USACE ERDC. Moderator: Courtney Chambers Julie Marcy AVM Webinar October 14, :58 am CT

Transcription

1 Page 1 USACE ERDC Julie Marcy AVM Webinar October 14, :58 am CT Courtney Chambers: Hello everyone I m Courtney Chambers from the ERDC Environmental Laboratory and I d like to welcome you to this Webinar series hosted by the US Army Corps of Engineers Invasive Species Leadership Team and the Aquatic Nuisance Species Research program. This series is intended to share topics of concern on invasive species issues across the Corps. The Web meetings are recorded and our archive files will be posted to the EKO and the Natural Resource Management Gateway Invasive Species Web pages for your future reference or to share with other colleagues. Just a few reminders before we begin today. As a courtesy to all our other participants, please keep your phones on mute and also be aware that during the presentation, I m going to apply a listen-only feature that will mute all participants until we begin the Question and Answer session. Following our presentation, you can take your phone off of Mute and ask questions verbally or you re welcome to utilize the Chat feature that you ll see in the lower right hand corner of your screen. Please notice that you can select to send your question to everyone or if you have a question about some technical component of the meeting you can also select to send that message just to me Courtney Chambers and that option is available in that drop down box just below the Text box. If you re having trouble being heard at any time feel free to select the Raise Your Hand button

2 Page 2 and that s located just below the Participant window on the right hand side of your screen and that ll let me know to address you. Please do not put us on hold with background music at any point in time so we can limit our distractions. Lastly, if you re calling in with others we would appreciate you taking just a minute to identify your organization and the number in your group by sending me a Chat message. Thank you very much. Without further ado, I will introduce today s speaker Dr. Susan Wilde. Dr. Susan is an Assistant Professor at the University of Georgia in the Warnell School of Forestry and Natural Resources in Athens, Georgia. While previously employed as a Research Scientist within the South Carolina Department of Natural Resources Marine Resource Research Institute and University of South Carolina Barrack Marine Laboratory, she focused on merging management concerns and targeted research to optimize management and critical and immediate aquatic natural resource problems. Dr. Susan Wilde s research and teaching at the University of Georgia continues to focus on impaired Aquatic systems and interdisciplinary challenges with invasive plants, animals, nutrient pollutions, harmful algae blooms, emerging contaminants, and the implications for fish, wildlife and even humans depending on these valuable water resources. She s discovered a new species of cyanobacteria growing on invasive aquatic plants that is producing the neurotoxin responsible for death of bald eagles and other aquatic wildlife. She s already working with reservoir managers to eliminate the Hydrilla associated cyanobacteria in those affected reservoirs. She s working with Army Corps of Engineers, US Fish and Wildlife Services, Municipal Water Reservoir and Forest and Wildlife Conversation Commission Lakes. Susan,

3 Page 3 we are very thankful for you joining us today and sharing your work. We re excited to hear from you. I m going to give you the presenter rights and then you can share your desktop. Susan Wilde: Thank you for inviting me. I really appreciate (Linda Nelson s) invitation to talk about Avian vacuolar myelinopathy. I must admit one of my favorite topics. And I have a list of collaborators here but it s much longer and certainly many in the Army Corps of Engineers have collaborated on and contributed to this research over the years. So I appreciate the great group of people I ve been able to work with on this. I will cover the first documentation of the disease, the behavior of the birds and these unique brain lesions, hence the name, and the initial search for disease agents and toxins and then the focus on food chain transfer and the data from field and laboratory trials that support that and then finish with some expanding locations and groups of aquatic organisms affected by that. And finally I d like to talk with everyone on the call about some management collaborations and some things we ve initiated. Initially at DeGray Lake, Arkansas and surrounding lakes there was large eagle die off between the years of 1994 and 1997 in the late fall and winter affecting the bald eagle, but also the American coots in these locations. At the time, the Army Corps of Engineer s Web site stated that this was the most significant unknown cause of eagle mortality in the history of the US. The visual you have there is a bald eagle flying over a raft of very nervous coots they bunch up like that when the eagles fly over them. The birds were neurologically impaired during this initial epizootic. They noted first eagles were stumbling on the ground when they were found alive. They wobble in flight if they re perched they tend to have these drooping

