Category: Uncategorized

Adventures in Oaxaca 2021!

by Julie V Hopper, posted in Uncategorized

Hola! I just got back from an amazing 10 days in Oaxaca, Mexico – a treat to myself following a long year of zoom teaching @ USC and a break before the two-day intensive hackathon (that actually just happened) through the Scripps-Rady Ocean Plastic Challenge Program (I will detail that another post). Of course traveling has changed a bit since before the pandemic- but my friend, Traci, and I each were able to get fully vaccinated prior to traveling (Lucky us, I know!). We also wore masks when in public and stuck to mainly outdoor activities and dining. Overall we had a fantastic and safe time, complete w/ negative Covid19 tests upon re-entry to the States.

I highly recommend going to Oaxaca if you have the opportunity to do so, as we both felt like it was super underrated and such a gem of a place (the people are amazingly friendly and it was very safe also!). So here, I’ll try to discuss some of the logistics of our travels, including those pertinent in the Covid19 pandemic, as well as of course relaying some of our adventure stories! I am not making any income off of this post or from any of my suggestions- so rest assured !

First- I highly suggest flying in directly to the Oaxaca International Airport (OAX) if you can rather than through Mexico city. We left from LAX so had a quick and easy 4 hour flight via Volaris! If we had flown to Mexico City first- it would have been almost the same price and then we would have had to take about a 12 hour ride via bus to Oaxaca.. no thank you! Plus I’ve heard that Mexico city has crazy customs and lines right now because of Covid19.. .versus arriving to OAX was easy breezy (although coming back to the states is another story because of their confusing Covid19 checking process which I’ll explain in more detail @ the end of this post).

Day 1: Oaxaca de Juárez:Once we landed in Oaxaca and passed through customs (15 minutes), we went to the ATM, and used our US cards to get Mexican Pesos (MX) – which gets you a better exchange rate than going to a currency exchange spot.. especially at the airport. Then we grabbed a collectivo (shared taxi) via the airport transportation office for about 250 MX ($12 USD) each to get to the center of Oaxaca de Juárez- and to our first hotel where we stayed for two nights: Boca Del Monte (highly recommended B&B, although my favorite place was the AirBnB we stayed for our last two nights which was a bit cheaper and had a pool ! I’ll detail that one below). Once we were at the B&B, I went for a quick run around the city, we cleaned up and then we ate at my # 1 favorite restaurant of the whole entire trip: Las Quince Letras!!!! The author of the Oaxaca guide book, Cody Copeland, could not have more accurately described this restaurant or its infamous Chiles de agua a la vinagreta – where as soon as he took a bite he wanted to call his mom to rave about them! Basically this dish is chiles stuffed with pork and delicious seasoning. I typically don’t like pork and never order it… and so my friend got this- and once I tried a bite of it I forgot that I don’t eat pork and wished I had ordered some too (and wanted to call my mom and rave about them!). Anyhow- all of the food and drinks were amazing!

Day 2: Oaxaca de Juárez: The next day we had breakfast at our B&B and then walked to the Monte Alban Bus area (near the 20 Noviembre Mercado) and took a bus ($80 MX per person = $4 USD) to the Monte Alban Archeological site- where the Zapotecs once ruled! (~$100 MX per person including photographic permission = ~5$)- We had a blast walking around looking at the ruins and the amazing insects and plants all over the place!! Including the coolest locusts ever!!! That is.. an amazing time minus the blisters from new waterproof sandals we had bought prior to the trip because we thought it was going to rain every day which it never did.. whomp whomp… (lesson: never buy new shoes to wear on a trip….. try them out for a week before!).

Once we got back to the main city center, we stopped by the famous market: 20 de Noviembre- grabbed some lunch (Mole and Tlayudas) and then headed to the Bonito Juarez Market to grab some snacks such as nuts, Chapulines (grasshoppers), and ice cream (chocolate and coconut!).

