Teaching while ‘Sheltering-in-Place’

Wow.. has life taken a surprising turn.. not just for me but for the whole world. With the onset and spread of the COVID19 pandemic we have seen people across the whole world have to adjust and make changes in both their personal and work lives.  It’s been interesting for myself and other teachers (K-12 and Higher Education) to try to quickly readjust and attempt to deliver a premium education for our students. Its also been comforting to see the rebirth of ‘community’  such as folks helping out their elderly neighbors and teachers banding together and sharing online teaching resource and tips.

Thus the focus of this post is to share what others have shared with me, as well as some of my lessons I have learned in the first week of online teaching.

However before I go into the meat of  online teaching.. let me back up a bit to BC (Before Coronavirus) time period. As you may know – I recently started a teaching postdoc position at the University of Southern California (USC) , which has been absolutely amazing so far. In the Fall semester I taught an upper division Ecology class and a lower division Env. Studies GE course, and now in the Spring Ive been teaching two sections of the lower division Env. Studies GE course (no labs this semester thank goodness!)

It all seems like a blur but just two weeks ago USC was still proceeding business as usual… as were most schools in California. Then as we got closer to our Spring Break, USC announced a ‘trial-period’ of online teaching for 3 days before Spring Break so that we can adjust our methods as needed “IF” we needed to extend to online teaching. Then as the # of COVID19 cases rose (see here a live tracking website made by 17 year-Avi Schiffmann)- USC quickly changed the plan to teach remotely after Spring Break until April 16th… and then two days later extended online teaching to the end of the semester. Ha.. people’s plans have been changing so fast.. but not as fast as the virus spreads and mutates: https://nextstrain.org/ncov

Immediately I had mixed feelings about online teaching for the rest of the semester: Cons: Im not very good at this online teaching thing yet and now I better get good at it fast! Pros: I can teach in my workout clothes (with makeup on and a nice top) and foster or adopt a dog since Ill be home all day! (..already made progress on latter- see my foster-fail/adopted dog- Yesenia) from the North Los Angeles Animal Shelter. I still have to officially fill out her adoption paper work.. but haven’t been able to go to shelter yet due to COVID19 restrictions.

Anyhow, now that I have my trusty dog by my side, I have been focusing on how to improve my online classes. I also have received lots of helpful links that I am pasting below this blog post to pass on the shared resources and knowledge.  

At USC most of the teachers and myself have started using Zoom as well as Blackboard, the latter which most of us have regularly used to post announcements and assignment instructions, as well as a platform for students to turn in assignments which we can grade online. So far: I have definitely learned some dos and don’ts with Zoom and remote teaching and testing:

ZOOM Dos:

  1. DO RUN A PRACTICE SESSION FIRST! I suggest practicing with your actual students in the physical classroom if possible prior to going to online only (this is what I did and we sorted out some issues on the student’s side of things this way, as well as how to share the powerpoints for them in slide show not presenter mode), but if not possible – get together w/ some colleagues and practice with each other (my mom actually did the latter and I thought that was a great idea!)
  2. Do set the settings to put everyone on Mute when they enter the room
  3. Do tell the students to unmute themselves when they have questions and feel free to speak whenever (much more engaging and feels more like a real class than written chat-room based questions/comments)
  4. Do TURN YOUR VIDEO ON and encourage students to turn their videos on if they feel comfortable-I noticed it makes me feel like Im actually talking to someone than just a green light on my laptop
  5. Do use the ‘polls‘ in lectures to mix things up and encourage participation
  6. Do use youtube videos or other documentaries to break things up, but be sure to adjust the settings to ‘optimize for full-screen video clip
  7. Do assign in-class activities, put students in ‘breakout rooms’ and have them report their results in their breakout rooms.
  8. Do record your lectures in the cloud in case you have international students that are in different time zones.

ZOOM Don’ts: 

  1. Don’t just lecture at the students and forget about encouraging participation and interaction (I actually am still working on this one.. it is more difficult than in-person classes because you can’t see all your students…)
  2. Don’t assume that all of your students are on the same time zone. I know for me it took me a couple days to realize some of my international students had gone back home. Thus I needed to adjust my expectations for them and let them watch recorded lectures and makeup any in-class activities w/in 24 hours .
  3. Don’t talk too fast (oops.. Im also working on this one…).

Blackboard tips and protocols for online testing: So I actually had in-class midterms planned for this week (BC) and I had to adjust to remote testing formats. I knew I didn’t want to use the ‘honor system’ since some students might cheat and that wouldn’t be fair to others… so I decided just to make it completely open-note, but I expanded on the # of questions in the exam so that it is fast paced enough that the students won’t have time to look up every question. Thus they still have to prepare for the exam a decent amount. I also decided to go with the “Respondus Lock Down Browser” app available in Blackboard for Online Exams. It basically locks out everything on a student’s computer except for the exam. For me the only reason why I decided to go this route is so that students can’t copy and paste answers from their lectures or from internet sources. But I did tell them they could use their ipad or iphones or other computers and notes for information. The key with online testing, just like with online classes, is to have the students PRACTICE! I offered my students 2 pts extra credit to try a practice exam with sample questions and the lock-down browser so that we could get all the kinks out of the way ahead of time. 

I also told me students to have a plan for what computers and internet sources they were going to use, and a backup plan in case they had an internet or computer malfunction. As for the actual testing day/time I will be online and can answer questions my students have via email in live-time.

For my international students and DSP students, blackboard testing tools lets you make make exceptions and adjustments to the test time period, date and time of test on a person-by-person basis. So for my students in countries with very different time zones, I adjusted the time of the exam to be at a more reasonable time for them. For my DSP students, I was able to provide them with extra time to take the exam. 

As far as how to make exam questions- I used the Blackboard ‘question poolsand then used questions from those pools to make the exams. The students are not able to see the question pools or the tests until the available date/time that you dictate in the settings. The tests are this week.. so fingers crossed they go smoothly. If not then Ill have to adjust and just make a take-home essay-based assignment for the final exam. The key about teaching and adjusting to a global pandemic is to be flexible, adapt and stay healthy.  To stay healthy, again I totally recommend fostering or adopting a pet, as well as getting more into cooking and working out everyday. (Lots of great online videos to stay in shape while sheltering-in-place, such as this oldie but goodie with Billy Blanks!: https://www.youtube.com/watch?v=jOaartzSX6A)

With this said, Im leaving you with some great resources that have been forwarded to me from many great teachers: You got this!

Climate Mitigation and Adaptation E-Learning Site from AESS https://camelclimatechange.org/index.html

Shared Resources for Online Teaching Received from Professor Chelsie L. Romulo (Univ. of Northern Colorado):

  1. Shared googlesheet resource
  2. Geoscience Materials for Teaching Onlinehttps://docs.google.com/spreadsheets/d/1-R6THvCIcAjGrWRspCN915SIzItdZ95ziwiF8BmQrYc/edit#gid=0
  3. Ecology and Environmental Science Materials for Teaching Onlinehttps://docs.google.com/spreadsheets/d/16K6bGTf-wGjxxi6aGi_v6vlLQSpsOgl1zq3tXLHWweg/edit#gid=0
  4. Dr. Romulo’s lecture videos for her Introduction to Environmental Studies course https://www.youtube.com/playlist?list=PLo6SyhPc8vba11Il5PBFeA-Uc4bCYPvb8
teaching meme
From Jazzmemes: https://twitter.com/jazzmemes_

A Beautiful Day for BioControl of Cape Ivy

Last Thursday on a typical sunny Californian day, I met up w/ researchers, Kirsten Sheehy (UCSB RIVRLAB), Bill Neill (Riparian Repairs) and Noa Rishe (California Department of Parks and Recreation) to romp around Topanga State Park in search of the invasive Cape ivy, Delairea odorata. As soon as we stepped foot onto the Los Leones trailhead we could see the vast entwined vines of this relentless invasive ivy climbing over and smothering all of the native species in its path, hence its nickname- the ‘California kudzu’.

