Hello everyone and welcome to today’s broadcast of the Monarch Joint Venture National Conservation Training Center Monarch Conservation Webinar Series My name is Tracy McCleaf and I’m a biologist with the Fish and Wildlife Service at the National Conservation Training Center We’re glad you could join us today for our March installment of the series Now I’d like to introduce you to Cora Lund Preston, the communications specialist at Monarch Joint Venture She’s going to tell you more about today’s topic and our presenters Cora Thanks Tracy and hi everyone Thanks again for joining us As Tracey mentioned, I’m Cora with the Monarch Joint Venture I’m also joined by Shelby and Wendy in the chat box Today Carl Stenoien and Dean Elmquist are joining us to give a talk about the fascinating topic of Monarch parasitoids Carl Stenoien began pursuing a PhD in ecology, evolution, and behavior with the Monarch lab at the University of Minnesota in 2012 and he’s defending his dissertation this summer Carl uses butterflies and their parasitoids to study how animal behavior and defense plant chemistry influence insect diet He is a graduate of Gustavus Adolphus College where he majored in biology, minored in neuroscience, and competed in track and field Carl is passionate about many things, including teaching and mentoring undergrads, advocating for long-term environmental sustainability, and outdoor adventures Dean Elmquist currently works for the USDA Agricultural Research Service in Washington state Prior to moving out west seeking adventure, Dean worked in the Monarch lab at the University of Minnesota as an undergrad researcher before graduating in 2014 After graduation, Dean continued in the Monarch lab as the assistant program coordinator During his time at the monarch lab, Dean was involved in aspects of the Monarch Larva Monitoring Project, education, outreach, and conservation Dean has a passion for entomology and enjoys working with educators to cultivate students’ interest in insects and the outdoors If you have any questions during today’s presentation, Shelby, Wendy and I will be monitoring the chat box, and we encourage you to put your questions in there throughout the webinar We will save your questions and have an answer period at the end of the webinar with Carl and Dean where we will get to as many questions as we can with the time we have So without further ado, I’ll turn it over to Carl to get started Thanks Cora and thank you to everybody out there listening We’re excited to share what we think is a pretty fun and interesting topic Just as a quick outline of the talk, I’m going to introduce you to Monarch chemical defenses, and monarchs as prey, and give you a brief introduction to parasitoids Tell because I know a lot of people aren’t that familiar with parasitoid life history strategies Then Dana is going to talk to you about tachinid flies and new knowledge that we’ve gained through the Monarch larva monitoring project And finally I’ll wrap up by telling you about some of my dissertation research which addresses Monarch’s interactions with a little known parasitoid wasp So Monarch butterflies are well known for at least two things One is their extraordinary migration, and the other is their chemical defense Monarch butterflies are toxic, or at least distasteful, to potential predators because they steal cardenolides from their milkweed plants and put them into their own tissues Cardenolides bind to essential cellular machinery of most animals and in a lot of mammals would cause heart failure if ingested in high enough doses This famous series of photos of a blue jay learning not to eat Monarchs was taken by Lincoln Brower in of his pioneering experiments to understand Monarch defense chemistry Monarch eggs larvae and pupae are also chemically defended by cardenolides, which they derive from their milkweed host plants Different asclepias, or milkweeds species, have different types and concentrations of cardenolides And these differences are reflected in the chemistry of the monarchs as well This figure, from a paper by Steve Malcolm in 1994, shows that Monarchs that feed on more toxic plants become more toxic themselves And the curve in the graph shows that monarchs are very good at sequestering even from relatively less toxic plants I’ve highlighted a few well-known species for your reference We know that Monarch cardenolides have a general defensive function, yet some animals are able to feed on Monarchs There are two bird species in the Mexican overwintering grounds that feed on Monarchs in large numbers, the Black-backed Oriole and the Black-headed Grosbeak The Oriole preferentially eats the fat bodies,

while the grosbeak feeds on just the abdomen and prefers the less toxic males over the female butterflies Another predator that was recently noticed comes from Connie Masotti in California This winter she noticed the Fox squirrel that was eating the abdomen of Monarch butterflies and leaving the wings on the ground It’s still not clear if there was only one offending squirrel or if multiple have learned how to eat Monarch butterflies Other anecdotal observations of predators of immature monarchs include soldier bugs, paper wasp, assassin bugs, garden spiders, and praying mantises And Monarchs are also the host to detrimental protozoan parasite, commonly known as OE And last but not least, Monarchs are attacked by parasitoids Dean will tell you about these tachinid flies in a few minutes, and I’ll describe what is known about Pteromalus cassotis after that Before that, however, I want to take a moment to introduce you to parasitoids, which lies somewhere on the continuum between predators and parasites Predators are typically larger than their prey items They consume entire organisms and they’re always lethal Parasites on the other hand, are typically smaller than the host that they feed on, they often live on that host, and by definition, they do not directly kill their host Although secondary things can can kill the host Parasitoids are something in between They’re similar to parasites in many ways, but like predators they kill the organism that they feed on Parasitoids are insects that feed on other insects, usually placing their eggs on or inside of that host Their offspring then consume the host eventually killing it These parasitoids then complete development, become adults and fly off to find a host for their offspring There are some groups of insects that have parasitoids, but most parasitoids are the wasps followed by the flies This is a caterpillar that looks like it has a bunch of Q-tips poking out of its body, but in actuality