Vascular architecture in the bacteriogenic light organ of Euprymna tasmanica (Cephalopoda: Sepiolidae).

Symbiosis between southern dumpling squid, Euprymna tasmanica (Cephalopoda: Sepiolidae), and its luminescent symbiont, the bacterium Vibrio fischeri, provides an experimentally tractable system to examine interactions between the eukaryotic host and its bacterial partner. Luminescence emitted by the symbiotic bacteria provides light for the squid in a behavior termed "counter-illumination," which allows the squid to mask its shadow amidst downwelling moonlight. Although this association is beneficial, light generated from the bacteria requires large quantities of oxygen to maintain this energy-consuming reaction. Therefore, we examined the vascular network within the light organ of juveniles of E. tasmanica with and without V. fischeri. Vessel type, diameter, and location of vessels were measured. Although differences between symbiotic and aposymbiotic squid demonstrated that the presence of V. fischeri does not significantly influence the extent of vascular branching at early stages of symbiotic development, these finding do provide an atlas of blood vessel distribution in the organ. Thus, these results provide a framework to understand how beneficial bacteria influence the development of a eukaryotic closed vascular network and provide insight to the evolutionary developmental dynamics that form during mutualistic interactions.

Evolutionary perspectives in a mutualism of sepiolid squid and bioluminescent bacteria: combined usage of microbial experimental evolution and temporal population genetics.

The symbiosis between marine bioluminescent Vibrio bacteria and the sepiolid squid Euprymna is a model for studying animal-bacterial Interactions. Vibrio symbionts native to particular Euprymna species are competitively dominant, capable of outcompeting foreign Vibrio strains from other Euprymna host species. Despite competitive dominance, secondary colonization events by invading nonnative Vibrio fischeri have occurred. Competitive dominance can be offset through superior nonnative numbers and advantage of early start host colonization by nonnatives, granting nonnative vibrios an opportunity to establish beachheads in foreign Euprymna hosts. Here, we show that nonnative V. fischeri are capable of rapid adaptation to novel sepiolid squid hosts by serially passaging V. fischeri JRM200 (native to Hawaiian Euprymna scolopes) lines through the novel Australian squid host E. tasmanica for 500 generations. These experiments were complemented by a temporal population genetics survey of V. fischeri, collected from E. tasmanica over a decade, which provided a perspective from the natural history of V. fischeri evolution over 15,000-20,000 generations in E. tasmanica. No symbiont anagenic evolution within squids was observed, as competitive dominance does not purge V. fischeri genetic diversity through time. Instead, abiotic factors affecting abundance of V. fischeri variants in the planktonic phase sustain temporal symbiont diversity, a property itself of ecological constraints imposed by V. fischeri host adaptation.

Phylogeographical patterns among Mediterranean sepiolid squids and their Vibrio symbionts: environment drives specificity among sympatric species.

Bobtail squid from the genera Sepiola and Rondeletiola (Cephalopoda: Sepiolidae) form mutualistic associations with luminous Gram-negative bacteria (Gammaproteobacteria: Vibrionaceae) from the genera Vibrio and Photobacterium. Symbiotic bacteria proliferate inside a bilobed light organ until they are actively expelled by the host into the surrounding environment on a diel basis. This event results in a dynamic symbiont population with the potential to establish the symbiosis with newly hatched sterile (axenic) juvenile sepiolids. In this study, we examined the genetic diversity found in populations of sympatric sepiolid squid species and their symbionts by the use of nested clade analysis with multiple gene analyses. Variation found in the distribution of different species of symbiotic bacteria suggests a strong influence of abiotic factors in the local environment, affecting bacterial distribution among sympatric populations of hosts. These abiotic factors include temperature differences incurred by a shallow thermocline, as well as a lack of strong coastal water movement accompanied by seasonal temperature changes in overlapping niches. Host populations are stable and do not appear to have a significant role in the formation of symbiont populations relative to their distribution across the Mediterranean Sea. Additionally, all squid species examined (Sepiola affinis, S. robusta, S. ligulata, S. intermedia, and Rondeletiola minor) are genetically distinct from one another regardless of location and demonstrate very little intraspecific variation within species. These findings suggest that physical boundaries and distance in relation to population size, and not host specificity, are important factors in limiting or defining gene flow within sympatric marine squids and their associated bacterial symbionts in the Mediterranean Sea.

Salinity and temperature effects on physiological responses of Vibrio fischeri from diverse ecological niches.

