Source: AUBURN UNIVERSITY submitted to NRP
MICROBIAL ASSOCIATIONS WITH GELATINOUS INVERTEBRATES IN MOBILE BAY AND COASTAL ALABAMA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0217327
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Biological Sciences
Non Technical Summary
The gelatinous predator Mnemiopsis leidyi is the most common ctenophore in the coastal gulf of Mexico. Ctenophores are eaten by many medusae such as the sea nettle, the spotted jellyfish, the moon jellyfish and various common fin fish such as spadefish, mullet, tidewater minnows, butterfish, and silversides. Mnemiopsis bears a very consistent and distinctive group of microbes, found all the way from the Gulf of Mexico to Cape Cod. The diversity of the microbial populations varies within a limited range. We can correlate the occurrence of microbes with different coastal locations with varying marine health, but have little understanding of the impact of these microbes on the ctenophore, fish stocks, human endeavors such commercial and tourist fishing, and on human food sources. Marine and brackish water invertebrates and microbes respond quickly to environmental change. The looming impacts of global change and the continued heavy dependency of Alabamians on the Gulf force us to become better informed about the relationships between invertebrates such as ctenophores, and marine microbes. We have only a poor understanding of jellyfish/microbe interactions. For instance, in the ctenophore, the ciliate Trichodina ctenophorii is not very harmful, yet it is related to notorious fish parasites that have devastating impact on fish raised in high density fish farms; moreover, ctenophores often co-inhabit marine fish farms - T. ctenophorii could become a pathogen under such circumstances. While nonpathogenic to human the ctenophore gut microbe, Vibrio splendidus, is very damaging to shellfish and finfish stocks. Vibrio cholerae and V. fulvinicus (the marine `flesh eating bacteria') are well-known, notorious human pathogens associated with oysters and crabs, the larvae of which are consumed by ctenophores. To date, V. cholerae and V. fulvinicus have not yet been detected in Mnemiopsis; however, we predict that they will show up in more complete analyses of ctenophores from the Mobile Bay. The ctenophore could therefor be a host for bacterial and eukaryotic microbes that could significantly impact the Alabama economy and Alabamian's health. It is of critical importance to Alabama, then, both from the viewpoint of biomedical safety as well as tourism, to know more about the nature of the microbes associated with these jellies. In this study we will determine 1) what microbes are found on jellyfish and ctenophores throughout the year, 2) the microbial types and quantities throughout Mobile Bay (correlated with land usage, seasonality and/or with coastal disturbance such as tropical storms.) We will also ask: 3) Does the microbial assemblage transfer up the food chain into fin fish We will use modern microscopy and molecular analyses to determine the quantity and variability of Mobile Bay ctenophore and jellyfish microbes, correlate their occurrence with environmental factors and study their transfer through the food chain. Through our work, we will be able to state the impact of the microbes on Mobile Bay invertebrates and fish, and will be able to provide information relevant to developing a better, more microbially-informed coastal management approach.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1353199110040%
1353199110330%
7233199110030%
Knowledge Area
135 - Aquatic and Terrestrial Wildlife; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
3199 - Invertebrates, general/other;