Native to South Africa, Cape ivy was originally introduced into the USA in the 1850s for ornamental purposes due to its pretty green color with lush yellow flowers. However, looks can be deceiving as in some areas this invasive weed has reduced native plant species richness by 36%, and decreased native seedling abundance by 88% (see Alvarez and Cushman 2002). In addition to its detrimental impacts on native plants, this invasive weed also produces many chemical defense compounds (eg. pyrrolizidine alkaloids and xanthones), which make it toxic and unsuitable for foraging by resident mammals; and potentially detrimental to fish survival if substantial amounts of these chemical compounds end up in waterways. Aside from its toxins, this weed can interfere with nesting sites by many riparian-dependent birds. This invasive weed is also quite the ecosystem engineer due to its shallow root system contributing to serious soil erosion problems on hillsides; as well as potentially forming a serious fire hazard due to its dried out foliage hanging over native trees during the dry season.

Thus, there is a dire need to control the spread and growth of this menacing invasive vine. Invasive weeds can be controlled in several manners including herbicide chemicals, mechanical removal (via hand-labor or machines), and biological control. In classical biological control, a pest or weed’s natural enemies (for example, the insect herbivores of Cape ivy) are collected from its geographic place of origin, tested for target specificity and efficacy, and then released into the invaded region.

Successful biocontrol agents can reduce pest populations below threshold levels that cause problems for humans and native species. Once established, biocontrol agents can provide a sustainable, long-lasting management option as biocontrol agents are self-reproducing and self-distributing. Biocontrol agents will not eradicate every target pest or weed individual but this is actually a positive feature as it prevents population crashes of the biological agent and promotes the long term control of the weed. In sensitive or protected regions, biological control and hand-removal of invasive weeds are often the preferred method of control in order to reduce any negative impacts to the surrounding native ecological community.

 

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Kirsten Sheehy holding up a vial of the biocontrol agents, the gall-forming fly: Parafreutreta regalis

Hence, last Thursday was quite a monumental day as it marked the first release of the biological control agent for the control of the invasive cape ivy in the greater Los Angeles Region. The biological control agent in this case is the gall-forming fly, Parafreutreta regalis Munro (Diptera: Tephritidae), that has already been approved for release after undergoing intensive testing through the USDA-ARS to ensure that it only targets the invasive cape ivy, in order to prevent any non-target effects on local plants. Similar to its host plant, this gall-forming fly is native to the Cape Province of South Africa and is known to stunt the growth of Cape ivy in both the laboratory and in the field. Thus, it is expected that this biocontrol agent will reduce cape ivy’s ability to spread and climb, both which would reduce the smothering impacts of this invasive weed on native vegetation.

Cape_Ivy_biocontrol_release1
Kirsten Sheehy releasing the super-hero gall-forming flies (Parafreutreta regalis) into a cage for biological control of the invasive Cape ivy

In two weeks from now Kirsten Sheehy and the UCSB RIVRLAB will come back to make sure that the galls are forming on the ivy before removing the cage. Once the galls have formed, these flies are pretty much on their own, continuing the cycle of injecting their eggs into new unsuspecting cape ivy hosts, and forming new galls that promote generation after generation of weed-controlling superheroes. Of course Kirsten will continue to make periodic new releases of adult flies in the SoCal region to increase the genetic variation of this fly to ensure the success of these new populations in the Los Angeles Region. The goals are to establish these super-hero flies in at least one site per coastal county in California to serve as ‘nursery’ sites for future regional releases. San Diego Co. is next up on this lucky-list of biocontrol study sites.

In addition to this beneficial fly, further biological control research on a stem- boring moth, Digitivalva delaireae Gaedike & Krüger (Lepidoptera: Acrolepiidae), is underway via the USDA-ARS (see Mehelis et al. 2015) and is likely to be approved for release in the near future. This stem-boring moth is actually expected to have an even greater impact on controlling Cape ivy, especially if it is combined with the impacts of the gall-forming fly. Once approved, we hope to add this additional superhero biocontrol agent in the SoCal Region in order to reduce the ecological crimes of the invasive Cape ivy villain. Stay tuned for the sequel.

In the mean time, if you would like to learn more, see the contact information, links and research articles below.

Contact Information regarding UCSB RIVRLAB Biocontrol Research:

Dr. Tom Dudley: tdudley(at)msi.ucsb.edu

Kirsten Sheehy: kirsten.sheehy(at)lifesci.ucsb.edu

 Relevant Articles:

Alvarez and Cushman (2002). Community-level consequences of a plant invasion: effects on three habitats in coastal California. Ecological Applications. 12(5): 1434-1444. http://marbles.sonoma.edu/users/c/cushman/pdf/alvarez%20&%20cushman%2002.pdf

Mehelis, C.N., Balciunas, J., Reddy, A.M., Van Der Westhuizen, L., Neser, S., Moran, P.J. 2015. Biology and host range of Digitivalva delaireae (Lepidoptera: Glyphipterigidae), a candidate agent for biological control of Cape-ivy (Delairea odorata) in California and Oregon. Environmental Entomology. 44(2):260-276. doi: 10.1093/ee/nvu030.

Relevant Links:

California Department of Parks and Recreation: http://www.parks.ca.gov/?page_id=21576

UCSB RIVRLAB: http://rivrlab.msi.ucsb.edu/biocontrol/cape-ivy

USDA-ARS, Albany CA:

Acknowledgements: Additional thanks to Dr. Tom Dudley and Dr. Adam Lambert from the UCSB RIVRLAB and Danielle LeFer from California Department of Parks and Recreation for coordinating this momentous day, and to Dr. Patrick Moran, Dr. Scott Portman, Dr. Angelica Reddy, Dr. Chris Mehelis and additional researchers from USDA-ARS in Albany, CA for all of the rigorous testing of this weed and its biological control agents.

 

New Teaching Gig at USC: Ecology and Env. Studies!

So, I have some exciting changes and news! Starting Monday I will be teaching two undergraduate classes at USC this Fall! I recently changed positions at USC as a postdoctoral researcher (studying parasite-host interactions in the marine phytoplankton community) to a new postdoctoral teaching fellow position. As much as I love research, I really missed teaching and mentoring and wanted to dive deep into teaching to gain more experience as an instructor of record.

USC CAMPUS PHOTO
Photo of USC campus from: https://dworakpeck.usc.edu

I will be teaching an upper division Ecology course with a lab (BISC 315), and a lower division Environmental Studies course (ENST100). I’ll also be participating in USC’s CET New Faculty Institute , a faculty development program. I’m super stoked, and have been hustling for the past month to modify and design the curriculum and get all of the materials ready for the Ecology lab course. I thought most of my time would be modifying and designing new lectures and labs…. and I’ve definitely had some surprises along the way regarding where all of my time goes:

  • It takes a lot of time to prep a lab room/facility if by chance you are ‘lucky’ enough  to have a lab that does’t have a lab manager…. The benefits are that I don’t need to share the lab w/ too many other classes.. so I can leave things set up from time to time. Plus my TA can use the lab as her office hours! Cons- it is up to me to fix everything and get everything ready for the semester! So I definitely spent some time on tasks like getting rid of that old whiteboard that doesn’t erase anymore and fixing the new one that somehow arrived broken (thanks to the hubby for the latter!). On the same note- all of that old hazardous waste in the fume hood.. yup- need to condense it and call EH&S to whisk it away. ….Those old dead snails that have been rotting in the back of the room for over 6 months.. .yea I put those in the dumpster. That cart with some strange devices that look like broken microscopes.. turns out nobody knows who it belongs to.. but it can’t be thrown away. I think that one will just get put in storage….  Oh and then the broken Monitor that we need to use for presentations…thank goodness for IT support…

 

