CN-770000-22136-TA2405.pdf

FFWCC Multi-Year Project Request Date: May 11, 2023 Project Title: Field Molecular Testing and Validation of a Species Distribution Model for a neurotoxigenic cyanobacterial species, Aetokthonos hydrillicola, in Florida watersheds.

Contact Information:

PI Susan B. Wilde, Co-PI Cristian Montes, Co-PI Caterina Villari, Erika Klar, Wesley Gerrin and Katherine Callaghan.

Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602- 2152. Cell phone: 706-247-5912 swilde@uga.edu

Need for Research/Outreach

Cyanobacterial harmful algal blooms (cyanoHABs) are a threat to aquatic ecosystems worldwide a trend that is expected to increase with eutrophication, the spread of non-native species, and climate change (Sukenik and Kaplan 2021). Cyanobacterial harmful algal blooms are common in inland freshwater ecosystems, and while most of the attention has focused on the risks of toxic cyanobacteria to water quality and human health, cyanoHABs also present a threat to wildlife including rare, threatened, and endangered species (Carmichael and Boyer 2016). Epiphytic cyanobacteria present an emerging threat to species and community dynamics through the introduction of novel toxins, with potentially far-reaching effects across taxa and trophic levels within aquatic communities (Paerl et al. 2018; Haram et al. 2020).

Vacuolar myelinopathy (VM) is a disease which has been documented across the southeastern United States since its discovery in DeGray Lake, Arkansas in 1994. The disease causes lesions in the myelin of birds, fish, reptiles, and amphibians, resulting in a loss of motor function and fatalities.

We have field and laboratory evidence that this fatal disease is induced by a novel neurotoxin produced by cyanobacterium Aetokthonos hydrillicola, Aetokthonotoxin (AETX), growing on highly invasive aquatic plants, specifically Hydrilla verticillata.

The molecular detection of A. hydrillicola is currently limited to recently developed Polymerase Chain Reaction (PCR) assays that target the AETX biosynthetic gene cluster et al. 2023).

Conventional PCR, however, is time consuming, requires very specific lab equipment, and high-quality DNA extracts (i.e., free of inhibitors). Loop-Mediated Isothermal Amplification (LAMP), on the other side, offers a valuable tool for addressing these limitations. This isothermal molecular technique allows rapid, sensitive, and highly specific detection of a target region from crude DNA extracts, thus allowing for in-field testing capability with minimal equipment (Aglietti et al. 2021; Hamilton et al. 2020; Jenkins et al. 2011; Notomi et al. 2000; Villari et al.

2013; Williams et al. 2017). Quantitative PCR (qPCR) is a form of PCR used to quantify DNA within a sample using standard concentration curves (Kralik and Ricchi 2017). Testing samples both in field with LAMP and quantitatively with qPCR will allow us to test the validity of the species distribution model to confirm testing regions that are at high probability of having suitable habitat characteristics for A. hydrillicola.

Florida currently has 3 lakes which have had cases of VM confirmed by the histological analysis of brain tissue from field collected birds and/or avian feeding trial using invasive plants collected onsite. Now that we have verified AETX in Florida panthers with VM symptoms and histology, this number is likely much higher. Florida also has 11 reservoirs in which the presence of A.

hydrillicola has been confirmed, we propose focusing our collections in locations with Snail Kite/Eagle nest failures and FLM panthers/bobcats.

Recent Discoveries

Breinlinger et. al 2021 successfully described and structured a novel neurotoxin, aetokthonotoxin (AETX), which is produced by A. hydrillicola. AETX was confirmed as the cause of vacuolar myelinopathy via a chicken trial, in which chickens were exposed to a purified form of the toxin.

The study also found that the structure of aetokthonotoxin was such that A. hydrillicola would require a source of bromide to produce the toxin (Figure 2) (Breinlinger et al. 2021).

Gerrin et. al (manuscript in preparation) investigated environmental conditions that could trigger production of aetokthonotoxin. Using toxin concentration data from multiple years of hydrilla samples and numerous environmental variables (temperature, precipitation, lake elevation), models predicted that a decrease in maximum daily water temperature was the key environmental factor contributing to increases in toxin concentration. A species distribution model (Figure 3) was developed to further understand the distribution of the cyanobacteria and to also predict where potential suitable habitat areas are for A. hydrillicola colonization. This model highlighted several parameters negatively impacting the habitat suitability including increased soil cation exchange capacity, land cover dominated by hardwood trees, and urban land cover. There was one main factor positively impacting habitat suitability:

land cover dominated by conifers, which in the tested areas correspond to pine species. This has vast implications since a large commodity within the southeastern U.S. is commercial loblolly pine forests.