Field Of Science
1103 - Other microbiology; 1100 - Bacteriology;
Goals / Objectives
The overarching goal of this project is to determine the impact of microbial associations and parasitism of the free-swimming gelata known as ctenophores and medusae (socalled medusazoa) on the ecology and ultimately the fisheries, of the Mobile Bay estuary. This will also have implications for human endeavors such as the tourism industry in Baldwin County and Mobile Bay. We will: 1. Characterize microbial populations associated with the major medusazoa and ctenophore species . 2. Determine the spacial variation of microbes as a function of freshwater and agricultural inputas a function of season and watershed outflow. 3. Determine the extent of trophic transfer of microbes to fish predators. Expected output This will be the very first effort, to the best of our knowledge, to attempt to characterize the microbial assemblages of the mobile, gelatinous predators of Mobile Bay. Our work will answer the following questions: 1) What microbes are found on jellyfish and ctenophores throughout the year We expect to see that the population of microbes observed on the ctenophore will be similar to the microbes observed on the larger medusazoa, fish and beroids that prey upon Mnemiopsis. However, it is very likely that the microbial consortia associate with each species could be at least subtly different, reflecting the strength of the symbiotic associations and the specificity of the cell-cell interactions with each host. 2) How do the microbes vary spacially along the coast How might this relate to land use And how does the assemblage vary seasonally and/or with coastal disturbance such as by tropical storms A correlation has been shown to exist between nutrient loading and parasite infestation intensity, and so we predict that the infestation intensity will increase with the bay water nutrient loading. We will sample ctenophores throughout the season, and collect microbial DNA which we wil subject to molecular analysis. We will examine both species range, and simple microbial diversity, both in the eukaryotic assemblage described above and prokaryotic assemblages. 3) Does the microbial assemblage transfer up the food chain into teleost fish We will collect small fish that we can find in the vicinity of the ctenophore, and will target fish that appear to be directly feeding on ctenophore. Using light and electron microscopy, and molecular analysis, we will determine if the microbial assemblage found on the ctenophore is also found on the fish. In fish that appear to bear the same organsisms, we will test for transfer between the ctenophore host and the fish using labelled (fluorescently or conventionally stained) microbes.
Project Methods
We will visit the coast at Ocean Springs, all along Dauphin Island and at Fort Morgan, on the west and east shores of Mobile Bay, and at sites as far north in the Bay as as we are able to find ctenophores and other jellies. We will collect ctenophores from all sites on a year-round basis. We will coordinate our efforts with colleagues at the coast to more reliably set effective collection dates. We will collect animals by gentle plankton net tows and by surface dipping from the Biological Sciences Marine Biology Club boat. At all sites, salinity, temperature, dissolved oxygen will be determined and samples will be taken to determine the algal concentration. Ctenophores gut microbes will be collected by quickly swabbing their gut with sterile plastic loop. We will culture the microbes on 1/10 LB broth marine medium, and will also use TCBS broth and variants to enrich for Vibrio species, which we have observed to be associated with the ctenophore gut. We will also collect samples for nonculture analysis of the diversity and identity of the microbes, by immediately fixing all samples in acetone for DNA extraction back at the lab. We will use small cast nets to capture small fish observed to be in the location of the ctenophore or jellyfish catch site. Fish will be quickly anaesthetized and dissected on the spot and/or fixed in acetone or formaldehyde / glutaraldehyde until analysis at the lab. Samples will be returned to the lab for culturing, as well as for the noncultured initial screening analysis of microbial taxa using the small subunit of the ribosomal RNA gene, followed up with additonal sequencing where needed and appropriate. Where possible, we will use automated analysis of ribosomal DNAs called ARISA to systematically analyse the microbial rDNA genes. ARISA will be performed in Dr. Covadonga Arias' lab in the Dept. Fisheries, and will use her LiCor 4300 gel analysis system. We will also use a simple DNA fingerprinting method, called Denaturing Gradient Gel Electrophoresis (DGGE) to determine the diversity of the microbes. We will analyse the ARISA and DGGE methods using specialty software (Bionumerics) in Dr. Yucheng Feng's lab in the Agronomy and Soils Dept, Auburn University. All samples will be examined by light and electron microscopy in order to identify morphologically the eukaryotes and prokaryotes collected on site. All water samples will be analyzed for phosphate and nitrate content according to standard methods already used in the lab. All results will be placed into our existing database to build on our information on coastal microbial communities and correlated with the molecular data. We will directly test to see if the microbes will transfer from the ctenophore host to a fish predator.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: College level and elementary and middle-school students through outreach efforts, using information derived in part from the studies. Presentations were made to cell and microbiologists who were interested in the molecular biology and cellular function of the prokaryotic groups presented in the work. Changes/Problems: The primary change has been to move from gel electrophoretic (strictly) analyses of microbial identification and diversity using DGGE to use of the Illumina work station based NGS methods, which provide considerably more detailed (and time consuming) analyses, but which are more complete, valid and appropriate for whole community analyses. What opportunities for training and professional development has the project provided? MS training for Shilpa Waduwawara (left project early) PhD training for Erin Donovan (left project at its inception, only a few week's into project) MS training for Jeffry Daniels who left project early. PhD training for Limin Gong, who switched labs after two years of support with this program and additonal funding from my GoMRI-I project. PhD training for Richard Mariita, who will complete the work and whose efforts in the past year have moved the project substantially forward with products now appearing (see earlier pages). Training for at least 6 undergraduate students in microscopy, physiology and molecular biology (molecular ecology work) over the funding period and beyond. One of the students, Miriam Ojima, was a Florida State University Summer CMB Undergraduate Research Scholar; Matthew Klinka and Wesley Wallacewere Auburn University students who both won the summer CMB-USRS How have the results been disseminated to communities of interest? Yes; basic science as indicated in the publication section. What do you plan to do during the next reporting period to accomplish the goals? This is the final reporting period for this project. I will however continue to pursue completion of all of the goals indicated above, and will seek additonal extramural funding to push the project ahead to complete the ctenophore symbiont studies indicated above. We will pursue the metals/antimicrobial resisitance problem and viral impactin the Bay in particular, but will addiontally attempt to find funding to find the source of the invasive ctenophore and additionally characterize it; also monitor the bay for additional invasions through alternative federal and possibly USDA funding sources (re: the antibiotic resistance components and the viral load).