Vibrio fischeri is a bioluminescent bacterial symbiont of sepiolid squids (Cephalopoda: Sepiolidae) and monocentrid fishes (Actinopterygii: Monocentridae). V. fischeri exhibit competitive dominance within the allopatrically distributed squid genus Euprymna, which have led to the evolution of V. fischeri host specialists. In contrast, the host genus Sepiola contains sympatric species that is thought to have given rise to V. fischeri that have evolved as host generalists. Given that these ecological lifestyles may have a direct effect upon the growth spectrum and survival limits in contrasting environments, optimal growth ranges were obtained for numerous V. fischeri isolates from both free-living and host environments. Upper and lower limits of growth were observed in sodium chloride concentrations ranging from 0.0% to 9.0%. Sepiola symbiotic isolates possessed the least variation in growth throughout the entire salinity gradient, whereas isolates from Euprymna were the least uniform at <2.0% NaCl. V. fischeri fish symbionts (CG101 and MJ101) and all free-living strains were the most dissimilar at >5.0% NaCl. Growth kinetics of symbiotic V. fischeri strains were also measured under a range of salinity and temperature combinations. Symbiotic V. fischeri ES114 and ET101 exhibited a synergistic effect for salinity and temperature, where significant differences in growth rates due to salinity existed only at low temperatures. Thus, abiotic factors such as temperature and salinity have differential effects between free-living and symbiotic strains of V. fischeri, which may alter colonization efficiency prior to infection.

ULTRASTRUCTURE OF LIGHT ORGANS OF LOLIGINID SQUIDS AND THEIR BACTERIAL SYMBIONTS: A NOVEL MODEL SYSTEM FOR THE STUDY OF MARINE SYMBIOSES.

The class Cephalopoda (Phylum Mollusca), encompassing squids and octopuses, contains multiple species that are characterized by the presence of specialized organs known to emit light. These complex organs have a variety of morphological characteristics ranging from groups of simple, light-producing cells, to highly specialized organs (light organs) with cells surrounded by reflectors, lenses, light guides, color filters, and muscles. Bacteriogenic light organs have been well characterized in sepiolid squids, but a number of species in the family Loliginidae are also known to contain bacteriogenic light organs. Interest in loliginid light organ structure has recently arisen because of their potential as ecological niches for Vibrio harveyi, a pathogenic marine bacterium. This also implies the importance of loliginid light organs as reservoirs for V. harveyi persistence in the ocean. The present study utilized transmission and scanning electron microscopy to characterize the morphology of loliginid light organs and determined the location of bacterial symbiont cells within the tissue. It was determined that the rod-shaped loliginid symbionts lack flagella, as similarly observed in other light organ-associated bacteria. Also, the interaction of individual cells to light organ tissue is not as defined as reported for other squid-Vibrio systems. In addition, SEM observations show the presence of two pores leading to the bacterial chamber. Data presented here offer support for the hypothesis of environmental transfer of bacterial symbionts in loliginid squids.

BIOLOGICAL PROPERTIES (IN VITRO) EXHIBITED BY FREE-LIVING AND SYMBIOTIC VIBRIO ISOLATES.

Adhesion and biofilm forming ability of symbiotic bacteria play a crucial role in host colonization and tissue infection. Bacteria benefit by adhering to their host in a manner that allows them to successfully maintain contact for the exchange of nutrients, hormones, or other necessary products. This study examined pili morphology, motility, and biofilm formation exhibited by Vibrio fischeri strains (free-living and symbiotic). Since these symbiotic factors contribute in some fashion to the interaction between V. fischeri and their squid host, variation between strains may be a contributing factor that leads to specificity among different hosts. V. fischeri strains examined in this study demonstrated considerable variation in their biological properties when observed in vitro. In addition to differences observed between strains isolated from several different host species, we observed variation between strains isolated from the same host species from diverse geographical locations. This study suggests that subtle differences in the biological properties of closely related V. fischeri strains may influence the nature of the interaction among V. fischeri and their sepiolid hosts.

Gene sequences of the pil operon reveal relationships between symbiotic strains of Vibrio fischeri.