Then in the evening-my friend Traci joined me on a walk to a Capoeira Class (Afro-Brazilian Martial Arts) !! whoop whoop- and yes of course I found a capoeira group to train with while on vacation because why would you not?! One of the capoeira groups in Oaxaca is led by Instructor Golias. I had a blast training w/ all of them- and even ran into my old friend- Nemo from UCA Capoeira in Berkeley! I also showed them a couple of my favorite moves passed down to me from some instructors and Mestres here in the USA- shout out to Mestre Xango, Instructor Macaco and Mestre Paulo Batutua! The gym was open-air- so that was awesome (re: being safe during Covid19) and after training we all headed to a famous chain known for their great Tlayudas: Tlayudas El Negro. Tlayudas in my opinion are basically like a mexican version of pizza-, and in Oaxaca they are complete w/ Oaxacan cheese-and fun toppings like chapulines (grasshoppers) and mole (depending on what you want)- yum! Although they are great – I think I burned myself out from Tlayudas – since I had 2 Tlayudas in one day! lol..

Day 3: Oaxaca de Juárez to Huatulco:The next morning we had an early breakfast, and then to a taxi to the Lineas Unidas transportation area in Oaxaca (Bustamante 601, OAX_RE_BENITO JUAREZ, Centro, 68000 Oaxaca de Juárez, Oax., Mexico) to make our way down to Huatulco on the coast! First we took a ~6 hour ride via Lineas Unidas to San Pedro Pochutla ($250 MX/person = $12.50 USD /person) and then from there we grabbed a different collectivo by Transportes Rápidos de Pochutla to Huatulco ($35 MX/person, 1 hour ride). Note- very important: Many people warned us about how windy the roads would be, so we each took one dramamine prior to taking the first collectivo. We both felt fine… so I guess I recommend taking a dramamine just to be cautious and to avoid getting nauseous. The ride was super pleasant and Lineas Unidas has a tv so I got to watch three movies in Spanish including ‘Finding Nemo’! Lineas Unidas was also super comfy and the driver was great!! Also, all of the collectivos make sure to stop for bathroom breaks about every 1.5 hours or so. Eventually, we got to our Airbnb in Huatulco.. had a couple of issues w/ trying to get into our place.. but soon succeeded and settled in, topping the evening off w/ a swim in the pool, a couple strong cocktails @ “Maz + Mezcal” and a delicious dinner at Terra Cotta in La Crucecita, Huatulco.

Day 4 Huatulco: This was probably one of the days I was most excited about! We set out to explore, relax and snorkel @ Playa La Entrega ! One of the themes of our trip also might have backfired a bit on us this day- where we pretty much decided to skip taking taxis when we thought we could walk somewhere …. So we looked at a map briefly, talked to some locals and set out on our walking way to the beach with all of our beach gear and luckily plenty of water (I brought a camelback full of water which I highly recommend using for adventure traveling!). The path we were aiming for was supposed to take about 30 min… but somehow we missed our turn-off (likely because we were distracted by a group of people right at the turn off that were trying to get us to take a taxi..lol)- and ended up walking on a hilly path for almost 2 hours in the hot sun.. yikes (should have taken that taxi!) Anyhow, eventually we found our way to the beach and managed to waive off all the people asking us if we would like to pay for umbrellas/tables/chairs etc.. and found a great natural shady spot on the beach under a tree- and next to a guy playing some good mexican music. One funny thing however is that by the time I got back from my snorkel, I came back to find that another family had set up a full blown stereo system just 10 ft away from the guy next to us- and so two different groups were playing music on two different sound systems.. lol. Funny enough.. they just each kept turning up their music louder and louder… like a music war! So eventually we relocated to another shady spot by some folks selling oysters. After we settled in our new spot – I took my friend (Traci) for her first snorkel! The snorkeling was great- and I just wore my goggles while she borrowed my gear (but there was also gear there you could rent if you don’t bring your own). We weren’t too worried about our stuff on the beach because we didn’t bring much of value w/ us-aka didn’t have our phones- hence why I don’t have photos of this day (below are photos of playa santa cruz) After snorkeling, reading and working up an appetite, we ate at one of the local beach restaurants.. which I’m not sure I recommend because we both got really really sick the next day… I think it was the fish tacos because they tasted off (and because the tap water in Huatulco is drinkable… so unlikely it was the water ).