Impacts
What was accomplished under these goals? We collected nearly two full years of seasonal samples at the 0.2 and 7 micron scale, and alsomicrographic data (usinga FlowCAM) from the free waters of the Bay and Mississippi Sound. WE have been given a set of samples that go back to 2009 to collect additional DNA so that the project will eventually extend significantly backwards in time to provide a larger longitudinal data set. In addition. We collected Mnemiopsis leidyi(a type of ctenophore) for microbial analysisbut unfortunately was unable to reliably collect animals throughout the two year collecting priod, at all time of the year because we often did not collect animals (despite a rigorous and repeatable small-boat collection protocol that included on average 1 boat trip per month. The problem was exacerbated by weather AND the invasion of a second, previously undescribedctenophore (for which we collected extensive DNA samples, established EM samples and have collected an etensive library of images) which will be published later this year (see previous abstracts indicated in this report). The invasive animals competed with Mnemiopsis and reduced food, killling off the native Mnemiopsis for nearly a full 5 months. Thus that data set is limited because of varying availabilty of the animals. We however have completed the NGS analysis of the free living microbes, and have isolated 50 ctenophore gut-located strains of microbes, which we are extensively characterizing (no abstracts or papers yet). Finally we will make a final one or twoctenophore collection soon to capture the Spring and possibly later on to obtain early Summer seasonal microbial populations assocated with the ctenophores, will analyse by Next Gen Sequencing (not mentioned in the original work) and will close the project, with probably 4 or more papers. the work will be published as a thesis (Richard Mariita, PhD, in my lab). We were not yet able to establish trophic transfer to fish, although we have examined fish ctenophore predators. We may need to return to that project in the future.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: R.M. Mariita, S. Bhatnagar, K. Hanselmann, M. Hossain, M. Liles, A.G. Moss, J.L. Leadbetter, D.K.Newman (2014)Characterization, Comparative Genomics and Genome Mining for Antibiotics and Secondary Metabolite of two Actinomycetales isolates. Annual Meeting of the American Society for Cell Biology, Phil., PA. 12/6-10/2014. Mol. Biol. Cell 25:1752 Abstr. #P2378.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: R. M. Mariita, M. J. Hossain, M. R. Liles, A. G. Moss. 2015. Metagenomic Assessment of an Estuarine System Reveals High Microbial and Mobile Genetic Element Diversity during Spring. Annual Meeting of the American Society for Microbiology, New Orleans, LA., May 30th-June 2nd 2015, Abstr. 15A1637GMASM
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: R. M. Mariita, S. Bhatnagar, K. Hanselmann, M. J. Hossain, S. Dawson, J. Korlach, M. Boitano, R. J. Roberts, M. R. Liles, A. G. Moss, J. R. Leadbetter, D. K. Newman. 2015. Mining for Secondary Metabolites and Epigenomics for Two Soil Actinomycetales co-Isolates. Annual Meeting of the American Society for Microbiology, New Orleans, LA. Abstr. May 30th-June 2nd 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Richard M. Mariita, Mohammad J. Hossain, Mark R. Liles, Anthony G. Moss. Seasonal Variability in the Diversity of Microbial Assemblages and Antibiotic Resistance Determinants of an Estuary System. Alabama NSF EPSCoR Science and Technology Open House. Renaissance Hotel Montgomery, AL.: Jan. 30-31, 2015.
  • Type: Other Status: Other Year Published: 2014 Citation: R. M. Mariita 2014. Isolation and characterization of species affiliated with family Actinomycetaceae. Invited presentation at the MBL, 18th August, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Richard M. Mariita, Clayton Lester, A. Ryan Hoskins, Jacob Britt, Matthew Klinka, Clifford Wood and Anthony G. Moss 2014. Molecular characterization of an invasive novel cydippid ctenophore. Auburn University Research Week presentation, June, 2014. Platform Presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Richard M. Mariita, Clayton Lester, A. Ryan Hoskins, Jacob Britt, Matthew Klinka, Clifford Wood and Anthony G. Moss 2014. Molecular characterization of an invasive novel cydippid ctenophore. Annual Meeting of the Alabama Academy of Science, Auburn, AL, March, 2014. Platform presentation.


