 Well thank you everybody for joining us today. Another great turnout for another Hydrotera webinar. Today we're talking all about invertebrate species identification made easy by DNA barcodes. And our presenter today is Melissa Karoo, who is an entomologist and geneticist from the University of Melbourne and Capham, as well as the PEARG. How do you pronounce that, Melissa? PEARG. PEARG. I should have checked that before we started. So, just a little bit about the topics today and a little bit about our speaker. So, Melissa has been an entomologist and a geneticist for over 20 years. She is primarily interested in applying DNA approaches to help understand invertebrate biodiversity. Her current research has involved creating the DNA barcodes needed for routine identification of Australian invertebrate species and developing high throughput DNA meta coding for the rapid cost effective monitoring and assessment of invertebrate biodiversity. Melissa has worked extensively with industry and research partners, particularly in fresh waters towards understanding the response of invertebrates to environmental change and to improve their use in monitoring programs. Now, what does all that really mean? It means that Melissa is passionate about invertebrates and there's a lot of invertebrates out there. There's millions and millions of them. So, Melissa's been working on a way to keep track of all those different species of invertebrates. And obviously, they're an incredibly important part of our biodiversity. Now, my own practice, I think the areas that we're really challenged as I guess managers of the environment is coming up with good biodiversity indicators. And certainly the stuff that Melissa's talking about today is going to be part of the answer to how we can truly quantify these impacts and get better at tracking the improvements to the environment that we need to make. So, a couple of administrative matters before we get started. So, we love your questions and thanks to those who've already sent through their early bird questions. We've got eight of those today. Obviously questions arise as we go through the presentation. You can lodge your questions by using the Q&A button at the top there. Why does HydroTerror undertake these webinars? Look, we like to share knowledge. Actually, I think, you know, personally, Australia does a lot of great research, but I think we haven't been fantastic at applying it traditionally. And part of the reason for this forum is to share some of the great research, particularly around monitoring that HydroTerror is lucky to become aware of through the various contexts we have in our day-to-day business. We like to help facilitate education, and really that's part of sharing knowledge as well. But also, there's some, you know, good for you to get an understanding of who is out there who are experts in these various fields. Finally, we'd like to be a bit of an industry leader. The presentation we've got on today, really, I think, you know, is one of those examples where monitoring needs to go. You know, monitoring of natural systems is a lot more complex than we've traditionally given it credit for. So we need technologies like the ones that Melissa is sharing with us today. All right. So the program for today, Melissa's going to talk about invertebrates. Why should we care about them? Why diversity? How to classify and measure? And DNA barcoding and metacoding for species identification. I'll then take over to read out your questions that you've lodged during the webinar, and Melissa will do her best to answer it. For those of you wondering how Melissa got to this position of expertise, I just thought I'd give you a little bit of a background of her early time. So she did a Bachelor of Science at La Trobe University specializing in biology related subjects. And then she progressed to do her PhD at Melbourne University, where she really started to understand the benefits of this DNA approach that we're talking about today. So there's an example of a career that you could emulate. All right. Without further ado, I will hand over to Melissa. Many thanks, Melissa. Thank you, Richard. So I'll go to the next slide. So I'll be speaking today about invertebrate species monitoring and how we can make that easier using DNA approaches like DNA barcoding and meta barcoding. So next slide. So I really want to make a case to you as to why we should bother looking at invertebrates. So for those of you that aren't familiar with invertebrates, these can include groups such as insects, worms, clams, snails, crustaceans, so our yabbies and crayfish, arachnids, spiders and mites, sponges, corals. It's an incredibly diverse group of organisms and basically includes anything that doesn't have a backbone. So globally, invertebrates represent approximately 97% of the animal species we have on Earth. And the most diverse or the most important group within this are the insects and they represent up to 80% of the diversity. So to put it in a local context, in Australia, invertebrates make up around 55% of the total known biodiversity. So this includes the higher animals. So the mammals, for example, fungi and plants. But when we go down to animal diversity, we have around 97 or greater than 97% of the animal diversity is represented by the invertebrates. So next slide. Invertebrates are pretty much everywhere and we can find them in most environments on Earth. So this can include things like deep sea vents where there's really unique species that colonize around these vents, all the way through to deserts, the Arctic regions, our oceans, forests and rivers. So next slide please. And invertebrates are a vital part of ecosystems. So they perform on a number of important ecological and economic functions. So they provide food for a wide range of other animals. They're important in the breakdown of organic matter and nutrient cycling. They're also quite important for filtering water. And economically, without pollinators to pollinate crops, we would not have a lot of the food that we have today. And they're also important in pollinating other flowering plants as well. So without invertebrates, ecosystems would collapse. And monitoring changes to invertebrate communities can tell us a lot about changes within ecosystems. So next slide. So invertebrate biodiversity and composition make really excellent indicators of environmental change for a number of reasons. This is because different invertebrate species can show quite specific responses to different environmental factors. Some invertebrates can be affected by particular plants or pollutants in a really specific way. A lot of invertebrate species can be sedentary. And this means they stay very close to the area and stay within a localized area. So this can give us a really good amount of information about local conditions. They're highly abundant, which means we can collect them easily. And as I mentioned before, they're highly biodiverse. So if you look at the figure on the right-hand side of the slide, this just gives you an idea of what you would typically see. So we've got a number of streams here. The top one is fairly untouched and we get a really high amount of invertebrate biodiversity at those particular sites. As things become more modified or deteriorate, we, for example, the middle site, we have a lack of riparian vegetation, a lot of erosion and turbidity. We start seeing a reduction in invertebrate biodiversity. And by the time we get to something that's highly polluted, we may have no invertebrates at all. Or one of the common ones we find in aquatic environments is worms. There are some worms that can withstand really, really poor environmental conditions. So next slide. So I think it's really important to understand what biodiversity is as part of this presentation. So you get a bit of a feel for what it means. So put simply, biodiversity is the variety of lifeforms that we have on Earth. And it can be measured in using different taxonomic groups. So insects or wider, more widely invertebrates, mammals or plants. And we can also look at it at different taxonomic levels. So things like the number of orders we find at a site or the number of families or the number of species. So just to give you an idea of how taxonomic classification works and why we use these different levels. I've taken one of my favorite non-invertebrate examples. So the domestic dog here. And you can see if you look, we can classify the dog all the way down from kingdom to species. And one thing you'll note as you move up the classification. So when we go to the level of genus, we'll have eight species of canis in there. And that includes a lot of wolves and other few other dog species. I think when we get to the level of canida day, that includes a lot more species up to 35. And that also brings in things like foxes and jackals and coyotes. And then we get up to the carnivores. We've got around 270 species all the way up to the mammals where we've got around 5,400 species of known mammals. So if we go to the next slide. So this slide, we're comparing it to what you see when you get to invertebrates. So this particular example is the mosquito, Edie's Egypti, which is a rather horrible mosquito that transmits a lot of diseases we don't like. Things like yellow fever and dengue fever. So if you have a look in comparison to our Labrador, you'll see that when we get to the genus level, we have around greater than 700 species just within that genus. When