  •  Then there is the ordering and organizing of all of the supplies for each lab. Which means you have to modify or design labs and fully prep all of the lab instructions and hand outs before the semester starts (which is a good thing anyhow). I did this… and I even made sure we had phase-contrast microscopes before I designed a lab. However I learned a big lesson- always physically check out the equipment before you spend all of the time designing a lab and handouts! Turns out – the phase-contrast microscopes  that we have available for our teaching labs don’t have 20X objectives. Later I learned it is standard for most microscopes to only have 4X 10X 40X and 100X.. and I guess 20X is rare.. who knew!?! Unfortunately, one of the labs I spent a couple days designing (including all the handouts, instructions and reading materials)- requires 20X objectives lens… oops! So had to scrap that one and cut my losses. In the end it worked out and I extended a lab on insect diversity instead which I think will be better anyhow. On this note- I am super grateful to the Entomology Curator, Brian Brown, at the Natural History Museum for donating some of their no-data insects for my class insect collection. Im planning on putting them on a backdrop of the phylogeny and evolution of insects (Misof et al. 2014, Science) so students can see the different adaptations that have evolved through time in the Class Insecta.
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In a couple weeks I will mount these onto a phylogeny backdrop w/ an evolutionary timescale.. new photos to come later. Insects courtesy of the Natural History Museum’s Entomology Collection in Los Angeles.
  • Lastly there are the little things- like moving into a new office and setting up the space so that it is beneficial for office hours; learning how to use the departmental printer/copier; going to all of the classrooms and making sure my computer connects properly (and organizing those chairs in the classroom since it looks like a rave recently happened!); and meeting with teachers and TAs that taught the classes in previous years so that I can get the run-down on what worked and what didn’t work.

 

Then when I do actually have time to work on my lectures.. I find myself going down rabbit holes of finding cool documentaries for my Environmental Studies lectures, such as my new one on the interactions of society, culture and the environment- (Check out this cool documentary series on Native Americans and their stewardship of the land and waters); or going through all of the scientific literature on interesting topics that I am incorporating into my lectures such as sex-changing fish for my ecology lecture on mating behavior and sexual selection (which by the way is how I got interested in ecology in the first place over 20 years ago!.. oh geez Im getting old!)

Screenshot 2019-08-24 10.58.16
Figure 1: Todd et al. 2017. Female Mimicry by Sneaker Males Has a Transcriptomic Signature in Both the Brain and the Gonad in a Sex-Changing Fish. Mol. Biol. Evol. doi:10.1093/molbev/msx293

Anyhow… Monday is right around the corner.. so with that Im going to start uploading everything onto our online BlackBoard system… here we go!

Ps- if any of my students are reading this— don’t worry- I got this! You are in good hands… ha ha ha. 

Spring Bloom Sampling in California

One of the reasons I haven’t posted for a bit besides the normal-busy routine is that it is Spring Time! What’s that got to do with anything you ask?

BLOOMS! BLOOMS OF EVERYTHING!

Me in the Anza-Borrego Desert next to an Ocotillo plant
Me in the Anza-Borrego Desert in California, next to a flowering Ocotillo plant

Besides blooms of flowers in the desert  (such as those in the Anza-Borrego Desert), we also get blooms of phytoplankton along the coast in Southern California.

Here, in the spring we get very high winds that can result in upwelling events in the coastal ocean, pushing waters offshore and bringing up cold, nutrient rich water from the bottom ocean layers to the top layers.

This increase in nutrients (such as nitrogen, phosphorous, iron, etc.) can result in massive ‘blooms’ or increases in specific phytoplankton species (diatoms, dinoflagellates, etc.), since typically their densities are limited by nutrient availability. During these blooms, whoever wins the space and resource competition will dominate… until they get run down by grazers, parasites or viruses.. or run out of their limiting nutrient. Once these species decline this then provides space/resources for the next dominating species.

Upwelling Diagram from Sanctuary Quest 2002, NOAA/OER
of Upwelling (Image from Sanctuary Quest 2002, NOAA/OER)

These upwelling events also offer AWESOME opportunities for scientists to examine the species dynamics, and the mechanisms that result in some species or functional groups of phytoplankton to dominate over others.

This year, our laboratory  (the Caron Laboratory at USC) decided to start our sampling period after we noticed strong winds on April 9th.. and I mean Strong! I was biking to my circus class that evening, and a branch literally flew and hit me.. luckily I was wearing a helmet 🙂  During lab meeting that week, we were all telling each other the horror stories of the strong wind, and realized.. ‘woah!’… we should start our spring sampling asap! So we quickly contacted the amazing Santa Monica Pier Aquarium (Heal The Bay) and received permission to use some of the space there to do our sample processing for three weeks. Then we finalized our schedules, rotating each daily to sample and process the water off of the Santa Monica Pier. Each day at 8:30am, we get to the aquarium, load up our cart with the RBR (an oceanographic instrument that measures temperature, salinity, chlorophyll and dissolved oxygen), a bucket and container for loading up sea water, and a 20 micron plankton net to collect a concentrated water sample. Then by 9am, we are loading up water into our collection container, and then rolling the water back to the aquarium to filter some of it down as fast as possible onto filters that we flash freeze for DNA/RNA extractions. We also preserve some of the whole sea water for relative abundance counts of the different organisms via microscopy, and we  sample the water for extraction of chlorophyll and domoic acid (toxin produced by some diatoms). Once we get back to the lab, we inspect the concentrated samples from the plankton net to get a quick overview of who is in the water, and who is the dominating species.

 

This year the sampling has been super interesting! It started off with a diatom and dinoflagellate bloom, and it looks like the diatoms have been CRUSHED by a parasitoid, Cryothecomonas spp.! Once the diatoms crashed, the dinoflagellates  increased more, in particular two species are currently dominating: Akashiwo sanguinea and Cochlodinium spp. (species will be determined after we get our molecular sequences back). I also found some tintinnid ciliates parasitized by Eudoboscquella parasitoids.. so beautiful.

 

In addition to using molecular sequences for identification of the different taxa, our laboratory also analyzes the RNA sequences (using bioinformatics) to examine gene expression of the different taxa that are increasing and decreasing during the bloom. These methods can help us determine when species are taking up specific nutrients, when they are multiplying, when they are stressed, and even if they are being attacked by parasites. Lastly, my work in particular during this spring bloom will examine the dynamics of these species and their parasites through time using qPCR (quantifies the relative number of the hosts and parasites by comparing samples to standard curves).

We have five more days left of daily sampling, and I will be sure to follow up with another blog on the results of this spring bloom sampling period. I will also post soon about the exciting results from a massive laboratory experiment that I just finished. Stay tuned!

Inspiring the Inner Parasitologist in Youth through Science Workshops

This weekend I had the awesome opportunity to spread my enthusiasm for parasites with  middle schoolers at a science workshop through the ‘LABs’ series at The Institute for Educational Advancement (IEA) in Pasadena, CA.

IEA12
Some of the students and myself and the IEA LABs parasitology workshop that I gave this Saturday. I think all of us were super excited to detach the parasitic female and male isopods from the host mud shrimp!

IEA in Pasadena, CA, is an inspiring non-profit organization, that helps to identify and foster the individual talents and abilities of gifted students from all backgrounds, and works to serve and support them and their families. It was super neat to interact with these students and to reflect on the world of parasites with them.

My main learning objectives for the students in the workshop were to: 1) Define different types of ecological relationships, including the different types of symbiotic relationships; 2) differentiate among parasites, parasitoids and pathogens; 3) get acquainted with different parasite lifestyles- including direct transmission, vectored transmission, trophic transmission, parasitic castrators, host behavior modification, etc. 4) revel in the sheer shock and aw of diverse parasites; 5) become familiar with using microscopes; and 6) gain experience with some basic dissection techniques.