Aquatic plant management is still the only known mitigation for reducing wildlife mortalities caused by the plant infestations supporting A. hydrillicola and novel neurotoxin.

Florida has conducted extensive monitoring and management of aquatic plant invasions, water quality and ecosystem health. A key factor in determining risk for VM is estimating the potential distribution of A. hydrillicola. Notable differences have been documented in both landscape characteristics and water chemistry of lakes and reservoirs where SAV does or does not support A. hydrillicola.

In the current project, we will ground-test the previously created species distribution model to determine its accuracy in predicting A. hydrillicola risk by using LAMP to detect presence and absence of A. hydrillicola. We also plan to use qPCR to quantify A. hydrillicola present among samples from infested reservoirs. These analyses will be performed on low and high probability reservoirs predicted by the species distribution model.

Previous research has been focused on the causative neurotoxin for VM, however, due to its apparent seasonality, detection of AETX and presence of A. hydrillicola is limited. Utilizing molecular techniques will allow for detection of the cyanobacteria regardless of the season. By partnering with FFWCC we can expand monitoring and work together to optimize plant management strategies that are effective at controlling dense plant infestations supporting the epiphytic cyanobacteria before AETX presence.

Florida Lakes

Known Aetokthonos hydrillicola lakes

East Lake Tohopekaliga, Lake Tohopekaliga, Lake Cypress, Lake Hatchineha, Kissimmee, Lake Istokpoga

These lakes have the following classification:

Soils: Podzols. Dominant Topsoil. pH 4.50. Soils with a pH between 4.5 and 5.5 are very acidic.

While some crops such as tea and pineapple may tolerate these conditions aluminum toxicity is often a problem. Calcium Carbonate 0.00%, None to Very Low. Gypsum 0.00%, None to Very Low. Electrical Conductivity 0.10 dS/m, Very Low. Dominant Subsoil, pH 4.90.

Watershed forest type. Longleaf pine, Scrub pine

Additional lakes with similar soil and watershed forest type

Fish Lake, Alligator Lake, Lake Lelia, Lake Glenada, Lake Gibson, Lake Parker Lake Thonotosassa, Lake Lizzie, Coon Lake, Trout lake, Lake Center, Lake Gentry, Lake Arbuckle, Lake Jackson, Lake Marian, Lake Rosalie, Lake Trafford, Lake Caloosahatchee

Florida Panthers are endangered apex predators in the Corkscrew Swamp and Everglades. We hypothesize that the panther/bobcats food web has been altered by a range of invasive species from primary producers, invertebrate consumers, scavengers and a voracious top predator (non-native python).

We propose additional sampling in this southwest Florida ecosystem, including Hydrilla + additional SAV, Apple snails, Python, python eggs, Feral hogs. We know that native mammals that panthers and bobcats rely on are in decline including; Raccoons, White tailed deer, Fox squirrels, Opossum, Red fox, White ibis, Coots, Marsh rabbits, and Cottontails.

Figure 4. Map of South Florida illustrating sampling locations in relation to python distribution. Road surveys for mammals were conducted in the 1990s and 2000s along the Main Park Road (MPR) in Everglades National Park (ENP). Areas recently invaded by pythons and surveyed for mammals in 2009 2011 include Big Cypress National Preserve (BCNP), Collier-Seminole State Park (CSSP), Chekika (CHK), and Key Largo. Immokalee and Corbett Wildlife Management Area (CWMA; north of the map) are two sampled sites where pythons have not yet become established. The purple region represents the area of ENP where pythons were found in the 1990s and where reproduction was first reported (16). Red triangles represent localities of pythons found during 2008 2009.