Day 5 Huatulco-Ugh.. so this day was a bit rough… I woke up in the middle of the night/aka the early morning w/ montezuma’s revenge (TMI?) and my friend also felt sick so we had to forgo our originally planned 7am jungle bike ride, but were lucky enough to be able to reschedule it to 4pm later that day. So to try to recuperate we went back to bed and self medicated by alternating charcoal tablets and pepto bismal tablets (two key medications I recommend for everyone to pack in their international travel bags!!) Charcoal binds to what ever crap is making you sick, and then well it comes out of you later (magic..) and then Pepto Bismol treats your symptoms and actually the problem sometimes- aka it actually kills the bacteria!! We took it easy in the morning at our favorite local cafe in Huatulco: Cafe Huatulco! and watched the birds, read and ate some very very plain toast (good for when you are sick!).

By 2pm we were feeling better so floated in the pool a bit, and then got ready for our jungle bike ride with our guide Antonio through the ‘Descubre Huatulco’ company. I highly recommend them! The guides are bilingual and know the Parque Nacional Huatulco pretty well. Plus we were advised by many people to not try to explore the park by ourselves. Anyhow Antonio was great, he picked us up w/ bikes and helmets in hand and then drove us to the park entrance where we explored the trails leading through the jungle to the beach and then to a cactus area and a bird watching area! After the bike excursion, he even took us to see a nearby lighthouse with some cool views! By the end of the night we almost felt almost back to ourselves so were able to eat some great fusion food at the Mercader in Santa Cruz Huatulco.

Day 6 Huatulco to San José del Pacifico: The next day we were about 80% normal and feeling pretty good for our ~3.5 hour ride up to the mountains in Sierra Sul to San José del Pacifico. So of course we decided to walk w/ our luggage about 30 min to an awesome brunch spot overlooking a harbor and the ocean Cafe Juánita prior to catching a ride to the mountains.

Prior to the long drive, we took some pre-emptive pepto bismol in the morning, followed by some dramamine as soon as we got on our van/collectivo. For this part of the journey, we ended up catching a ride w/ the company ‘Huatulco Dos Mil‘, (Transportes Huatulco 2000) but you can also use Expresos Colombo as well. Both of these companies go from Huatulco to Oaxaca and stop at San José del Pacifico,on the way (~$240 MX/person). We safely arrived in San José del Pacifico, just in time to get a tour of the town, check out the place we were staying @: La Puesta del Sol (highly recommended!) and then eat @ the La Puesta del Sol restaurant!

Day 7- San José del Pacifico:

The next morning, we woke up feeling 100% back to ourselves, and so of course planned an epic hike through the mountains (Traci has the AllTrails App... and so she found this trail!). I highly recommend the trail we did, but just a note we didn’t get all the way to the vista point because there were some guys and trucks on the road about mid-way along the trail and they didn’t want us to continue hiking. There was definitely something weird going on because when we got back to town, other people were asking us about the guys and the trucks and it definitely seemed like they weren’t supposed to be stopping us from our hike… but oh well- we didn’t want to cause any problems so of course we just complied when they told us to turn back around – whomp whomp! It also wasn’t that big of a deal because we still were able to hike for about an hour prior to being stopped, so tons of cool flora and fauna, and then we followed this hike up with lunch at the Café Express (huevos rancheros, complete w/ a mocha and a Oaxacan artesian chocolate bar!)

After lunch we walked around town and into the mountains again (but on a side road rather than finding a nature trail) and got to see some really cool artwork and vistas. For dinner- we wrapped up the day with some amazing Italian food @ La Taberna De Los Duendes (some of the best I’ve had.. and I’m part Sicilian!). The owner was super nice and the ambience had chic-hippy vibe.