I found some super awesome and educational parasite videos during the process  of getting my interactive lecture together-including videos of: 

The tongue-eating parasitic isopod of fish

The sexual parasitism of the female deep sea anglerfish by the male deep sea angler fish

And of course parasitic wasp larvae developing inside of their host caterpillar: 

This was also a great opportunity to become more acquainted with local parasitologists and the common parasites and hosts in the Los Angeles area. This meant connecting with my local parasitologist colleagues (many of whom are part of the Southern California Society of Parasitologists) and finding some parasite ‘hot-spots’ so that I could bring in ample numbers of snails, crabs, shrimp and protozoans for some hands-on activities including dissections and mounting slides on the microscope.

Below was one of the students’ favorites – the parasitic isopod couple (yes.. male and female showing their love for each other all while parasitizing the host shrimp). ‘Couples that parasitize hosts together.. stay together!.. awww’

They were also amazed by the marine protistan parasitoid Parvilucifera sinerae, that they happened to catch in the act of bursting out of its dinoflagellate host (and thus killing its host!). I am particularly fond of this parasitoid right now.. since I’m working with marine plankton communities every day at USC. You can read about some of my current postdoc work here

The dinoflagellate, Lingulodinium polyedra, parasitized by the perkinsid parasitoid, Parvilucifera sinerae
The dinoflagellate, Lingulodinium polyedra, parasitized by the perkinsid parasitoid,    Parvilucifera sinerae

This event  reminded me that one of the main reasons why I love science is actually  the amazing support from other scientists.. and getting to know these scientists as people! For instance, I could not have done this workshop, without the support of Dr. Kevin Lafferty (USGS, UCSB) and Dr. Ryan Hechinger (Scripps-UCSD) who provided me with some hot spot localities for collecting highly-parasitized populations of the California Horn Snail, and how to access those hot-spots. These snails are parasitized by many different species of trematodes (Platyhelminthes), which are trophically transmitted parasites. This means that these parasites use multiple hosts to complete their life cycle and thus require their first set of hosts to be eaten by their final ‘definitive’ host. These parasites are also great for show-and-tell purposes since the cercariae (parasitic stage that searches for the next host) are larger than 100 um, move around quite a bit and can often have charismatic features, such as eye-spots.

I also got more great advice from Dr. Kimo Morris, a Professor at Santa Ana Community College and Dr. Ralph Appy regarding what other critters I could collect, and how to alter my workshop and lectures to appropriately target middle schoolers. Dr. Ralph Appy actually invited me to his laboratory to learn how he digests crabs and shrimps with an acidic solution to fool the parasites (in these hosts) into thinking that they are in the stomach of the next host of their life-cycle.. with the ultimate goal of collecting the parasites into a pool of liquid for research or show-and-tell. Dr. Ralph Appy provided me with a ton of ghost shrimp, mole crabs (sand-crabs) and the really cool mud shrimp that was parasitized by two ectoparasitic isopods (one female and one male).

All in all… I definitely learned a ton from this great opportunity.. and look forward to giving another parasitology workshop to K-12 students in the future (next time with fresher snails.. so they don’t smell so bad!).

 

 

 

 

Into (and Out of) the Weeds: Lessons Learned from my Newest Publication

Woohoo!… finally my newest publication is available via Early View in Evolutionary Applications

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This study was a product of my Delta Science Postdoctoral Fellowship to investigate the mechanisms for effective biological control of the invasive water hyacinth in the Sacramento-San Joaquin River Delta (hereafter “Delta”).

In a nutshell:  Two weevils (insects) are currently used all over the world for the biological control of the invasive water hyacinth, including the Sacramento-San Joaquin River Delta, California. They have had variable success, with notable reduction of biomass and cover of this invasive aquatic weed in warmer climates compared to more temperate climates such as the Delta. Although temperature plays a large role in their success, I also investigated the role of genetic variation in the success of these weevils and whether there is lower genetic diversity and heterozygosity in the Delta compared to the native origin of these weevils (South America). To do this, I used polymorphic microsatellite markers  (repeating regions of DNA in the genetic blueprints of a species) to detect differences between individuals and between populations. Additionally, as myself and others noticed weevils from the field that appeared to be hybrids of these two species, I examined whether these hybrid-like weevils are genetic hybrids (meaning that they have genetic patterns representative of the genetic blueprints from both species)

In my opinion, the most important findings from this study were:

  1. We found hybrids! This is huge! These two weevils are introduced all over the world for the control of invasive water hyacinth. So now that we know hybridization occurs, it is critical since to understand how hybridization affects their success. For instance, sometimes hybrids can outperform non-hybrids (hybrid-vigor) whereas other times hybridization can decrease performance, as well as population growth (hybrid-breakdown). I am very excited however that Dr. Julie Coetzee’s laboratory in South Africa is now starting to look into the effects of hybridization between these two weevil species.. so stay tuned (I know I will!) .
    Demonstration of hybridization between the two weevils: Neochetina bruchi and N. eichhorniae
    Typical elytra markings characteristic of (a) Neochetina bruchi and (b) N. eichhorniae; compared to atypical elytra markings for (c) N. bruchi and (d) N. eichhorniae. Microsatellite markers confirmed that specimens (c & d) are hybrids. A weevil (c) from the study site in California resulted in 100% amplification of markers for N. bruchi and 80% amplification of the markers for N. eichhorniae, whereas a weevil from Texas (d) resulted in amplification of 25% of the markers for N. bruchi and 100% of the markers for N. eichhorniae.
  2. We found that low genetic variation from demographic bottlenecks (small populations of the weevils being introduced over and over again through the biological control programs), can sometimes be buffered by genetic admixture from multiple introductions. This was one of several findings from this study that was made possible through the unique combination of documented historical records from biological control programs and population genetic analyses, such as those we made with the program, FLOCK.

    Importation history and the Introduction Processes of Two Biological Control Agents of the Invasive Water Hyacinth
    Partial importation history (a, b) compared to the introduction processes predicted by FLOCK genetic analyses (c, d) of Neochetina bruchi and Neochetina eichhorniae, two weevils native to South America. Arrows depict the direction of the biological control releases and the date initially released, but do not point to the exact release site in that locality. Black lines and yellow‐filled regions represent the routes of importation history that were tested with microsatellite markers.Abbreviations are detailed in Table 1 (Hopper et al. 2019, Evolutionary Applications). Numbers next to abbreviations indicate the number of genetic sub‐clusters found from FLOCK analyses (c, d)
  3. Through combining this genetic study with a temperature performance study, we found that low genetic variation does not always hinder population adaptation or performance. This finding has been observed in other study systems, such as with the invasive Argentine ant, which has lower genetic variation in the introduced region, but is more successful than in the native range due to reduced intraspecific aggression among separate ant nests in the introduced populations. 

I also think that the lessons I learned from the process of writing this manuscript were very important, and I detail these below. 

Lesson 1: Know when to ask for help

This study culminated out of work that I did at UC Davis, advised by Dr. Ted Grosholz, and in collaboration with researchers, Dr. Paul Pratt and Dr. Kent McCue (USDA/ARS), Dr. Ruth Hufbauer (Colorado State University) and Dr. Pierre Duchesne (Université Laval, Quebec, Canada). The latter two coauthors of whom I actually contacted out of the blue during the analysis and writing portion of the study, since I felt like I needed more guidance from experts in the population genetics and data analysis field. I think knowing when to ask for help is really critical in science (no matter what your academic standing is), and it almost always improves the study to get additional opinions and critique. Think of it as a preliminary peer review before the ultimate peer review!

I also asked several folks that are experts in population genetics for advice on the collection, processing and analysis of the data before and during the start of this project, including:  Dr. Jeremy Andersen (UC Berkeley), and Dr. Rick Grosberg and Brenda Cameron (UC Davis) and Dr. Neil Tsutsui (UC Berkeley).