it was attacked by a parasitoid that had laid many eggs into it Know that most insects have a life cycle that goes from egg, to larva, to pupa, to adult, and each stage looks very different from the last Parasitoids have these life stages as wll The wasp eggs hatched into larvae, feed on the inside of the host, and when they had their fill they burrowed out and spun their cocoon where they’ll complete development And soon they’ll emerges as adults If this life cycle sounds out of this world, the makers of these movies thought so too That’s right, the aliens in these movies were modeled after parasitoids that live right here on Earth However, an important difference is that the parasitoids we have on Earth only attack insects and other arthropods, not humans In fact, parasitoid wasps, unlike the wasps most people are familiar with, can’t sting humans The wasps that sting are mostly social wasps that sting in self-defense or in defense of their colony, and have different type of stinger than parasitoids Intra parasitoids attack eggs, others must burrow through the bark of a tree to get the wood boring larva several centimeters below the surface of the tree Other parasitoids attack adult honey bees and in this case, these bees remain alive for several days after the attack and begin to exhibit strange behaviors as the larvae feed from the inside As such, these bees that are infected, but still alive, have been termed “zombees”, which is quite a clever pun, I think And this is one of those flies burrowing out of the bee Other parasitoids are masters of behavioral manipulation In this case these cocoons on the stem have already fed upon and emerged from this caterpillar The thrashing caterpillar remained alive, but the parasitoids have altered its behavior so that it will stay near these cocoons, thrashing at any potential predators of the parasitoid pupae until it starves to death There are also parasitoids of aphids In some cases, this aphids can even reproduce after being stung, but the parasitoid inside will be fatal Once an aphid has been stung, it will

become what’s called a mummy It will become brown and papery, and eventually the wasp will emerge from the hole on the backside of that aphid So far we have talked about aliens, zombies, slaves under mind control, and mummies, which I think is safe to assume is a unique combination of nouns for a Monarch Joint Venture webinar Believe it or not there are even parasitoids that’s specialize on those parasitoids of aphids This small black wasp is a hyperparasitoid of a parasitoid that’s already attacked this aphid and it’s become a mummy So I’ve shown you that there is incredible life history diversity among the parasitoids There’s also extreme species diversity Just for comparison sake, in one superfamily of parasitoid wasps, there are about 80,000 species In all the vertebrates combined there are only 64,000 species So share that with your friends and family Parasitoids are also really important for their ecological services They are the top source of mortality for bivorous insects, which may help us explain why the world is green when we look out the window, and not just a bunch of defoliated plants overrun by herbivores They’re often important to protecting crops, releases biological control agents Especially from aphids, moth and butterfly caterpillars, and beetle larvae So now that I’ve introduced you to Monarchs as food and the wild world of parasitoids, I’m going to turn it over to Dean All right Thank you, Carl My name’s Dean Elmquist As Cora said in the introduction, I used to work at the Monarch lab as the assistant program coordinator and I was involved with a lot of the work with the Monarch Larva Monitoring Project or MLMP for short And So Carl just discussed chemical defenses in Monarch butterflies, and the parasitoids that subvert these chemical defenses I’m here to tell you some more about the parasitism of Monarch butterflies by tachinid Tachinids are one of the most well-studied Monarch parasitoids out there Much of what we know about the relationship between tachinids and Monarchs has been discovered with the help of the Monarch Larva Monitoring Project and the citizen scientists that contribute to the MLMP Today we’ll discuss some recent discoveries resulting from the work of researchers and the citizen scientists that have contributed such data that we were able to use So the Monarch lab recently conducted a large scale analysis of tachinid fly data with the help of some experts in the field of tachinidae So were lucky enough to work with Dr. John Stireman and Juan-Manuel Perilla Lopez from Wright State University Like I mentioned, they’re the tachinid experts so a lot of the work that we did would not have been possible without their help so we’re very grateful for their contributions Elsa Gibhardt right here, she’s a longtime MLMP volunteer, and contributed a lot of fly specimens that we used in this analysis And Dr. Karen Oberhauser, Laura Lukens, and myself made contributions from the Monarch lab So the Monarch Larva Monitoring Project, I’m just going to describe a little bit about that so you guys get a sense of how some of this data was collected and how important volunteers were to this entire process So the MLMP was created out of a need for large scale spatial and temporal data on breeding Monarchs And this was not obtainable by one, or even a few researchers, so this led the use of citizen science Since its inception, the MLMP has gone on to recruit thousands of volunteers citizen scientists to collect data on Monarch butterflies, and there are over 1,200 registered sites in the United States, Canada, and Mexico So the goal of the MLMP is to collect data that documents spatial and temporal differences in Monarch abundance, which essentially means where and how many Monarchs there are at different times during the season So you might have gotten an idea from Carl’s talk, tachinid flies and other parasitoids contribute to Monarch population dynamics So it’s very important that we understand the impact these parasitoids have on the general Monarch population Especially currently, how they might affect or contribute to current Monarch conservation goals So results in data generated from the MLMP has driven Monarch tachinid research efforts, and much of what we’ve discovered about the relationship between monarchs and tachinid flies would not be possible without the MLMP wide volunteer base In fact, six citizen scientists from 16 different states contributed specimens to the recent tachinid analysis conducted by the Monarch lab and collaborators at Wright State University So the MLMP has a variety of approaches to Monarch research