Face-off: Novel depredation and nest defense behaviors between an invasive and a native predator in the Greater Everglades Ecosystem, Florida, USA

Figure 5. Selected photo sequence at a Burmese python (Python bivittatus) nest site from June 01, 2021 August 30, 2021, starting at top left (a) and ending at bottom right (o) camera deployment initiation photograph with clipboard where python is seen brooding her nest in center of frame (a); bobcat (Lynx rufus) discovers unguarded nest (b) and proceeds to depredate, cache, and uncover the eggs over several days (c h); the bobcat returns to find the female python back on the nest (i) and later proceeds to swipe at the snake (j l); post-nest salvage attempt by biologists, the bobcat returns to scavenge discarded, inviable eggs over several weeks (m o). Entire photo sequence can be viewed in MP4 video format through the ScienceBase Catalog at:

https://doi.org/10.5066/P97ZDQHY (Currylow et al., 2022). Photographs were captured in Big Cypress National Preserve within the Greater Everglades Ecosystem, Florida, USA. https://www.sciencebase.gov/catalog/item/62044bb2d34ec05caca1cfd4

Study Goal and Objectives

Determine risk of Florida lakes for VM disease outbreaks based on presence and severity of A.

hydrillicola colonization. We will develop an efficient protocol for detection of A. hydrillicola by designing a specific field-portable LAMP test and a quantitative laboratory assay obtained by adapting to use with qPCR technology an already published PCR primer set. We propose to sample A. hydrillicola from Florida watersheds encompassing previously confirmed positive reservoirs as well as potential A. hydrillicola -free ones. This protocol will be made accessible to labs throughout the U.S. to support early detection and eradication efforts. Specific steps for the achievement of this objective include:

i. Developing LAMP primers that detect specific genes responsible for producing AETX within A. hydrillicola.

ii. Adapting the current PCR primers for use in quantitative assays (i.e., qPCR)

iii. Assessing the specificity and sensitivity of the LAMP and qPCR assays using pure DNA extracts of A. hydrillicola, non-target cyanobacterial species, and plants from infested reservoirs.

iv. Determining the parameters needed for application of in-field LAMP testing.

v. Validating the versatility of the LAMP assay by using it to detect A. hydrillicola on different substrates (such as Hydrilla or other plants) in multiple reservoir locations within Florida watersheds that are at high and low probability of colonization by A.

hydrillicola based on the species distribution model.

vi. Evaluating the level of A. hydrillicola colonization on plant samples collected from infested reservoirs using qPCR.

Methods

Field Data Collection

The proposed project will be a collaborative effort between University of Georgia, University of Florida, and Florida Fish and Wildlife Conservation Commission. UGA will provide FFWCC with a list of lakes that will need to be sampled as a part of this project, as well as a field protocol to be followed for data collection consistency. We prioritize Hydrilla verticillata screening as this species most commonly supports A. hydrillicola, however, other plant species as well as soil samples will also be tested for cyanobacterial presence to test the versatility of our LAMP assay.

We have also documented A. hydrillicola growing densely on additional invasive species (Hygrophila polysperma, Limnophila sessiflora) as well as native SAV (Bacopa, Cabomba caroliniana, Ceratophyllum demersum, Utricularia spp.).

Samples will be collected by FFWCC and shipped to UGA to be screened for A. hydrillicola presence and molecular analysis.

Aquatic plant sample collection protocol

IMPORTANT: Prior to collecting and shipping specimens, please contact us to obtain a copy of our APHIS permit that MUST be included with your shipment.

Plant sample collection: Collect hydrilla or other plant sample from at or below surface, squeeze excess water out, and place in a ziplock bag. A 1-quart freezer style bag is plenty for screening purposes. Please be sure to only include vegetation. Label the bag with site and date information. Please include with submission: The GPS location of each site, and any water

Soil Sample Collection: At time of plant sample collection, collect additional soil sample in the same manner as plant sample collection.

Shipping: Place the sealed sample bag(s) in a cooler or waterproof packaging with the APHIS permit, a coldpack, tape the cooler/package shut, place it inside a box and mail* it to UGA Wilde Lab. Please call or email to let us know to anticipate your shipment and include the tracking number.

Submerged Aquatic Vegetation FIELD DATA COLLECTION FORM

DATE:

RESERVOIR:

Location Description:

GPS coordinates:

Water column depth:

Plant height:

Depth where plants were collected:

Temperature:

Oxygen:

pH:

Conductivity:

Alkalinity/Hardness: (any data on these parameters from your reservoirs would be valuable)

Turbidity: (please comment on water clarity, if instrumentation is not available)

Secchi depth:

Comments: (Was hydrilla actively growing, dying back? Treated? Were birds present? Any sign of impaired waterfowl?)

