Illuminating the Microbe Associated with AIS in the Search for their Achilles Heel

Transcription

1 Illuminating the Microbe Associated with AIS in the Search for their Achilles Heel Chan Lan Chun and Michael J. Sadowsky & September 16, 2015

2 Aquatic Invasive Species in Minnesota Waterways Aquatic invasive species (AIS) pose a common threat to the health, structure, and function of aquatic ecosystems. These include: - Zebra mussels (Dreissena polymorpha) - Quagga mussels (D. rostriformis) - Eurasian Watermilfoil (Myriophyllum spicatumas) Only limited methods presently exist to control AIS.

3 Microbes in Aquatic Ecosystems Microbes are present in large quantities and are highly diverse in aquatic ecosystems. Microbes in waterways play foundational roles as primary producers and through their roles in nutrient cycling. Some bacteria have evolved to live in close association with other aquatic organisms (e.g. macrophytes, bivalves, fish etc) Bacteria Archaea Animals Fungi Plants Eukarya Protists

4 Microbial Association with Aquatic Organisms These associations are often dynamic (symbiotic, pathogenic, parasitic) and are based on nutrition and habitation provision. Some bacteria are found in association with mussels, clams, and tubeworms some providing the majority of nutrients. Aquatic plants are capable of supporting the growth of a variety of microorganisms, including some pathogens.

5 Example: Bacterial Epiphytes of Macrophytic Aglae Large mats of the macrophytic alga, Cladophora harbor enteric bacteria, including some pathogens and are of public health concern: Higher levels of enteric bacteria are found in floating mat of macrophytes than other media. C. botulinum These bacteria can detach and be released into the surrounding water, influencing water quality, health, and recreational activity.

6 Microbial Interactions with AIS Only limited information is available. Bacterial toxins have been discovered which induce mortality in zebra/quagga mussels - Soil-water bacterium, Pseudomonas florescens (Molloy 1998) - Four bacterial isolates from stressed mussels (Ji-Dong et al. 1994) However, many other microbial species produce toxins or factors that may be specifically lethal to AIS. These microbes themselves or their toxins may be effective in controlling AIS populations at very low concentrations

7 Studies on AIS-Microbe Interactions Culture-based method - Identification and quantification of fecal indicator bacteria (FIB) - FIB signal the possible presence of human pathogens

8 Studies on AIS-Microbe Interactions Microscopic observation - Visual observation of AIS-microbe interactions - Fluorescent-labeling techniques

9 We propose to use DNA sequencing approaches to identify microbes associated with AIS.

10 Hypothesis Healthy AIS Sick or dying AIS Identification of AIS specific microbiota

11 DNA Sequencing (Metagenomics) The study of the totality of genetic material (genomes or their fragments) recovered directly from environmental samples. Why use metagenomics - The majority of microorganisms in environmental and animal samples remain uncultured (<<1%) or non-culturable. - Cultivation-based approaches for discovery of diversity and microbial functions bear the risks of missing novel genes and microbial activities.

12 Advantages of Metagenomic Approaches Versatility: suitable for both sequence- and function-based discovery. Adaptability: various environments can be prospected for desired microbes.

13 General Metagenomic Approaches DNA Extraction Sequencing Sequence Analysis Identification of AIS specific microbiota

14 Objectives Understand the microbiota associated with AIS in Minnesota waterways, across time and space, using metagenomic approaches. Develop molecular methods which allow us to be able to ascertain the presence/absence of AIS and quantify their abundance in lakes. Develop effective and perhaps selective microbiological control agents for management of AIS.

15 Overall Approach Metagenomic characterization of microbial communities associated with AIS Sample collection Identify microbial community associated with AIS. Correlations of AIS microbial community to biological characteristics of AIS.

16 Prediction AIS support large populations of microbes (bacteria and fungi) which can be extremely distinctive to AIS depending on their lifecycle and particular environmental conditions. Naturally-occurring microbes may produce toxins which could be used as lethal agents to specifically control AIS. Naturally-occurring AIS symbionts may be manipulated to control AIS under specific conditions.

17 Target AIS Eurasian Water Milfoil Zebra Mussels Quagga Mussels

18 Eurasian Water Milfoil (EWM) Invasive aquatic plant that proliferates in Minnesota due to its ability to grow at wide ranges of temperature (even low) under a variety of nutrient concentrations. EWM fragments can re-grow into dense floating mats. EWM displaces native aquatic plants, lowers dissolved oxygen content, and causes eutrophication due to nutrient loading from vegetation decay. Traditional methods of control include mechanical harvesting and application of chemical herbicides.

19 EWM Infestation in Minnesota EWM was introduced to North America from Europe and spread primarily by boats and water birds. From MN DNR EWM was first discovered in Lake Minnetonka in EWM can limit recreational activities on water bodies and alter aquatic ecosystem by displacing native plants. EWM has difficulty becoming established in lakes with well-established populations of native plants.

20 Microbiota Associated with EWM Sample Collection Multiple sites based on anthropogenic influence, water quality, and milfoil abundance Benthic, epiphytic, and endophytic regions of EWM in various state of decay Water and sediment samples near EWM will be sampled EWM Microbiota Total microbial community associated with EWM Distribution of rare and abundant microbes 16S and 18S rrna/its metagenomic analysis for both prokaryotic and microbial eukaryotic lineages Sequencing to a depth of 200,000 OTUs Correlation EWM microbial community to biological growth stage of EWM and various state of decay EWM microbial community to water quality Understanding of microbial biodiversity and ecology of EWM

21 Zebra and Quagga Mussels Temperate, freshwater invasive filterfeeding bivalve mollusks. Extraordinary ability to reproduce - Lay over 1 million eggs in a season - Spawning catalyzed by temperature and food availability. Costly macrofouling and ecological pests. Extinction of some native clam species Clog water-intake pipes and negatively affect tourism.

22 Infestation of Zebra and Quagga Mussels in MN First found in Duluth/Superior Harbor in Zebra mussels have now spread into ~100 inland lakes, causing great ecological disruption. Limited options to reduce the number of these invasive mussels.

23 Microbiota Associated with Invasive Mussels Sample collection and processing Minimum 50 adult mussels from each site Mantle cavity fluid will be obtained via syringe Gill and gut samples dissected aseptically Water and sediment samples near mussels will be obtained Metagenomic Analysis Microbiota associated with cavity fluid, gills, and guts of adult mussels Distribution of rare and abundant microbes Analyses Microbial community in relation to mussel age Microbial community associated with larvae veligers Molecular markers for key microbes associated with invasive mussels.

24 Summary Understand the microbiota associated with these invasive species. Provide a distribution map of microbes associated with AIS and create molecular tools to identify and quantify their abundance in lakes. Allow us to eventually develop effective, specific, and cost effective biological control agent of AIS. That is, we can identify bacteria that are unique to AIS and use them to develop biocontrol strategies

25 Thank you for your attention! Questions?