Long-term laboratory culture of symbiotic coral juveniles and their use in eco-toxicological study

Reef-building (or hermatypic) corals live in mutualistic symbiosis with the dinoflagellates Symbiodinium spp. (Alveolata, Dinophyceae, Gymnodiniales), and contribute to the accretion of coral reefs. Due to the difficulty in culturing them in laboratories, these ecologically important cnidarians have not been characterized extensively in physiological, biochemical, molecular and toxicological experiments. The present study was conducted to develop a model symbiosis system for long-term experimental analyses of a symbiotic coral. Aposymbiotic (symbiont-free) juveniles of the hermatypic coral Acropora tenuis were infected with three Symbiodinium strains, and the resulting symbiotic corals were examined for growth and maintenance of the symbiosis for approx. three months. Of the tested Symbiodinium cell lines, CCMP2467 (clade A1) inhabited the host the most densely, and the population in hospite did not decline over the period of three months in laboratory culture. The CCMP2467-inhabited juveniles outgrew the populations infected with the other two strains and aposymbiotic specimens. The A. tenuis juveniles in symbiosis with CCMP2467 cells were used in eco-toxicological tests to study long-term effects of two commonly used biocides (tributyltin-chloride and diuron). Delay in growth was observed after exposing the symbiotic juveniles to the two chemicals for approx. 50 days at the nominal concentrations of 0.4 and 1 μg/L, respectively.     

Distribution in relation to life history in the directdeveloping gastropod Batillaria cumingi (Batillariidae) on two shores of contrasting substrata

Population structure and spatial distribution with growth ofthe direct-developing gastropod, Batillaria cumingi, were investigatedon two shores of differing substrata. Sand-mud shore and rockyshore populations differed in size structure; first-year snailswere ca. 7 mm in shell length (SL) in both populations, whereassecond-year snails, merging with older cohorts, measured 15–25mm SL in the sand-mud shore population and ca. 15 mm SL in therocky shore population. Egg distribution matched adult distributionin the sand-mud shore population, but was more restricted thanthat of adults in the rocky shore population. The distributionof newly-hatched juveniles (0–1 mm SL) was restricted inboth populations, but the growth stage at which snails extendedtheir distribution differed between the two populations; 1–2.5mm SL on the sand-mud shore and 5 mm SL on the rocky shore. Floatingachieved by early juveniles (ca. 2 mm SL), was commonly observedin the sand-mud population, but rarely in the rocky shore population.The sudden expansion in distribution of the 1–2.5 mm SLgrowth stage in the sand-mud shore population is consideredto have been caused by floating, while expansion of the distributionof older growth stages (>5 mm SL) in the rocky shore populationprobably occurs by crawling. (Received 13 May 1998; accepted 25 August 1998)     

Size-related habitat use and schooling behavior in two species of surgeonfish (Acanthurus bahianus and A. coeruleus) on a fringing reef in Barbados, West Indies

Surgeonfish (Acanthuridae) are prominent, herbivorous members of coral reef communities that occur as dispersed individuals and small, loose groups ('non-schooling fish') or as members of large, highly aggregated, mixed-species schools ('schooling fish'). We examined the relationships among fish size, habitat use and schooling in two species of surgeonfish on a fringing reef in Barbados, West Indies. Both ocean surgeonfish, Acanthurus bahianus, and blue tangs, A. coeruleus, appeared to show ontogenetic habitat shifts. The density of juvenile ocean surgeonfish was highest in the back reef (inshore), lower on the reef crest (intermediate) and lowest in the spurs and grooves (offshore) zone, but schooling adults were most abundant in the spurs and grooves zone. In a multiple regression considering the effects of depth, algal cover, rugosity and distance from shore, the density of non-schooling ocean surgeonfish was positively associated with percent algal cover on the substratum and negatively with distance from shore. Newly settled blue tangs occurred only in the reef crest and spurs and grooves zones, but larger juveniles were more common in the back reef, while adults were more evenly distributed across zones. The density of non-schooling blue tang was positively associated with rugosity, distance from shore, and percent algal cover. In both species, schooling occurred primarily in adults; small juveniles never participated in the large, dense schools. The proportion of adults that were schooling increased from the back reef to the reef crest to the spurs and grooves zone. These results are consistent with the hypothesis that schooling permits adult surgeonfish access to higher quality food in the territories of damselfish (Pomacentridae) that predominate on the reef crest and spurs.     