Day 8- San José del Pacifico to Oaxaca de Juárez: The next day, after a quick jog through town (dodging some territorial dogs along the way!), some Oaxacan coffee, huevos rancheros and fruit- we started walking toward town to grab a van through Lineas Unidas to head to Oaxaca de Juárez. But- the van grabbed us before we could grab them!! We seriously were walking towards the bus area at the Café Express, and were about 10 minutes away (slowly dragging our luggage ), when a Lineas Unidas Van saw us and stopped right in the middle of the road in order to help us get on board w/ our luggage- what great luck?!! It actually happened to be the earlier van that we weren’t planning on catching, so it was great to get to Oaxaca de Juárez a bit earlier than initially planned- especially since we had to take Covid19 tests on this day! We were also lucky in the fact that once we arrived to Oaxaca de Juárez, we happened to be just about a 5 min walk from Salud Digna where we had planned to get tested for Covid19 via their antigen tests to meet the requirements of testing negative for covid19 within 3 days of re-entry into the United States (even if you are vaccinated!). You can take the antigen or the PCR test, and we chose the antigen test because it was a lot cheaper (260 MX versus 950 MX for the PCR test)! We didn’t have to have appointments as US citizens, but we did have to bring our passports and waited for about 15 min to be seen (not bad!). I think though if I had to redo it, I might have paid more for the PCR test because the antigen test was super uncomfortable- basically a really really long probe that they stick up your nose and practically all the way up into your brain (jk.. but felt that way…). Anyhow -it was good to get all of it out of the way and we received our results w/in 2 hours and were both negative- so hooray! After we got tested, we headed to our new AirBnB.. which was maybe my favorite place we stayed during the whole trip aside from our cabin in San Jose Del Pacifico. I cannot recommend this AirBnB enough! José, the host, was so kind and pro-active, and took time to talk to us about our plans and with great advice for our last two days in Oaxaca. After settling in, we headed to dinner at the highly recommended Casa Oaxaca El Restaurante. The views and the food were amazing! My favorite were the stuffed squash blossoms- which inspired me to cook with my squash blossoms @ home!

Day 9- Oaxaca de Juárez:The next morning we woke up to an amazing breakfast at our AirBnB and great conversation w/ the other guests and José (the AirBnB host). I had a goal to seek out some artesanal oaxacan chocolate bars, so we got some great direction to visit Mama Pacha’s chocolate shop (owned by a chocolate-making goddess and mother, her son was even there when I visited). I bought a ton of her chocolate-it was so good!). José also advised us to explore the Barrio de Xochimilco– one of the oldest neighborhoods in Oaxaca de Juárez and well known for its traditional textiles. If you are lucky enough to get an invitation like we were- you can actually watch the experts weaving the textiles on looms. After watching and meeting the artists.. how can you not want to buy some of the beautiful pieces? -So- yes, of course we bought some small souvenirs! (We couldn’t fit the large pieces in our carry-on-luggage.. womp womp! I also loved this neighborhood because there was so much beautiful street artwork! I mean in general, the street art is frikin incredible in Oaxaca, and so thus I’ve put more photos at the end of this post, in addition to the art we saw in Barrio de Xochimilco. After we finished exploring the chocolate and textiles around town, we headed to an art collective ‘ARIPO (Instituto de Artesanias Oaxaqueños)‘ which had some amazing pieces ranging across 3D sculptures, ceramics, paintings, textiles and clothing. There was even an amazing artistic replica of the Loteria game, that we saw being put together by the artist himself in real time (see photo below)!

By the time it was 1:30 pm- we were starving from all of the walking we did! So we got lucky and found a great lunch spot: La Popular w/ some amazing tacos and a guest star- a hairless dog! I’ve never seen one in real life before- and she was adorable!! After lunch, we managed to find another awesome chocolate spot (specializing in truffles) and then headed back to our AirBnB where I immediately plunged into the amazing pool for an afternoon swim. After cleaning up, we walked around town for a bit, and caught a massive local political convention/campaign -complete w/ a concert! For dinner- we managed to finally get a table at Los Danzantes……. a gourmet five star restaurant that did not disappoint! I highly recommend their mole sampler!

Day 10 – Oaxaca de Juárez to Los Angeles, CA: Womp womp.. this was our last morning in Oaxaca, and we had a flight at 10am out of OAX. So we didn’t even have time to get coffee or breakfast before heading to the airport. We ended up being glad we left so early as we got to the airport > 2hours ahead of time. It was definitely necessary due to all of the Covid19 related policies and checks. It was actually a huge mess, and even though we both had already checked-in online, had our vaccination information, our passports, our covid19 negative test results, and the online covid19 questionnaire from Volaris airlines— we still had to double back to a crazy swarm of people all trying to take photos of these tiny QR codes (all on the same bulletin in the same spot) in order to answer yet another online questionnaire. We then had to show we had completed this particular questionnaire to an agent at the Volaris desk… which meant we still had to wait in the line w/ all of the other confused passengers. Then we had to go through an immigration line, and a security line…so needless to say we didn’t get to our gate until 9:15 AM! Luckily they had a coffee spot, and so that helped us settle in and wait for our flight to take off. Lesson learned: give yourself lots of time for these international flights because the Covid19 related procedures definitely slow things down!