Progress 10/01/09 to 09/30/14

Outputs
Target Audience: College level and elementary and middle-school students through outreach efforts, using information derived in part from the studies. Presentations were made to cell and microbiologists who were interested in the molecular biology and cellular function of the prokaryotic groups presented in the work. Changes/Problems: The primary change has been to move from gel electrophoretic (strictly) analyses of microbial identification and diversity using DGGE to use of the Illumina work station based NGS methods, which provide considerably more detailed (and time consuming) analyses, but which are more complete, valid and appropriate for whole community analyses. What opportunities for training and professional development has the project provided? MS training for Shilpa Waduwawara (left project early) PhD training for Erin Donovan (left project at its inception, only a few week's into project) MS training for Jeffry Daniels who left project early. PhD training for Limin Gong, who switched labs after two years of support with this program and additonal funding from my GoMRI-I project. PhD training for Richard Mariita, who will complete the work and whose efforts in the past year have moved the project substantially forward with products now appearing (see earlier pages). Training for at least 6 undergraduate students in microscopy, physiology and molecular biology (molecular ecology work) over the funding period and beyond. One of the students, Miriam Ojima, was a Florida State University Summer CMB Undergraduate Research Scholar; Matthew Klinka and Wesley Wallace were Auburn University students who both won the summer CMB-USRS How have the results been disseminated to communities of interest? Yes; basic science as indicated in the publication section, to other scientists. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We collected nearly two full years of seasonal samples at the 0.2 and 7 micron scale, and also micrographic data (using a FlowCAM) from the free waters of the Bay and Mississippi Sound. WE have been given a set of samples that go back to 2009 to collect additional DNA so that the project will eventually extend significantly backwards in time to provide a larger longitudinal data set. In addition. We collected Mnemiopsis leidyi (a type of ctenophore) for microbial analysis but unfortunately was unable to reliably collect animals throughout the two year collecting priod, at all time of the year because we often did not collect animals (despite a rigorous and repeatable small-boat collection protocol that included on average 1 boat trip per month. The problem was exacerbated by weather AND the invasion of a second, previously undescribed ctenophore (for which we collected extensive DNA samples, established EM samples and have collected an etensive library of images) which will be published later this year (see previous abstracts indicated in this report). The invasive animals competed with Mnemiopsis and reduced food, killling off the native Mnemiopsis for nearly a full 5 months. Thus that data set is limited because of varying availabilty of the animals. We however have completed the NGS analysis of the free living microbes, and have isolated 50 ctenophore gut-located strains of microbes, which we are extensively characterizing (no abstracts or papers yet). Finally we will make a final one or two ctenophore collection soon to capture the Spring and possibly later on to obtain early Summer seasonal microbial populations assocated with the ctenophores, will analyse by Next Gen Sequencing (not mentioned in the original work) and will close the project, with probably 4 or more papers. the work will be published as a thesis (Richard Mariita, PhD, in my lab). We were not yet able to establish trophic transfer to fish, although we have examined fish ctenophore predators. We may need to return to that project in the future.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: R.M. Mariita, S. Bhatnagar, K. Hanselmann, M. Hossain, M. Liles, A.G. Moss, J.L. Leadbetter, D.K.Newman (2014)Characterization, Comparative Genomics and Genome Mining for Antibiotics and Secondary Metabolite of two Actinomycetales isolates. Annual Meeting of the American Society for Cell Biology, Phil., PA. 12/6-10/2014. Mol. Biol. Cell 25:1752 Abstr. #P2378.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: R. M. Mariita, M. J. Hossain, M. R. Liles, A. G. Moss. 2015. Metagenomic Assessment of an Estuarine System Reveals High Microbial and Mobile Genetic Element Diversity during Spring. Annual Meeting of the American Society for Microbiology, New Orleans, LA., May 30th-June 2nd 2015, Abstr. 15A1637GMASM
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: R. M. Mariita, S. Bhatnagar, K. Hanselmann, M. J. Hossain, S. Dawson, J. Korlach, M. Boitano, R. J. Roberts, M. R. Liles, A. G. Moss, J. R. Leadbetter, D. K. Newman. 2015. Mining for Secondary Metabolites and Epigenomics for Two Soil Actinomycetales co-Isolates. Annual Meeting of the American Society for Microbiology, New Orleans, LA. Abstr. May 30th- June 2nd 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Richard M. Mariita, Mohammad J. Hossain, Mark R. Liles, Anthony G. Moss. Seasonal Variability in the Diversity of Microbial Assemblages and Antibiotic Resistance Determinants of an Estuary System. Alabama NSF EPSCoR Science and Technology Open House. Renaissance Hotel Montgomery, AL.: Jan. 30-31, 2015.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: R. M. Mariita 2014. Isolation and characterization of species affiliated with family Actinomycetaceae. Invited presentation at the MBL, 18th August, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Richard M. Mariita, Clayton Lester, A. Ryan Hoskins, Jacob Britt, Matthew Klinka, Clifford Wood and Anthony G. Moss 2014. Molecular characterization of an invasive novel cydippid ctenophore. Auburn University Research Week presentation, June, 2014. Platform Presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Richard M. Mariita, Clayton Lester, A. Ryan Hoskins, Jacob Britt, Matthew Klinka, Clifford Wood and Anthony G. Moss 2014. Molecular characterization of an invasive novel cydippid ctenophore. Annual Meeting of the Alabama Academy of Science, Auburn, AL, March, 2014. Platform presentation.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: I reported on the invasion of a newly discovered ctenophore in Mobile Bay and Mississippi Sound at the Association for Limnology and Oceanography, at the annual meeting in New Orleans during February of last year. The presenation was given by undergraduate student Clayton Lester An initial report was given for both the effects of oil on microplankton, and for seasonal impacts on microplankton, at the same meeting. The presentation was given by undergraduate student A. Ryan Hoskins, who substituted for graduate student L. Gong. Changes/Problems: The project was very slow starting because the investigator had to locate repair and implement the BioSciences research boat, which was an instrumental component of the work. Once implemented and repaired it functioned well until late november 2013 whereupon it has needed a steering assembly repair, to be implemented very soon. Unfortunately only a single collection point, the winter 2013-2014 data could be collected for this reason. Other than that, no major problems. What opportunities for training and professional development has the project provided? Trained two graduate students: Limin Gong and Mariita Richard, both on molecular microbial biology. Gong is Chinese; Mariita is Kenyan. Trained undergraduates: Clayton Lester (discovered invasive ctenophore with AGM), A R Hoskins (DNA extraction, purification and quantification), J. Britt and M. Klinka worked on ctenophore biology and parasitic amoebae on the ctenophore; C. Paul Wood and AR Hoskins worked with the newly discovered dinoflagellate, which seems to pass between ctenophore species. How have the results been disseminated to communities of interest? Results have been partly disseminated at scientific conferences and are being developed for publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Complete writing up manuscripts on: 1. the invasive ctenophore discovery 2. the molecular biology of the invasive ctenophore 3. the molecular ecological microbial impact of the ctenophore on Ms Sound and Mobile Bay 4. the seasonal variation in molecular microbial ecology of Mobile Bay, comparing with previou FOCAL data (material provided by A Ortman from U. South Alabama and DISL); this may include the impact from summer storm Isaac 5. complete the oil impact manuscript with R. Condon and Ortmann (and Yueh?) of Univ. AL. Tuscaloosa