Symbiosis between the bobtail squid Euprymna scolopes (Mollusca: Cephalopoda) and Vibrio fischeri bacteria has been a well-studied model for understanding the molecular mechanisms of colonization and adherence to host cells. For example, pilin expression has been observed to cause subtle variation in colonization for a number of Gram-negative bacteria with eukaryotic hosts. To investigate variation amongst pil genes of closely related strains of vibrios, we amplified pil genes A, B, C and D to determine orientation and sequence similarity to other symbiotic vibrios. The pilA gene was found to be upstream from all other pil genes, and not contiguous with the rest of the operon. The pilB, pilC and pilD loci were flanked at the 3' end by yacE, followed by a conserved hypothetical gene. DNA sequences of each pil gene were aligned and analysed phylogenetically using parsimony for both individual and combined gene trees. Results demonstrate that certain pil loci (pilB and pilD) are conserved among strains of V. fischeri, but pilC differs in sequence between symbiotic and free-living strains. Phylogenetic analysis of all pil genes gives better resolution of Indo-west Pacific V. fischeri symbionts compared with analysis of the 16S rRNA gene. Hawaiian and Australian symbiotic strains form one monophyletic tree, supporting the hypothesis that V. fischeri strain specificity is selected by the geographical location of their hosts and is not related to specific squid species.

THE EVOLUTIONARY ECOLOGY OF A SEPIOLID SQUID-VIBRIO ASSOCIATION: FROM CELL TO ENVIRONMENT.

Mutualistic relationships between bacteria and their eukaryotic hosts have existed for millions of years, and such associations can be used to understand the evolution of these beneficial partnerships. The symbiosis between sepiolid squids (Cephalopoda: Sepiolidae), and their Vibrio bacteria (gamma Proteobacteria: Vibrionaceae), has been a model system for over 20 years, giving insight as to the specificity of the association, and whether the interactions themselves give rise to such finely tuned dialog. Since the association is environmentally transmitted, selection for specificity can evolve from a number of factors; abiotic (temperature, salinity), as well as biotic (host species, receptors, cell/cell interactions). Here, we examine the transition between these forces effecting the symbiosis, and pose possible explanations as to why this association offers many attributes for understanding the role of symbiotic competence.

Deciphering Evolutionary Mechanisms Between Mutualistic and Pathogenic Symbioses.

The continuum between mutualistic and pathogenic symbioses has been an underlying theme for understanding the evolution of infection and disease in a number of eukaryotic-microbe associations. The ability to monitor and then predict the spread of infectious diseases may depend upon our knowledge and capabilities of anticipating the behavior of virulent pathogens by studying related, benign symbioses. For instance, the ability of a symbiotic species to infect, colonize, and proliferate efficiently in a susceptible host will depend on a number of factors that influence both partners during the infection. Levels of virulence are not only affected by the genetic and phenotypic composite of the symbiont, but also the life history, mode(s) of transmission, and environmental factors that influence colonization, such as antibiotic treatment. Population dynamics of both host and symbiont, including densities, migration, as well as competition between symbionts will also affect infection rates of the pathogen as well as change the evolutionary dynamics between host and symbiont. It is therefore important to be able to compare the evolution of virulence between a wide range of mutualistic and pathogenic systems in order to determine when and where new infections might occur, and what conditions will render the pathogen ineffective. This perspective focuses on several symbiotic models that compare mutualistic associations to pathogenic forms and the questions posed regarding their evolution and radiation. A common theme among these systems is the prevailing concept of how heritable mutations can eventually lead to novel phenotypes and eventually new species.

Spatial and temporal distribution of the vibrionaceae in coastal waters of Hawaii, Australia, and France.

Relatively little is known about large-scale spatial and temporal fluctuations in bacterioplankton, especially within the bacterial families. In general, however, a number of abiotic factors (namely, nutrients and temperature) appear to influence distribution. Community dynamics within the Vibrionaceae are of particular interest to biologists because this family contains a number of important pathogenic, commensal, and mutualist species. Of special interest to this study is the mutualism between sepiolid squids and Vibrio fischeri and Vibrio logei, where host squids seed surrounding waters daily with their bacterial partners. This study seeks to examine the spatial and temporal distribution of the Vibrionaceae with respect to V. fischeri and V. logei in Hawaii, southeastern Australia, and southern France sampling sites. In particular, we examine how the presence of sepiolid squid hosts influences community population structure within the Vibrionaceae. We found that abiotic (temperature) and biotic (host distribution) factors both influence population dynamics. In Hawaii, three sites within squid host habitat contained communities of Vibrionaceae with higher proportions of V. fischeri. In Australia, V. fischeri numbers at host collection sites were greater than other populations; however, there were no spatial or temporal patterns seen at other sample sites. In France, host presence did not appear to influence Vibrio communities, although sampled populations were significantly greater in the winter than summer sampling periods. Results of this study demonstrate the importance of understanding how both abiotic and biotic factors interact to influence bacterial community structure within the Vibrionaceae.