we go to the family level, we've got over 3,600. By the time we get up to the insect group, the estimates kind of, we're really reliant on estimates, it kind of falls apart. It's anywhere between two and seven million species that fall into the group of the insects. So one thing that's important to point out, a lot of environment, a lot of biomonitoring using invertebrates today often uses the taxonomic classification of family. So I hope this gives you an idea when I mentioned family, exactly what I'm talking about. So next slide, please. So I'd like to use a bit of an example of some of the work that I've done previously. And this is looking at macroinvertebrates from freshwater environments. So just to give you an idea of what freshwater invertebrates are that we use in monitoring freshwater ecosystems, we call them macroinvertebrates because we're only looking at the section of the community that is visible to the naked eye. And these are usually animals that are greater than 250 micron in size. So there's a big history to invertebrate monitoring in freshwater ecosystems. It's been going on worldwide as a standard part of water quality monitoring programs for decades. So traditionally these programs identify the macroinvertebrates to the family level. And we have around 200 families of macroinvertebrates that are included in stream monitoring in Australia. Next slide. So to give you an idea of how this identification occurs, we use it doing a process called morphological identification. And this simply means just looking at the features of the animal as a means to identify it. So a typical freshwater biomonitoring survey would be going out and collecting the samples. And this involves kicking around in the stream through the different habitats. So these might be the riffles, the pools, the edge habitat, any submerged logs or anything like that to try and capture as much biodiversity that's present at that particular location. The samples are then taken to the lab. And because we collect a lot of debris when we collect aquatic invertebrates, we need to then pick those animals out for identification. So this can be quite time consuming. Those animals then go into a jar and go up to the taxonomist. And these are highly skilled people who can use a variety of different taxonomic keys to go through and identify every single specimen from that particular sample. Generally, with this sort of work, the taxonomic classification is checked where they re-identify around 5% of samples from a given study. So what your taxonomist will give you then is a list of the mostly families at that particular site with the abundances of the different animals. So the areas that I've got in orange and red with the arrows, these are greatly affected from sample to sample. So they can be affected by things like density. So how many animals you have compared to the debris in the sample? So a sample with a high amount of debris and a low density of animals can take a long time to pick the animals out. The overall abundance of the animals. Some sites can have huge abundances of animals. So you can be picking to get quite a lot of animals out. And that means more to identify as well. And also the diversity. So at the taxonomic side, it can take quite a long time if you get a highly diverse sample because you need lots of different taxonomic keys to identify the animals. So overall, a large study that was involving using these sorts of processes can take many months to complete all these tasks. So there are, you know, in saying this at family level, it's quite tedious at the moment to run these studies or can be. But I'm actually advocating to take it down to the lower level, which is looking at species. And I want to give you some reasons why that will be the case. So next slide. So one reason we should be moving to species is it's really the core measure of biodiversity. When we're talking about biodiversity, we're really talking about species. And if we're using anything above that, we're really only using a surrogate approach to measuring biodiversity. So if you look at the graph on the left, you'll see we have the number of families on the vertical axis and the number of species on the horizontal axis. And these are counts for the same 47 sites we measured in this particular survey. So whilst there is a relationship where you do often get more species in sites that have more families, it's certainly not a perfect relationship. So sites with the highest family diversity don't always have the highest species diversity. So we can be kind of missing out a little bit on how we might rank sites for conservation. Oops, sorry. Next slide. Another reason is that we can use specific responses to assess environmental change if we're identifying invertebrates to species. So I put an example here on the left that is from my PhD thesis a few years back. And what it actually is looking at is one single family that we get in freshwater streams and the variety of responses of species within that particular family. So what we have on the bottom axis is zinc concentration and this is used as a surrogate for pollution. So in urban systems, most of our pollutants are so correlated that you can just choose one and use that as essentially a guide to overall pollution in the environment. And on the other axis, we have the frequency of occurrence. So this is just whether these animals were there or not. So what you can see is some species disappear quite quickly as the zinc concentration increases, whereas others almost seem to like it. And we were able to cross-reference this data against ecotoxological testing and we found the sort of patterns roughly translated when we looked at the points at which they started to die off in our ecotoxicology tests compared to what we were seeing out in the field. Next slide. And the other thing is if we monitor at species level, we can look at specific species and their patterns or distributions. And this could be quite important if a species is threatened and under conservation significance. So we do have a couple of amphipods that fall under that category and some stoneflies in the Greater Melbourne area. And we can also look at the penetration of invasive or cosmopolitan species into different locations. So if you look at the map on the left, you'll see that the blue dots, dark blue dots that represent the invasive species are much larger in those close to Melbourne CBD. If you look out to the sort of, I guess the right of the diagram, these are where we have a lot of our forested catchments which are relatively untouched. And you can see the proportion of invasive or cosmopolitan species is far less in these environments. So they haven't penetrated as well as they have into many of the urban areas. So next slide. So as I mentioned, it's great to use species but there's a big problem if we want to use morphology to identify species. So it requires even more highly specialized taxonomic expertise compared to identifying families. So if you were going to take invertebrate samples and do it morphologically, you'd be sending samples off to a variety of taxonomists that specialize in different groups. A lot of species can't be distinguished on morphological features alone when you're looking at invertebrates and the butterflies from the paper on the left here I hope kind of illustrate that point if I was to give you a bunch of butterflies and ask you to ID these, some of them you would have a lot of trouble telling apart. Often when we sample animals, we might get damaged animals or animals that are immature and this might be things like larvae. So we may have taxonomic keys that enable us to identify say the adult life stage but not the larval life stage. So it can be quite difficult. It's really dependent species identification on the availability of information and it's just considerably time-consuming and costly to do this morphologically. So next slide. So this is where DNA barcoding comes in and it's a really great way to start to identify species. So what DNA barcoding is, if you define it, is the systematic DNA sequencing of a standard region of DNA for all life. And it's completed on individuals and if you look at the diagram to the left, you'll see we have our beetle specimen here. So the way a DNA barcoding works is we would take a leg or something and not destroy the specimen, extract the DNA, amplify the DNA up and then DNA sequence it. And what this produces is essentially a barcode that's based on the arrangement of the four different bases we have in DNA. And this DNA barcode, if it's a new one, we can send the specimen off to a taxonomist and they can then identify it. So then we have a DNA barcode with a species name attached to it. And once this is done, we have really a digital record for that particular species. So one great thing that DNA barcoding can do that morphology can't do is it can identify any species. We don't need morphological features to be able to identify them. Next slide. So once we start to go through and DNA barcode lots of different individual specimens, we can build up what's called a DNA barcode reference library. And this is really critical for us moving to using invertebrates for routine DNA-based species identification. So next slide. And there's a really awesome repository out there that's being developed around the early 2000s that contains all the DNA barcodes that are available currently. And this is the barcode of life systems database. So I encourage anybody who's interested in barcodes to go and have a look. It doesn't just have invertebrates. It has all animals, plants and fungi, although they use slightly different genes for plants and fungi than they used to animals. So next slide. So just to give you an idea of the scale of this database and the kinds of information that you can get off it. So most records that are submitted to the barcode of life database contain the geographical information, the DNA sequences, as well as in some cases photographs as well, which is really useful when you're just trying to eyeball specimens and look at barcodes that have close matches. So currently there are over 12 million DNA barcodes from over 3... 