Impacts
What was accomplished under these goals? We have learned that oil impact causes a distinct redirection of the trophic flow. This wes partly reported as part of a GOMRI-I funded grant that overlapped with this project. The work was published previously in PLOS-1:Ortmann AC, Anders J, Shelton N, Gong L, Moss AG, et al. (2012) Dispersed Oil Disrupts Microbial Pathways in Pelagic Food Webs. PLoS ONE 7(7): e42548. doi:10.1371/journal.pone.0042548. No molecular analyses were included in this work. Data for this analysis was entirely flow cytometric: the FlowCAM and a standard flow cytometer available at the Condon lab at DISL. We continue to analyse the data using both eukaryotic and prokaryotic molecular markersDNA collected from this project is being subjected to Next-Gen genomic sequencing methods to determine the diversity and quantity of different groups of microbes in the treatment conditions. We have learned that the invasive ctenophore is most likely Pleurobrachia globosa or a closely related species. This animal normally inhabits the Western Pacific throughout the Far East. Animals may have been transported to Mobile Bay and Miss. Sound in ballast water, esp considering that there is considerable shipping with the Far East via interactions with the Korean auto manufacturers in Alabama. FlowCAM (and direct visual analysis) of the Miss Sound plankton and the lower Mobile Bay reveal significant changes in the zooplankton of those bodies of water. The resident Mnemiopsis population was nearly wiped out for that winter as a consequence. We have purified and highly quantified mixed genomic DNA from the microbial plankton during the major summer storm Isaac and data and storm data and will compare that with the impact of the invasive ctenophore and the seasonal changes in the microbial populations. We have successfully performed SEM analysis of the amboeba and have determined that the cultured organism displays the same morphology as the ctenophore-attached cell (in situ). We have collected DNA from this organism and determined that it displayes significant lateral gene transfer from the host. This was not an expected result and underscores the importantance of studying this host-microbial association.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Hoskins, A.R.; Gong, L.; Ojima, M.; Moss, A. G. MESOCOSM-BASED ANALYSIS OF OIL AND DISPERSANT-INDUCED MICROPARTICULATES.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Lester, C.; Hoskins, A. R.; Mariita, R. M.; Britt, J.; Klinka, M.; Moss, A. G.: A NEWLY INVASIVE CYDIPPID CTENOPHORE IN MOBILE BAY AND MISSISSIPPI SOUND.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: With R. Condon, and L. Gong, we are developing new analysis of trophic energetics. Work is in progress and should be sent out published this year.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Molecular characterization of an invasive novel cydippid ctenophore. 2014. R. Mariita, C. Lester, A.R. Hoskins, J Britt, M. Klinka, C.P. Wood and A. Moss. Cellular and Molecular Biosciences & Department of Biological Sciences, Auburn University, Auburn, AL 36849
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Parasitic amoebae of the ctenophore Mnemiopsis leidyi. Matthew Klinka, Jacob Britt and Anthony G Moss, Biol. Sciences, Auburn University, Auburn, AL 36849