Biodiversity among luminescent symbionts from squid of the genera Uroteuthis, Loliolus and Euprymna (Mollusca: Cephalopoda).

Luminescent bacteria in the family Vibrionaceae (Bacteria: γ-Proteobacteria) are commonly found in complex, bilobed light organs of sepiolid and loliginid squids. Although morphology of these organs in both families of squid is similar, the species of bacteria that inhabit each host has yet to be verified. We utilized sequences of 16S ribosomal RNA, luciferase α-subunit (luxA) and the glyceraldehyde-3-phosphate dehydrogenase (gapA) genes to determine phylogenetic relationships between 63 strains of Vibrio bacteria, which included representatives from different environments as well as unidentified luminescent isolates from loliginid and sepiolid squid from Thailand. A combined phylogenetic analysis was used including biochemical data such as carbon use, growth and luminescence. Results demonstrated that certain symbiotic Thai isolates found in the same geographic area were included in a clade containing bacterial species phenotypically suitable to colonize light organs. Moreover, multiple strains isolated from a single squid host were identified as more than one bacteria species in our phylogeny. This research presents evidence of species of luminescent bacteria that have not been previously described as symbiotic strains colonizing light organs of Indo-West Pacific loliginid and sepiolid squids, and supports the hypothesis of a non-species-specific association between certain sepiolid and loliginid squids and marine luminescent bacteria.

Population structure between environmentally transmitted vibrios and bobtail squids using nested clade analysis.

Squids from the genus Euprymna (Cephalopoda: Sepiolidae) and their symbiotic bacteria Vibrio fischeri form a mutualism in which vibrios inhabit a complex light organ within the squid host. A host-mediated daily expulsion event seeds surrounding seawater with symbiotically capable V. fischeri that environmentally colonize newly hatched axenic Euprymna juveniles. Competition experiments using native and non-native Vibrio have shown that this expulsion/re-colonization phenomenon has led to cospeciation in this system in the Pacific Ocean; however, the genetic architecture of these symbiotic populations has not been determined. Using genetic diversity and nested clade analyses we have examined the variation and history of three allopatric Euprymna squid species (E. scolopes of Hawaii, E. hyllebergi of Thailand, and E. tasmanica from Australia) and their respective Vibrio symbionts. Euprymna populations appear to be very genetically distinct from each other, exhibiting little or no migration over large geographical distances. In contrast, Vibrio symbiont populations contain more diverse haplotypes, suggesting both host presence and unidentified factors facilitating long-distance migration structure in Pacific Vibrio populations. Findings from this study highlight the importance of how interactions between symbiotic organisms can unexpectedly shape population structure in phylogeographical studies.

Differentially expressed genes reveal adaptations between free-living and symbiotic niches of Vibrio fischeri in a fully established mutualism.

A major force driving in the innovation of mutualistic symbioses is the number of adaptations that both organisms must acquire to provide overall increased fitness for a successful partnership. Many of these symbioses are relatively dependent on the ability of the symbiont to locate a host (specificity), as well as provide some novel capability upon colonization. The mutualism between sepiolid squids and members of the Vibrionaceae is a unique system in which development of the symbiotic partnership has been studied in detail, but much remains unknown about the genetics of symbiont colonization and persistence within the host. Using a method that captures exclusively expressed transcripts in either free-living or host-associated strains of Vibrio fischeri, we identified and verified expression of genes differentially expressed in both states from two symbiotic strains of V. fischeri. These genes provide a glimpse into the microhabitat V. fischeri encounters in both free-living seawater and symbiotic host light organ-associated habitats, providing insight into the elements necessary for local adaptation and the evolution of host specificity in this unique mutualism.

Evolutionary relationships among squids of the family Gonatidae (Mollusca: Cephalopoda) inferred from three mitochondrial loci.