343... Sorry. Over 300,000 species. This includes the fungi, plants and the animals. So for invertebrates, we have over 200,000 DNA barcodes available. And in Australia, these barcodes represent around 28,000 species. So just to give you an idea of how many barcodes we have relative to the estimated number of species we potentially have in Australia, we're really talking about, you know, still a relatively small fraction of Australia's invertebrates that have been DNA barcoded. Next slide. So we can go through obviously and DNA barcode individual specimens, but this is going to also be pretty slow and cumbersome. So the process that we use when we go out into the field and we want to start surveying large numbers of sites and get an idea of the biodiversity at those sites is called DNA meta-barcoding. So essentially what this means is it's just the simultaneous DNA barcoding of many species within many samples. And this is, as I said, what we need for routine identification. So if you look at the figure on the left, you'll see that we in this case have some aquatic sites that we've sampled. We've got the material from those sites. We DNA extract that material, amplify up the barcode sequence them. And for each site, we'll end up with a essentially a list of all the composition of the species that we get at the particular site all divided up. Next slide. So just to give you an idea of how DNA meta-barcoding works in relation to DNA barcode reference library building. So obviously our reference library is our DNA sequences attached to our individual specimens. But when we get a sample back that's being DNA meta-barcoded, we get a whole little jumbled mixture of different DNA sequences. And these are represented in the circle by the different colors. So next slide. So what we can do is with our DNA meta-barcoded sample is we can start looking to see if any of the sequences in there match anything that is in our DNA reference barcode library. So you can see at this stage we've already found two species that appear in our library. And we can count the number of sequences as well. So when we do meta-barcoding, we're actually sequencing individual strands of DNA. So we can count each sequence, which is called a read, to get an idea of the number of sequences that were returned for that particular species. Next slide. So once we complete this process, one particular animal, which is represented by the yellow DNA sequence, that wasn't in our library. So this suggests that this quite commonly happens in DNA meta-barcoding studies where your libraries are incomplete. That means that if we have access to actual animals, we might go back and have a quick look and find out what we're missing and then go back and DNA barcode that animal to add to our reference libraries for next time. Right, next slide. So there are a number of benefits of DNA meta-barcoding that we don't have when we're trying to deal with things at a morphological level or actual specimens at hand. And that is that we can use a variety of different sample types. A particular type of meta-barcoding that people may have heard of is EDNA or environmental DNA meta-barcoding. And this is where we just essentially take samples that may have DNA contained within them. So from water, sediment, soil, and I've even heard people are now trying to do it from air as well. Another is we can do things to look at diet of particular animals. So this might be diet of larger animals or even very small things like spiders or mites where we can meta-barcode either the fecal material or the whole animal and get an idea of what that animal's been eating. And this could be really useful in looking at things like food webs or interactions of species or understanding just what species eat if we're interested in those particular animals. There's another one which is a wider one called tissue-based meta-barcoding. And this is where we're actually sampling the animals and meta-barcoding them. And this is something I've mostly been working on. So this is where we can use a variety of different trapping methods, whether they're active trapping methods, going out and actually collecting the invertebrates or passive methods where we're just relying on invertebrates falling into the chap trap. And we can then go and meta-barcode those particular samples to get an idea of the composition of the species within them. And the final one is one I've been working on quite a lot lately and that is trying to find a bit of a happy medium between EDNA and tissue meta-barcoding where we actually don't sort animals from the net collected material where we've got a lot of debris and we just directly DNA-barcode the animals and the debris together. Next slide. So I guess one thing to be aware of whenever you're doing meta-barcoding is that sample type can be quite important to what you want to actually find out at the end. So in the freshwater system or in the freshwater, as we found when we do DNA meta-barcoding, we can get quite different results compared net collected invertebrates that have been all sorted to invertebrates that are being assessed through samples of water, so an EDNA-based approach. So I've taken a couple of figures from some key papers in this area. So if you look at the one on the left, you'll see that the biodiversity that we found from the net collected sample is much higher than what we got from the EDNA samples. So we also see sort of shifts in the composition of a lot of different species when we look at EDNA from water compared to net collected animals. And this is largely because net collecting we're collecting from all environments. EDNA, it can be much more transient. Another is the type of the number of DNA sequences we get back or reads we get back when we meta-barco can vary. So if we get a net collected sample, most of the DNA sequences and the diversity in the sample EDNA, we pick up a lot of by-catch. So we can end up with a lot of algae bacteria and other things in the sample and the invertebrates can really represent quite a small portion of the pie. So next slide. So what I've done in this slide is I really just wanted to give you an idea of some of the work we've been doing looking at unsorted invertebrate samples. So these pie charts to the side are I've split all the invertebrates groups up because I was very much more interested in whether I was detecting freshwater invertebrates. So the dark blue indicates where I was getting freshwater invertebrates. So in terms of the number of sequences I was getting back or the reads, I was getting mostly DNA sequences from invertebrates. But when I was looking at the diversity it was slightly less. But what the two charts together sort of indicate is yes, I got a lot of other things being identified that they represented very few sequences. So most of the extra sequences I got weren't invertebrates. We're all different to each other. And the little table at the side just sort of brings together the information from the previous slide where we're looking at the diversity compared to the sequences. And it kind of shows that really the unsorted invertebrate samples represent a really good medium between EDNA and sorting samples where you sort of reducing the amount of effort in processing but getting really good returns on the biodiversity that you're seeing in the samples. So next slide. So another benefit is we just uncover so much more biodiversity if we're comparing families to species. So this chart on the side here just shows you when we ID stuff for the same sites to family and then to species we can get to two and a half times more biodiversity detected looking at the different sites. So we're kind of missing out on a lot if we only look at family and it's also showing just so many of the families we're detecting are made up with multiple species. Next slide. So one of the final sort of benefits I would put forward is really cost effectiveness. So if I go back and if you look to the figure to the right you'll see that I've sort of illustrated what we get when we use morphological approach and that's one from earlier on. If we then sort of tag in using DNA meta barcoding approaches we get a few shifts. So what we find is if we can take the sample directly from the field and do a bit of sieving and then meta barcoding it so not sorting the animals out we can save up to 30% on cost and a fair bit on time as well. If we're pretty keen to just still do a jar of invertebrates or the sample type we're doing doesn't come with a lot of debris then we can just go through and we can save around 15% on the identification cost and keep in mind this is comparing families to species. So our tox taxonomous will give us a list of families. Meta barcoding will give us a much, much longer list of species at