4 Page 4 wings as this bird does that s pictured here. The waterfowl also stumbled when they re walking and coots are a little awkward getting up off the water even when they re healthy. But when they re impaired sometimes they won t fly at all. If they fly they might wobble and fall back to the water, but sometimes they ll dive down to avoid a boat approaching them. And when they come to the surface they have trouble righting themselves. The coots that are pictured in this video are very sick. We don t always see them when they re this critically impaired. These birds have trouble even walking they re stumbling around indicating there s some damage to the central nervous system. So the trick with this diagnosing the disease is that you don t always see impairment in birds even when they have the characteristic lesion. Sometimes they appear pretty normal even when they have fairly extensive lesion development. But the sickest birds do seem like they re almost blind and don t respond even when you come up to them. So they re pretty easily collected by us and also by predators. The brain lesions that were documented during the first outbreak at DeGray Lake, Arkansas investigated by scientists and veterinarians at the National Wildlife Health Center were unique. These were not something they had seen in previous eagle histology work that they ve done as the eagle was being listed and there were a lot of investigations being done on any cause of mortality for eagles. But Nancy Thomas and other scientists described these lesions as open spaces in the white matter or sheath of the brain. It s really a swelling and opening that presents as white space on these histology slides. And it s actually pretty obvious even to someone who studies algae like myself but veterinary pathologists are still doing their final diagnosis on all of the AVM birds either

5 Page 5 at the National Wildlife Health Center or Southeastern Cooperative Wildlife Disease study here in Athens. Unfortunately this is the only way that you would know with absolutely certainty that the bird has AVM. Even if they present with the neurological impairment, the clinical signs of AVM; unless we have a freshly collected bird carcass and we get the brain in formalin and pretty quickly (within 24 hour and can do this histology, we don t know that this is definitely an AVM bird. And I ll talk later about the implications for being able to document disease because you can t freeze the carcasses because there can be some damage to the brain just from the freezing process. It has to actually be recovered fairly fresh and the brain then fixed in formalin. When the initial birds died at Arkansas, they investigated every possible source of mortality there. They looked at all of the different organs for any other abnormalities and didn t find any. Some of these birds present with fairly good body conditions. Some of the birds that we collect are still pretty fat and happy. They seem to have been eating even up to the point right before they die. They didn t find any infectious disease agents or known toxins and they were specifically looking for those that might cause that type of swelling or edema that they saw in the bird brain. Those lesions were the only consistent finding across those birds. And at the time when I got involved with AVM research in 2001, there were ten reservoirs that were known to have AVM birds. They had been documented at those sites and this was as a result of an extensive survey by Dr. Fisher and the folks at the Southeastern Cooperative Wildlife Disease Study.

6 Page 6 And a lot of work came out of North Carolina from the USGS Lab where they looked at some sentinel trials and determined that AVM was site specific, that the birds were coming in to that site and getting exposed to whatever that disease agent was, that it could happen fast (within as few as five days) and that it was occurring only seasonally during late fall and winter. So they assumed even at this point that there was probably a food chain transfer of the toxin. And Dr. Fisher and some of his students and technicians did some studies to look at non-releasable red-tailed hawks being fed some of the coots collected from these diseased locations and they were able to recreate the disease in the laboratory. The non-releasable red-tailed hawk developed the same region that the eagles had. So with that experiment they showed that it was possible for the toxin to be transferred from that prey item the water fell up to the eagles. But the question still remained as to where the waterfowl were getting this toxin. Other waterfowl were also documented with the same brain lesions and those included some herbivorous waterfowl; mallards, ring-necked ducks, buffleheads, American wigeons, Canada geese, and other predators. A great horn owl was found with AVM lesions at Thurmond. And they also found a killdeer that had the same type of brain lesions. I started these reservoir surveys in I actually saw an article in the paper that 17 bald eagles had died on J. Strom Thurmond Reservoir; this is where I had done my dissertation research and I was very interested in this. I was working down at the marine lab. And so I started working with some of the eagle biologists from South Carolina DNR and we went to the AVM sites that were close enough for us to sample. All the AVM sites were manmade

