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Nish/Castillo Squid Room Tour

lab tour

Landie and Amanda(Super Squid Techs) lead a tour of the Nish squid room in Foster Hall.

Nish Lab Tour

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









The Nishiguchi Laboratory

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Philosophy of Research Program
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 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.  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.  This 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.

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


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.  Working with Drs. Enrico Pontelli and Joe Song (NMSU, Computer Sciences), Dr. Michaela Buenneman (Geography), and Dr. Christine Laney (UTEP, we are combining computational and GIS 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.

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General modeling framework for explaining and predicting phenomena between abiotic (environmental) and biological (genes, gene products) data.



 


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 and 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.  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, 2010).  We have also begun to investigate other abiotic factors (such as UV light, oxygen depletion) and biotic factors (viral specificity to Vibrio bacteria and intra- and interspecific interactions between Vibrio species/strains) to determine if this contributes to colonization efficiency as well as distribution and abundance of symbiotic biovars.  We test this by experimentally evolving strains to the various stresses to determine whether bacteria that are capable of adapting to those stresses are better able to infect novel host species.

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Ecological specialization from one Vibrio fischeri strain into different morphotypes.




 


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. 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 (Soto et al., 2012; 2014).


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Schematic of how our experimentally evolved Vibrios are generated.



 

 


Comparison of environmental vs. symbiotic expressed genes in biofilm production
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 (Jones and Nishiguchi, 2006, Guerrero-Ferriera and Nishiguchi, 2010).  We have used several techniques (allelic exchange, gap repair) to determine how biofilm phenotypes change with respect to a particular host squid or to the environment/habitat from which they were isolated (Chavez et al., 2012, Chavez and Nishiguchi, 2011).  We have several ongoing projects focusing on specific mutant knockouts, to determine whether those specific genes, operons, or parts of the genomes (specific chromosomes or plasmids) have an affect on biofilm formation during colonization, competition, and persistence, both in the free-living and symbiotic state of these bacteria.  In addition, we are determining whether specific biofilm components help protect bacteria from protozoan grazing while in their free-living state, and if those components also help provide immunity while inside host squids (Chavez et al., 2013).  Using Vibrio shotgun genomic libraries, we hope to determine if specific patterns or clusters of genes are similar between the vast majority of Vibrio symbionts (symbiotic or free-living) that my laboratory has amassed over the past 15 years (over 9000 isolates; Howard et al., in review) and use this information to determine how phenotypes such as biofilm vary among Vibrio strains

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A)
Colonization assays 48-hour post-infection of juvenile Euprymna scolopes or

B. E. tasmanica by wild-type, mutant, and complement strains of the lux operon for Vibrio fischeri. Single strain infection experiments are represented when only a single bar is shown (□). Competition experiments are represented with two bars (□ is the first strain and ■ is the second strain), and each strain used is indicated below each competition.



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Grazing dynamics between 3 different protist species and biofilms from symbiotic and free-living V. fischeri.

 

 

 

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; Zamborsky and Nishiguchi, 2011). 



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Zamborsky and Nishiguchi, 2011











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.  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 (Guerero-Ferreira et al., 2012). 


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Jones et al., 2006











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; Nair and Nishiguchi, 2009).




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unpublished data










Summary
Overall, my laboratory has ongoing research projects from microbial ecology to the evolution of host-symbiont assemblages.  The significance of this work will help clarify whether recent insurgences of newly evolved infections are correlated to environmental changes, and that beneficial associations are much more amenable to the transient nature of these fluctuations.  Increasing awareness of the pivotal role that microorganisms have in ecology, antibiotic resistance, and bio-terrorism is becoming more evident through the study of both environmental and pathogenic microbiology.  Biologists may yet see the future development of a new era of microbiology that synthesizes pathogenic and environmental microbiology with immunology, bioinformatics, and disease prediction, in the context of evolutionary systems biology.  My track record with both minority postdoctoral, graduate and undergraduate students in their training towards research in the STEM disciplines has been a major influence of both my philosophy towards research as well as the complementation of both research and teaching.  Such an environment that involves diverse members provides greater critical analyses of problems and helps solve them in more innovative ways.