particular sites. So if we're doing this over really large surveys of lots and lots of sites we can save quite a bit of money and get a lot more detailed information. Next slide. So this is the one that I wanted to end on and I hope I've made a great case for you as to why you want to look invertebrate biodiversity at species level and why you should choose DNA approaches to do this. So if you're thinking of doing this kind of study or want to incorporate it I've put a few things here that I think that you sort of questions you should ask or things that you should think about. So the first one would be what group are you interested in? So what ones are going to sort of tell you about your environmental condition? Is this terrestrial, aquatic, aerial invertebrates or those in feces or groundwater it just depends on the type of study and what you're interested in looking at. What's the best way to sample biodiversity in your target group? So for example EDNA could be very good. We've been talking about looking at it for stycophorna where you don't get stycophorna in a single bore that you sample. So EDNA might make a really good screening tool to have a look at whether that stycophorna or a kid present without actually having to actively sample every well and if you find hits you may go back and actively sample the wells to get the animals and look at it in a little bit more detail. In other cases things like passive sampling may be a really good option opposed to say EDNA if you want to try and attract animals from a large area rather than take a bit of EDNA from a small area. So it's really sort of horses for courses in terms of what are your questions and which one of these sort of sampling approaches is the best one. EDNA is by far the simplest to collect but it's what information you really want to get out the other end as to whether it's a suitable thing for your particular study. And the final one I'd like to highlight is looking at whether there are DNA barcodes available to identify the species that you're interested in or the group of species that you want to target. So one of the first things I do whenever I enter a new environment is I check the bold database. Are there species available? In a study where we were looking at worm biodiversity, first thing I noticed is all the worms, terrestrial worms that we find tend to be on the databases that introduces very few barcodes for example for our native species. So this really makes them a bit of a priority to look at and start to gather this kind of information. So you sometimes need to be prepared for the idea that you may need to create your own DNA barcode reference database and this might seem daunting but it's actually not because if you're particularly wanting to do repeat sampling in a particular region there's some library will pay dividends and you're really adding to our understanding of invertebrate biodiversity in Australia. So I will leave it at that. Thanks Richard. Thanks very much Melissa. I have a little sneaky early question for you. So I guess quite a few of the people in the audience would be consultants that might be dealing with contaminated sites and on quite a few sites it's about trying to assess the vulnerability of the receptors and I know the Australian water quality guidelines and I apologize for my rustiness on this but they they had a they had a framework that would allow you to decide if it was disturbed or I can't remember exact term semi-disturbed or highly disturbed and that was always a very big deal right to try and work out whether or not it was in fact that and what remediation criteria would need to be put in place to protect that environment so you would sort of classify it in a very broad context like highly disturbed and then you would make this leap of faith that all that meant I could adopt this cleanup criteria. How do you see this approach that you've got tying into maybe a better way of classifying that and there's two parts to the question and then the second one is from your early slide you showed the impacts of you mentioned the diversity dropping as we went through that sort of picture ending with the drain saying well there's no invertebrates there how realistic is it to be creating those remediation criteria to protect those environments and that's a bit of a loaded question so two questions if we look if I could answer it from what I understand about invertebrates I guess the thing is that if you move from we can give much more detailed information if we look at species so very quickly once we start doing meta bar coding over large ranges we'll get a really good idea of what species are around and we can start to associate them with different environmental measures like chemical measures those sorts of things so we'll know certain species may respond to various things in the environment so rather than just have this thing of something being highly disturbed you might see that certain species are missing and you know those species respond to one particular attribute of the environment so you might give you a little bit of a clue or a priority list of maybe what you might like to target first sometimes it's obvious sometimes it's not I guess one thing I certainly found is I think with as a scientist you sort of see all the complexities in these sorts of questions and managers like to deduce everything down to a score or a figure or a ranking and yes that sort of can be quite loaded because there's often a lot of caveats that sort of come with that so I think one thing that we can do so much better is if we're understanding species and we're using invertebrates we've got this huge biodiversity to draw on and this huge ray of responses everything can just be more targeted so it might not be highly disturbed but it looks like the major thing impacting the population is the lack of flowering plants or it's the pollution or sometimes it's not the pollution it might be things that come alongside the environment like pollution it may be that it's been cleared as well or various plant species or anything I hope that kind of goes it was a pretty nasty first question we better move to the summary slide and people's questions but thanks very much for answering that one alright so just in summary what have we learnt from Melissa today invertebrates are an important group for environmental assessment they are the most diverse and dominant animal group invertebrates respond to environmental change monitoring invertebrates at species level can give far more information that using higher tax than using higher taxonomic levels ag family to understand biodiversity we really need to use species species responses can be used in monitoring the distribution of key species can be assessed DNA barcoding and meta barcoding methods are ready to go we have standardised approaches for DNA meta barcoding they are rapid cost effective and can be used for routine monitoring however more individual DNA barcoding is likely to be needed for some invertebrate groups without further ado we are going to move to the Q&A Melissa are you ready number one is there any technique or future technique on the horizon for continuous barcoding rather than taking distinct samples so I'm sorry I'm assuming that this question is really asking distinct samples meaning maybe individual samples so I guess this is really our transition from DNA barcoding to meta barcoding so with meta barcoding we can take samples and not necessarily have to look at individuals but we can look at communities within different types of samples so I'm hoping I'm kind of answering that if the questioner is online I'm certainly happy for them to kind of clarify if I haven't quite got that right let's assume that's right question number two do sampling techniques need modification for highly turbid records colloidal clays in land waters 10 mil through 2.45 micron filters is hard okay so this sounds like a person who's been looking at EDNA and this is one of the problems with EDNA in that the if the clay particles are quite numerous and the water samples really turbid those filters clog fast and you don't put a sufficient amount of water through it's not something I specifically looked at because usually when people tell me this I'm like instead of if you're for example it's looking at bore water and trying to capture styrofoam or I'd be saying put it through a sieve so I've been working on a project where we've done both where we've looked at collecting EDNA and collecting the animals which we use it's about a large entomological sieve and we just pump the water through the sieve so we can put a high volume of water and any animals that we capture in the sieve we then transfer so clogging is a lot less of an issue when you've got a larger sort of surface area to pump the stuff through all right thank you for that next question has this technique been used on marine sediments? answer is yes so I had some colleagues that work for Syro Anthony Charitan he's done quite a lot on estuarine sediments and I'm sure he's sort of most of these estuarine areas are almost verging on marine I guess it's a little bit more complex because we have a lot more but a lot more small invertebrates in the sediments in fresh waters I think things are a little bit different marine systems it's not my area of expertise so I can kind of really only loosely comment but there's generally not a place where people haven't tried to apply DNA meta barcoding or are about to kind of go