Biomimicry of symbiotic multi-species coevolution for discrete and continuous optimization in RFID networks

In recent years, symbiosis as a rich source of potential engineering applications and computational model has attracted more and more attentions in the adaptive complex systems and evolution computing domains. Inspired by different symbiotic coevolution forms in nature, this paper proposed a series of multi-swarm particle swarm optimizers called PS²Os, which extend the single population particle swarm optimization (PSO) algorithm to interacting multi-swarms model by constructing hierarchical interaction topologies and enhanced dynamical update equations. According to different symbiotic interrelationships, four versions of PS²O are initiated to mimic mutualism, commensalism, predation, and competition mechanism, respectively. In the experiments, with five benchmark problems, the proposed algorithms are proved to have considerable potential for solving complex optimization problems. The coevolutionary dynamics of symbiotic species in each PS²O version are also studied respectively to demonstrate the heterogeneity of different symbiotic interrelationships that effect on the algorithm’s performance. Then PS²O is used for solving the radio frequency identification (RFID) network planning (RNP) problem with a mixture of discrete and continuous variables. Simulation results show that the proposed algorithm outperforms the reference algorithms for planning RFID networks, in terms of optimization accuracy and computation robustness.     

Agonistic Behaviour and Development of Territoriality During Ontogeny of the Sea Anemone Dwelling Crab Allopetrolisthes Spinifrons (H. Milne Edwards, 1837) (Decapoda: Anomura: Porcellanidae)

Allopetrolisthes spinifrons is an ectosymbiotic crab of the sea anemone Phymactis clematis . As a consequence of low host abundance, these represent a scarce and limited resource for the crab. Additionally, the relatively small size of the sea anemone host suggests that few symbiotic crabs can cohabit on one host individual, forcing crabs to adopt a territorial behaviour. In order to examine the potential presence and ontogenetic development of territoriality, the agonistic behaviour between crabs of various ontogenetic stages (adults, juveniles, and recruits) was studied in the laboratory. Laboratory experiments demonstrated that adult or juvenile crabs aggressively defended their sea anemone hosts against adult or juvenile intruders, respectively, but both adult and juvenile crabs tolerated recruits. Adult crabs behaved indifferently towards juvenile crabs, sometimes tolerating them, sometimes expelling them. Recruits never showed agonistic behaviour among themselves. The agonistic interactions observed in the laboratory and the uniform population distribution pattern on sea anemones recently described for A . spinifrons indicate that this species exhibits territorial behaviour, which develops during ontogeny. Territoriality in this species and other symbiotic decapods may function as a density-dependent mechanism of population regulation, being mediated by the availability of hosts. Resource monopolisation behaviours may be common among other symbiotic and free-living marine invertebrates inhabiting discrete habitats that represent a limiting resource.     

Agonistic Behaviour and Development of Territoriality During Ontogeny of the Sea Anemone Dwelling Crab Allopetrolisthes Spinifrons (H. Milne Edwards, 1837) (Decapoda: Anomura: Porcellanidae)