7 Page 7 impoundments. They vary in terms of their nutrients from something fairly low to somewhat moderate. I didn t see any harmful algal blooms in the ones that I visited or where sent and none have been documented previously. But they all have these dense and non-native submersed aquatic plants. And at the time when I initiated the survey, there were ten sites and nine of those ten sites were dominated by hydrilla. Since that time, we ve documented a number of other sites and so predominantly hydrilla is present at the sites and generally the dominant vegetation. But we also have Egeria densa, which was the dominant vegetation in DeGray Lake, Arkansas during the first outbreak, and Eurasian watermilfoil is present in a number of sites in which AVM occurs. But what I was looking for was the cyanobacteria in those sites. I initially thought it might be in the water column because typically the cyanobacteria that produced dangerous neurotoxins or liver toxins are in the water column. But what I found was a new species of cyanobacteria that was growing on the invasive aquatic plants, primarily on hydrilla, but also on Egeria, Eurasian watermilfoil, and even some native plants that co-occur with them. And I hadn t seen this species before, and spent some time trying to characterize it morphologically and genetically to see what it was related to. We also initiated some feeding trials with materials with hydrilla from reservoirs where we had active disease and where I had documented the novel cyanobacteria and hydrilla without the novel cyanobacteria from control lakes. There are many lakes which have hydrilla which do not have the cyanobacteria on them (this particular species). From those trials we were able to recreate the disease in the laboratory but only in those mallards that

8 Page 8 consume the hydrilla with the novel cyanobacteria. So the working hypothesis at that point was that we had potentially toxic cyanobacteria on hydrilla and some other aquatic plants at AVM sites. Coots find hydrilla, they concentrate in reservoirs with abundance aquatic vegetation, but especially hydrilla. When they re sick and flopping around in the water they are easy prey for the bald eagles, and consequently the sickest waterfowl are probably targeted by these eagles. We tested that in a field trial as well. We had a small farm pond with the hydrilla infestation (a recent one) and found the cyanobacteria growing on it; so we thought this would be a good test of our theory and released farm raised mallards into that pond. Some of them got sick within a week or so and we euthanized them, but all of the birds had AVM lesions at the end of that trial. We wanted to extract the toxins from that matrix of hydrilla and cyanobacteria to be able to characterize the toxin. So we do this now with all of the material we collect. We freeze dry (lyophilize) it, grind it up, and then extract it with some solvent. And the solvents range from hexane to methanol. Hexane would take out something that is nonpolar or maybe more lipophilic that might be attracted to some fat and maybe bio-accumulate in the system whereas the polar compound methanol will take out those that are more water soluble--something that might disappear more quickly from the system and not necessarily accumulate in tissues as much. What we found is that the toxin was in that methanol fraction indicating that it was at least somewhat watersoluble. In this trial we used just the extract alone and gavaged it (that means squirt it into the mallard s mouth). We knew they were getting the extract either from the polar fraction or the non-polar fraction and found that only the birds that

9 Page 9 were given the methanol fraction from the hydrilla with the cyanobacteria developed AVM lesions. This was also confirmation that this was a neurotoxin and not an infectious disease agent because it s now in a beaker in a methanol extract. We also wanted to get away from having to test on birds. We didn t like to kill birds and we needed a faster essay to be able to detect this unknown toxin. So Faith Wiley as part of her Ph.D. down at the Marine Lab in Charleston developed this cell line bioassay. We can expose these cell line to the same extract causing lesions in birds and we found a characteristic response of those cell lines they was specific only to the extract from the hydrilla/cyano sites. We used it and compared it with the hydrilla sites without the cyanobacteria and found that the cell line was normal with our control sites, but if you use the hydrilla/cyano extract and exposed the cells to that you get these little rounded up cells that you re seeing now on the screen. And what happens is that it stops cell division. There s a cell cycle arrest that is diagnostic then of several algal toxins and many toxins can do that. But this one specifically causes a cell cycle arrest that we can see and you use as a screen for the AVM toxin at a new site. We see them stop dividing when we expose them to the methanol extract from an AVM site. This helped us to go on to a final characterization of the hydrilla cyanobacteria as well and I was able to complete the genetic and morphological characterization of this species and because it is so very different from other described cyanobacteria is not only a new species but a new genus. So I got to name it, and I named it eagle killer living on hydrilla (sort of dramatic). The cyanobacterial nomenclature is Greek for genus and then Latin for the species.