forth and give it a go okay so the answer is yes you can use it for marine sediments question number four sequences in gen bank indication of how broadly you have covered invert taxonomy geographic area by geographic area so I think I've half answered this one in the presentation so I would go directly to bold it's most up to date and sequences often appear in bold before they appear in gen bank and bold actually mines data from gen bank so someone does submit barcodes to gen bank they'll very quickly appear on bold the bold is really trying to make itself the repository for all DNA barcode data so you can very and obviously with the extra sort of pictures and maps and things like that it's a really useful interface to kind of gather whether you've got good taxonomic coverage or good coverage for a particular geographical area I'm going to sneak a question in so a lot of studies there are a lot of ways of I guess assessing whether or not the environments change to sort of benchmark at the moment I'm thinking if there's a new process where we look at what would the vegetation have been like pre-European settlement versus now and try to sort of benchmark that in a process to quantify change towards a better place and we refer to that as environmental accounting do you see an application of this sort of biodiversity measurement being applicable in that sort of scenario people who are pegging themselves to an environmental accounting standard could use this as a way of confirming whether or not they're heading in the right direction I can more speak from my area which is freshwater systems so there are some of the metrics that they use to measure or have used in the past to look at assessing freshwater ecosystems do rely on reference sites so there's a number of predictive model approaches that they use where you look at the attributes of the site and you compare it to a relatively untouched site and then you've got a sort of idea of aspiration where you would like to get to for that site so the problem that we often have is humans have pretty much touched every part of the globe so current sites don't exist I guess historically it's much more difficult because we need to kind of sometimes we just don't know what was there before we can only kind of guess what it was like or based on records like for Australia it might be what Indigenous people told us or what that painting suggests or something along those lines so yeah it looks it's a difficult one and it really kind of I guess depends on the environment if you can find some relatively untouched things it will tell you potentially what your more impacted sites could potentially look like if you could get them back sometimes that's not possible it'll be interesting to see though because a lot of the money to sort of clean up the environment is coming from investors who are shedding things home to like biodiversity credits or similar I would have thought it has a role to play potentially but that's probably enough on that one. Next question would this technology be able to determine differences in subspecies how accurate are the barcode markers in this way? Very good question it really just depends on the how medically divergent for the standard region where DNA sequencing the subspecies are so sometimes they might carry very similar identical sequences for that region which means they can't differentiate them sometimes it can vary so it's a little bit hit and miss DNA barcoding works for the vast majority of species but there's always exceptions in biology so there are some that are even different actual species that aren't easy because they represent large complexes with continuous genetic distributions and it just means you can't easily sort of aggregate and separate them out as a species group but overwhelmingly DNA barcoding works really really effectively for the vast majority this is very much a small percentage. So a subspecies is something that can make with a what is the definition of a subspecies? It's very much a taxonomic term so it's really just talking about the classification so a subspecies people aren't really sure if it's part of the same species or because it looks a bit different or it behaves a bit different they think it might be a separate species so you're really in that process of still gathering evidence to determine whether it is a separate species or they're all part of the same classification of animals particularly invertebrates is just notoriously difficult and particularly in the flies and the beetles which are the most diverse groups of animals on the planet. It does sound complicated alright next question clarify the development validation and application for meta barcoding to detect aquatic stream macro invertebrate assemblages Okay I've spent a lot of time on all three of those points development validation application so one really neat thing is I've had throughout developing this method access to a lot of taxonomically sorted materials so things that were done prior to me starting this work so I've been able to get samples where I know exactly what is in them and then go through and DNA barcode them and match up what's in the sample versus what the taxonomist found so in some cases I get really high overlap occasionally you get certain groups when you're meta barcoding that are a bit patchy in their detection so I for example had a lot of problems with these little guys called flatworms they have their own genetic code and they're very very different to most other macro invertebrates so some species I find I miss even if they're there but the key to kind of validating and applying this stuff is understanding that some assemblages may be more poorly detected compared to others but a lot of the validation I've done suggests that it's just incredibly powerful in terms of and picks up very much what we see in the sample so and there's very little divergence I'm usually getting upwards of over 90 plus percent overlap between what I see morphologically to what I see with meta barcoding. So once you've done all of that and you're just looking at your natural resource management and wanting to know whether it's improving or not do you have any sort of broad indicators that can be used by natural resource managers and can you give us a case study sort of example? Yeah look for example when we look at freshwater ecosystems there has been attempts at family level for example to integrate responses at family level into stream management. So there's a particular index that's called the signal index and people use that to kind of rank the condition based on the responses of families in terms of doing this it would be really good and part of the bigger aim is to start to incorporate species responses into into a similar way potentially as a signal index. So we can start to give that information to natural resource managers about these are the assemblages of species you're getting these are what are missing that we might expect to find here these are indicative or we've got getting lots of these particular groups that we know respond in a positive way to particular things. So this stuff's still very much in its infancy and it's something that people doing meta barcoding are really keen to kind of draw on information. So we're not just measuring biodiversity but we're also starting to overlay species responses to provide information that natural resource managers can use actively. So a project I'm currently working on is using invertebrates over a huge number of sites across Greater Melbourne that we're identifying to species level and we are using those in ecological models that encompass a range of different inputs. So things like forest cover impervious area which is indicative of pollution and runoff that we get into streams and we're using that to model and a lot of the models that the people I'm working with use enable the Melbourne water who we're working with to start to make decisions about how they can best change different environments to conserve biodiversity or to improve biodiversity. So there's lots of different ways it's not sometimes just looking at the species responses but it can also be using modelling approaches and using the meta barcoding data as an input for these approaches to really sort of give that sort of finer scale stuff and helping decision making. Do they use your measures to monitor the and confirm the results of the modelling? So at this stage we're still in the middle of the project we're right in the guts of collecting all the data so it'll be really exciting sort of towards the end of next year once we have all the data in the models. At this point out early sort of provisional analysis is indicating that the species data we get from meta barcoding adds a lot to how the models fit which means they're going to be more predictive as well in terms of changing various environmental parameters and seeing what kind of differences you get in the species. So yeah, it's still in progress I guess at the moment but these are sort of ways that these approaches can be used as real tools for water waterway managers. Yeah, that sounds exciting. Next question. What is the extent of DNA tools for assessment of Australian stygo former? Sorry at this point not a lot has been done in this space and I think it's one that's sort of gathering a lot of interest. So there have been a few studies where people have looked at how you can sample stygo fauna they're also trying to look at bacteria and things