Lesson 2: Be Flexible, and Adapt to let the Data tell the Story

The title of this manuscript felt very suitable to me as ecological data are not always clear-cut, and sometimes it can take some time to wade through the weeds of data and figure out how to tell the accompanying story.  This is especially true for when resulting data don’t match up with your original expectations and initial story you thought you would tell. The key to this issue, is don’t try to force your old story on the data… get a second opinion if needed, and be open-minded by letting the data ‘speak’ for itself.

Lesson 3: Work Hard, Be Patient and Persistent

I think with anything that you do, sometimes a final product comes easy… and other times it seems like a long drawn out process. This project fell in the latter category, as it was my first time learning about and implementing a population genetics study, and I was working on the analysis and write-up of this study all while starting a new postdoc in an entirely new study system. I think an important aspect to finishing this project was really persistence. I spent week nights and weekends working diligently on the data analysis and writing and re-writing the paper. I also had to be patient with myself as I had to give myself time to learn the new types of analyses (which means new R packages and code!) and time to read all of the important papers in the study field.

If by chance you are also just starting a population genetic study, and feel a bit lost, please see my three-part tutorial blog posts which hopefully will provide some assistance:

  1. How-to use microsatellites for population genetics, Part I: Study Design, DNA extraction, Microsatellite Marker Design/Outsourcing
  2. Population Genetics Part II: Tips and Tricks, Multiplex PCR and Workflow of Microsatellites- the cheap way
  3. Population Genetics, Part III: Data Wrangling and Analyses

Lesson 4: Implement Self-Deadlines and Advertise them to your CoAuthors

writing_phdcomicSometimes its hard to finish something if you don’t have a deadline. So make yourself a deadline, and tell everyone about this deadline, so that you are held accountable for this timeline. I actually had some coauthors that needed me to submit this article to the journal by October 1st in order to meet some of their workplace requirements for publications. Needless to say, I pulled an all-nighter and got it in to the journal by 5am that day.. true story….

Nothing like a little pressure to light up that writing-fire…

Lesson 5: Don’t cut corners

This goes with Lesson 3, on being patient. Towards the end of writing up a big study, you might find yourself just wanting it to be over. You would do anything to not have to think about that project or the data anymore. However, crossing that finish line is actually one of the most crucial components and can make or break your ability to get into a decent journal. Having co-authors often really helps solve this problem, as they will call you out on any cut corners (if they are doing their job), and will suggest critical improvements to the paper that maybe you were thinking about.. but were just initially too lazy to do. Also on this note.. Read the proof-version (final version before being published) of the paper word for word! You don’t want any typos in your finished product.. especially true in your Title, Abstract and Figure Legends!

Lesson 6: Celebrate at Each Stage of Completion

Be sure to acknowledge your accomplishments after you submit the manuscript the first time, after the revisions and acceptance, and after the manuscript goes In Press. After all- you worked hard to get to each of those stages, and celebration will help motivate you for the next time you have to do it all over again!

writing god

 

Population Genetics, Part III: Data Wrangling and Analyses

So some good news!- My population genetics study on the two herbivorous biological control agents of water hyacinth: Neochetina bruchi and N. eichhorniae, was finally accepted for publication w/ minor revisions in Evolutionary Applications. I will certainly post it once it is In Press! This was one of the projects I did for my Delta Science Postdoctoral Fellowship research 

So with that, I will fulfill my promise on posting Part III of my ‘how-to’ series for population genetics using microsatellites.  To recap, Part I of this series explained what microsatellites are, and how to develop microsatellite markers, and Part II was on how to amplify and genotype these markers (the cheap way with universal fluorescent labeled tails, and multiplex pcr).

Part III (right here!) is my attempt to guide you through the jungle of population genetic analyses. I will discuss the main programs and analyses I used and how to properly format your data to make these packages and programs work!

NB_admixture_K2_K6
STRUCTURE analysis of N. bruchi across eight populations and eight loci

I am not going to go into nitty-gritty detail because the tutorial for the ‘poppr’ package in R, does a FANTASTIC job on guiding newbies (including my former self) through the process of how to import data into R, exploring the data, and then how to conduct some basic and advanced analyses. The link is here  http://grunwaldlab.github.io/Population_Genetics_in_R/index.html

Honestly- this is how I started learning how to conduct population genetic analyses in R.. I kid you not. I literally followed the above tutorial step by step and did almost all of the analyses just to get a feel for the data and how to run population genetic stats.

So- Where to start you ask?


Well, one of my collaborator/coauthors (Dr. Ruth Hufbauer-CSU) emphasized that before you analyze the data, a good first step is to know what your question is, and why you are asking those questions. Then you should base your analyses on those questions.

Here are some example questions:

  • Where did these samples/individuals originate from?
  • How many populations are there?
  • What is the genetic diversity in these populations, and are some populations more diverse than others? Genetic diversity is often based on one or more of the following: heterozygosity, allelic richness and diversity indices such as the Shannon, Simpson, or Nei)
  • Are there population genetic bottlenecks?
  • Is there inbreeding?
  • Are there hybrids (crosses between two species)?

Then of course you have to report some general marker- and population-based stats (Deviation from HWE- Hardy Weinberg Equilibrium, Linkage Disequilibrium (LD), overall expected and observed heterozygosity, (He and Ho), null alleles..etc).


Load the Data: Before you do anything, you have to load the data in a format that the programs recognize!

  •  GenAlex- Excel Based Program-useful to check data formatting, and reformat data for import into R or other programs. However the main thing I found useful was understanding just what your dataframe should look like, which the Poppr tutorial emphasizes nicely: here
  • Adegenet package in R- (Jombart et al., 2010) Converts any type of data frame or matrix or txt file to a format that you need for a specific type of analysis
    • For most of my data analyses, I used the following two formats, converting my csv to data that the packages could recognize, or that I could convert further:
      1. newdataname <- read.genalex(“datafile.csv”,genclone = FALSE)
        • you can convert this to a genepop format with the following code-
      2. newdataname2=read.genalex(“datafile.csv”)
        • #need genclone for gytpes conversion, hence don’t use genclone=FALSE
          • gtypesdata=genind2gtypes(newdataname2)
Screenshot 2018-12-08 12.48.53.png
Example dataframe for import and analysis with the Poppr R package. Areas selected in blue represent the Loci, Samples and Populations, see poppr tutorial for further examples

Basic and Advanced Stats- I suggest to use:

  • Poppr– (Kamvar et al., 2015; Kamvar et al., 2014) this package depends on loading a lot of other packages and guides you through analyses in the tutorial. One example- is as a wrapper for the ‘vegan’ package- poppr calculates genotype accumulation curve (see if you sampled enough loci and individuals),
  • Pegas-(Paradis, 2010) -calculate Linkage Disequilibrium (LD) and HWE across populations for each locus
  • PopGenReport-(Adamack et al., 2014)- calculate null-allele frequencies pairwise FST and Jost’s D analyses, compare total and average allelic richness (accounting for sample size) and the number of private alleles among populations
  • diveRsity– (Keenan et al., 2013)-Estimate the average observed (Ho) and expected (He) heterozygosity, deviations from HWE (exact test) and the average ‘inbreeding coefficient’ (FIS) for each population across all loci.  In my paper I distinguish FIS as a measure of increases in homozygosity due to genetic drift caused by a larger population being separated into sub populations, rather than due to consanguineous mating (Crow, 2010)
  • InbreedR- (Stoffel et al., 2016)-calculate g2 as a measure of inbreeding.

Hypothesis testing: 

  • Linear Mixed Models, or Generalized (GLMMs) depending on which is more suitable for your data- with the lmer function in the lme4 package (Bates et al., 2015): I used this to test for the effects of population (collection site) on genetic diversity. Implementing an LMM accounts for the variability of the microsatellite loci by modeling locus as a random effect, and collection site as a fixed effect with allelic richness or expected heterozygosity as the response variables in separate models. Stepwise model simplification (Crawley, 2013) can be performed using likelihood ratio tests. Differences across collection sites can be compared, based on 95% CI, using Tukey’s post-hoc test in the ‘multcomp’ package (Hothorn et al., 2008). Read more about mixed models here. 