and one of these approaches involves volunteers rearing wild caught monarch butterflies So these dedicated Monarch rearers collect larvae from the wild and rear them to adulthood And a lot of the rearing outcomes are submitted to the MLMP database, and that has revealed a lot about how tachinidae interact with the Monarch population Basically, we’ve discovered that the tachinidae are a primary parasitoid of Monarch butterflies They are very efficient parasitoids and evolved a successful parasitic way of life Success is almost an understatement as experts estimate that there are actually 10,000 described species of tachinidae, and that’s actually kind of a low balling it They think the actual number may be between 15,000 and 20,000 species making the tachinid family one of the most specious families of flies out there So they have a very successful life strategy So tachinids are a major environmental factor that influence Monarch population dynamics, and this has prompted researchers to study them Tachinids affect the survival rates of immature monarchs, and additionally, the seasonal Monarch populations in certain localities may be intertwined with tachinid populations As we discussed earlier, citizen scientists play a huge role in making this research and data analysis possible And we expect that there will be a summary publication that includes this work in 2017 This would actually be the fourth published research project that used MLMP rearing studies to document tachinid fly parasitism rates in the wild So let’s talk briefly about the tachinid’s life cycle So you got an idea of the parasitoid life from Carl’s previous presentation, but there are some pretty unique qualities that tachinid exhibit So I’ll just touch on those a little bit right here So this first picture is of an adult tachinid fly Most tachinids are larger than your average housefly and noticeably more bristly, as you can see right here I’ve actually read about them affectionately referred to as the bristle butt family So across the family, there’s a large variety of shapes, colors, sizes and types of bristling and we’ll see another picture later on here that’ll show that a little better Tachinids have a global distribution, they’re most diverse in the tropics, and part of the reason for their success at inhabiting a variety of habitats across the globe is their parasitoid life strategy Female tachinids possess many strategies for parasitizing an unsuspecting caterpillar peacefully munching away on its milkweed Sometimes females will seek out a caterpillar host and lay her eggs on the host, sometimes they’ll actually inject eggs inside the host, and even sometimes females will search for specific habitats, like a plant species, in hopes of finding a host there So they’ve got some pretty amazing strategies for actually parasitizing their hosts They have equally amazing strategies for actually seeking out these hosts As we discussed above, they have a variety of ova position or egg laying strategies And some hosts even pick up on olfactory cues called volatiles, which are given off by plants that a host caterpillar is feeding upon So it actually sensing cues from the plants to find their host Pretty cool Others tachinids search out a host food source or even the borough of a hidden host and conveniently lay their eggs there Some tachinids are even able to recognize host pheromones, which host caterpillars use pheromones to communicate with each other And these tachinids are able to recognize those pheromones of those hosts and actually use that as a host finding ability So they can be pretty nefarious in the ways that they seek out their hosts As you can probably tell, there are a ton of different strategies used by tachinid flies So once the egg, or the larvae is inside the host, they undergo that parasitoid lifestyle, eating the host from the inside out before they eventually need to leave their host and pupate Usually they leave the host and pupate on the ground or in the soil Some species of tachinidae, as you can see in this picture, actually will eject themselves from the host And they come out on these long strings, that you might be able to see here, that are called gelatinous tendrils They’ll fall to the ground Here’s an example of some tachinids that have just emerged from this Monarch chrysalis here So they fall to the ground and then they’ll pupate safely in the soil and the whole process will begin again So monarch caterpillars happen to be one of the most popular tachinid hosts And the MLMP uses one of its five activities

to study the effect of tachinid parasitoids on monarch Populations So MLMP volunteers participating in activity number three called Monarch survival And you access this on the Monarch Larva Monitoring Project website if you’re interested after this So MLMP volunteers participating in activity three collect wild caught larvae from milkweed patches, and rear those Monarchs to adulthood in their home Sometimes those Monarchs don’t reach adulthood so we always ask the volunteers to record the outcome of the Monarch that they reared Sometimes you’ll get that beautiful adult Monarch butterfly However, you might end up with this picture And you see that’s likely a fifth instar monarch with some tachinidae that emerged from it So volunteers record monarch survival, or whatever the outcome they may see like these tachinids that you see here A lot of times people will be a little bummed out when they’ve gone through the whole process of rearing that Monarch caterpillar sometimes from an egg and then they never get to see that nice orange butterfly, but rather, they get to see these kind of nasty looking larvae here However, tachinid researchers and people at the MLMP here get kind of excited because it gives us a unique opportunity to examine some cool ecological relationships Just going to skip over the slide here So we make it easy for our volunteers to enter their data online in the MLMP parasitism data entry database so it’s really easy The MLMP needs a variety of information from the volunteers regarding the monarchs that they have collected from the wild We’d like the date that the monarch was collected, very important is the instar at collection, and eventually after that monarch has been reared, you’re going to want to record the result, that outcome, and even the number of adult flies that have emerged from the monarch There’s a very easy way to do this, about three steps you can see right here Entering everything into these fields and it goes into an online database that allows