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Outputs Personnel (the PI and his undergraduate and graduate students) have carried out nearly a full-year of collection of ctenophores and microbial plankton samples. All freely suspended microbial plankton less than 200 microns diameter were sampled with a FlowCAM (procured via a previouis NSF RAPID project, 2010) to indicate particulate size and to identify freely swimming ciliates, flagellates, invertebrate larvae and bacterial aggregates in addition to oil droplets. Water samples were additionally collected for the extraction and analysis of DNA from microbes associated with the ctenophores when available and from swimming and suspended particulates throughout the entire period using a dual-level (7 um and 0.2 um) vaccuum filtering technique. DNA has been extracted from approximately 1/2 of the samples (there are 12/collection date X 12 months). We have tested for DNA quality and yield: quality is excellent as determined by A260/A280 ratios. We continue to extract DNA and test DNA quality via PCR. Samples will be prepared for next gen analyses in March of this Spring. We also have extracted DNA from an invasive ctenophore discovered in the Fall of 2012 as a primary invader of Mississippi Sound. We have done preliminary sequence analyses of this animal but while the results clearly show that the DNA source is greatly enriched for the ctenophore, there is yet (Feb 2013) no definitive demonstration of the ctenophore ID, although morphology supports the claim given in Outcomes (below). Dissemination The general quality of the changes in microbes have been discussed in marine biology classes on campus. An abstract on the occurance of an invasive ctenphore has been submitted to the ASLO annual meeting for February 2013 (to be held in New Orleans 2/17-2/22) and will be presented at that meeting in a platform session. PARTICIPANTS: Individuals: Anthony G. Moss, PhD. is primary PI on this project. Mark Liles PhD. did not receive remuneration but was advisor on this stage. Training and professional development. Graduate Students: Shilpa Waduwadara, Jeffrey Daniels, Limin Gong, Mariita Richard. Undergraduate Students: Matthew Klinka, Clifford (Paul) Wood, Wesley Wallace, Ana (Ryan) Hoskins, Clayton Lester, Jacob Britt, Alyssa Eidbo. TARGET AUDIENCES: Target audiences will include coastal managers with regard to the cycle of changes in response to bay perturbation, and additionally state legislators with regard to the invasive ctenophore. The work will be published in academic journals, and will be included in classroom work for the public, and there will be an attempt to target the shipping trade to warn them of the dangers of uncontrolled release of ballast water. Currently, while there is no evidence to support this mode of transfer of the invasive ctenophore but it is unlikely that there would be another mode of transfer from the Far East to the Gulf of Mexico. Increased Far Eastern and Gulf business traffic may a root cause, if ballast water regulations are not stringently followed. If the ctenophore appears to have established itself as of December 2012; if so this organism could have major impact on the Gulf oyster, shrimp and crab industry since it will most likely feed on larvae of those important economic fish stocks. PROJECT MODIFICATIONS: There was initially considerable difficulty in procuring the departmental boat; it had to have many repairs which rendered it seaworthy (there wre significant problems with the motor controls and power output but these were slowly discovered and corrected. Repairs were paid out of Departmental overhead but were lengthy and frustrating. By March of 2012 the boat was fully functional, and now only needs regular upkeep and the usual repairs to keep it safe and fully operational.

Impacts
(am listing only the outcome type that is relevant) Changes in knowledge We are still generating internal data sets that have not yet realized changes in knowledge with regard to the microbial plankton. A critical change in knowledge that unexpectedly occured with this project is that there has been invasion of Mobile Bay and Mississippi Sound by a small cydippid ctenophore that we tentitively identify as Pleurobrachia globosa based on morphology described by Moser (1903) from Indonesia; range is known from S. Japan to W. India. We observed P. globosa by accident; it came up in the plankton tows used for the usual ctenophore collections. We observed that this ctenophore eats small zooplankton. Where the new ctenophore is prevalent, there tend to be very few of the native ctenophore, Mnemiopsis leidyi. We expect that this animal intercepts trophic flow, preventing energetic support of the larger zooplankton that are preferentially 'fished' by M. leidyi. We suspect that this animal is an Asian invasive, possibly as a ballast animal invader.

Publications

  • Lester C., Hoskins A., Mariita R., Britt J., Klinka M., Moss A. 2013. A newly invasive cydippid ctenophore in Mobile Bay and Mississippi Sound. Annual Meeting of the Association for the Sciences of Limnology & Oceanography: Aquatic Sciences, New Orleans, LA, February 17-22.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Personnel have collected microbial samples from water collected during the year at shoreline locations, from Dauphin Island northward to Mobile Harbor. Samples have also been taken for a MESC project that specifically analyses the impact of oil on suspended plankton populations. Samples have been analysed to date by FlowCAM for the presence of oil and suspended eukaryotic microbes (protists and algae). Samples have been filtered at the 7 um and 0.22 um level and frozen. We have been working with the Halanych lab to use enhanced preparation techniques to provide greatly improved DNA extraction, which will then be used as template to provide next-gen (microbial population shotgun sequencing using 454 and/or Illumina technique (the Mollette lab has recently obtained an Illumina sequencer). We are at the beginning of this phase of the research. We will also attempt to quantify overall viral populations by fluorescence microscopy of frozen samples, and will also seek to amplify out antibiotic resistance plasmids by PCR; all of this needing to be done yet. DNA extraction optimization from the filters however is now complete, and we will move into that phase now for the rest of the project, while samples continue to be collected. PARTICIPANTS: Limin Gong, PhD student, Jeff Daniel, MS student, Shilpa Waduwawara, MS student, Miriam Ojima, visiting CMB Summer Scholar from Florida State University, Steven Broadway, AU undergraduate in microbiology. These students received training by learning how to do the FlowCAM analyses, and the sample filtration protocols, as well as DNA extraction methods. Work was partnered on the MESC project with Dauphin Island personnel, most notably Drs. Monty Graham, Rob Condon and Alice Ortmann. TARGET AUDIENCES: Presentations made to Marine Biology Students at Auburn University. A Science Cafe was given early in the project and another one is planned for this spring. PROJECT MODIFICATIONS: The only significant change is that the samples will be subjected to next-gen sequencing (this had not been originally planned); this is possible through cost-savings, and the dramatic reduction in the cost of such services in the past year. Additional samples will be provided for the project by Dr. Ortmann, named elsewhere; this will extend the sampling backward in time to 2009 or earlier.

