proteobacteria
Doubilier et al. 1995
-Proteobacteria
Haddad et al. 1995
-Proteobacteria
Polz et al. 1994
-Proteobacteria
Haygood & Distel 1993
-Proteobacteria -Proteobacteria
-Proteobacteria
Methanogens Anaerobic photosynthesis Sulfate reducing
tion, can in E. coli be catalyzed by several enzymes: the genes for these enzymes are not included on the plasmid. A similar plasmid has also been found in Buchnera from R. maidis, S. graminum and two other species in the same family of aphids (Bracho et al., 1995). The chaperone, GroEL, is overproduced in Buchnera All prokaryotes produce the essential proteins GroEL and GroES which are members of a class of proteins known as ‘chaperones’, whose major role is the prevention of misfolding that might occur during protein synthesis, translocation across membranes and recovery from stress. Many of these proteins are present in high levels within cells under laboratory conditions of
Archaea
-Proteobacteria -Protebacteria
Amann et al. 1991; Du et al. 1994a 1994b; Gauton & Fritsche 1995; Hookey et al. 1996 Jeon 1992; Springer et al. 1992, 1993, 1996; Viale et al. 1994 Embley & Finlay 1994 Fenchel & Bernard 1993, Fenchel and Finley 1994 Fenchel & Ramsing 1992
cultivation. Since a variety of deleterious conditions, including an increase in temperature, results in their increase, these proteins have also been called ‘stress’ or ‘heat shock’ proteins. Buchnera from the aphids A. pisum and S. graminum have groES-groEL arranged in the same order as in E. coli. These genes are also preceded by a putative promoter containing nucleotide sequences similar to those recognized by 32 (Baumann et al. 1995; Ohtaka et al. 1992). In Buchnera from S. graminum GroEL constitutes about 10% of the total protein (Baumann et al. 1996). This level is only slightly less than that in E. coli at 46 C, a temperature near the maximum at which growth occurs and one at which many of the E. coli stress proteins are present in high amounts. Many intracellular pathogenic bacteria
46 as well as several endosymbionts also have increased or high levels of GroEL (Baumann et al. 1996). Although the function of this increased level is not known, it is probably an adaptation to the intracellular association. High levels of GroEL also have been observed in endosymbionts of tsetse flies (Aksoy 1994).
Symbiotic associations of prokaryotes with other eukaryotes In many mutualistic endosymbioses involving prokaryotes and insects it is likely that the host is able to take advantage of the biosynthetic capability of the endosymbiont which provides the host with essential nutrients required for growth. Many prokaryotes are also unusual with respect to their ability to utilize substrates as sources of energy which are not utilized by eukaryotes. Table 2 lists the symbiotic associations between a variety of non-cultivable prokaryotes and animals. Many of these associations involve the exploitation of a uniquely prokaryotic metabolic attribute by the eukaryotic host allowing the composite organism to live off substrates which cannot be metabolized by the host. One of the most extensively studied symbioses of this type involves bacteria which are able to utilize reduced-sulfur compounds as a source of energy and fix carbon dioxide into organic carbon. These associations are widespread in marine environments (Table 2) and have been recently reviewed (Cavanaugh 1994; Nelson & Fisher 1995). Distal et al. (1994) found congruence between the phylogeny of such chemoautotrophic endosymbionts of bivalve molluscs and their hosts. Some bivalve molluscs also have been found to have methanotrophic endosymbionts alone or in combination with sulfur-oxidizing chemoautotrophs (Table 2). A variety of protozoa also have endosymbionts. In the case of anaerobic protozoa many of these are methanogens (Archaea) although one is an anaerobic photosynthetic bacterium (Table 2). Many of the endosymbionts of aerobic protozoa cause the death of protozoa that lack these endosymbionts. One mutualistic association between Amoeba proteus and a non-cultivable bacterium has been studied in some detail (Jeon 1992). This organism belongs to the Proteobacteria (Viale et al. 1994) and appears to be related to Legionella, a group of organisms which inhabit protozoa (Barker & Brown 1994). It has been shown that the endosymbiont makes a number of proteins at least one of which enters the host cytoplasm
while others are associated with the host-derived vesicle in which the endosymbiont is found and may protect it from digestion by the host (Jeon 1992, Kim et al. 1994). In a separate category from these nutrition-based symbioses are the symbiotic associations between noncultivable luminous bacteria and marine fish (Table 2). In these cases, the hosts exploit an unusual property (bioluminescence) found in some bacteria.
Conclusions The phylogeny and genetics of the associations involving non-cultivable symbionts have only recently begun to be studied. It is, therefore, probably hazardous to attempt to derive general principles, which may, in any case, not be found due to the diversity of the associations. Nevertheless, the currently available information suggests that many mutualistic endosymbioses of animal hosts are a consequence of a single infection of an ancestral host by a free-living bacterium. Following the establishment of the association, endosymbiont transmission is exclusively vertical and host and endosymbiont coevolve with no genetic exchange between lineages. This interpretation is strongly supported by studies of diverse endosymbiotic associations of animals, including a variety of insects and marine animals. In these associations, the endosymbiont inhabits cells separate from the germ line, and it would be of interest to know whether its genome size is reduced or remains similar to that of free-living bacteria. Most genes appear to be retained in endosymbionts of aphids, suggesting that these endosymbioses are not representative of a an early evolutionary stage in the origin of organelles. Additional studies are needed before other trends can be established.
Acknowledgements Research from the author’s laboratory was supported by NSF grants IBN-9201285 (PB), MCB-9402813 (PB), DEB-9306495 (NAM), DEB-9527635 (NAM and PB), Entotech Inc. (Novo Nordisk) (PB) and the University of California Experiment Station (PB).
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