researchers to analyze this data like we’ve done in this study here So in 2011, MLMP actually started collecting parasitoid samples for identification So we encouraged volunteers that reared monarchs and ended up with parasitoids to actually send us those adult parasitoids, usually tachinid flies, and even some of their pupae and the actual caterpillar But we’re really interested in the adult flies So many people got involved in this project and eventually we ended up with too many tachinids The monarch lab freezer was overflowing and eventually we decided that it was time to categorize and catalog these flies, identify them ideally down to the species level And what actually resulted out of this were some pretty cool discovered So in order to do this, we needed the help of some tachinid experts We were lucky enough to collaborate with the Stireman lab at Wright State University Dr. Stireman specializes in the tachinid family and his lab studies the evolution and ecology of insects with a focus on the phylogeny and evolution of the tachinids, speciation, co-evolution between plants, insects, and parasitoids as well as diversity, community ecology and tritrophic interactions So we were very lucky to have them on board to help us complete some of the goals that we wanted out of our collection And so here you can really see a great example of the diversity in that the tachinid family right here, some of the colors, the shapes, and even the bristles There’s some great pictures, if you want more I’d suggest a Google search You can come up with some really neat stuff So I was lucky enough to be the one to actually visit Wright State University in March 2016 where we set out to identify the species of every specimen in our collection And it was ambitious goal, but we were able to accomplish it The major objective of the trip was to evaluate the diversity of the collection, identify the flies, but we also wanted to create a characteristic matrix, and later on an identification protocol that would allow the MLMP to continue identifying to tachinid specimens so this important project could carry on You see here that this is a little rough draft of what eventually became the characteristic matrix, which evolved into the actual guidebook And actually in 2016 and 2017, Laura Lukens, who’s currently in charge of the project, successfully use the manual to identify a number of flies from monarchs submitted by volunteers The characteristics that actually separate these flies can be really hard to distinguish, and they often require the use of a microscope and a trained expert like Laura who knows what to look for To illustrate this, here’s a close-up of one

of the tachinid fly species in our collection I just want to show you how fine the characteristics that MLMP staff have to look for to identify these flies So here you can see on a part of this fly called the facial ridge that’s heavily bristled You can really get a close-up look at these bristles on tachinidae And that’s actually an identifying characteristic to distinguish between species As you can see on this tachinid fly, which is actually a different species, there is no bristles on the facial ridge So obviously requires a microscope, a keen eye, and some training to know what you’re looking for This is another example of how sometimes involved and difficult it can be to identify these flies Sometimes species are distinguished by measuring the bristle size on a certain portion of the fly compared to the bristle size on another portion of fly So we’re definitely grateful to have the Stireman lab’s help in creating this tachinid ID manual, and we’re fortunate that it was able to identify flies even after we had moved on with the project here So with the information that we were able to gather from our large scale analysis of the tachinid fly specimens from monarchs that we’d received over the years, we’ve made some pretty interesting discoveries So this study cataloged and described a community of tachinids that attacked monarchs in the Eastern United States Examined their relative frequency, their over position strategies, and the use of different monarch larval host stages during parasitoid development The majority of the analyses focused on the outcomes of rearing reported by citizen scientists to the MLMP data portal you just saw, and the identity of the parasitoid specimens received from volunteers These volunteers put in quite the effort too From 1999 to 2016, over 20,000 monarch butterflies were collected, and reared, and reported to the MLMP parasitoid portal Using this data, the study revealed that overall tachinid parasitism across all monarch life stages was 9.8% by tachinid flies Additionally, out of our specimen collection, 1,146 flies were identified to the species level Flies came from 16 states, ranging from California to Maine, but they were mostly concentrated right in the middle of the country in the upper Midwest Out of 460 three different monarch hosts, we found seven different species tachinid flies that attack monarch butterflies, including one possibly undescribed species, which I’ll discuss in just a few slides here So we can see from this figure here that the frequency of parasitism increased as larval instar increased Which essentially tells us that monarchs are more vulnerable to tachinid parasitism as they develop And you can see here at the fifth instar level maximum parasitism rate of about 17% Tachinids often emerge from their caterpillar host before they can pupate, making the monarchs that survive to be pupation less likely to be parasitized, as you can see here in this figure So the four most common tachinid species we examined showed a lot of variation in which host stages they appeared to attack, and we’re looking at the first four bars here on this graph For example, you can see that one common species, probably the most common species in monarch, Lespesia archippivora is under-represented compared to the total number of monarchs collected as eggs See that there’s no real black bar here And this is because they deposit their egg directly into the host The same pattern emerges when you look over here at Compsilura concinnata Because again, they deposit the egg directly into the host However, Hyphantrophaga virilis and Leschenaultia species, two other tachinids that were identified from our collection, show a substantial parasitism from eggs This is likely due to the reason they lay microtype eggs, which are thousands of small eggs that they would scattered across a potential host habitat, which would then be ingested by the monarch caterpillar What this indicates likely is that the instars that were being reared by volunteers were actually fed milkweed infected with microtype eggs, which is probably why we’re seeing these representations here Overall you can also see that the conclusion that larval instars are parasitized more often as a fifth instar holds true, right here, with our most represented species in our collection