So- all in all -an amazing trip – full of adventures, beautiful art, friendly and amazing people, coffee, cocktails and food that you will keep coming back for! Get yourself to Oaxaca!

Holiday Updates + Progress on Fighting Marine Plastic

by Julie V Hopper, posted in Uncategorized

Happy Holidays! If you are reading this congratulations- you’ve almost made it through 2020! (knock on wood! keep on going, be healthy and try to be happy… you got 29 days left!). After this zoommester-I feel like myself and all of my students should win an award at this point, and looks like a lot of people feel that way based on this plaque I just found on Etsy:

In addition to surviving the 2020 zoommester- I recently blogged about how the last paper from my Delta Science Postdoctoral Research Fellowship was accepted to the Journal of Biological Control. Now it is hot off the press (Hopper et al. 2021) in all of its glory! You can access this free link and download the free scientific article for about 45 more days.. so have at it (and share it if you like it). I also want to thank California Sea Grant and the Delta Science Program for helping to fund this project and give a shout out to all of my coauthors from USDA and UC Davis. This includes my long-time undergraduate mentee (Somanette Rivas) who worked for me both when I was a grad student and when I was a postdoc, and is now at the USDA as a Research Technician! (I’m a proud mama bear… what can I say..)

Graphical Abstract from Hopper et al. 2021 (Biological Control)

If you want to learn even more about this postdoctoral research, check out my earlier blogs from 2016-2018.

Beach cleanup I organized in Oct. 2020: Photo by Maurice Roper

I also have some really exciting news related to my last blog post, where I discussed the huge problem with marine plastics and potential solutions, including beach cleanups. I promise I will soon post a Part II that details how we can all decrease our waste (including single use plastics) and how we can be more sustainable, even in these weird pandemic times. However, for now I’m excited to share that I will officially be part of the 2021 Scripps-Rady Ocean Plastic Pollution Challenge! I will be on the data mapping team working with other researchers, students and activists to help solve our ocean plastic pollution problem. This program is a 6-month program focused on identifying effective, evidence-based approaches to curb the flow of plastic into the ocean, with a specific focus on marine cultural preservation and marine conservation areas along California’s coast. 

Aside from these two bits of news, I just plan on using my teaching break to chip away at all of my Fusarium spore-suspension samples from my research in collaboration with Tom Dudley’s group at UCSB on an invasive fungus-beetle team that invaded Southern California in 2004, as well as taking a road trip to Sedona w/ my hubby, mom and pitbull rescue pup- Yesenia (lots of hiking planned), and then spending some time with my in-laws! Stay heathy and happy y’all!

The Invasive Polyphagous Shothole Borer Beetle, Its Fungal Symbionts and the Fastest, Most Intense Summer Experiment Ever…

by Julie V Hopper, posted in Uncategorized

One thing I’ve learned while in a teaching focused position: is that you have to collaborate with others, piecemeal together workspaces, and work fast and hard if you want to do research during the academic holidays. The fast part means designing experiments that will start and end within a reasonable time frame.. say- how about less than 2 weeks?

So I tried this approach.. and just wrapped up an intense 11 day experiment (12 days if you include clean-up time) that included three car trips back and forth between USC and UCSB, countless hours spent in a laminar flow hood, and the trips speckled with adventures and good times w/ friends and my collaborators.

In a nutshell, I collaborated with the UCSB Rivr Lab group to investigate the growth of the pathogenic invasive fungus, Fusarium euwallaceae, on different host plants- including two native willow species, two native cottonwood species and three avocado cultivars (Zutano, Bacon and Hass). (See funding and acknowledgements below this post!)

This invasive fungus causes Fusarium dieback disease on a lot of native plants in California, and is known to infect avocados (luckily there are management practices available to control the spread and pathogenicity in avocado orchards).

Our exploration stage of the study back in June, collecting beetles and traveling around to the different field sites w/ the field expert- Shelley Bennett and my trusty science-savy dog- Yesenia! (Pit bulls do science too!)