LAMP Design

LAMP primers will be designed utilizing the programs Geneious Primeã and Primer Explorer v5ã (Eiken Chemical Company). The complete DNA sequences of each AETX gene cluster (aetA, aetB, aetC, aetD, aetE, and aetF) will be retrieved from the National Center for Biotechnology Information (NCBI) repository and assessed in Geneious Prime. Candidate target region sequences will then be extracted and uploaded to Primer Explorer to generate candidate LAMP primer sets. The loop-binding primers will be designed with a fluorescent probe attached for visualization during amplification. The current methods of DNA extraction for A. hydrillicola, NucleoSpin Soil Mini kit and CTAB buffer extractions, require clean DNA that is difficult to obtain in the field due to the requirement of lengthy and laborious procedures and several pieces of equipment et al. 2023). Using LAMP, which is less sensitive to inhibitors, there are many crude DNA extraction methods that may enable detection of A. hydrillicola without extensive processing (Aglietti et al. 2021; Hai-sheng et al. 2012;

Hamilton et al. 2020; Hodgetts et al. 2015; Mason and Botella 2020; Nagai et al. 2012;

Stehlíková et al. 2020; Zou et al. 2017). Our study will assess these different methods to determine which extraction technique best prepares A. hydrillicola-contaminated samples for amplification. Different visualization techniques will also be tested to determine which is most accurate and suitable for field testing. A dipstick extraction method and fluorescent probe-based visualization will be the first pair of extraction and visualization methods tested (Mason and Botella 2020; Meinecke et al. 2023)). Sensitivity and specificity testing will also be performed with several other species of bacteria, including other cyanobacteria.

qPCR Design

We will adapt a conventional PCR assay developed to detect A. hydrillicola AETX biosynthesis genes et al. 2023) for use in qPCR by the addition of fluorescent probes or dyes for visualization of amplification. Quantification of A. hydrillicola DNA will be based on comparison to standard curves, generated by amplification of known quantities of DNA from cultured freeze-dried A. hydrillicola. Field samples will be tested using LAMP to screen for the presence of A. hydrillicola and then with qPCR to quantify the amount of A. hydrillicola DNA present in each sample. This will allow to assess the relative level of colonization among different substrates.

DNA extraction protocol identification

Along with freeze-dried cultured A. hydrillicola, samples from Hydrilla leaves that have the cyanobacteria growing on them will be used to identify the most suitable extraction protocol for both in-field and laboratory settings. The Qiagen DNeasy Plant Kit, a dipstick extraction, boiling, Chelex, or a combination of several methods will be tested (Aglietti et al. 2021; Hai-sheng et al. 2012; Hamilton et al. 2020; Hodgetts et al. 2015; Mason and Botella 2020; Nagai et

al. 2012; Stehlíková et al. 2020; Zou et al. 2017).

Application of LAMP and qPCR testing to ground-truth a Species Distribution Model

To test both A. hydrillicola from field samples and ground-truth the species distribution model, Hydrilla samples from 15 different sites in Florida will be collected.

Each sample will first be analyzed using a portable LAMP device in the field (Hamilton et al.

2021). If positive, 3-5 additional samples will be collected and shipped to the lab for qPCR analysis to confirm the field-based results using pure DNA extractions. High probability watersheds from the species distribution model where A. hydrillicola has not yet been detected will be tested for presence of the cyanobacteria. Low probability watersheds will also be sampled to determine how accurately the model can predict the presence of A. hydrillicola in reservoirs.

The upper Mississippi River basin shows a relatively low probability to house suitable habitat for A. hydrillicola, based on the model, however, the distribution of Hydrilla encompasses the region and reservoirs have not yet been testing for cyanobacterial presence.

Other Substrate Testing

Besides Hydrilla, A. hydrillicola has also been observed growing on leaves, stems and root hairs of invasive and native aquatic plants including; Egeria densa, Myriophyllum spicatum, Najas quadalupensis, Potamogeton illinoiensis. Interestingly, we have also found Aetokthonos growing on water willow root hairs and on submerged loblolly pine needles. A. hydrillicola found growing on American water-willow was detected using PCR assays targeting the toxin gene cluster and the internal transcribed spacer 1 (ITS1) portion of ribosomal DNA. However a primer set specific to the ITS2 region did not detect the cyanobacteria on this new substrate, suggesting there might be some variability within the species et al. 2023). Moreover, there was no toxin production, indicating that different substrates may influence AETX synthesis. This is the first indication of genetic and phenotypic diversity among colonies of A. hydrillicola. To better delimit the habitats of A. hydrillicola within an infested reservoir, we will collect samples from other substrates, such as soil, other aquatic vegetation, and submerged pine needles.