the Lespesia archippivora So the calculated parasitism rate of 17% for monarchs collected as fifth instars is similar to previously calculated rates using tachinid data 75% of the parasitism in our collection was due to the most common parasitoid of monarchs called Lespesia archippivora And it was actually found out of 13 out of 16 states that submitted flies So the next two specimens that were responsible for about 25% of parasitism in our collection were Hyphantrophaga virilis and Compsilura concinnata We’ll come back to Compsilura concinnata in a little bit to discuss the particular ecological significance of this species Other described species were Nilea erecta, Madremyia saundersii and a Lespesia species, but based on our sample size, they were a very small representation of the actual the tachinids in our collection So we think that they might be incidental monarch parasitoids, but again we would need more data to really confirm that Another very interesting discovery that resulted out of this was the discovery of a Leschenaultia species, which is a genus of tachinidae And when we looked at this Leschenaultia species it actually has not been reported to parasitize monarchs, and it actually looks like it may be an undescribed species as it has failed to match, or key to any recognized North American Leschenaultia species This is an exciting discovery the MLMP hopes of follow up on, and it’s very possible that citizen scientists may have contributed to the discovery of a new species So one interesting thing that this study reports is the possibility of that Lespesia archippivora that is highly represented in our monarch tachinid parasitoid collection is potentially monophagous species, which means that it only eats one kind of host This is interesting because archippivora are typically thought to be generalists, but preliminary morphological examination of the archippivora from our collection by the Stireman lab suggests that specimens reared from monarchs may actually be different from other archippivora specimens reared from different Lepidoptera Interestingly enough, as you can see in this figure here, the year to year variation in parasitism frequency of archippivora and monarchs suggests that the archippivora actually track the monarch population densities with a one year lag time Further work is needed to determine if this Lespesia archippivora in our collection reared from monarchs represents a specialized or cryptic tachinid species, but it’s an exciting discovery with a future track for research nonetheless So just to touch on the ecological importance of the Compsilura concinnata That tachinid has a very broad host range and is recorded to parasitize over 180 species, and we actually had relatively high frequency of this parasitoid reared from monarchs And this is actually notable and alarming because most of us know the monarch population is in a bit of a struggle right now So the high frequency of this parasitoid from monarchs is something that it is good to be aware of And in addition to actually learning about the relationship between the Compsilura and the monarch butterfly, our records were actually able to expand the known range of the Compsilura concinnata And we’ve got records in Texas and Iowa that formerly were not known So again I just really want to acknowledge the citizen scientists that contributed to this project and made this research and answering these questions possible The volunteer ability to collect data over broad areas and times allows us to document new monarch parasitoids and it may have even contributed to the discovery of a new species The value of engaging citizen scientists in natural history research seems to endless, and we’re very grateful for their contributions to this research Now I’ll turn it over to Carl who’s going to tell you about another monarch parasitoid that we are currently learning more about All right I’m back Thanks, Dean So this last part of the talk I’m gonna talk about Pteromalus cassotis This is sort of a rediscovered parasitoid, and I’ll tell you more about that story I’m going to show you a couple of videos if you’re interested in playing them again or showing someone else, here are the YouTube link to my channel If you want to get in touch with me you can email me here or tweet at me This is a male and a female specimen of Pteromalus cassotis

that I took in the lab And I’ll tell you more about them in a second, but first we’re going to watch these videos This is a video I took in the lab This is a female Pteromalus cassotis encountering a very newly formed monarch chrysalus She’s investigating it using her antennae to chemo sense and eventually will decide that this is a good place to put her offspring These parasitoids likely bind these host as pupae, but maybe also as larvae They’ll find the larvae based on [INAUDIBLE] cues or a plants cues and they’ll ride that larva until pupates So at that point, there’s really not much that a monarch pupa can do to defend itself But if you check out the rest of that video you’ll see the larva is able to knock the wasp away from it About 15 days later, those wasp offspring will have completed development and will begin to emerge as adult wasps We can check that video out now One female wasp can produce a brood size of over 100 In my lab studies the mean is about 70 offspring, but I’ve seen pupae from the field with over 400 dead wasp larvae inside What probably happened in that situation was that multiple females ovaposited into the same host and over exploited it These wasps often have female bias sex ratios, and they can control the sex ratio by laying either fertilized or unfertilized eggs Unfertilized eggs become males so they’re parthenogenetic in a sense And they’ll meet with their siblings immediately upon emerging, then the females will fly off to find a host of their own The males are much smaller, much less robust, and have a much shorter lifespan than the females But the females survive in the lab very easily over three weeks So for some historical perspective on this project, our lab in 2008 decided that we wanted to better understand the causes of monarch mortality In order to figure out what might be killing pupae, they put pupae outside for a few days, came back