This fungus is spread by the invasive polyphagous shothole borer beetle which was first discovered in Southern California in 2003, and is thought to have been accidentally introduced via products and/or shipping materials from SouthEast Asia. By 2010, this beetle was recognized as being the main cause of death of several street trees in Long Beach and by 2012 it was found in a residential backyard avocado tree. If you like your guacamole.. you can understand why the alarm bells started to ring!! This beetle has now been found in almost all Southern California counties and has been found as far north as Santa Cruz county. Furthermore, this beetle has invaded many other countries all around the world.

 Invasive Polyphagous shothole borer beetle Euwallacea aff. fornicatus
Photo credit: Jiri Hulcr, Ph.D.)
Entry holes from the shothole borer beetles – 
Photo credit: Monica Dimson, University of California Cooperative Extension

The spread of this beetle is problematic because it carries several mutualistic fungi (including Fusarium euwallaceae) that help it digest the tree tissues as it bores into its host trees. As the beetle bores into the tree and chews away- the spores of these fungi get released into the tree (beyond the protective bark) and can cause pathological symptoms in the tree, including ultimate death. Meanwhile the little beetles keep munching away and reproducing as much as they can.. and their offspring continue the cycle along with their mutualistic fungi …

Hence for management purposes- if we want to understand what tree species will be most impacted by this invasive beetle and its fungal symbionts- we need to know: 1) what species of trees are most likely to attract and be attacked by the beetle- and of these, which ones result in the highest survival, growth and reproduction of the beetle (and why)? and 2) what species of trees promote or prevent the growth of the different fungal symbionts (and why?), with a focus on Fusarium euwallaceae, which is particularly pathogenic. These questions are intertwined because the fungus needs the beetle to drill the holes for it, and to introduce the spores into the tree tissues; and the beetle needs the fungus to be able to digest the tree tissue to obtain nutrients and to keep growing, reproducing, etc.

There are many different research groups in California (including Akif Eskalen’s group at UC Davis, Shannon Lynch at UC Santa Cruz and Richard Stouthamer’s group at UC Riverside) and all around the world working on answering these questions. Our work this summer was focused on a small part of the larger picture, specifically investigating the growth of Fusarium euwallaceae on native California trees that grow in riparian habitats, as well as on several avocado cultivars. In regards to the native riparian plants, a graduate student at UCSB -Shelley Bennett, sampled from different parts of the Santa Clara River so that we could examine how proximity to the river impact the moisture, density and nutrients in the tree tissues and whether this impacts the fungal growth rate on these different host tree species.

Even though this was an 11 day experiment- it actually took a lot of prep work, a lot of troubleshooting and a lot of reconfiguring and pilot studies prior to execution of the actual experiment (what experiment doesn’t?!). Also complications due to Covid19 definitely put a huge slowdown on the research with massive supply and shipping delays and errors in June through July- which then meant that we no longer had time to cultivate the fungus from the beetles themselves (which requires cultivating, isolating and then propagating .. and ensuring that we had the right Fusarium isolates)- so we were very fortunate enough to receive fungal isolates from Richard Stouthamer’s lab at UCR just in time to start and finish our experiment before I started teaching classes (next Monday- eek!).

Fusarium euwallaceae isolate that we received graciously from Richard Stouthamer’s laboratory at UC Riverside.. which I propagated like woah to prepare for our experiment.

In the end we settled on two experimental methods:

1) using a saw to create saw dust (very inefficient I know) from over 140 branches of different host plants in order to produce unique host-plant autoclaved agar-based solutions that we could pour into petri dishes (along w/ just PDA agar replicates as a positive control); and 2) sawing cross-sections of the different branches, and bleaching, and rinsing these thin cross section pieces before putting into petri dishes (not-autoclaved). Then we put a 6 mm agar plug of Fusarium euwallaceae on the center of each of these samples (or a 6 mm agar plug without the fungus as a negative control).

The idea was to test the impacts of nutrient differences among these different host tree species (present in the agar-based solutions and in the cross-section pieces), and chemical defenses (which would ideally still be present in the cross-section pieces) in respect to the growth of the invasive fungus, Fusarium euwallaceae. Originally we did not want to use cross-sections, we wanted to create longitudinal slices.. but turns out that it is very difficult to do that safely and to get it to fit into petri dishes. So hence we had to change our experimental design on the spot. During the set up of this experiment, I also found out that I get very excited about the idea of consolidating nature from large sizes to small consolidated items that fit into a petridish.. is that weird?