Allopetrolisthes spinifrons is an ectosymbiotic crab of the sea anemone Phymactis clematis. As a consequence of low host abundance, these represent a scarce and limited resource for the crab. Additionally, the relatively small size of the sea anemone host suggests that few symbiotic crabs can cohabit on one host individual, forcing crabs to adopt a territorial behaviour. In order to examine the potential presence and ontogenetic development of territoriality, the agonistic behaviour between crabs of various ontogenetic stages (adults, juveniles, and recruits) was studied in the laboratory. Laboratory experiments demonstrated that adult or juvenile crabs aggressively defended their sea anemone hosts against adult or juvenile intruders, respectively, but both adult and juvenile crabs tolerated recruits. Adult crabs behaved indifferently towards juvenile crabs, sometimes tolerating them, sometimes expelling them. Recruits never showed agonistic behaviour among themselves. The agonistic interactions observed in the laboratory and the uniform population distribution pattern on sea anemones recently described for A. spinifrons indicate that this species exhibits territorial behaviour, which develops during ontogeny. Territoriality in this species and other symbiotic decapods may function as a density-dependent mechanism of population regulation, being mediated by the availability of hosts. Resource monopolisation behaviours may be common among other symbiotic and free-living marine invertebrates inhabiting discrete habitats that represent a limiting resource.     

Equilibrium between the "genuine mutualists" and "symbiotic cheaters" in the bacterial population co-evolving with plants in a facultative symbiosis

The mathematical model for evolution of the plant-microbe facultative mutualistic interactions based on the partners’ symbiotic feedbacks is constructed. Using the example of rhizobia-legume symbiosis, we addressed these feedbacks in terms of the metabolic exchange resulting in the parallel improvements of the partners’ fitness (positive feedbacks). These improvements are correlated to the symbiotic efficiency dependent on the ratio of N₂-fixing bacterial strains (“genuine mutualists”) to the non- N₂-fixing strains (“symbiotic cheaters”) in the root nodules. The computer experiments demonstrated that an interplay between the frequency-dependent selection (FDS) and the Darwinian (frequency-independent) selection pressures implemented in the partners’ populations ensures an anchoring or even domination for the newly generated host-specific mutualists (which form N₂-fixing nodules only with one of two available plant genotypes) more successfully than for the non-host-specific mutualists (which form N₂-fixing nodules with both plant genotypes). The created model allows us to consider the mutualistic symbiosis as a finely balanced polymorphic system wherein the equilibrium in bacterial population may be shifted in favor of “genuine mutualists” due to the partner-stipulated selection for an improved symbiotic efficiency implemented in the plant population.     

Impediment to Symbiosis Establishment between Giant Clams and Symbiodinium Algae Due to Sterilization of Seawater

To survive the juvenile stage, giant clam juveniles need to establish a symbiotic relationship with the microalgae Symbiodinium occurring in the environment. The percentage of giant clam juveniles succeeding in symbiosis establishment (“symbiosis rate”) is often low, which is problematic for seed producers. We investigated how and why symbiosis rates vary, depending on whether giant clam seeds are continuously reared in UV treated or non treated seawater. Results repeatedly demonstrated that symbiosis rates were lower for UV treated seawater than for non treated seawater. Symbiosis rates were also lower for autoclaved seawater and 0.2-µm filtered seawater than for non treated seawater. The decreased symbiosis rates in various sterilized seawater suggest the possibility that some factors helping symbiosis establishment in natural seawater are weakened owing to sterilization. The possible factors include vitality of giant clam seeds, since additional experiments revealed that survival rates of seeds reared alone without Symbiodinium were lower in sterilized seawater than in non treated seawater. In conclusion, UV treatment of seawater was found to lead to decreased symbiosis rates, which is due possibly to some adverse effects common to the various sterilization techniques and relates to the vitality of the giant clam seeds.     

Aspects of the thermal ecology of the rusty crayfish Orconectes rusticus (Girard)