to see how many had survived, and try to figure out what had killed the others Most survive these tests, but several were taken or eaten likely by paper wasps and ants, maybe some birds and mammals And after returning to the lab, instead of enclosing as an adult butterfly, one of the 340 pupae that were placed that year produced over 100 tiny wasps instead Our lab didn’t know what species they were so they sent them to the Smithsonian Institution And the folks at the Smithsonian were also not confident about the identification, but they told us it looked like Pteromalus puparum, which is a parasitoid known to attack several other species of butterfly pupae When I heard about this I thought that this was very strange because generalists typically can’t reproduce in chemically defended hosts Usually it’s only specialists that attacked chemically defended hosts because these generalists haven’t evolved the cellular machinery to detoxify or avoid the toxin They just don’t have that much of an evolutionary history with the toxic species So to make a long story short, I collected some Pteromalus puparum, the generalist that the folks at the Smithsonian suggested our wasps might be, and exposed monarchs to them in the lab Many of these wasps eagerly ovaposited into the monarchs, but none of them were able to produce offspring It was at this point that we realized that P. puparum was probably not a species attacking monarchs After some biological sleuthing, I found this paper from 1888 in which the author describes a monarch pupa, which he had found and reared, only to find parasitoids emerge instead

of a butterfly This search was made more difficult because the names of both the host and the parasitoid had changed in the over 125 years since publication And this is still the only paper I’m aware of that mentions parasitism of monarchs by this parasitoid So why is it that no one had published on this species interaction between 1888 and 2008? I think it’s probably because finding monarch pupae in the field is hard If you spent much time looking for monarchs larvae in the field, you can be pretty sure that you’ll find them on milkweed plants, but the pupae could be anywhere The larvae wander away from their host plants when they finish feeding, and may be high up in a tree, or on a fence or, in the understory And they’re quite cryptic, they blend in Furthermore once a pupa is discovered it must be collected and monitored and the collector must resist the urge to freeze the pupa, or throw it out if it turns brown and it becomes clear that butterfly will not emerge, which is what happens when parasitized by these wasps That’s maybe one reason why we don’t know much about this species interaction And so probably the most basic place to start in studying these organisms is to ask when and where are monarchs parasitized by their Pteromalus cassotis So far we just know that in 1888 there was one record and in 2008 there was one record from Wisconsin So I set to work collecting data searching through museum records as well as working with citizen scientists, and all the states that are shaded gray on this map are known to be the home for at least one specimen of Pteromalus cassotis collected Now many of these are museum specimens that do not have host records so we don’t necessarily know that they reproduced in monarchs Those states with stars, we do know that they came from monarchs And you can see that they’re covering much of the eastern U.S., but part of what’s limiting this is sampling effort You can’t find the wasps in places that you’re not looking, or that citizen scientists aren’t looking So since 2008, we’ve continued the same experiment where we place the pupae out into the field, we leave them for a week in the wooden and mesh cages Mesh is small enough to keep out things like these polistes wasps, or other potential predators, but it’s large enough to let these tiny parasitoid wasps in After seven days we bring these cages back inside, separate the pupae, and monitor them to see if butterflies or wasps emerge As you can see, parasitism from year to year is highly variable reaching 60% of the pupae we placed in 2010 The sample sizes are on the bottom In 2013 and 2014, we had zero parasitism, and this was actually pretty frustrating for me, someone who’s trying to study this species interaction and finding no parasitism Zeroes our data but they’re not terribly informative data sometimes And the last two years we’ve had parasitism rates hovering right underneath 10% or so Of course this is a very limited scope in telling us anything about monarch population biology or parasitoid population biology, and we really need more data from other places and continued long term studies in order to determine if there are population level consequences, and potential conservation implications for the monarch Another question that I was really interested in that I introduced at the beginning of this webinar is the question of monarchs’ chemical defenses, and how they might influence the survival of the host, or the performance of the parasitoid wasps So in order to address this question, I reared caterpillars in a greenhouse on two different species of milkweed One is very high and cardenolide concentration and the other quite low We then had these caterpillars pupate inside of the deli containers, which you can see on the right side of the screen,

and we exposed each pupa to one female wasp We let that wasp hang out in the container with the pupa for two to three days, and during that time we’d make observations to see if it was laying eggs These are the two plants we used Asclepias incarnata is lower toxicity milkweed down here So presumably the pupae become less toxic, and we contrasted it with Asclepias curassavica, a higher toxic milkweed There’s a lot of information on this slide so I’ll walk you through it slowly On the x-axis, we have the host plant So these bars represent hosts that were reared on the low toxicity milkweed, and these bars represent hosts that were reared on the high toxicity milkweed And then the colors represent the outcome of those trials So green bars are where wasps emerged, blue bars are where we found wasps dead inside of the host, orange bars represent a host that died, but we couldn’t necessarily say why, we couldn’t find parasitoids inside, and black bars are situations where the butterfly successfully emerged I’ve mentioned Pteromalus cassotis is the one that is attacking monarchs Pteromalus puparum is the one I mentioned that is unsuccessful at attacking monarchs And using Fisher Exact Test, which is similar to a Chi-square