Analyses of Population Structure

I suggest using several programs to see how they compare. I used:

  • STRUCTURE -as it is one of the most popular programs-(Pritchard et al., 2000). I used Clumpak (Kopelman, Mayzel, Jakobsson, Rosenberg, & Mayrose, 2015) to analyze the Best K, and to visualize and produce plots based on all of the runs from STRUCTURE outputs. Please see data-wrangling section below for more details on how to get your data into STRUCTURE, and also into Clumpak.
  • FLOCK- great program in excel (Duchesne & Turgeon, 2012), to see which populations are genetic sources for other populations, as well as determining ‘K’ the number of genetic clusters within a given population or site (useful to compare to output ‘K’s from STRUCTURE
  • ‘adegenet’– to conduct Discriminant Analysis of Principal Components (DAPC) (Jombart, Devillard, & Balloux, 2010). There is a great tutorial here:
DAPC analysis on microsatellite data (eight loci) from eight populations of N. bruchi
Used the Adegenet package in R, and the Adegenet DAPC tutorial

Of course life is never easy.. especially when you have a MacOSX and for some reason the world revolves around PCs.

Here are some Data-wrangling tips for getting data into STRUCTURE and ClumpaK 

  • To get my data into the STRUCTURE format, I used the function ‘genind2structure’ that I found online here. Then in R, I used: genind2structure(inputdata, file=”outputdata.txt”, pops=TRUE).
  • Following this , you will need to:
    • DELETE THE GENALEX HEADERS
    • GET RID OF ANY ‘_’ IN THE TEXT FILE
    • GET RID OF LETTERS IN POP FILE, REPLACE WITH #S
    • DELETE IND AND POP HEADER
    • SAVE AS TXT FILE (TABS DELIMINATED)
    • RUN PERL SCRIPT Below..
    • since I have a MacOSX, I had to convert from DOS to UNIX with terminal program before loading in STRUCTURE by using similar code to this: while($_ = <>){s/\r\n|\n|\r/\n/g;print “$_\n”;}
      and you can find more info here .
    • DON’T TOUCH FILE AFTER THIS.. TA DA!
    • To get my files into the Clumpak web processor, I had to use a different zip-program (Zipfiles4PC) than what the MacOSx does, as for some reason Clumpak couldn’t process- Mac-zipped files.

Ok.. I think that is enough for now.. but really.. If I can emphasize one thing it is to go through the whole Poppr tutorial to get a handle of how to analyze data in R, and a feel for YOUR data!

 

 

Hot off the Press: Cool Temperature Performance of a Biological Control Agent of the Invasive Water Hyacinth

Wow.. I can’t believe May was my last post.. ugh! Ive been swamped with starting my new postdoc, moving into a new place, and writing an NSF-OCE grant! Anyhow, I am back and will try to be more regular again!

I am excited to announce a new article that is hot off the press! I coauthored this article with Dr. Angelica Reddy (first author) and Dr. Paul Pratt at the USDA, along with researchers from Argentina and Uruguay. You can read it here with free access for 50 days: Article in Biological Control.

This study was in conjunction with some of the work I did as a Delta Science Postdoctoral Fellow to investigate the mechanisms limiting the current biological control of invasive water hyacinth (Eichhornia crassipes) in the Sacramento-San Joaquin River Delta in northern California, USA (hereafter ‘Delta’). Classical biological control uses natural enemies (predators/herbivores, parasitoids and parasites) to control invasive populations of weeds, pests and disease vectors in the introduced range. 
Successful biological control agents can reduce pest populations below threshold levels that cause problems for humans and native species. Once established, biological control can provide a sustainable, long-lasting management option.

In a previous study, my coauthors and I conducted a one-year field survey 34 years after the initial releases of several biological control agents of water hyacinth in the Delta (Hopper et al. 2017). We found that two biological control agents, the herbivorous weevils, N. bruchi and N. eichhorniae (Coleoptera), were still present in the Delta and the associated tributaries. Although N. bruchi was broadly distributed throughout the Delta, N. eichhorniae was only found in the southernmost tributaries. Densities of N. bruchi during the warmer months in the Delta are comparable to densities in other regions with successful control of water hyacinth, but were not high enough year-round to reduce water hyacinth biomass and cover. Thus one idea to improve control is to re-introduce the more rare weevil, N. eichhorniae, in order to increase its abundance and distribution in the Delta and compliment the impacts of the existing weevil populations.

ChillyWeevilOne theory for the difference in the current abundance and distribution between these two weevil species is that the present biotype of N. eichhorniae in the Delta is less cold-tolerant than N. bruchi. Thus, the researchers from the USDA and myself were interested in determining whether a cold-temperature biotype of N. eichhorniae is present. If a cold-tolerant biotype exists, then the goal will be to screen it in the quarantine (host range tests and pathogen screening), access the necessary permits, and then release it into the Delta to improve the performance of the current population of N. eichhorniae and ultimately enhance the control of the invasive water hyacinth in the Delta. 

To achieve these goals, we (Reddy et al. 2019) examined the cool temperature performance and cold tolerance of four populations of the biological control agent, N. eichhorniae. These populations consisted of N. eichhorniae from: the Delta (California: USA), a population within the native range (Uruguay), and two temperate populations (Kubusi River, Stutterheim, South Africa and Jilliby, Australia). The geo-locations of these populations are noted as red markers in the green-highlighted regions on the map.

NE_NB_Map_for BLOGIn this study, we measured life history parameters of these weevil populations under temperatures occurring in the Delta during the cooler seasons (Fall and Winter). These life-history parameters included: Egg survivorship and development, juvenile (larval and pupal) survivorship and development, adult fecundity and adult longevity. I then used these parameters to construct stage-structured matrix models and calculate the intrinsic growth rates, doubling times, generation times and reproductive potential of each of these populations (as I have detailed in a previous blog and linked here).

In summary, Reddy et al. (2018) found that the population from Jilliby, Australia had the highest intrinsic rate of increase under conditions simulating Fall temperatures in the Delta due to the fact this population had the highest fecundity compared to all of the other populations (including the existing population residing in the Delta). Permission is thus being sought to release the Australian population into the Delta to improve the biological control of the invasive water hyacinth.

Please read the published paper for more details! 

And I will be back to update you with much more soon, especially on the results from my study on the population genetics of both of these weevils (N. bruchi and N. eichhorniae), using these same populations pictured above and many others! I finally submitted this manuscript for review.. so stay tuned!

 

New Post-doc Position at USC!

I am taking a small break from my blog tutorials on using microsatellite markers in population genetic studies to make an exciting announcement: I recently started a new 1-year Post-doc position at the University of Southern California in Dr. Dave Caron’s laboratory (more time pending funding from fellowships)!

USC-Dornsife-Cardinal-Black-on-White-RGBAlthough it is sad that my Delta Science fellowship is over, as it was a wonderful opportunity, I will still be working/writing hard to finish up my publications from this work and I will of course share these with all of you as they are published.phd011817s

In the mean time- I am moving back into marine study systems to examine the diversity and function of protists in the marine phytoplankton community!  Click here to check out the fascinating research in Dave Caron’s lab.  In addition to dabbling in several different ongoing projects in Dave’s lab- I am also very excited about starting up some of my own projects (pending funding) on the abundance, diversity and consequences of parasite-host interactions in the phytoplankton community.  As some of you might already know- I am an extreme parasite enthusiast, and only recently have researchers started to examine the potential abundance and importance of parasites in the marine phytoplankton community!