Impacts
An extension from this project was the oil impact project with MESC/Dauphin Island. The two projects will work synergistically to give the final outcomes. The most significant change in the approach and conditions arises from a change in plan to perform next-gen sequencing of the samples, rather than using PCR-based amplification of the DNA for sequencing; this will produce a more reliable end product because there is far less sample bias in the next-gen approach.

Publications

  • No publications reported this period


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Activities included: collection of ctenophores from the Alabama and Florida coasts during the Spring, Summer, early Fall and early Winter. Animal were treated for the removal and archiving of surface and gut microbes, and parallel water samples collected from each site to compare with the ctenophore samples. Events included: 1. As part of outreach to Middle-school and high school students, approximately 7 college students and graduate students received instruction in microbial assocations with ctenophores and marine microbes in general. Also, 2) approximately 50 middle-schol and high school students had hands-on demonstrations with microbial ciliary activity in the same outreach program. Finally, 3) as part of the NSF-EPSCoR program, high school students attending a marine sciences program at Tuskegee were trained in molecular microbiological techniques, oil pollution and oil drilling, and the BP Deepwater Horizon Spill. 4) Middle- and high-school students and their parents attending a special Auburn University weregiven instruction in marine microbiology and the BP oil spill. In addition AGM attended the MESC-GRI program in Mobile, to seek extended funding for the oil pollution component of the project. Products: Collaborations were formed with faculty at DISL, Univ. S. Alabama, and Univ. Alabama as part of the MESC-GRI that was related to this perturbation-based project. My research lab web page is being updated and redesigned to include work and descriptions from work associated with this project. This is only just being started. The web site is http://www.auburn.edu/~mossant. PARTICIPANTS: Initially, Khristian Smith and Erin Donovan were active on this project, however they left suddenly in late spring and midsummer, 2010. Graduate Students Shilpa Waduwawara and Jeff Daniel helped during the Fall, as did Katherine Hotop and Cameron Welch. Limin Gong joined the lab in the winter of 2011. As part of the MESC-GRI collaboration, Alice Ortmann, Rob Condon, Monty Graham, and Yuehan Lu are part of the collaboration on the BP-MESC-GRI project, which is related to the Hatch Project. In addition, a Hatch MultiState award was provided to Dr. Mark Liles and myself; that project involved collaboration with Dr. Larissa Parsley at University of Mobile; this project is related to variation in the microbial assemblage and antimicrobial activity of microbes, in response to seasonal and acute weather perturbations. This work, funded 10/1/2010, is in startup phase currently (2/2011). In addition, Liles, Alan Wilson, Kenneth Halanych and myself were awarded a NSF-MRI RAPID to obtain and operate a FlowCAM to examine primarily eukaryotic micro and holoplankton in Mobile Bay and offshore of Alabama and Florida, in response to the BP Oil Spill. The instrument will also be used in collaboration with the DISL and Univ. S. Alabama group in the MESC-GRI project. TARGET AUDIENCES: Target audiences and efforts: As mentioned above, students were trained; they were target audiences. Of these, the students who attended the Tuskegee University session were all minority students. Students at the Opelika Middle School location were largely minority African American students, also. Students in AUExplore outreach were partly minority students. PROJECT MODIFICATIONS: The only major change in approach involves the inclusion of the MESC-GRI program regarding the effects of the oil spill; the main funding came from the USDA as part of the multi-state Hatch project to examine seasonal and acute weather events. these programs did less to change the fundamental nature of the project, than they did to extend and apply it. In addition, toghether with co-PIs Drs. Kenneth Halanych, Alan Wilson and Mark Liles, as I received as main PI, major funding from the NSF, as part of their NSF-RAPID MRI program, to obtain a FlowCAM, to accurately and rapidly describe primarily protozoan populations in seasonally and acutely impacte marine loctions, mainly the Mobile Bay. The instrument arrived late fall, and was given a shakedown run in pristine waters of the Eastern Gulf of Mexico while on board the R/V Pisces from 11/15-11/23/2010

Impacts
We are still compiling the results. Unfortunately two key students in the marine microbiology program: Erin Donovan and Khristian Smith, left the program suddenly, and I have had to recover my research group during the past Fall program. Early work have shown that microbes are associated with the ctenophore only in the gut and in particular organ systems, microbes represent the local environmental diversity in some parts of the host, while other parts have quite different assemblages of microbes.