like that that are in the bores along with the stygo fauna but at this stage we don't have a lot of probably DNA reference barcodes for a lot of stygo fauna it's only been specific studies people have done in specific locations and the stygo fauna are notoriously difficult because a different bore can have a whole different assemblage of species as one 20k down the road if the aquifers aren't connected. So they can be incredibly what we call locally endemic. So there are the tools available so the methods for developing a DNA barcode reference library for stygo fauna we have them I'm doing that at the moment with a variety of invertebrates and also the meta barcoding approaches we can sample and detect DNA from stygo fauna it's more going to be the issue is how well covered are they so when we pick them up in a sample can we identify them using DNA or do we need to do a lot of reference database construction to do that accurately. Okay now we have a resident yabby person passionate yabby fisher I suspect is this method suitable for monitoring yabby populations in streams. Okay so look this is probably more one where eDNA is a great way of doing this so for example with eDNA you can go out and sample water and see if you find yabby DNA present in it in terms of managing populations you might look at the genetic diversity within the DNA that you're getting which might give you an idea of how many individuals you might be detecting for example in a particular stream so there are tools available for doing that that require don't require you to necessarily sample the animals in my work every now and again we get yabbies when we go out and do our aquatic testing or our aquatic surveys and we usually just take a small piece of tissue and pop that in with our normal samples so we can work out what yabby is. There's a lot of different freshwater crayfish and some of them are endangered and some of them are of significance and that just enables us to kind of gather a bit of a map why we're doing surveys of other aquatic invertebrates as to whether they're there or not. Excellent all right now we're on to the questions from today. We've got about four minutes so you have to go a bit longer. Okay first question from Tony Squires hi how do we go with ground truthing especially with unidentified unknown unexpected species in a maple body I presume that's sample body maybe. Is the ground truthing well underway or still in its early days? Do you know what I'm saying? I have to confess I don't know what ground truthing is this is a new one for me I guess if Tony's there it's sort of benchmarking benchmarking. Oh okay so it's very much an industry term look if we're talking about how we're using this stuff it is still early days I think this is stuff that we're really trying to get people to see so using species using invertebrates more while people have been using in some fields as I mentioned freshwater invertebrates a lot and there are benchmarks around them using them at family there's really nothing at species level at the moment so it's a case of we're going through a bit of a technological advance and we're starting to build a picture of invertebrate biodiversity we need to almost then start looking at how we sort of set benchmarks that incorporate that Okay here's an interesting question for you from Andrea Hill is there a way for citizen science to get involved in species barcoding? Yes and it currently is in a couple of ways so certainly there are a lot of programs that are run where people use get citizen scientists to go and collect EDNA from various environments and barcode that we would really hoping to try and at one point get scientists involved in sending us invertebrates that we could sequence from various environments so I'm still potentially keen to see if we could work with citizen scientists to go out there and start to get the invertebrates from really environments where we just have such little understanding and enable us to start building those DNA barcode reference libraries for those environments and as I mentioned things like terrestrial worms are a great example trying to get some of our native species of worm barcode it would be great and citizen scientists sending in samples would you know be a great way to facilitate that for example So just in terms of that I don't mean there might be a few people on the call who are involved with that the collection and preservation of the samples and shipping them and how would that happen like how could we help you to link up with citizen science like is there a easy methodology that they can follow and Yeah oh look I haven't probably thought of it that far I'm thinking more about how I've dealt with people who aren't DNA based because obviously everything we want to do is to try and preserve the specimens but I guess the idea is really they need to make it from the citizen scientists to the lab fairly quickly so ideally samples are being stored for longer times away from the lab we need to preserve them in ethanol and refrigerate but for example if we had one way I could see it working with earthworms is if we had citizen scientists going out collecting samples and they'd pop them in their freezer and then we very quickly within a course of a week get them into the lab and preserve them properly for longer term use and it is a really important thing and it's something that you would probably end up coming up with a list of instructions and various sort of ways depending on how quickly you could get the samples what equipment you would use and how you store them to keep the DNA in good nick and the specimens in good nick too so that we can ideally get a taxonomist to take a look at them and ID them if they're new species and we're routinely finding new species that we haven't seen before and they've existed. Have you done any work with the Yarra River Keeper Association? No, no most of the stuff I've done with the Yarra is being with Melbourne Water so using invertebrate samples that have been provided by them might be good to see if you can link up with them because they've got quite a lot of volunteers and they've done various surveys on polystyrene and things in the river I'm happy to link you up with them All right, next question from Michael Curran really enjoyed the talk when metabarate coding the bulk invertebrate samples how long is the fragment you are amplifying and sequencing? Okay, so this has been part of my sort of goes back to the development and validation question so I've been looking at a few different fragments some quite long that are around so this won't mean anything to anybody without a genetics background but around 420 base pairs but currently I'm using two amplicons because this gives me better coverage of the species one's 320 base pairs and the other one's 220 so this seems to recover a good number of species so the fragments we use when we're doing tissue-based metabarate tend to be quite a bit longer than the ones that are typically used for EDNA tends to be more degraded so I hope that answers your question Very good Next question from Sonja Sharma How do you sort through the noise with so many different context generated whilst processing a whole heap of DNA through metabarate coding? Okay so this really comes down to how good your reference libraries are so the first thing I would do when I get a whole bunch of different samples that have been DNA metabar coded is compare them to my reference database and where I get matches that's great if I've got a match that is a species level match then I can move that sample kind of to the side then we kind of have to deal with it at different levels of matches so if matches are very close to a species so sometimes DNA sequences match close there's not a species in the database she'll get different levels of matches so in general my barcode library is pretty complete so the majority of my specimens do match but it's a case of then I start looking for what it might be if it's a close match but not a perfect match it might be something within the same genus then there are level of matches that might mean it's within the same family and so we can still sort of loosely classify things in some cases we can work out if a sequence we get is an invertebrate sequence or it's from fungi in the sample fairly quickly so usually we just go through this process of what we call taxonomic assignment of the metabar coding data and that sort of gives us a bit of a picture of what's in there, how we kind of deal with it and also for me it highlights areas where I need to go back and DNA metabar code or DNA barcode individual specimens to kind of fill gaps in the library and be spending a lot of time on this year where I've finished my metabar coding and now I'm going to be looking at that data and starting to work out are there particular animals that I'm missing I'm lucky enough to have access to samples that were collected along with my samples that have been morphologically ID so I can go through and get those individual specimens and backfill any gaps in my library so I'm hoping by the end of my current project I'll have all the common macroinvertebrates around the greater mountain area covered with individual barcodes so comprehensive reference library that's the dream Before we move to the next question so the lab that's actually doing the analysis is that part of Melbourne UNE Metabar coding Yeah so I do most of the setup in our lab so the PAG lab and the DNA sequencing