test if you’re familiar with that, I found that the proportions of the outcomes of the trials were different when facing Pteromalus cassotis, but not different when facing Pteromalus puparum The Pteromalus puparum will ovaposit into the host, and can kill the host but they can’t reproduce in them So it’s really a lose-lose situation for the wasp and the butterfly And in Pteromalus cassotis, the trend is as we’d expect but pretty weak, really There is a higher proportion of wasps emerging when facing lower toxicity hosts, and more butterflies emerging when they’re fed on higher toxicity plants To investigate this one step further, I mentioned that we take observations during the trials It seems like what’s happening is those wasps facing the lower toxicity hosts are simply more likely to attack those hosts than the wasps facing the high toxicity hosts Again, there’s no difference with a few puparum wasps, but there is a difference with the cassotis wasps and it seems like this is what’s driving the differences in [INAUDIBLE] If we look only at those hosts only at those trials where the wasp attempted to lay eggs into the host, and we plot, in this case, the number of wasps emerged on the y-axis There is slightly higher average brood size with a 95% confidence interval indicated here than Asclepias curassavica, the high toxicity plants But this is not a significant difference Similarly, if we look at the number, or the proportion of wasps that survived to adulthood, this is also not significant And finally, I just couldn’t believe that there was almost no effect of host plants and so I decided to look at the lifespan of these wasps I kept some around and didn’t feed them any food or water and the ones reared on the low toxicity plants survived almost exactly the same length as those reared and high toxicity plant So it seems like the difference we’re seeing in the face of the wasps is really driven by the behavior, the acceptance of these wasps And what I’m interested to dig deeper into my data and see if I can understand why So just to wrap up, I’ve shown you that our knowledge of monarch pupa parasitoids is limited probably because detection is so difficult And we’re doing all we can to learn from this rediscovery of parasitoid I’ve shown you when me where in the United States we’ve seen parasitism It’s across much of the eastern U.S., but of course this is limited by sampling effort And parasitism in the field is highly variable as I’ve shown you from our field experiments in Minnesota But it could have implications for monarch conservation And finally the cardenolides derived from milkweed that the monarchs sequester seems to have little effect on the performance of these parasitoid wasps, but may be the factor that’s

preventing these generalist wasps, Pteromalus puparum, from developing in the monarch So I want to thank my collaborators in the Monarch lab, and my department, as well as at other institutions Again, if you want to get in touch with me, or check out those YouTube videos, the links are down here All right Thank you, Carl and Dean We are going to switch over to the question and answer period now Thank you to everyone who’s been entering in your questions We will get to everything that we have time for Before we quick dive in there, I just want to do a couple of housekeeping items here We did record today’s webinar So if you want to share it with your friends or come back and watch it again, it will be available on the Monarch Joint Venture and NCTC websites as soon as it is available And we will follow up after today’s webinar with a short survey for you to complete, sharing any feedback you have about this presentation, or about the webinar series overall And we would really love to hear from you on that so please take our survey Finally, we just might not get to all of the questions that were asked throughout the webinar, but we’ll get to as many as possible So let me pull up my list here, and then we’ll get started with some questions for Dean about the tachinid flies Dean, once a tachinid lays its egg, do we know how long before the egg hatches and starts eating the caterpillar? Yeah That’s a great question So it kind of will depend on the type of egg that’s laid So sometimes the egg will be laid directly onto the caterpillar, and sometimes it’ll hatch almost immediately and burrow into the caterpillar and begin feeding Others times, the eggs are injected into the caterpillar, and sometimes they will take a little bit of time to hatch But the typical tachinid life cycle kind of follows along with the host that they are inhabiting So it definitely varies quite a bit throughout species, and depending on the egg type that they actually lay Interesting Thank you A similar question, we’ll keep going with that theme Do you have recordings of multiple flies laying eggs in the same larva? Oh, that’s a great question Yeah, and actually I didn’t have time to touch on that today But multi-parasitism is what you’re describing there and that’s actually a pretty common in tachinids And so they can actually lay multiple eggs inside a host and sometimes even two different species can lay multiple eggs inside a host And we actually did see that in our collection So tachinids that parasitize monarchs, we had, I believe it was seven cases of multi-parasitism so we had two different species emerging from one monarch host Wow That’s definitely possible Carl, does that happen with the wasps as well? I’ve seen the parasitoid wasps attempting to lay eggs into pupae infected with tachinids Wow And they’re completely unsuccessful The tachinids emerged just a day later or so So they were too late and not perceptive enough to know that In terms of multi-parasitism with wasps, we don’t know of any other parasitoid wasps that could do multi-parasitism in monarchs, but I’ve seen Pteromalus cassotis multi-parasitize with Pteromalus puparum in cabbage white butterflies So it’s possible with these guys as well Interesting We have another tachinid biology question Dean, do the adults tachinids eat anything, and if so what do they eat? Yes So the adult tachinids they will feed during their life And you know I’m not exactly sure and it will kind of depend on the species and the habitat that they occupy But they are kind of hard to rear in the lab because of their specific dietary requirements, or so I’ve heard, I’ve never tried it So I don’t have an exact answer for that, but they’re feeding definitely depends on the habitat that they occupy and I’m not sure Maybe Carl might be able to add some input, but again it would depend on the habitat that they occupy OK

