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Nish/Castillo Squid Room Tour
lab tour
Landie Romero and Erin Pearcy lead a tour of the Nish/Castillo squid room in Foster Hall.

Nish/Castillo Squid Room Tour

Dr. Nishiguchi takes you on a tour of the Marine Symbiosis Lab.

The Nishiguchi Laboratory

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Research Program


Krista and Alba getting samples from
the freezer.
My primary research interests examine the mechanisms and evolutionary processes that are fundamental for establishing how bacteria affect the population structure, life history, and molecular specificity in symbiotic associations, and how these associations can be used to examine modes of infection and pathogenicity between animals, bacteria, and the communities/habitats where they are located. The sepiolid squid-Vibrio light organ association provides an
Clayton and Bong working up their data on the computers.
experimentally tractable model system for the study of the mechanisms underlying bacterial colonization of animal tissues, and exploring the population dynamics, ecology and community dynamics between squid hosts and their bacterial symbionts. In this animal-bacterial system, there is no contact between host and symbiont throughout embryogenesis; the symbiosis is established within hours (environmentally) after the juvenile squids are hatched into the surrounding environment.


Alvaro and Alba working up Vibrio samples.
During this infection period, specific strains of Vibrio from the water column are able to recognize and colonize the sterile light organs of the newly hatched juveniles. Bacteria that are capable of persisting in the developing light organ after this infection period represent a successful association by one specific subset (or subspecies) of Vibrio strains. This specificity is maintained throughout the squid’s life history; however, other symbiotic strains of Vibrio isolated from different host squid’s are able to cross-infect juveniles of closely related species. Competition experiments have proven that there is a hierarchy among these Vibrio strains that is congruent to the phylogenetic relationships of the host squids, and that some level of molecular recognition or specificity is involved in the establishment of each sepiolid species partnership.

Ed separating out his Euprymna
populations.

This information provides the basis for further investigation of the molecular differences between closely related strains and species of competent vibrios and whether these differences are the result of the complex interactions (both biotic and abiotic) that have led to the evolution and radiation of several species of Vibrio bacteria. This information has led to understanding microbial/host community structure and speciation overall, and has led to further questions regarding how populations are maintained in ecologically sensitive areas (seagrass beds, coastal tropical reefs).

The following paragraphs are summaries of some of the ongoing research projects in my laboratory.

slide 1DNA microarray analysis of 12 different V. fischeri strains (courtesy of C.P. Lostroh).   View large

Determining overall ecological patterns driving microbial speciation
My laboratory has been interested in the overall mechanisms that drive bacterial speciation. Specifically we are using both abiotic and biotic measurements, to predict models of infection, colonization, and persistence in our squid-Vibrio model. We have begun to utilize computational approaches to predict how bacteria are capable of adapting not only to new host species, but also to different environments over large geographical distances. By building these interconnecting networks, we hope to provide a more detailed picture of how bacterial infections are spread throughout the world, a phenomenon that is highly dependent upon bacterial adaptation.

slide 2Two fully sequenced V. fischeri genomes (ES114 and MJ11).
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My laboratory has recently sequenced an additional three genomes of Vibrio fischeri, one from an evolved Hawaiian strain adapted to an Australian host (through 400 generations), its ancestral lineage (non-evolved Hawaiian V. fischeri), as well as a wild-type Australian V. fischeri. Using a bioinformatics approach (in collaboration with Dr. GongXin Yu, Boise State University), we are determining which genes have been selected for over a period of ~400 generations, and whether this newly evolved strain is more similar to those existing in present Australian hosts.


Microbial ecology and abiotic factors influencing bacterial speciation
Previous results have demonstrated that the stratigraphical distribution of V. fischeri and V. logei, two species capable of colonizing Mediterranean sepiolid light organs in the genus Sepiola, was determined by temperature and not host squid (Nishiguchi, 2000). Numerous Sepiola species exist sympatrically in the Mediterranean, and all of these host species simultaneously co-occur with the symbionts V. fischeri and V. logei. Contrary to the competitive dominance observed in Euprymna, Vibrio symbionts in Sepiola are host generalists. Previous studies (both in my laboratory an in others) have made it apparent that salinity and temperature influence all life cycle stages of Vibrio species, including the biogeography of free-living cells, host attachment with subsequent proliferation during symbiosis, and the alternative evolutionary trajectories available to different host ranges.