Publications

  • Donovan, E.W. and A.G. Moss, 2009. Organization and bacterial assemblage of the glandular papillae of the comb jelly Mnemiopsis leidyi. Molecular Cell Biology 20:2320 (abstr)


Progress 10/01/09 to 02/21/10

Outputs
OUTPUTS: Activities: Ctenophores were collected from several sites along the Gulf coast for purposes of analysing the microbial populations, and also for purposes of carrying out molecular analyses of the ctenophores over time and space in different locations. In this phase, animals were collected from Texas to Northern Florida in mid-Fall and at Christmas; animals could not be found during this period in Alabama Bay. We also have continued to work on the microbial populations found differentially in different organ systems, including the gut and epithelium. Investigator has also initiated studies with new graduate student (see below) concerning bulk fluid flow in the near-benthic regime by ctenophores, using laser paticulate interferometry velocity analysis, a collaboration with B. Thurow of Auburn University Aeronautical Engineering. Although not originally a part of the design, this opportunity presented itself and we are following through. This aspect of the project is relevant to understanding how microbial populations transfer to the ctenophore when it encounters benthic interfaces. Three undergraduate students are involved in this process; a third is working on a different but related project regarding wound repair. Animals could not be procured for that student during this period. Events: Plans have been made to disseminate these and earlier results at a meeting of the American Society of Limnology and Oceanography; that meeting is planned for June, 2010. I will co-chair a session on the invasive characteristics of micorbial populations associated with ctenophores. Services: Investigator has conducted tutoring/training of 4 undergraduate (Cameron Welch, Daniel Joiner, Timothey Tatum and Caitlin Lassiter) and 4 Graduate students (Khristian Smith, Erin Donovan, Matthew Dodson and Shilpa Waduwawara) and one high school student (Perri Moeller) during this period. Moeller applied for two prestigious scholarship awards during this period. Products: Include a patent application on a tool to measure microbial movement, and a mount to support a micromanipulator; both relevant to the project because they provide means of monitoring/manipulating microbes. New collaborations during this period include work with Martin Zimmer and Thorsten Reusch of the IFM-GeoMar in Kiel Germany; their interest stems from the impacts of Mnemiopsis on Kieler Fjord, the Baltic, and the North Sea. I point out here that because no projects were funded in the HATCH program that molecular analyses are very limited in nature. Some of the work has had to be sent out (hence the collaborations with Zimmer and Reusch). PARTICIPANTS: Individuals who worked on the project include: Shilpa Waduwawara, Erin Donovan, Matthew Dodson and Khristian Smith, all graduate students. Daniel Joiner, Timothy Tatum, Caitlyn Lassiter and Cameron Welch, all of whom are undergraduate students. The efforts provided training and professional development for all of these students. Partners new to the project include Dr. T. Reusch, IFM-GeoMar, and Dr. M. Zimmer, also IFM-GeoMar, Kiel, Germany. TARGET AUDIENCES: Middle School and High School Students have benefitted from this project. I have given presentations to Opelika High School about jellyfish and ctenophores. We plan to discuss/display ctenphores at the upcoming AUExplore program, Spring 2010. The results will also eventually have positive impact on coastal management for the state of Alabama. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Through collaboration with T. Reusch's group of IFM-GeoMar (to whom we have turned for molecular analyses/support) we have determined that there are distinct populations of ctenophores along the US coastline. The populations roughly approximate original distributions described in the early 20th Century by Mayer. The data include other analyses that refer to invasiveness of local populations. These data are important because they can be used to track movements of ctenophores in the near-coastal environment. We have learned that there are distinct microbial populations in the ctenophore body, and that they reflect different aspects of the water column microbial distribution. This is relevant to coastal management because it implies that microbial variation and load resulting from agricultural runoff can directly affect the ctenophore populations, which grow and diminish as a result of coastal nutrient loading. We have learned from the PIV analyses described above that the ctenophore has enormous potential to provide a near-benthic water turnover, resuspending particulates from the benthos into the water column at astonishing rates; as much as 65% of the water can be turned over per day by small (<1 cm) ctenophores at their maximum reported population concentrations.

Publications

  • Thorsten B.H. Reusch, Soeren Bolte, Maximiliane Sparwel, Anthony G. Moss, Jamileh Javid, 2010. Molecular Ecology (submitted) title retained at this time to maintain security.
  • Shilpa Waduwawara, Cameron Welch and Anthony G. Moss. 2010. SIGNIFICANCE OF WATER DISPLACEMENT BY SEDIMENT-APPOSED MNEMIOPSIS (CTENOPHORA: LOBATA). Biological Sciences, Auburn University, Auburn, AL 36849 (submitted)
  • A.G. Moss, E.W. Donovan and M.B. Dodson, 2010. BACTERIAL ASSEMBLAGES OF THE LOBATE CTENOPHORE Mnemiopsis. Biological Sciences, Auburn University, Auburn, AL 36849 (submitted)