Summary Orconectes rusticus currently is undergoing an explosive range expansion in the midwestern U.S.A., but information on the potentially important effects of water temperature on the species' biology is lacking. The thermal ecology of O. rusticus in southwestern Ohio, U.S.A., was examined by determining 1) the effects of four water temperatures (16, 20, 25, and 29°C) on survival and growth of juveniles, 2) the responses of juveniles and adults to a thermal gradient (7–27° C), and 3) the thermal tolerances (critical thermal maximum, CTMax, and critical thermal minimum, CTMin) of free-living, field-acclimatized juveniles and adults on a biweekly basis throughout the summer. Month-long growth experiments predicted maximum growth rates of juveniles at water temperatures between 26 and 28°C, but greatest survival between 20 and 22° C. Laboratory-acclimated (22° C) adults and field-acclimatized (2.5° C) juveniles both had an acute preferred temperature of 22° C. CTMaxs and CTMins of juveniles were 0.5–2.6° C higher than those of adults throughout the summer, suggesting that juveniles were exposed to water temperatures 1.5–6.8° C warmer than those of adults. Juvenile and adult O. rusticus prefer habitats where water temperatures favor maximum survival, but they usually are not found together in the same habitat; adults apparently displace the juveniles into warmer habitats. Warmer temperatures can decrease survival of juveniles but improve their growth rates, leading to enhanced fecundity and competitive ability. The past and future success of O. rusticus in expanding its range may depend, in part, on the species ability to adjust to new thermal environments occupied by other species of crayfish.     

Assessing population increase as a possible outcome to management of invasive species

Some efforts to reduce invasive populations have paradoxically led to population increases. This phenomenon, referred to as overcompensation, occurs when reduced intraspecific pressures increase juvenile survival or maturation rates, leading to increased population size. Overcompensation in response to eradication efforts could derail management efforts, so it would be beneficial to evaluate the likelihood of overcompensation prior to removal. We conducted a series of experiments to examine the potential for overcompensation of a non-native population of the European green crab, Carcinus maenas, which was being removed in Bodega Harbor, California. First, we examined the impact of adults on juvenile survival by measuring adult cannibalism on juveniles in the presence and absence of alternative prey, and the survival of tethered juveniles at varying adult densities. Second, we examined how adult presence affected juvenile short-term foraging and growth rates. Although adult presence reduced juvenile short-term foraging, we detected only minimal cannibalism and found no evidence that adults greatly reduce juvenile growth or survivorship. These results suggest that overcompensation is not likely to occur in this population in response to removal. We assessed this prediction using pre- and post-removal surveys of juvenile recruitment in Bodega Harbor compared to nearby populations, testing for evidence of overcompensation. Relative juvenile abundance did not statistically increase in removal compared to reference populations, consistent with our conclusion from the experiments. This experimental approach which focuses on an organism’s population biology provides a tool to assess capacity for assessing the capacity for overcompensation in management strategies for invasive species.     

Comparative phylogeography, genetic differentiation and contrasting reproductive modes in three fungal symbionts of a multipartite bark beetle symbiosis

Multipartite symbioses are complex symbiotic relationships involving multiple interacting partners. These types of partnerships provide excellent opportunities in which to apply a comparative approach to identify common historical patterns of population differentiation and species-specific life history traits. Using three symbiotic blue-stain fungal species (Ophiostomatacea) associated with outbreaking populations of the mountain pine beetle (Dendroctonus ponderosae Hopkins) in western Canada, we applied phylogenetic, population genetic and demographic approaches to clarify phylogeographic patterns among the three fungal species. Broadly, the three species showed significant population differentiation, forming northern and southern populations, despite dramatic differences in haplotype diversity. Finer structuring and population demographic patterns were less consistent, showing some interspecific incongruence. By contrasting these species simultaneously, we were able to identify differences in recombination rate and ecological traits that can explain the observed patterns of incongruence among the fungal species. By applying a comparative approach to partners of a multipartite symbiosis, we were able to distinguish congruent population structuring and species-specific differences that help us to understand the complexity and evolution of this symbiotic system.     