Various V. fischeri growth curves over increasing salinities.
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My laboratory is presently studying the effects of salinity and temperature on Vibrio growth and competition in the environment to determine if abiotic factors have some influence on the distribution of both free-living and symbiotic strains. Presently, we know that under laboratory conditions, both temperature and salinity work synergistically to effect competition between strains of Vibrio isolated from both squid light organs and from surrounding habitats (Soto et al., 2009). We have also begun to investigate other abiotic factors (such as UV light, pollution) and biotic factors (intra- and inter-specific interactions between Vibrio species/strains) to determine if this contributes to colonization efficiency as well as distribution and abundance of symbiotic biovars.


Experimental Evolution of Vibrio bacteria
Because viable Vibrio bacteria are vented every morning at dawn, this leads to an increased amount of free-living Vibrio in the water column that are capable of infecting new juvenile squids, recombining with other free-living vibrios, or being transported to different habitats which may contain other populations or species of host squids. If temperature and salinity are important factors that select for Vibrio fitness in their free-living state, then chances of adapting to and colonizing a new host squid may be driven by how well symbiotic bacteria are capable of surviving between infections.

Schematic of how our experimentally evolved Vibrios are generated.
 View large

This, combined with the fact that specific genes are expressed in either environmental or symbiotic modes (see next section), provides support that both symbiosis and environment have key roles in symbiont fitness. Although in vitro experiments can demonstrate Vibrio adaptation to certain abiotic conditions, the combination with animal passage experiments (experimental evolution) can provide an intimate picture of what actually drives symbiont selection during colonization.


Comparison of environmental vs. symbiotic expressed genes
Identification of specific “determinants” that are the genetic signals between host-symbiont pairs, and determining whether they instigate beneficial or detrimental associations, has provided insight into the mechanisms that render virulence and pathogenesis as well as whether host reservoirs help stabilize the evolution of symbiotic vibrios from their free-living counterparts (Guerrero-Ferriera and Nishiguchi, 2009; Jones and Nishiguchi, 2006). We have recently used a comparative genomics approach to investigate changes between symbiotic and free-living states of the same strain, by using a technique termed SCOTS (Selected Capture Of Transcribed Sequences).

Comparison of genes solely expressed in the squid light organ or seawater environment using Selected Capture of Transcribed Sequences (SCOTS).
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This allows us to test the same strain in two different environments (in this case, light organ and seawater). Our comparisons have allowed us to isolate both light organ and seawater specific genes, and subsequently compare those expressed genes to ones previously isolated from a number of additional wild-type strains (using PCR microarrays). We are now in the process of making mutant knockouts, to determine if those specific genes, operons, or parts of the genomes (specific chromosomes or plasmids) have an affect on colonization, competition, and persistence, both in the free-living and symbiotic state of these bacteria. We are now planning to utilize this information to screen Vibrio shotgun genomic libraries, to determine if specific patterns or clusters of genes are similar between the vast majority of Vibrio symbionts that we have amassed over the past 12 years (over 6000 isolates), as well as Vibrio genomes that have recently be sequenced.

This not only includes mutualistic strains, but also those involved in pathogenic associations (i.e., V. parahaemolyticus, V. cholerae). In addition, we are beginning to analyze our data from both the comparative microarrays to the SCOTS data to determine whether specific patterns exist among gene networks in both environments (in collaboration with Dr. C. Phoebe Lostroh, Colorado College and Dr. Neil Sarkar, University of Vermont) to determine which trade-offs are more important in environmentally transmitted symbionts.


Population biology and phylogeography of host-symbiont associations
My laboratory also investigates the ecology and evolutionary history (population genetics using nested clade analyses, and environmental ecology using in situ hybridization of environmental isolates) of host acquisition from various symbiont biovars, as well as how both allopatric and sympatric populations drive bacterial biogeography and diversity (Jones et al., 2006). We have monitored sepiolid species from the Indo-west Pacific (primarily allopatric) and Mediterranean (sympatric) along with their bacterial symbionts, to determine degree of specificity, as well whether hosts and symbionts are evolving together.

V. fischeri haplotype network from strains isolated from Hawaii, Australia, and Thailand.
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Thus far, we have shown that Euprymna populations in the Indo-west Pacific are separated by host boundaries as well as temperature gradients, whereas Vibrio populations exhibit a higher degree of gene flow, not only among host populations of the same species, but also those from different species as well. This has also been observed in surveys of the environments in which the symbiosis occurs, where temperature plays a much more important role in determining host specificity than previously thought. We have also used Fluorescent In Situ Hybridization (FISH) techniques to determine if Vibrio bacteria are indeed present in higher concentrations where sepiolid squids are present, and whether these populations fluctuate over time (seasonally) in relation to salinity, temperature, and depth (Jones et al., 2007).