The oceanic squid family Gonatidae (Mollusca: Cephalopoda) is widely distributed in subpolar and temperate waters, exhibiting behavioral and physiological specializations associated with reproduction. Females of several species undergo muscular degeneration upon maturation; origins of this complex morphogenic change are unknown, hindering our understanding of ecological and morpho-physiological adaptations within the family. To provide further information regarding the evolutionary relationships within Gonatidae, three mitochondrial loci (12S rRNA, 16S rRNA, and cytochrome c oxidase subunit I) were analyzed for 39 individuals representing fourteen gonatid and six outgroup cephalopod species. In addition to elucidating relationships among gonatids, molecular data provided more information than morphological data for problematic specimens. Although some data sets are incongruent or have low nodal support values, combined molecular analysis confirms the presence of gonatid groups previously established by morphological characteristics (i.e., possessing radular teeth in seven longitudinal rows and muscular mantle tissue). These characteristics are basal to taxa possessing radular teeth in five longitudinal rows and less muscular mantle tissue, indicating that the derived forms are those species exhibiting physiological adaptation such as tissue degeneration upon maturation and egg brooding.

Enlightenment of old ideas from new investigations: more questions regarding the evolution of bacteriogenic light organs in squids.

Bioluminescence is widespread among many different types of marine organisms. Metazoans contain two types of luminescence production, bacteriogenic (symbiotic with bacteria) or autogenic, via the production of a luminous secretion or the intrinsic properties of luminous cells. Several species in two families of squids, the Loliginidae and the Sepiolidae (Mollusca: Cephalopoda) harbor bacteriogenic light organs that are found central in the mantle cavity. These light organs are exceptional in function, that is, the morphology and the complexity suggests that the organ has evolved to enhance and direct light emission from bacteria that are harbored inside. Although light organs are widespread among taxa within the Sepiolidae, the origin and development of this important feature is not well studied. We compared light organ morphology from several closely related taxa within the Sepiolidae and combined molecular phylogenetic data using four loci (nuclear ribosomal 28S rRNA and the mitochondrial cytochrome c oxidase subunit I and 12S and 16S rRNA) to determine whether this character was an ancestral trait repeatedly lost among both families or whether it evolved independently as an adaptation to the pelagic and benthic lifestyles. By comparing other closely related extant taxa that do not contain symbiotic light organs, we hypothesized that the ancestral state of sepiolid light organs most likely evolved from part of a separate accessory gland open to the environment that allowed colonization of bacteria to occur and further specialize in the eventual development of the modern light organ.

A combined approach to the phylogeny of Cephalopoda (Mollusca).

Cephalopoda represents a highly diverse group of molluscs, ranging in habitat from coastal regions to deep benthic waters. While cephalopods remain at the forefront of modern biology, in providing insight into fields such as neurobiology and population genetics, little is known about the relationships within the group. This study provides a comprehensive phylogenetic analysis of Cephalopoda (Mollusca) using a combination of molecular and morphological data. Four loci (three nuclear 18S rRNA, fragments of 28S rRNA and histone H3 and one mitochondrial cytochrome c oxidase subunit I) were combined with 101 morphological characters to test the relationships of 60 species of cephalopods, with emphasis within Decabrachia (squids and cuttlefishes). Individual and combined data sets were analyzed using the direct optimization method, with parsimony as the optimality criterion. Analyses were repeated for 12 different parameter sets accounting for a range of indel/change and transversion/transition cost ratios. Most analyses support the monophyly of Cephalopoda, Nautiloidea, Coleoidea and Decabrachia, however, the monophyly of Octobrachia was refuted due to the lack of support for a Cirroctopoda + Octopoda group. When analyzing all molecular evidence in combination and for total evidence analyses, Vampyromorpha formed the sister group to Decabrachia under the majority of parameters, while morphological data and some individual data sets supported a sister relationship between Vampyromorpha and Octobrachia. Within Decabrachia, a relationship between the sepioids Idiosepiida, Sepiida, Sepiolida and the teuthid Loliginidae was supported. Spirulida fell within the teuthid group in most analyses, further rendering Teuthida paraphyletic. Relationships within Decabrachia and specifically Oegopsida were found to be highly parameter-dependent.

Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism.

Associations between environmentally transmitted symbionts and their hosts provide a unique opportunity to study the evolution of specificity and subsequent radiation of tightly coupled host-symbiont assemblages [3, 8, 24]. The evidence provided here from the environmentally transmitted bacterial symbiont Vibrio fischeri and its sepiolid squid host (Sepiolidae: Euprymna) demonstrates how host-symbiont specificity can still evolve without vertical transmission of the symbiont [1]. Infection by intraspecific V. fischeri symbionts exhibited preferential colonization over interspecific V. fischeri symbionts, indicating a high degree of specificity for the native symbiotic strains. Inoculation with symbiotic bacteria from other taxa (monocentrid fish and loliginid squids) produced little or no colonization in two species of Euprymna, despite their presence in the same or similar habitats as these squids. These findings of host specificity between native Vibrios and sepiolid squids provides evidence that the presence of multiple strains of symbionts does not dictate the composition of bacterial symbionts in the host.