that I do to generate the metabar codes and the barcodes I outsource to a company called AGRF and they're a DNA sequencing company amongst other things that they do so at this stage it requires to do this sort of work it requires access to a particular equipment in a DNA based lab to kind of get everything prepared and sent off It seems like there's going to be a lot of samples coming in is there sufficient capacity to keep up? Yeah look at this point I would pretty much say I'm not doing research in terms of method development now I'm looking at application so I can take I've got a number of different sample sources or sample types I can process and everything's done routinely in a standardized way so I can make sure I've done all the background work to make sure that I'm comparing different skies so I'm comparing apples with apples so we've worked on getting everything standardized to enable that to be done Okay So in a given year what are you doing thousands of samples? No, not for this project so the scope of the project I'm currently working on we're going to end up with probably around 300 samples invertebrate samples and that was what we had in the budget to do for the particular project but that's just me doing it nobody else you know with a team of people you can just easily you know if I added an extra person I could double that capacity so it's just okay so it depends how many people have working on it but certainly much faster than doing it manually or morphologically Yeah Next question What methods are you using to extract DNA from collected samples be it EDNA or physical specimens There was work years ago University of Canberra on producing a field based instrument that would allow you to add a water sample and then get back a list of species using meta barcoding has there been any developments on this front? I'm not involved in that particular study but I'd be highly skeptical at how good the detection of biodiversity would be if you're putting one drop of water on into a device you're going to get what DNA is in one drop of water at this stage if you really want to get a very good idea of biodiversity you need to sample all the habitats in the particular environment that you're looking at EDNA can only give you really a very small section of the picture in terms of using these approaches in the field it can work quite well with EDNA for detecting small numbers of species or individual species so for example I've got I know someone who's been working on technologies where you can take a device out into the field and you can detect DNA in the water so people are working on these sorts of things but at this stage I'd be very skeptical as to how good they'd be for monitoring biodiversity I think that they're probably more going to be useful in the first instance for single species or just small groups of species that may be a target once people really want to know about It sort of starts to come back to that whole designing your sampling plan and methodology for the application by the sounds of it Next question Adam Harmon It is possible to derive relative abundance data from meta barcoding or only taxonomic richness This is the million dollar question that we get so there are a lot of people and a lot of research that seems to be going on so this is still very much the research stage looking at whether we can get abundance and I've probably put that in quotation marks because what we're really talking about would be biomass because different animals are different sizes and contribute different amounts of DNA so that kind of means you're not really getting abundance but overall the amount of DNA contributed by a particular species so if I look at my fresh water example if I got a dragon fly or a yabby and I processed the whole animal along with a tiny little water beetle I'm not really getting I would think the yabby was hugely abundant and the water beetle was barely there when in actual fact I had one of each so it is problematic the way one thing that we can kind of do is look at individual samples and look at the relative abundances between them because we're never going to be able to look at the abundance between different sites no matter what the DNA technique you do I think it would be incredibly difficult to kind of say that there were more animals at this site or less animals at that site so no it's more richness at the moment and that's certainly where I'm targeting it I'm closely watching the research in the area of looking at whether we can start to get quantification I'll be keen to see where that sort of heads it's certainly something I haven't written off but I'm just more overwhelmed with the amount of biodiversity trying to get barcodes up and running then kind of really playing that side of the research yeah it's a good question though isn't it like you're going down to subspecies and then the question is can you go down to individuals to a certain extent you can look at diversity so I've kind of oversimplified it in that one species doesn't have an identical sequence there's a little bit of variation in that and we can look at how many types of sequence for a particular species with a few DNA bases that might be different between them and get an idea so people are using it to a certain extent to look at how much variation but of course some individuals can have exactly the same DNA sequence so you can't really use it as a counting method but you can use it to kind of get an idea of the variation of genetic variation of a species within a particular site that's a good question next question Andrew Christie can you comment on the use of DNA to determine the approximate timeframes for the arrival of marine pest species is this a realistic endeavor and has it really been tested to the required extent so far we have recently had the Asian shore crab turn up in the waters of Port Phillip Bay there are some attempts underway to use the technology to work out an approximate date of invasion look the problem with using eDNA alone is we're looking at very very short sequences of DNA so what you would really need to do is try and look at longer sections if you want to do tracking or tracing of where these animals are potentially come from so what's the source and how many of them so is there genetic diversity present there eDNA you need to be incredibly careful using it as a tool for biosecurity or screening you need to it needs to be properly validated and tested it's very susceptible to contamination and various other sort of problems if people don't know how to take samples properly those sorts of things but I think yeah eDNA alone no but I think it's a good part of the toolkit to kind of start to understand invasions but it really requires a lot of more specialized research and people who are really proficient in tracing and tracking and using these techniques so okay we've been going for an extra 15 minutes we'll if you're happy we'll push on through there's 15 more questions to go are you happy to hang around a little bit longer Melissa that's fine I think it might be was there 15 in total I know you're taking them off yes so you're obviously generating some good questions next question from Diana Montano Navarro would the readings give you an accurate indication of abundance of that species alright so I guess that's a little bit similar to the previous question is do we get abundance information or do we just get richness as I said you could get proportional kind of bio massive within a sample but as I said this is still an area of very much sort of under research at the moment and we don't really have an easy way of doing that and I think we'll be able to find that out probably in the next few years as people are really investing in that because we constantly get asked can you provide abundance information with DNA data and it's obviously tricky and it can be more tricky with groups where you have a really large size variation as I mentioned in my example before okay next question DNA analysis can replace the presence measure of manual sampling I think it's can it sorting and IDing but can it replace the abundance measures probably very similar to the previous question so at this point I would say no but it really depends how detailed information you want about abundance we do often get of course more DNA sequences from animals that are more abundant in the sample but it's not a perfect relationship either it's not a predictive relationship at this stage so you'll have to just wait and see where the research takes you and where the people can come up with really robust ways of doing this that answers that abundance question is a good one from Edward Sirlan what happens when you uncover a presumed species not listed in the DNA libraries Eddie should know that one I know Eddie try to obtain an individual and barcoded so certainly that's the philosophy I take so that's why I also like working with tissue based samples because I have the animals at hand and it means that if I find something I haven't seen before I've got an animal to barcode and doing that kind of work really helps people in the DNA field because they don't have the animals so they rely on people like me to produce the barcodes that they can use to then identify their sequences or meta barcodes from their DNA samples okay next question what is a typical cost to barcode or meta barcode a sample it's a good question and it's sort of a difficult one to answer sorry rough number we'll do yeah look