10 Page 10 Aetokthonos hydrillicola and sometimes I call it cola for short. And we found it in expanding locations across the Southeast. Initially, as I said, we found it in the ten sites where AVM lesions had been documented. But we found it in additional locations and then also collected birds from the locations to check for AVM lesions or collected materials from those locations to test on birds back in the laboratory and confirmed additional sites. So at this point we have 20 sites where we have AVM birds with lesions, hydrilla, Aetokthonos hydrillicola, and we have lost a lot of eagles. This number is probably an underestimate because you certainly don t recover all of the birds that die at a location. I now have a total of ten more sites (well actually 15, including those new ones from Florida). But I want to investigate whether or not there is bird disease that s gone undiagnosed at this point. I would suspect that there are additional sites that I would consider at risk for the disease. And we ve seen an expansion in the food chain. We know there are additional pathways beyond hydrilla being consumed by coots and affecting eagles. We ve also documented lesion formation in grass carp, seen toxin transferred through apple snails, and some effects on additional herbivorous waterfowl species. I ll talk to you about some of the results with turtles. Recently we tested tilapia that also developed these same vacuolar lesions and we saw tadpole mortality when they were feeding on the toxic hydrilla. Another concerning (but that we still don t know really the final word on this) is the recovery of a lethargic beaver from Strom Thurmond during late fall. This beaver was asleep underneath a dock and very lethargic. We delivered him to SCWDS and they did an immediate euthanasia and necropsy and found that it had the same vacuolar lesions that the birds developed. We did recover

11 Page 11 the gut contents and found hydrilla and the cyanobacteria present and tested positive on the cell line for toxicity. They just are still unable to confirm the AVM lesions under an electron microscopy, so I think we ll hear more from Dr. Fisher about that in the future. We have done some recent studies with turtles because certainly reptiles and amphibians are becoming a group of concern. We have a lot of diversity in Georgia and the Southeast is considered a hot spot for fresh water turtle diversity and a lot of them do consume aquatic plants. This trial was conducted in a laboratory where ten turtles from a pond were then fed hydrilla with the novel cyanobacteria (and without) over a course of 90 days then they were euthanized and the brain fixed in formalin and an EM fixative and then histology conducted to check for those characteristic lesions. I think you can see this turtle stumbling now across the screen. All of the turtles in the toxin positive hydrilla treatment trial developed some weakness and lethargy, anorexia, sometimes even ataxia; they weren t even moving at the end of the trial. So definitely a behavior change as well as the brain lesion development that looked very similar to those that are seen in the birds so we should now call this vacuolar myelinopathy as opposed to AVM. I ll have to get used to that change and the lesions were confirmed with electron microscopy and saw this splitting along the myelin sheet. In addition we conducted some trials with tadpoles which are consuming some aquatic plant directly, but they also spend some time just nibbling at the biofilm on the surface of the leaf. They are certainly abundant in a lot of water bodies across the Southeastern US. We found that the tadpoles were actually pretty sensitive to the hydrilla and novel cyanobacteria complex. The bullfrog

12 Page 12 tadpoles went from an average of 5 per tank at the beginning of the trial to all of them being dead by the end of the first 27 day trial. The second trial was bullfrog tadpoles. We used a little smaller size and ten bullfrog tadpoles per treatment and still saw a very high mortality in those that were consuming the hydrilla/aetokthonos hydrillicola extract. The leopard frog tadpoles were also sensitive to the toxin. We lost a lot of the tadpoles in those treatment tanks. The open circles are the control tanks; we had very good survival in all of those control tanks. The green tree frog doesn t seem to be as sensitive. We had some initial mortality within the first five days of the trial but then it sort of tapered off. So it may be informative to look at differential sensitivity of these different species. This schematic is from Dr. Maerz just to point out the relatedness of these different groups of aquatic organisms. Some of these taxa that are sensitive are closely related like say the birds and the turtles relative to the mammals and then frogs and fish. We would not necessarily expect any of these groups to have immunity to the toxin necessarily. There may be species within this group that would be immune but there would be no reason evolutionarily to expect that they would be excluded from sensitivity. But the other piece of that is not only that they are sensitive to the toxins but that they would be exposed to it in the natural environment. The bullfrog tadpoles over winter, and they would be consuming aquatic vegetation in the late fall to winter when we have seen toxicity in the field sites. They would definitely be exposed. It s not as clear if the other species that are only consuming vegetation in the summer would necessarily be at risk.