  • In the beginning.. there was wood.. a lot of wood…
  • Then there was lots of sawing- extra props to Zoe Wood and Shelley Bennett for being super stars on the saws!
  • My role was to weigh the saw-dust and make agar w/ each unique treatment
  • Pouring of the autoclaved saw-dust agar mixes into petridishes
  • Method 2: Chopping up the wood for cross-sections, as well as samples for moisture, density and nutrient analyses
  • Ah.. so satisfying.. everybody is tidy in their little dishes..

Anyhow- we just took this experiment down this past weekend- and now we have probably about 1000 images to process with imageJ (measuring the area of fungal growth from days 0-11) and over 140 spore suspensions to count with a hemocytometer (they are safe and sound in the freezer at this point .. I hope they are safe anyhow…). Wish us luck!

Since everything is now in a ‘do it later’ state- this experiment model gives me time now to finish prepping for my two classes (Env. Studies and Ecology) that start on Monday (eek!).. all online, with guided outdoor-distanced projects for my Ecology lab section- some as distanced as Taiwan!. I had to make an insect collection care package for that student.. Anyhow- stay tuned for my ideas on fun, distanced/remote hands-on learning for Ecology and Env Studies!

Funding and Acknowledgements

This work was funded by the Thelma Hansen Fund of UC ANR and additionally supported by Annemiek Schilder, the director of the Hansen Agric Research & Education Center in Santa Paula, CA. We want thank Dr. Richard Stouthamer (UC Riverside), his postdoc: Valeh Ebrahimi and lab technician Taha Farooqi for providing beetles and fungus during the initial phase of this research, as well as USC for use of their unused teaching lab space to conduct pilot studies, Ryoko Oono (UCSB) for her advice and use of her lab’s laminar flow hood, Carla D’Antonio for use of her lab space, and Dr. Akif Eskalen (UC Davis) and Annemiek Schilder for their advice. Huge shout out to Dr. Tom Dudley and Dr. Adam Lambert for this research opportunity and to Zoe Wood and Shelley Bennett for being awesome rockstar teammates on all of the field and lab work (Adam helped in the field too!)! I think their thumbs are still hurting from all of the sawing… lol (funny but maybe not funny).

Spring Bloom Sampling in California

by Julie V Hopper, posted in Uncategorized

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.

 

Me on the Santa Monica Pier sampling water
Look how brown the filter is! So much biomass from all the phytoplankton!
Our filter rig to filter the water from the pier
Our set up in the back of the aquarium

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.

 

Guinardia sp. with Cryothecomonas sp. parasitoid
Dead Guinardia sp with Cryothecomonas sp. parasitoids
Tintinnid (Eutintinnus sp.) with parasitoids (Euduboscquella sp.)
Cochlodinium sp. (dinoflagellate)
Akashiwo sanguinea (Dinoflagellate photo by Jennifer Beatty)

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

by Julie V Hopper, posted in Outreach and Education, Uncategorized

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’

Photo: 2017 @HakaiInstitute Bioblitz Team
Ectoparasite demonstration at my workshop for IEA-LABs on Feb.16.19

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.

California Horn Snails, Photo: http://nathistoc.bio.uci.edu/Molluscs/Cerithidea.htm
Typical lifecycle of parasitic trematodes that use the California Horn Snail as their first intermediate host. Figure from: Torchin et al. 2005, Biological Invasions “Differential parasitism of native and introduced snails: replacement”of a parasite fauna

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

by Julie V Hopper, posted in Uncategorized

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

screenshot 2019-01-12 20.58.25

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

by Julie V Hopper, posted in Uncategorized

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

by Julie V Hopper, posted in Uncategorized

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!

by Julie V Hopper, posted in Uncategorized

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.

Alacid et al. 2015: Life Cycle of the parasitoid, Parasitoid Parvilucifera sinerae
Alacid et al. 2016: Life cycle stages of Parvilucifera sinerae, a generalist parasitoid, in several of its hosts

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!

 

Tomont of Euduboscquella parasitoid in Eutintinnus ciliate host
Sporocytes of Euduboscquella in Eutintinnus ciliate host
Sporocytes of Euduboscquella in tintinnid ciliate host, Eutintinnus host
sporocytes of Euduboscquella in Eutintinnus host

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

by Julie V Hopper, posted in Uncategorized

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.