Forest fragmentation genetics in a formerly widespread island endemic tree: Vateriopsis seychellarum (Dipterocarpaceae)

Habitat fragmentation and changed land use have seriously reduced population size in many tropical forest tree species. Formerly widespread species with limited gene flow may be particularly vulnerable to the negative genetic effects of forest fragmentation and small population size. Vateriopsis seychellarum (Dipterocarpaceae) is a formerly widespread canopy tree of the Seychelles, but is now reduced to 132 adult individuals distributed in eleven sites. Using ten microsatellite loci, a genetic inventory of all adult trees and a sample of 317 progeny, we demonstrate that despite its restricted range, overall genetic diversity was relatively high (H(E) : 0.56). The juvenile cohort, however, had significantly lower allelic richness (adults R(S) : 3.91; juveniles R(S) : 2.83) and observed heterozygosity than adult trees (adults H(O) : 0.62; juveniles H(O) : 0.48). Rare alleles were fewer and kinship between individuals was stronger in juveniles. Significant fine-scale spatial genetic structure was observed in remnant adults, and parentage analysis indicated that more than 90% of sampled progeny disperse <25 m and pollen dispersed <50 m. The molecular data confirmed that two populations were derived entirely from self-fertilized offspring from a single surviving mother tree. These populations produce viable offspring. Despite this extreme genetic bottleneck, self-compatibility may provide V. seychellarum with some resistance to the genetic consequences of habitat fragmentation, at least in the short term. We discuss our findings in the context of other rare and threatened dipterocarp species which are vulnerable to miss-management of genetic resources and population fragmentation.     

Toxicological effects of biocides on symbiotic and aposymbiotic juveniles of the hermatypic coral Acropora tenuis

Reef-building (or hermatypic) corals harbor the symbiotic dinoflagellates Symbiodinium spp. (Alveolata, Dinophyceae, Gymnodiniales), and contribute to the accretion of coral reefs in tropical and sub-tropical zones. In this study, toxicological effects of three commonly used biocides (dichlorvos (DDVP), a commonly used insecticide; diuron (DCMU), a herbicide; and tributyltin chloride (TBT-Cl), an anti-fouling agent) on the hermatypic coral Acropora tenuis (Anthozoa, Hexacorallia, Scleractinia) were studied using juveniles in both aposymbiotic (symbiont-free) and symbiotic conditions. After exposure to the chemicals, abnormalities such as detachment of soft tissues from the skeleton and/or death were observed, as well as lowered uptake of symbiotic algae (in aposymbiotic juveniles) or reduction of the symbiont population in tentacles (in symbiotic juveniles). Significant reduction of the symbiont population in tentacles of symbiotic juveniles exposed to DDVP, DCMU and TBT-Cl was observed at the concentrations of 100, 10, and 1 μg/l, respectively. Results of this study suggested that symbiotic juveniles of A. tenuis are more sensitive than in the aposymbiotic condition to DDVP and DCMU, but not to TBT-Cl.     

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria , a freshwater protozoan, typically harbors hundreds of symbiotic algae ( Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria . Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross-breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria , F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.     

Multiple bacterial symbionts in two species of co-occurring gutless oligochaete worms from Mediterranean sea grass sediments

Gutless oligochaete worms are found worldwide in the pore waters of marine sediments and live in symbiosis with chemoautotrophic sulfur-oxidizing bacteria. In the Mediterranean, two species of gutless oligochaete worms, Olavius algarvensis and O. ilvae, co-occur in sediments around sea grass beds. These sediments have extremely low sulfide concentrations (< 1 microM), raising the question if O. ilvae, as shown previously for O. algarvensis, also harbours sulfate-reducing symbionts that provide its sulfur-oxidizing symbionts with reduced sulfur compounds. In this study, we used fluorescence in situ hybridization (FISH) and comparative sequence analysis of genes for 16S rRNA, sulfur metabolism (aprA and dsrAB), and autotrophic carbon fixation (cbbL) to examine the microbial community of O. ilvae and re-examine the O. algarvensis symbiosis. In addition to the four previously described symbionts of O. algarvensis, in this study a fifth symbiont belonging to the Spirochaetes was found in these hosts. The symbiotic community of O. ilvae was similar to that of O. algarvensis and also included two gammaproteobacterial sulfur oxidizers and two deltaproteobacterial sulfate reducers, but not a spirochete. The phylogenetic and metabolic similarity of the symbiotic communities in these two co-occurring host species that are not closely related to each other indicates that syntrophic sulfur cycling provides a strong selective advantage to these worms in their sulfide-poor environment.     