Recently, researchers in the Tara-Oceans Expedition found that parasitic interactions were the most abundant pattern in the global marine phytoplankton interactome (Lima-Mendez et al. 2015). Results from the V9-18S tag-sequence processing revealed parasite-host associations that included the copepod parasites: Blastodinium (Dinophyceae: Blastodiniaceae), Ellobiopsis (Marine Alveolate Group I: Ellobiopsidae), and Vampyrophrya (Ciliophora: Oligohymenophorea: Foettingeriida) and alveolate parasitoids of dinoflagellates and ciliates (Lima-Mendez et al. 2015). The alveolate parasitoids in particular were recognized for their top-down effects on zooplankton and microphytoplankton (Lima-Mendez et al. 2015).

 

Screenshot 2018-05-12 12.38.19
Figure from: Lima-Mendez G, Faust K, Henry N, et al. (2015) Ocean plankton. Determinants of community structure in the global plankton interactome. Science 348, 1262073.

Parasitoids are parasites that kill their host in order to complete their development (Lafferty and Kuris 2002) and increased abundance of alveolate parasitoids have been linked to declines of dinoflagellate blooms (Coats et al. 1996, Coats 1999, Chambouvet et al. 2008, Mazzillo 2011, Jephcott et al. 2016) and have been shown to regulate their dinoflagellate host populations in laboratory experiments (Noren 2000, Coats and Park 2002). The most researched alveolate parasitoids include several strains of Amoebophrya ceratii (Marine Alveolate Group II: Syndiniales) . These parasitoids have small flagellated infective stages that penetrate and multiply inside the dinoflagellate host cell, and produce numerous infective flagellates after killing and exiting the host (Cachon & Cachon 1987; Jephcott et al. 2016). For example A. ceratii can produce 60-400 new infective dinospores from its host in less than 48 hours (Chambouvet et al. 2008; Mazzillo 2011), and the generalist parasitoid, Parvilucifera sinerae, can produce 170 to > 6000 zoospores per sporangium, depending on the species and size of its host (Garces et al. 2013), with zoospore release within 72 hours of infecting a host (Alacid et al. 2015).

Below for your viewing pleasure is an example of these parasitoids- the life cycle diagram and life-cycle stages from Alacid et al. 2015, and Alacid et al. 2016 (respectively) of the generalist parasitoid Parvilucifera sinerae, in its host dinoflagellates.

So now of course the question you might have is: “why do we need to research these parasites/parasitoids further?” Well, we simply do not  know enough about these amazing parasite-host interactions, and most of our knowledge is currently limited to the photic zone of the ocean, and concentrated on just a few of these parasite species (there are many parasites out there just waiting to be discovered!). For those of you that don’t think ‘not knowing enough’ merits more work- my reply to this is that: mortality rates in the phytoplankton community have an incredible significance regarding the total primary production and biogeochemical processes in the ocean. However, how can we account for the mortality rates in the phytoplankton community and consequences for primary production if we are not accounting for a large % of contribution to mortality due to parasites that have not yet been characterized? And this folks.. is the reason why this research should be funded (aside from the obvious fact that parasites are absolutely fascinating, and the evolution and ecology of parasites can tell us a lot about related free-living species as well (that is another blog topic I will save for the future).

Of course my new Post-doc research in this field is still a bit tentative as it depends on gaining further funding- but in the mean time I am posting some lovely photos of parasites (Euduboscquella spp.) in tintinnid ciliate hosts (Eutintinnus spp.) that I have been finding from some local net tows (marine sampling nets that concentrates organisms of different size classes). So exciting- it is like a treasure hunt every time!

 

References (highly recommended reads also!)

Alacid E, Rene A, Garces E (2015) New insights into the parasitoid Parvilucifera sinerae life cycle: the development and kinetics of infection of a bloom-forming dinoflagellate host. Protist 166, 677-699.

Alacid, E., Park, M. G., Turon, M., Petrou, K. & Garces, E. (2016) A game of Russian roulette for a generalist dinoflagellate parasitoid: host susceptibility is the key to success. Front Microbiol 7, 769.

Cachon J, Cachon M (1987) Parasitic dinoflagellates. In: Biology of dinoflagellates, pp. 571-610. Blackwell, New York.

Coats DW (1999) Parasitic life styles of marine dinoflagellates. Journal of Eukaryotic Microbiology 46, 402-409.

Coats DW, Adam EJ, Gallegos CL, Hedrick S (1996) Parasitism of photosynthetic dinoflagellates in a shallow subestuary of Chesapeake Bay, USA. Aquatic Microbial Ecology 11, 1-9.

Coats DW, Park MG (2002) Parasitism of photosynthetic dinoflagellates by three strains of Amoebophrya (Dinophyta): Parasite survival, infectivity, generation time, and host specificity. Journal of Phycology 38, 520-528.

Chambouvet A, Morin P, Marie D, Guillou L (2008) Control of toxic marine dinoflagellate blooms by serial parasitic killers. Science 322, 1254-1257.

Garces E, Alacid E, Bravo I, Fraga S, Figueroa RI (2013) Parvilucifera sinerae (Alveolata, Myzozoa) is a generalist parasitoid of dinoflagellates. Protist 164, 245-260.

Jephcott TG, Alves-De-Souza C, Gleason FH, et al. (2016) Ecological impacts of parasitic chytrids, syndiniales and perkinsids on populations of marine photosynthetic dinoflagellates. Fungal Ecology 19, 47-58.

Lafferty KD, Kuris AM (2002) Trophic strategies, animal diversity and body size. Trends in Ecology & Evolution 17, 507-513.

Mazzillo FFM (2011) Novel insights on the dynamics and consequence of harmful algal blooms in the California Current System: from parasites as bloom control agents to human toxin exposure PhD dissertation, University of California, Santa Cruz.

Lima-Mendez G, Faust K, Henry N, et al. (2015) Ocean plankton. Determinants of community structure in the global plankton interactome. Science 348, 1262073.

Population Genetics Part II: Tips and Tricks, Multiplex PCR and Workflow of Microsatellites- the cheap way

No this blog is not a belated April fools joke… there really is a method to save thousands of dollars on microsatellite marker multiplex and genotyping! If you are just reading my blog for the first time, this is Part II following up on my last blog: How-to use microsatellites for population genetics, Part I: Study Design, DNA extraction, Microsatellite Marker Design/Outsourcing.

When I first set out to work with microsatellites, I was on a budget and I had never had experience with multiplex PCR, genotyping or fluorescent markers before- so there was definitely an uphill learning curve.

To start (assuming you already have your microsatellite markers- see previous blog for this)- the next step is to order your fluorescent markers to see how things work in multiplex PCR. FYI-Don’t order your Liz Size Standard until you are done troubleshooting and checking things on gels because it expires kinda quick! Plus it ships really fast (at least if you are in the USA). 

What are fluorescent markers you ask? If you look at the microsatellite genotyping peaks above, the different color peaks correspond to the different fluorescent tails that are ‘attached’ to the microsatellite primers. FAM= blue, PET = red, VIC =green, and NED = yellow. This way, you can see the different colors and know what markers they correspond to. The orange peaks are from the 600 Liz size standard which enables you to actually calibrate the size of the peaks. This is very easy if you use a program like Geneious as they have a microsatellite plugin.  I will detail more of the data handling in Part III of this blog series.

Why multiplex PCR? Simple.. it is faster (& cheaper if you troubleshoot things quick).PCR Reaction for Multiplex PCR of Microsatellite Markers

Many folks use multiplex genotyping, where you do singleplex PCR with all the separate microsatellite markers, and then you add for example 4 non-overlapping microsatellite marker amplified products (from your singleplex PCR) into a well together for downstream genotyping (more on that process later). This process saves money, since instead of genotyping a single marker for 1$.. you can genotype 4 markers for 1$! However- singleplex PCR takes forever if you have a lot of samples and markers!!!! If you have 400 individual DNA extracts, and each sample requires 10 markers of genotyping.. this means 4000 PCR REACTIONS- YIKES- that would result in carpal tunnel in a heartbeat!