13 Page 13 Depending on whether or not they have a winter stage they may or may not be effected by this toxin. We wanted to work on management solutions as soon as possible even if we had remaining questions because we had felt that we had pretty clearly demonstrated that exposure and consumption of the hydrilla complex was dangerous to aquatic organisms. Triploid Chinese grass carp have been used pretty effectively to control hydrilla in other lakes and reservoirs. We wondered if they would be susceptible to the disease and Rebecca Haynie s research for her Ph.D. did demonstrate vacuolar lesions in grass carp that were fed hydrilla. But those grass carp didn t die in the trials in either the laboratory trials or in our field exposures and they did eat all of the hydrilla. And then she took those fish and fed them to chickens and the chickens didn t develop AVM lesions. We were able to demonstrate with this series of experiments that while the fish are vulnerable to the toxin and that they do develop lesions it doesn t seem to kill them and they can still control aquatic vegetation and they don t appear to transfer the toxin (at least from these first trials). We scaled up to a larger situation where we had a pair of lakes in Henry County these are managed by the Henry County Water Authority. These drinking water reservoirs were built about 15 years ago and got hydrilla in them about eight years ago and within a few years became problematic. Then I went down to check out which species of aquatic vegetation they had and I found cyanobacteria growing in both locations on the hydrilla. We stocked grass carp in Towaliga but not in Long Branch so that we would have a control site for our management. During the first year, we released sentinel mallards and during the first year we didn t expect to and didn t see

14 Page 14 much control of the hydrilla from that initial stocking of grass carp and we had AVM lesions in those sentinel mallards in both locations. During the second year of our sentinel trial we had seen a removal of the hydrilla from the lower end of the reservoir and all of those sentinel mallards that were released into that zone and restricted to that zone were (AVM) negative. But the sentinel mallards in the upper end of the reservoir with access to hydrilla and in the Long Branch reservoir that was untreated were AVM positive. This is a video of the sentinel mallard from the untreated reservoir of Long Branch in year two and you can see that this bird has both tremors and paralysis. Extensive neurologic impairment and we certainly euthanized the birds when they reached this stage. As we continue to investigate new locations we were contacted by the Florida Wildlife Commission to investigate whether the snail kite might be at risk as well as any eagles in Florida. The snail kite we were concerned about it of course because of it s endangered status in Florida and they consume apple snails for most of their diet. They have switched to the invasive apple snails since this larger invasive apple snail has invaded these new locations. The location where we have snail kites nesting and extensive hydrilla beds that support the invasive snail is Lake Tohopekaliga. This is one of the first locations in Florida where I found the cyanobacteria, Aetokthonos hydrillicola, growing on hydrilla and specifically it was fairly dense in one particular cove of Lake Toho. This is a critical snail kite nesting area so we did some research to see if the hydrilla matrix was toxic at this location. We also wanted to test to see whether or not the apple snail would be able to transfer the toxin up the food chain to bird predators. The apple snails eat hydrilla they eat a lot of hydrilla. It s amazing in a tank setting how much they can consume. We tested them on

15 Page 15 material that was known to be toxic from Lake Thurmond and some control material with none of the toxic cyanobacteria at the site. The snails were fed the hydrilla and then those snails fed to chickens which are really our current bioassay at the bird level for AVM lesions. We have conducted three separate feeding trials, the initial one which was just to test whether or not if you fed the chickens the hydrilla from Lake Toho if it would cause lesions and it did in all ten of those chickens. Interestingly, none of those chickens developed neurologic impairment. We did not realize until we finished the histology that they had AVM. But they had extensive lesion development in their brains. We also saw severe AVM lesions in all of the chickens that were fed snails that had consumed that hydrilla/a. hydrillicola matrix. We found lesions in five of the ten coots that were collected from that region of Toho that I showed you on the map where we had documented the A. hydrillicola. We actually did a coot collection from the open water and all of those coots appeared normal. The strange thing about coots is they actually appear to be pretty cove specific once they arrive and over winter on in a reservoir. So we had demonstrated at least in theory that the snail kites are at risk because the toxins can transfer through the food chain by way of these apple snails. It may also be true with the native apple snails but the invasive apple snails are so much more abundant, reproduce very rapidly and they consume such a large amount of hydrilla that it is even more relevant that the snail kites are now eating this larger prey item. And we have also demonstrated that the toxin is present on site. This kind of opens up a whole new area where the disease hadn t been documented previously. And some additional surveys that I conducted in