Trophic interactions among native and introduced fishes in a northern Patagonian oligotrophic lake

In summer the littoral zone of Lake Rivadavia contained almost all the juveniles of the native fish species present in the lake together with juveniles of the introduced salmonid Oncorhynchus mykiss . In particular, a high degree of foraging interaction was found among juveniles of the native species Aplochiton zebra and Percichthys trucha and juveniles O. mykiss. Differences in the juvenile assemblage composition, both in the presence and in the relative proportion of the species were associated with areas with different densities of vegetation. The adults of nearly all species were generalized benthic invertebrate predators. Only adults of A. zebra were absent in the littoral. Juvenile and adult O. mykiss were the only fish that preyed on planktonic organisms. Oncorhynchus mykiss was the only species that performed an ontogenetic diet shift between juveniles and adults, changing main prey categories from benthic to planktonic species.     

The recent evolution of a symbiotic ion channel in the legume family altered ion conductance and improved functionality in calcium signaling

Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wild-type and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.     

Trans-generational specificity within a cnidarian–algal symbiosis

Ocean warming and other anthropogenic stresses threaten the symbiosis between tropical reef cnidarians and their dinoflagellate endosymbionts (Symbiodinium). Offspring of many cnidarians acquire their algal symbionts from the environment, and such flexibility could allow corals to respond to environmental changes between generations. To investigate the effect of both habitat and host genotype on symbiont acquisition, we transplanted aposymbiotic offspring of the common Caribbean octocoral Briareum asbestinum to (1) an environmentally different habitat that lacked B. asbestinum and (2) an environmentally similar habitat where local adults harbored Symbiodinium phylotypes that differed from parental colonies. Symbiont acquisition and establishment of symbioses over time was followed using a within-clade DNA marker (23S chloroplast rDNA) and a within-phylotype marker (unique alleles at a single microsatellite locus). Early in the symbiosis, B. asbestinum juveniles harbored multiple symbiont phylotypes, regardless of source (parent or site). However, with time (~4 yr), offspring established symbioses with the symbiont phylotype dominant in the parental colonies, regardless of transplant location. Within-phylotype analyses of the symbionts revealed a similar pattern, with offspring acquiring the allelic variant common in symbionts in the parental population regardless of the environment in which the offspring was reared. These data suggest that in this host species, host–symbiont specificity is a genetically determined trait. If this level of specificity is widespread among other symbiotic cnidarians, many cnidarian–algal symbioses may not be able to respond to rapid, climate change-associated environmental changes by means of between-generation switching of symbionts.     

Tyrosinase from Rhizobium etli is involved in nodulation efficiency and symbiosis-associated stress resistance

Tyrosinase (EC 1.14.18.1) is a monophenol oxidase responsible for the synthesis of the black pigment known as melanin. The tyrosinase gene (melA) is plasmid-encoded in many rhizobial species. In Rhizobium etli CFN42, the genetic location of melA in the symbiotic plasmid (p42d) and its RpoN-NifA regulation suggest an involvement in symbiosis. In this work, we analyzed the symbiotic phenotype of a streptomycin-resistant derivative of CFN42 (CE3), a melA mutant (SP2) and a complemented strain (SP66), demonstrating that melA inactivation reduced nodule formation rate and diminished total nodule number by 27% when compared to the CE3 strain. The nitrogen fixation capacity of the mutant strain was not affected. Also, in vitro assays were performed where the resistance of CE3, SP2 and SP66 strains to H(2)O(2) was evaluated; the melA mutant strain was consistently less resistant to peroxide. In another series of experiments, Escherichia coli W3110 strain expressing R. etli melA displayed enhanced resistance to p-hydroxybenzoic, vanillinic and syringic acids, which are phenolic compounds frequently found in the soil. Our results are the first to demonstrate a specific role for tyrosinase in R. etli: this enzyme is required during early symbiosis, apparently providing resistance against reactive oxygen species and phenolic compounds generated as part of the plant protective responses.