Carl, do you have anything to add to that? I’m also not sure exactly what tachinids eat The Pteromalus cassotis will feed on nectar in the field, and so in the lab I keep them alive on honey water That’s a common way that we do keep a lot of insects alive in the lab So I’m assuming that the tachinidae are probably pretty similar Interesting Let me see Let’s find another question here for you Carl, are the wasps easily visible or found in the wild very frequently? They are not Really the way I’ve had success collecting them in the wild is by putting hosts out and hoping that they get parasitized? They’re probably at very low densities and can’t necessarily know where they might be if they’re not searching for hosts On that train of thought, what do you think happened in 2013 and 2014 when you were putting hosts out there that resulted in no parasitism for your study? One idea that we’ve suggested is a similar one to what Dean presented in that the density of monarchs in the previous year may have big implications on the density of those parasitoids in that year that you’re interested in So there were very low numbers of monarchs in 2012 and 2013, which may have put a damper on the parasitoid populations as well, especially if they’re specialist parasitoids They’re relying on monarchs Yeah Do you know, either of you on tachinids or wasps, if environmental factors like the weather affect tachinids or wasps? Dean, do you want to go first? Yeah You know, I’m not exactly sure on that I think what we what we definitely do know, going back what to Carl said, is that the monarch population is a main driver of some of those parasitoid dynamics And you know we do know that the monarch population is at least affected by certain weather patterns, which would in turn have an effect on that parasitoid population So you could kind of say, yes Kind of getting around to it, I would say that the weather probably would have some type of effect on parasitoid populations due to the effect it would have on their hosts That makes sense It’s really fun to think about what it’s like to be a tiny little insect in a big world These parasitoids are the size of a drop of water or less so you can imagine that venturing out into a windy or rainy situation is not a risk they want to take So nice, sunny days are much more likely to be foraging, and you can see that in their activity levels in the lab too But I can’t say much more beyond that Yeah Well that makes sense Dean, so do tachinids have a useful purpose in the ecosystem other than controlling monarchs that we know about? Oh yeah, definitely So actually they have been introduced as a beneficial insect for pest control Like Carl kind of mentioned, you don’t look outside and see a bunch of defoliated trees, and that’s because of these parasitoids And the tachinid flies definitely play a large role in that Actually, the Compsilura concinnata species, that’s kind of a concern for its high frequency of non-target hosts in monarchs, was an introduced species to control the gypsy moth, I believe And the Lespesia archippivora, that really highly represented species in monarchs, was actually also introduced as a biological control species So they have a lot of great benefits in pest control, but as we can see some of the introduced species are also having some non-target effects So they definitely do play a large role, the tachinids do, in the ecosystem Interesting Yeah Dean, another question for you Are there management strategies that people can use in their habitat, or when they’re rearing monarchs, to reduce parasitism? That’s a really good question I’m not sure if you necessarily want to reduce parasitism I guess on a personal note that to keep everything as natural as possible is probably what you’d want to do because if you do happen to get parasitism, you can report that to the MLMP, and that’s really important data that we can use to assess monarch-parasitoid relationships But one thing that people that bring monarchs inside and rear them they can do, they can look for them as the younger instars So if you recall that graph showed,

the chances of monarchs being parasitized increased linearly as the instar stage grew So those fifth instars were likely to be parasitized more than, say, a first instar So if you do bring in those instars at an early stage, they stand less of a chance of being parasitized Also, it’s always good to wash your milkweed leaves very carefully Again you can recall back to that earlier talk about those microtype eggs being laid on the milkweed plant and then being fed to caterpillars, likely indoors, that are already being reared So again, washing those milkweed plants leaves very carefully and collecting the earlier instar larvae would be a great way to hedge your bets against your monarch being parasitized Great Thank you There was also a question that I’m going to take a stab at answering here about how talking about toxicity Carl mentioned that the milkweed toxicity would make it less likely that the monarchs would be parasitized But a conundrum there is that tropical milkweed has higher toxicity levels than a lot of other species And the different monarchs parasite, OE, is more prevalent on tropical milkweed So to address that, OE is more prevalent on tropical milkweed where the tropical milkweed grows year round And so that’s in areas in the south and especially along the Gulf Coast, and on the Pacific coast and so being very careful to choose native milkweeds is going to be your best bet Especially in those areas where tropical milkweed could grow year round and foster OE, which is a different monarch parasite But there are different toxicity levels within the different native milkweeds And Carl, do you have any thoughts on what kind of native milkweeds have a higher toxicity than others? There are in general more diversity of highly toxic milkweeds in the southern United States than in northern United States We’re sort of limited to just a handful of less toxic species in the northern United States Yeah Great Well that is all the time that we have for questions Thank you all so much for answering asking so many great questions And I want to say a huge thank you to Carl and Dean for presenting with us today And also, thanks to NCTC for hosting our webinar We hope to see everyone for our next webinar, which is going to be Solar with Monarch Habitat, a win-win in the land of milkweed and honey, which will be on Thursday, April 6th at 2:00 p.m Eastern time And you can visit the MJV events page to register So just a huge thank you to everyone again today and we will see you next time Thank you