16 Page 16 January of 2015, I found the cyanobacteria growing in East Lake Toho which is slightly upstream from Toho and then down the Kissimmee chain of lakes in Kissimmee, Cypress, and Hatchineha. In all of those locations, I found at least one cove, sometimes several coves with fairly dense colonization by the cyanobacteria. But some of the highest densities were down in Istokpoga, I did not find it in the surveys that we did on Okeechobee although they do have hydrilla there near the canal that connects to the Kissimmee chain of lakes. We did test hydrilla on the cell lines to look at relative toxicity and the Kissimmee Cove where we found the highest densities of A. hydrillicola had the highest toxicity and unfortunately this is also a location that where a number of the snail kite nests are. Then Istokpoga, Toho, Hatch and Cypress also had toxicity associated with them. Very recently, I received some samples from North Carolina so extending the geographic regions a little bit further North now. I found some colonies on samples I received in mid-september from (Mayo) Lake and from Kerr Reservoir which is a COE reservoir that s on the border of North Carolina and Virginia and the highest density of colonies were actually on Mayo Lake in North Carolina. I ll be going there in a couple of weeks to do a large collection so that we can do a chicken feeding trial to see whether or not the material is toxic in this location. I have marked with pens (I know this is sort of the busy map) to point out all the sites where we have confirmation of AVM through histology on birds and then some of the additional sites where I ve detected the cyanobacteria but we don t have evidence of bird disease yet. So we just have the one pen in Florida in Toho where AVM lesions have been confirmed. But we are concerned

17 Page 17 about J Strom Thurmond Reservoir. This is the location where we have the highest mortality now. There have been 83 bald eagles recovered from the site and considered AVM suspect. As I mentioned, it is not always possible to do the diagnosis for AVM because you have to collect a fresh bird carcass and you have to be able to get that brain into formalin within a 48 hour time window. If not, there is too much decomposition to do an accurate assessment of the brain tissue. We are worried that this is an ecological trap at this point; coots are attracted to this site, and eagles are attracted to this site because it appears to be good habitat. As I mentioned, we now have higher mortality at Thurmond than at the initial outbreak in Arkansas. Brigette Haram working with the biologist and rangers at Thurmond to track the coot densities on the reservoir. The folks at Thurmond have been collaborating with us for a long time trying to keep track of all the locations where we have the cyanobacteria. They keep a very good records of all eagle mortality and eagle nests. And this past season, we worked with them to get some of the nestlings that did survive in the upper end of the reservoir fitted with satellite transmitters so that we could track their movement for the rest of the year. I ll show you where they are now. This schematic of Thurmond shows where the hydrilla is along the shoreline the Pink symbols are eagle sightings and the high coot densities are shown with the little Green circles and the eagle nests with the Yellow houses. With some help from (Libby Mohica) and (Craig Koppie) of US Fish and Wildlife Service we were able to get these little Thurmond nestlings fitted with satellite transmitters and they are sending their locations to (Argos) and those get reported down to us and we can see their locations once a day. The

18 Page 18 two male nestlings flew up to Canada after they fledged, but they re starting to move back down. One of them is all the way down to Lake Ontario and another one has made his way back into Maine. The little female nestling from the Broad River has been kind of staying close to home. She flew back to the Savannah River site for a little while but now she s back near Bussey Point. We are going to be working with the Corps of Engineers this fall to start a preliminary grass carp stocking with some radio tagged grass carp so that we can see how far they moved from the point where we are initially stocking them. We want to select some coves using sonar to map the hydrilla density that have very high density of hydrilla and some of lower density hydrilla to see how much movement we get away from the point where they are stocked. These grass carp will have radio transmitters that we can follow with a yagi antennae from a boat or even potentially from an airplane when we fly to check on the eagles. We ll collect some of these fish to check for vacuolar lesions and we will also collect some fish to test for toxin transmission. We don t want to introduce another means of risk for fish-eating predators. I have a lot of questions remaining and I suspect some of you on the call may also have questions. So I ll finish with some of the questions and some thoughts I have on what we could do to answer those questions. But I would really like to hear what folks think are the most important next steps in this research. We would certainly like to know the chemical structure of the neurotoxin. We would like to be able to have an analytical technique that we could tell you exactly how toxic the sample is. But we have some bioassays that give you indication of whether toxicity is present but (Brigette Haram) is working with some scientists at EPA here in