Figure 6. Most of the Florida lakes where Aetokthonos hydrillicola has been documented are within the Kissimmee Chain of Lakes. We further divided these lakes into subwatersheds based on the location of inflows, and this figure indicates those documented with A. hydrillicola using red hatching.

List of Florida sites to prioritize for plant collection

(* indicates Aetokthonos hydrillicola documented previously at this site)

Lake Tohopekaliga*

East Lake Tohopekaliga*

Lake Hatchineha*

Lake Cypress*

Lake Istokpoga*

Lake Okeechobee

Lake Trafford

Mariah McInnis will be our primary Contact for plant collection coordination. She will ensure that biologists follow collection protocols and submit forms. She will set up a meeting for Wilde lab and FWC biologists to go over the protocols and answer any questions. We propose conducting this meeting in August 2023.

Deliverables: A mid-point status report for the first year of funding will be sent by December 15, 2023 and a final report provided to the FFWCC by June 30, 2024. We will continue to add this Florida AVM monitoring and research to our website hosted at the University of Georgia in conjunction with the Warnell School of Forestry and Natural Resources. This site will be linked to UGA Bugwood network: Center for invasive species and ecosystem health and Army Corps of Engineers: Invasive Aquatic Nuisance Species Program. We will also link to the Invasive Plant Management Section: Florida Fish and Wildlife Conservation Commission.

http://www.warnell.uga.edu/swilde/ . We will attend the 2023 FFWCC APM annual research meetings to present our findings and discuss potential management strategies to reduce the risk that this invasive associated epiphyte presents to fish, wildlife, and potentially human health. We will also present our findings at additional scientific conferences (FAPMS, NALMS) and submit papers for publication in journals and outreach publications. A complete molecular guide for detection of A. hydrillicola as it pertains to VM risk throughout the U.S. will be published for additional research and use throughout affected areas.

Timeline of Activities

TIMELINE OF KEY ACTIVITIES YR 1

Jul- Dec

YR 1

Jan- Jun

YR 2

Jul- Dec

YR 2

Jan- Jun

YR 3

Jul- Dec

YR 3

Jan- Jun

Develop and validate in-field and quantitative molecular detection methods

Collection of Hydrilla and other substrates from throughout Florida watersheds

Test LAMP detection on field samples

Conduct qPCR detection to verify LAMP detection and quantify DNA

Write and disseminate results

Budget Justification 2023-2024

1. PERSONNEL

A. Salaries: The personnel for this project include:

Wesley Gerrin, Research Professional (200 hrs @ $25/hr) ($5000) Katherine Callaghan, Research Professional (200 hrs @ $25/hr) ($5000) Erika Klar MS graduate student (500 hrs/$25/hr ) ($12,500)

The research professional and graduate student will assist PI Dr. Wilde in field sampling, algal evaluation, toxin screening, analyses, and reports. The research professional and graduate students will work with PI Wilde, Co-PI Montes, and Co-PI Villari to develop risk assessment analysis, modelling, mapping, and writing peer-reviewed publications.

B. Fringe benefits

51% for the research professionals. ($5,100) 5% for the graduate student on 50% research assistantship. ($625)

Total Personnel $28,225

2. SUPPLIES

Supply charges include consumable materials for laboratory analysis including counting slides, cover slips, extraction glassware and chemicals necessary to complete the project ($250).

Laboratory supplies will be required for microscopic screening for the cyanobacterial species ($500). Cyanotoxin analysis will require supplies for chemical extraction including specialized glassware, pipets/tips, solvent chemicals, and filters ($1500)

Total supplies: $2250

3. TRAVEL

Travel funds are requested to collect samples from Florida lakes. Funds are requested for 2 persons @ $40/day per diem for 5 days and ~$100/night lodging for 4 nights ($1,200).

Total travel: $

7. OVERHEAD/INDIRECT Indirect Costs are calculated at the rate of 10% of Total Direct Costs ($ ). This is the rate allowed by the Florida Fish and Wildlife Conservation Commission. Total Budget: $3 ,84 . 0

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