Evolution of light organs in sepiolid and loliginid squids (comparative evolutionary development)
One of the interesting aspects of cospeciation, is whether symbionts (in this case, Vibrio bacteria) influence the development and evolution of host squids and the light organs where the bacteria are housed. Previous studies investigating the developmental biology of E. scolopes, the endemic sepiolid from Hawaii, has demonstrated that symbiotic bacteria cause a series of developmental changes once symbiosis has begun. My laboratory is now beginning to investigate how different species of sepiolid squids have evolved using both phylogenetics and an evolutionary developmental approach (Nishiguchi et al., 2004) to determine how different genera of host squids have evolved the light organ, and if this morphologically complex organ is developmentally controlled by genes turned on by symbiotic infection.

We have begun to initiate in situ studies comparing 4 different genera of host squids; Rondeletiola and Sepiolina, two genera with round light organs (supposedly the ancestral state, Naef ,1921) and Euprymna and Sepiola, two genera with bilobed light organs (dervived state). In a separate project, my laboratory is investigating the evolution of bacteriogenic light organs in loliginid squids (Guerrero-Ferriera and Nishiguchi, 2009), the other family of cephalopods that have light organs that bear bioluminescent bacteria (Uroteuthis and Loliolus).

Phylogeny of representative Sepiolidae species and selected outgroups (for reference). Sepiolid taxa in red have no light organs. After a combined analysis of four loci (mtCOI, mt12S, mt16S rRNA, and partial sequence of the nuclear 28S rRNA, total approximately 2.4 kb) using direct optimization as implemented in POY (Wheeler et al. 2002). We analyzed six parameter sets varying the relative weights of insertion/deletion events and of base transformations. The tree on the left represents the best hypothesis using the parameter set that minimizes incongruence among all gene partitions (for all transformation receiving equal weights). On the right, a more conservative hypothesis, the strict consensus of all parameter sets examined, is presented, with all the nodes in this tree indicating parameter independence. Numbers on branches (left tree) indicate jackknife proportions (branches with no value have jackknife valueso50%). View large






















Origins of molluscan evolution (systematics of Cephalopoda and their relationship to other molluscs and their Vibrio bacteria)
Part of the foundation for understanding the evolution of cospeciation, is using phylogenetics to determine the evolutionary histories of both host and symbiont. My laboratory has used a multi-gene/morphological combined analysis approach to determine both host and symbiont phylogenies (Guerrero-Ferriera and Nishiguchi, 2007; Nishiguchi et al., 2004; Nishiguchi and Nair, 2003; Nishiguchi et al., 1998). Some of our research (particularly with Vibrio bacteria) has demonstrated the evolution of symbiosis, with respect to the origins of pathogenicity and those bacteria that have evolved as mutualists with squids and fish. Much of this work is on the premise that many of the genes present in different Vibrio species are co-opted to infect and colonize hosts either passively (as in mutualists) or aggressively (as in pathogens). This has important implications for medical microbiology and the interactions between eukaryotic hosts and microbial fauna.

Phylogenetic tree depicting the relationships of Monoplacophora to other molluscs based on the combined analysis of all molecular loci. Shown is strict consensus of two most parsimonious trees at 64,679 weighted steps (gap opening cost of 3, gap extension cost of 1, all base transformations cost 2) for the analysis of all data under direct optimization with tree fusing. Numbers on branches indicate jackknife support values. Gastropods (in red) and bivalves (in blue) appear diphyletic. Polyplacophora and Monoplacophora form a well supported clade (95% jackknife support). The monoplacophoran species (purple) appears nested within chitons (dark green), but nodal support for its exact position is low. The tree shows monophyly of molluscs, as well as that of Scaphopoda, Cephalopoda, Caudofoveata, and Solenogastres. View large



























Conclusion
We have also been interested in the overall evolution of cephalpods and their placement in the overall molluscan tree of life (Strugnell and Nishiguchi, 2007; Giribet et al., 2006; Lindgren et al., 2004). Since cephalopods represent one of the most derived molluscan classes, it is important to investigate morphological features that are synapomorphies between the groups, particularly those that contain bacteriogenic light organs. I am also interested in how molecular data, along with various types of phylogenetic analyses affect the systematics of cephalopod evolution, and whether various genes (or the addition of multiple loci) increase the resolution of closely related families within the Cephalopoda.

Overall, my laboratory has ongoing research projects from microbial ecology to molluscan biodiversity. My philosophy has been to not only focus on the main research plan that my laboratory has, but also to allow collaborations that are related to the overall themes of the lab, which has helped broaden my perspective on symbiosis and the interactions between animals and their bacterial partners, from molecules to ecology.