19 Page 19 Athens through a fractionation process that will make that extract clean enough to use some analytical techniques and eventually get to the point where we can characterize that toxin. I d like to know what is the mechanism in which that toxin causes these open spaces in the brain tissues. We re going to use the zebrafish as a bioassay also. They have been used a lot in human disease research because they share a lot of our genome and can give you some indication of how it might affect a mammal cell. I d like to know where the cyanobacteria came from. I d like to screen some hydrilla from Japan and Asia and look at the genetic heterogeneity of the cyanobacteria at all the different locations where we see it currently. I d like to know what triggers the toxin production. It doesn t appear to be toxic year-round. We see that the spike in toxicity in the late fall. I think it s important that we maintain monitoring for the sake of the birds but also for the clues that it provides for us in knowing what environmental conditions are conducive to toxin production. We have some cultures in the lab which appears to be toxic and some that are non-toxic and we are trying to work with the viability of using laboratory cultures to answer some of these questions. We have seen some native plants with the cyanobacteria on them on so we re going to be doing some feeding trials with native plants to see if we would see vacuolar myelinopathy associated with natives. And finally I think we all would like to know that there is a risk to human health. I think we have mostly negative information so far on that concern. But I m not satisfied, I feel that we still need to further investigate whether there s any concern for fisherman or waterfowl hunters in these locations.

20 Page 20 We have had some feeding trials using the meat from chickens from previous AVM positive trials that cause mild lesion formation. I don t think that we can say that there is absolutely no toxin into the tissues. I think it may be in low concentration, but I think more needs to be done on that issue. And I ll just finish with acknowledging a few of the many agencies that have collaborated but these are the main players. Certainly the Army Corps of Engineers was there at the beginning and very pleased to be on this Webinar but Army Corps of Engineers Group that can be a big help in trying to correct the problem in all the locations where we know it occurs. And with that I think I want to turn everyone s microphones on so somebody can talk besides me. Courtney Chambers: Excellent all right. Thank you Susan. Hey participants you are welcome to chime in with your questions for Susan or just discuss the potential implications. And again you re welcome to use the Chat box if you d rather that. You ll still need to un-mute your phone line although we are in interactive mode now. (Ian): Hey yes I have a question. So do you - have you seen the cyanobacteria on the other bio type of hydrilla, the monoecious type? Susan Wilde: Oh yes in fact most of what that Thurmond is monoecious... (Ian): Okay yes...

21 Page 21 Susan Wilde:...and most locations in North Carolina are monoecious. We have both actually at Thurmond but yes it s on both. (Ian):...okay yes that s what I assumed since I knew there were both there. Okay. Susan Wilde: Yes. (Ian): Thank you. (Linda Nelson): Susan this is (Linda Nelson) I have a question for you. So traditionally some of the aquatic plant management strategies that are frequently implemented when you have high infestation of hydrilla, of course, is the use of aquatic herbicide. Is there any information or data or about whether, you know, plants that are decaying after herbicide use would maybe cause a toxin release if the cyanobacteria is growing on hydrilla, is that a concern at all? Susan Wilde: (Linda) I think that s a reasonable question and certainly something to keep in mind. But I tend to think it s much like when you treat a harmful algae bloom that s producing toxins. You may release some of the intercellular toxins but you re killing the bloom. So you might elevate risk in the short term, but certainly in the long term that would be a good correction. I do think that the cyanobacteria is more resistant to algaecides and herbicides than is hydrilla. I have tried to kill it. I m much more successful at killing hydrilla than the algae with algaecide. This kind of bacteria has such a strong sheath, it s fairly resistant. But I think if you drop the hydrilla out of the water column with herbicides then you take away a substrate and you remove it from it being a source of food for the waterfowl and other aquatic organisms that might really be affected by it.