Temperature affects species distribution in symbiotic populations of Vibrio spp.

The genus Sepiola (Cephalopoda: Sepiolidae) contains 10 known species that occur in the Mediterranean Sea today. All Sepiola species have a light organ that contains at least one of two species of luminous bacteria, Vibrio fischeri and Vibrio logei. The two Vibrio species coexist in at least four Sepiola species (S. affinis, S. intermedia, S. ligulata, and S. robusta), and their concentrations in the light organ depend on changes in certain abiotic factors, including temperature. Strains of V. fischeri grew faster in vitro and in Sepiola juveniles when they were incubated at 26 degrees C. In contrast, strains of V. logei grew faster at 18 degrees C in culture and in Sepiola juveniles. When aposymbiotic S. affinis or S. ligulata juveniles were inoculated with one Vibrio species, all strains of V. fischeri and V. logei were capable of infecting both squid species at the optimum growth temperatures, regardless of the squid host from which the bacteria were initially isolated. However, when two different strains of V. fischeri and V. logei were placed in direct competition with each other at either 18 or 26 degrees C, strains of V. fischeri were present in sepiolid light organs in greater concentrations at 26 degrees C, whereas strains of V. logei were present in greater concentrations at 18 degrees C. In addition to the competition experiments, the ratios of the two bacterial species in adult Sepiola specimens caught throughout the season at various depths differed, and these differences were correlated with the temperature in the surrounding environment. My findings contribute additional data concerning the ecological and environmental factors that affect host-symbiont recognition and may provide insight into the evolution of animal-bacterium specificity.

Competitive dominance among strains of luminous bacteria provides an unusual form of evidence for parallel evolution in Sepiolid squid-vibrio symbioses.

One of the principal assumptions in symbiosis research is that associated partners have evolved in parallel. We report here experimental evidence for parallel speciation patterns among several partners of the sepiolid squid-luminous bacterial symbioses. Molecular phylogenies for 14 species of host squids were derived from sequences of both the nuclear internal transcribed spacer region and the mitochondrial cytochrome oxidase subunit I; the glyceraldehyde phosphate dehydrogenase locus was sequenced for phylogenetic determinations of 7 strains of bacterial symbionts. Comparisons of trees constructed for each of the three loci revealed a parallel phylogeny between the sepiolids and their respective symbionts. Because both the squids and their bacterial partners can be easily cultured independently in the laboratory, we were able to couple these phylogenetic analyses with experiments to examine the ability of the different symbiont strains to compete with each other during the colonization of one of the host species. Our results not only indicate a pronounced dominance of native symbiont strains over nonnative strains, but also reveal a hierarchy of symbiont competency that reflects the phylogenetic relationships of the partners. For the first time, molecular systematics has been coupled with experimental colonization assays to provide evidence for the existence of parallel speciation among a set of animal-bacterial associations.

Temperature- and concentration-dependence of compatibility of the organic osmolyte beta-dimethylsulfoniopropionate.

The effects of the organic osmolyte beta-dimethylsulfoniopropionate (DMSP) on the structural stability of three model proteins were examined to determine whether DMSP, like the structurally similar solute dimethyl sulfoxide (DMSO), is compatible with native protein structure at low, but not elevated, temperatures. DMSP stabilized phosphofructokinase under conditions of cold-induced denaturation. Thus, DMSP, like DMSO, may be an effective protein cryoprotectant. However, DMSP was not an effective stabilizer of protein structure under conditions of heat denaturation. Whereas low (0.2 M) concentrations of DMSP stabilized lactate dehydrogenase against inactivation at 50 degrees C, higher DMSP concentrations were ineffective. DMSP favored the denaturation of glutamate dehydrogenase at all DMSP concentrations tested. DMSP may be a compatible osmotic solute only under conditions of moderate temperature and low, yet physiological, concentrations. The mechanistic basis of DMSP's temperature- and concentration-dependent effects and the possible roles played by adaptation temperature and severity of osmotic stress in the evolutionary selection of organic osmolytes are discussed.