Multiplex PCR in contrast allows you to add 2-4, (or more) microsatellite markers with fluorescent tails into the PCR mix, making sure that markers with the same fluorescent tails don’t overlap in size (ie- a FAM marker amplified product of 100-250 bp compared to another FAM marker amplified product of ~300-400 bp size range- should be fine to put together in a mixture). Whereas you don’t need to worry as much if they are similar size but have different fluorescent markers (such as FAM (blue) versus NED (yellow)). There are pull-up issues, and inhibition issues.. but that is why you will need to test everything out first anyhow before running your final assays.

As for myself, two papers were key in learning how to streamline microsatellite multiplex and genotyping: Blacket et al. 2012 and Culley et al. 2013. Both studies utilized four universal fluorescent tails (different ones in the different studies)- so that all you need to do is to add the non-fluorescent tail (just the ATCGs) of the corresponding fluorescent tail (the ATCGs + the FAM, VIC, NED or PET fluorescent marker) to your forward primer, and then use pig-tails on your reverse primers (such as GT, GTT, GTTT- depending on your reverse primer). Cullen et al. 2013 has an appendix which actually walks you through every step, including the reaction concentration of each Forward primer +tail, Reverse primer +pig-tail, and Fluorescent marker +tail in the final multiplex mix. I ended up using the four universal tails in Blacket et al. 2012, and then used Culley’s reaction mixes.

Screenshot 2018-04-01 12.55.20
Universal Tails used with PCR fluorophores, from Blacket, M.J., Robin, C., Good, R.T., Lee, S.F., Miller, A.D. 2012. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence. Molecular ecology resources 12, 456-463.

Below is a great pictorial image of how this works (from Blacket et al. 2012)

Screenshot 2018-04-01 12.54.34
Multiplex PCR with universal tails: Process from Blacket, M.J., Robin, C., Good, R.T., Lee, S.F., Miller, A.D. 2012. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence. Molecular ecology resources 12, 456-463.

In addition to reading these papers (and their supplementary material) thoroughly, I recommend the following: 

  1. Talk to as many people as you can before you start/ while you are getting started -you always learn fabulous tips and tricks as well as what not to do!
  2. Use the multiplex manager program -this will help you simulate what markers are compatible with each other based on the estimated product sizes, the melting temperatures (Tm) and the specific tails and flourophores you want to use. This will help you think about the different multiplex reactions that you can use.
  3.  Order the Qiagen Multiplex Plus Kit– this will streamline everything! I accidentally ordered the regular multiplex kit.. which is an older version and slower- so I had to stick with it once I got started. However- the Plus version enables you to use a faster PCR protocol! This kit is the same thing as their old ‘Type-it kit”, just better.
  4. Order a set of just the universal tails without the fluorophores attached first, in addition to the forward primers with the universal tails, and the reverse primers with the pig tails. This will let you try running all the multiplex reactions out and test them on a gel to make sure you have everything working before you waste your precious flourophores which are expensive. This is also a cheap thing- 4x $6 max.. 24$ to try out a bunch of stuff before spending the big money is well worth it! By the way  you will order all of your flourophores (reporter dyes) attached to the universal tail or the forward primer (the latter is a more expensive technique) from Thermo Fisher Scientific as they have patents on all of them except for FAM, which you can buy cheaper from Sigma or other companies (IDTdna, Elim BioPharm…etc)
  5. When you have everything working (bands are where you expect them to be, and no large gaps between bands which indicate that the msat markers are targeting multiple regions of the DNA sequence rather than one region)- Then order black, sterile micro centrifuge tubes (this link is just an example- but any sterile brand will work)- this will be what you make all your fluorescent primer ‘party’ mixes in, which will protect the fluorescence from degradation- preventing you from having to order more and save you money and time!
  6. Color code everything -from the tops of your individual fluorescent forward-reverse-fluorescent primer mixes (FRT: Forward-tail +Reverse-pigtail +Fluorophore-Tail) to your tubes with primer party mixes (all four FRT), to your  multiplex pcr reaction mixes and to your excel files for the pcr-plates and genotyping plates.
  7. Be organized when you pipette things onto your pcr-plate, and into your genotyping plate, see below image on how I organize pipetting into a pcr-plate, with the samples in the large tubes being moved in the order that I add things to the plate. I close the lid of each tube and move it to the upper tray after I add it to the plate so I don’t lose my place. I also use my tips from the box in order so that I can look at my tip-box as well to see where I should be. IMG_7752
  8. Talk to the genotyping facility about if they permit a ‘troubleshooting’ run (free-of-charge) so that you can test the amount of final pcr product to add into each genotyping well. I used 0.5 ul pcr product (that had amplified products of four markers) with 11 ul genotyping mix (Liz size standard in Hi-di formamide)

Random Tips/Interesting findings:

  • The Liz size-standard has strict “keep in dark and don’t-freeze’ instructions when it is shipped to you. Be sure to put it in the fridge at 4 degrees and NOT in the freezer! Unfortunately it comes in a styrofoam box w/ ice-packs with no-outside labels instructing to not freeze…and so the mailing department or your lab technician might accidentally put it in the freezer (speaking from personal experience…)- The company (Thermo Fisher Scientific) was very nice in shipping me a replacement because of this issue.  However… due to this occurrence I had the opportunity to test whether or not the Liz -size standard would still work when frozen for 6 hours, and when frozen for 24 hours….Results: The size standard still works great when frozen for 6 hours and for 24 hours ! With that said.. obviously don’t purposely test this, but if it is accidentally frozen- chances are you are still ok!
  • I also found that the Liz size-standard works great and is consistent for at least 2 months beyond its written expiry date…
    • For both of these tests I had unexpired and unfrozen Liz-size standard to compare these tests to. No p-value available.. just my experience 😉
  • As for the genotyping- I used less Liz Size Standard than recommended (and so did everyone that I talked to). My specific reaction mixes were the following: 0.5 ul PCR product + 0.5 ul Liz Size standard, and 10.5 ul Hi-Di Formamide per genotyping reaction well. I know a lot of folks that use 0.5 ul PCR product + 0.2 ul Liz-size standard+ 9.3 ul Hi-Di Formamide.. and they have great success as well. I tried the latter mix  and it worked, but because my pcr products had such high fluorescence (and I was over troubleshooting my primer fluorophore mix concentrations- I decided to  instead increase my size-standard so that I could better separate the noise from the signals). My genotyping mix and final primer ‘party’ mixes result in the initial genotype peaks image of this blog- so you can see what I mean by high sample peaks compared to the size-standard.
  • As for the Hi-Di Formamide- I noticed that this does not have good results when you leave it in the fridge overnight and try to use it the next day for a second genotyping plate. However- I had good results with using Hi-Di Formamide that underwent 1-3 freeze-thaw cycles. Thus my advice is that you never leave it at room temperature or in the fridge if you have extra, but also to avoid too many freeze-thaws.
  • Additionally- Im sure you will find this out- but NEVER freeze your pcr-products after the multiplex pcr with the flourescent markers, or after you add the liz-size standard and hi-di formamide. If you can’t get your samples to the genotyping facility right away, then be sure to do a quick denature (95 degrees for 5 min) post combining the pcr product w/ the Liz size standard and hi-di formamide and then just keep in the fridge (and in the dark!) until the next day or two.

References

Blacket, M.J., Robin, C., Good, R.T., Lee, S.F., Miller, A.D. 2012. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence. Molecular ecology resources 12, 456-463.

Culley, T.M., Stamper, T.I., Stokes, R.L., Brzyski, J.R., Hardiman, N.A., Klooster, M.R., Merritt, B.J. 2013. An efficient technique for primer development and application that integrates fluorescent labeling and multiplex PCR. Appl Plant Sci 1.