22 Page 22 (Linda Nelson): Yes thank you. I was just curious how do we manage this problem? Because it seems to be an increasing problem on Corps reservoirs so thank you I appreciate it. Susan Wilde: I think we should use both, I didn t really talk about the use of chemicals in addition to using the grass carp on Thurmond. We re actually going to try both and I think that the folks at the COE at Thurmond would like to be able to keep (if we go with) the grass carp stocking as conservative as possible. And I think that coupling that with herbicides would allow you to do that. So I think that s a good idea. Courtney Chambers: Very good thank you. Are there any other questions this afternoon? (Mike Vischelli): Hey this is Mike Vischelli. I have a quick question. I saw you had said somewhere along the line that this was only found on reservoirs. Is there any reason to believe it doesn t exist in areas that are non-reservoirs that are just naturally formed lakes? Susan Wilde: I can t imagine why it couldn t. I think this is more a question of just where it s occurred we don t have any natural lakes really in the Southeast except for like Oxbows or Carolina Bay and sinkhole lakes. So I think it s more the nature of where we are but I think its possible as we see hydrilla extending further North. This is also a plea for samples. If there are folks who are further North, or anyone who has a natural lake with hydrilla, I would certainly be very glad to screen it. It would be interesting to find out. (Mike Michelli): Thanks. (Julie Marcy): Susan this is (Julie Marcy) from ERDC. Do you want to talk a little bit more about sending Hydrilla samples or what folks might be able to do to help?

23 Page 23 Susan Wilde: Yes, I think I have that the contact slide at the end. But if you just send an to me I will send you a data sheet sampling instructions protocol and then our APHIS permit for shipping the hydrilla. And yes it s pretty simple you really can just go out and cram a zip-lock bag full of hydrilla and ship it to me. It s nice to get it overnight, if possible, but this cyanobacteria is fine even if it takes a couple of days to get here. So and if you have to collect hydrilla and store some for a few days so that you have like all the samples you want to ship to me that s fine too. Then I guess the good part about this type of cyanobacteria is it s pretty hard to kill so even in older samples I find it. Courtney Chambers: And again thank you and thank you (Julie). All right are there any other questions this afternoon or anybody maybe that thinks this might be a problem where they re at or able to ship some samples? If you re planning to do that and want to share I d love to hear. Okay... (Julie Marcy): Courtney and Susan...this is again just to let you know I m getting some updates from the folks in the Vicksburg District of the Corps. They are going to pull some more definitive information for me but they were on the front lines of the initial AVM outbreak in the early 90 s. They report they have seen significant reduction in their populations of Hydrilla and Egeria densa using bio control flies, triploid grass carp and herbicide applications. We are checking, but I don t believe we ve had a confirmed Bald Eagle die from AVM at those Arkansas Lakes since about So there is some hope. Courtney Chambers: Oh wow. Susan Wilde: That is good...

24 Page 24 Courtney Chambers: Very good. So they re using flies as well as part of their treatment plan? (Julie Marcy): Yes they started the Pakistani flies at DeGray and Ouachita in Courtney Chambers: (Very good) is that something you all have used at all your control area in your studies Susan? Susan Wilde:...no we haven t initiated that yet. I don t think it s been shown to be as effective on the monoecious biotype they ve certainly tried it up in some of the lakes up in North Carolina. I m always in favor of trying to use biocontrols and consider this an option the folks at Thurmond can look at. Courtney Chambers: Any last questions today? Susan it s been a pleasure hearing from you today. Do you have any closing comments for us before we wrap up? Susan Wilde: No I just wanted to thank you all for giving me this opportunity and I would like to continue to expand our monitoring efforts. So any help that you all can give me on letting folks know that we are still interested in continuing to investigate any new locations where hydrilla is, or egeria, or watermilfoil. Thank you very much. Courtney Chambers: Susan thank you again for sharing your information with us today. And we will be sure to spread the word that you re looking for additional plant samples to contribute to the effort. And on that note, participants we re glad to have you with us today. Please continue to join us for future meetings. Watch your for notices on upcoming meetings and we ll look forward to learning together with you all again soon. Have a good day.

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