we're probably talking in a few hundred roughly it depends on the type of sample you're processing it can be more or less than that the skill of the person performing the work the size of the study more samples often means you can scale down the cost per sample it varies but it's certainly a lot cheaper than paying a tax on an ID a vial of animals so or a person to sit there for 10 hours and pick all the animals out of your sample so there there's certainly our loose sort of comparisons indicating that it's quite cost effective okay Tony is there any chance of getting out of historic samples ah yes depends how historic you're talking about so I had gone through some of the macroinvertebrate samples collected or at least 10 years ago by EPA Victoria and we were able to kind of get an idea of the biodiversity in those samples the only thing we found is it was a little more patchy so there were certain species we just weren't detecting as the samples got older but there is the opportunity to do that so it can be pretty cool if you want to compare something that happened a decade ago just something happening today using historically collected material it also depends a little bit on how that material was stored so I was quite fortunate with EPA their material had been constantly kept in ethanol so even though it wasn't 100% which is what we prefer for DNA and it wasn't coal storage which is what we also prefer are still able to get some meaningful biodiversity measurements all right next question Diana Montano Navarro could you talk briefly on the detection limit of species that could be in very limited abundance yeah so this one's a tricky one and certainly it it's not easy to set a limit for these sorts of things because it also is dependent on the overall abundance of other animals and the composition of the sample as well so I have found when I've been doing a lot of the validation and testing very small animals so for example things like aquatic mites which in my freshwater samples probably represent one of the smallest groups occasionally I don't detect them even though I know they're in there so it would be a case of if we're looking at biodiversity in a sample we just need to understand that if there are mites the detection of them might be patchy and how we look at the data after that might need to take that into account so overall the detection is really good across a broad range of invertebrates but yeah there are some groups so small or as you said very limited abundance can be problematic next question from John Morrison what is the half life of an EDNA signature therefore how far back in time can you detect that an organism styrofoam how do environmental conditions affect the longevity of the sigma barcode really good question and this is something that came very early on when people started looking at EDNA so obviously sunlight is really bad for EDNA for example it breaks it down much more quickly hence why you put out something in the sun to sterilize it in terms of the other thing that's really bad for DNA is water so DNA does actually break down a lot faster in water than what it does in say very dry pot environments so it's really very it's really difficult I couldn't really answer that question in terms of specifically with styrofoam and how long ago or how far back you'd have to actually do styrofoam control experiments and they may vary even from bore to bore when you're looking at styrofoam as to how long the EDNA persists in the environment okay next question Michael Curran jumping in on the styrofoam question there is an extensive styrofoam EDNA research program running out of universities in western Australia and south Australia consultants are rolling out these methods currently and are reporting on these results to clients to support environmental impact assessments the regulators are keen to see these techniques continue to develop thank you for the addition there no that's great and I'm glad that that's occurring I wasn't aware excellent Michael you might want to watch out and work together next question Rowan I need to go so thank you for the presentation today it's been great to hear advance and opportunities I think that one's an easy one Melissa next question would EDNA meta barcode give us an accurate estimation of what's in the environment in that moment in time would we expect EDNA to be identifiable in sediments could it be very long that's similar to the previous one about how long DNA persists in styrofoam or DNA might persist in ball water again it's variable so for example with water it's quite difficult because EDNA of course could be washed from upstream we're talking about in streams a transient environment EDNA might not even give you an idea of the local foreigner living at that particular location so it's always sort of something to keep in mind it might be better at giving you more a catchment scale everything happening there and upstream EDNA can be incredibly variable spatially so people often subsample take multiple samples at a particular site but yeah it can also vary quite a lot at a temporal level too so whether you go one or five days later you may get quite different results so in terms of how long it persists in the environment again there's a lot of literature out there and it varies depending on how much sunlight penetration to bitity a range of different factors so I hope that sort of helps answer yeah that's good Michael Curran and which genes why also have you started DNA longevity and shedding I think we haven't covered the shedding bit I don't think the shedding is very much an important part of more the EDNA side of things so as I've kind of mentioned EDNA is something I've not really done a lot of work on there's probably others better to answer that question than me in terms of the genes for animals we use a particular gene called the mitochondrial 1 gene and that's what the bold database is built on and it's a great gene because this just as you saw 12 billion sequences and most of those are animal ones and so it means we just have a huge repository of information and that genes been chosen because it has really good species signals so it's one of the better genes we have for separating different species and we use slightly different ones so we've got a lot of information about the gene and a couple of chloroplast genes for plants okay we've got three questions to go and I think we're nearly there you're doing well Melissa feel free to email me if you have any other questions yes and look just before we read these if anyone wants to so please feel free to see the recording there okay Tony is back again thanks Richard and Melissa with the ground truth in question I saw Melissa suggesting that continuous upgrading of own databases and sharing via libraries of DNA sequences is in its early days with less than 10% of known Australian inverts currently coded work to do yes and work we should be doing too Tony cracking Melissa absolutely very passionate about keeping it cracking that's the one alright next one that's an anonymous one thanks for the talk great presentation and discussion good work Melissa do the sequences in the library are defined well enough to make sure that they incorporate the natural genetic diversity in a species really good question and largely yes and usually when we create DNA barcodes we try and create a barcode from multiple individuals within a species across different geographical sites so that's sort of your ideal world sometimes we don't have that but I've not really had too much issue when I've been trying to do taxonomic assignment between my DNA barcode reference which to my meta barcoded samples in doing that it does line up reasonably well so I would say it's not a huge problem okay last question firstly great presentation it's certainly a very interesting angle to waterway management so as I understand it currently this sounds fantastic as a diversity measure at any taxonomic level do you feel at this stage it's as good as family level taxonomy and all the standard biotic indices used by industry today such as abundance richness signal scores and Osrevis modelling if not do you force it being on par or better in the short term mid term or long term I think that it has a lot of different things to offer that we don't get when we look at family level so I think it's going to be a bit of a reinvention of how we do bioassessment and biomonitoring and it just opens a lot of doors that we couldn't kind of do before so signal scores for example in urban areas tend to flat line we get very similar signal scores across a range of different environments because they're all impacted by urbanization if we look at species we might get a lot more information about how different urban sites for example vary and what are the predominant things affecting the biota well done Melissa so I think thank you very much to you but also thank you very much to the audience and some fantastic questions there today I've certainly got a couple of applications that we became to look at for this technology Melissa like with the Maloon Institute like I mentioned earlier but also I would have thought the Yarra River Keeper Association would be interested in how we can use citizen science to get you more samples but thank you very much it was a fantastic presentation really really interesting and we'll leave it there so thanks for coming to Hydra Terra's webinar yes and thank you everybody much appreciate the opportunity