A Year in Review - What Worked & What To Change Next Year

We’re coming up on the end of the year and the beginning of the year-in-reflection season in blogs across the internet. In the spirit of this, and because I’m currently on a plane with some time to fill, here is a quick overview of my whirlwind of a year, specifically focused on the results thus-far of research project. Some of this information may be helpful for other field ornithologists.

Total blood samples from SF birds – 78

Birds by location:

            Eastshore State Park - 2

Golden Gate Park – 27

Lake Merced – 28

            Lobos Dunes – 5

            McLaren Park – 6

            San Francisco State University – 3

San Francisco Zoo – 7

Total number of birds infected – 7

Infected birds by location*:

            Eastshore State Park – 2

            Golden Gate Park – 2

            Lake Merced – 1

            Lobos Dunes – 1

            San Francisco Zoo – 1

Total usable** audio recordings – 37

Recordings by location:

            Golden Gate Park – 19

            Lake Merced – 13

            McLaren Park – 2

            San Francisco State University – 2

            San Francisco Zoo – 1

Things That Worked – Field

• Target netting white-crowned sparrows is easy – they respond well to playbacks and for the most part can be caught in less than 5 minutes
• Only one net that is needed at a time – more than that gets too difficult to manage because more than likely, by the time I had a second net up I had caught a bird in the first one
• Customized data sheets – data sheets tailored to my project kept me on task and thorough with a bird in the hand
• A backpack vs. an over-the-shoulder bag – having my hands free and weight equally distributed is fantastic
• A well-organized banding/bleeding kit – a place for everything and everything in its place – makes setting up and breaking down so much easier
• Labeling GPS points of banded birds with their color code – this helped when I returned to record audio – I knew which color combinations to look for in which area
• Immediate changing of audio files with the bird’s color combination – it was a pain to cycle through the whole alphabet on the simple interface of the recorder but was worth it when sorting the songs on the computer later on – a pro for the recorder though was that it logged the date and time of each recording

Things That Worked – Lab

• Having a well-organized and detailed lab book – this is a must! Even though it gets tedious to write the same ingredient list for PCR over and over it is so important in case something goes wrong (or right!) and can be referenced in detail at a later date
• Concise but clear labeling – there is very little room on strip tubes so having a short alias for a sample is essential
• Lab aliases and samples are organized in a table in my lab notebook
• Labeling each strip tube, even though they’re attached, is important in case my mind wanders while pipetting (it inevitably does)
• Testing protocol with 6 samples at first then scaling up – there are 8 tubes on one strip – this makes room for one positive control, one negative control, and 6 samples – once I ran the whole nested PCR cycle and gel through and proved the protocol works, then scale up 
• Setting aside time for lab work and breaking step up between days – it always took longer than I thought and I would be worn OUT after a long day in our window-less lab – running the nested PCR one day and then the gel the next helped 
• Having great lab mates willing to share skills and bounce ideas off! Dave, Brad, Allison, and Brett were such a great help this year

Modifications for the 2015 Field Season

• *Incorporate more field sites, emphasize those that worked, and phase out ones that didn’t
• When I initially began this project Golden Gate Park, Lake Merced, and McLaren Park were priority sites. Eastshore State Park, the San Fran Francisco Zoo, and SFSU were added on opportunistically – these ended up being fantastic sites and had infected birds! This year I’ll place a greater emphasis on this area 
• McLaren Park was tough to work in – the ground was hard, there weren’t many great netting locations and there were many off-leash dogs – I may come here to augment my sample size if it’s low but it will be a lower priority this year
• Take many more notes during audio recording – It would be great to have a field assistant or a bribed friend for this. One person will record audio while the other takes notes on the bird’s behavior/current environmental conditions. 
• **Many birds had compact, high-density territories and some of the audio recordings had to be thrown out because it was impossible to tell which singing bird was the target bird.
• Some birds flew to alternate perches during the recording – it was difficult to distinguish between a background bird and one that had simply flown further away 
• A 6-meter net! Our lab has many 12m nets but you really don’t realize how long they are until you’re trying to stretch one out in a small pathway between two shrubs in Golden Gate Park – part of the budget for my crowdfunding campaign will go towards the purchase of a couple 6m nets and lightweight poles

Birds!

So this post isn’t left without pictures:

I visited the San Francisco Zoo the other day to try seed trapping white-crowned sparrows. Since it was warm this summer we thought maybe there would be an increase in infection prevalence but it’s difficult to target net outside the breeding season because the birds aren’t territorial. I was told it’s best to pre-bait the area to get the birds used to coming to that particular spot. Even better is to pre-bait and leave the trap out to get them acclimated. Unfortunately my field sites are in public parks so leaving the trap out is impossible.

However, the site at the Zoo is in a back area that is not accessible to the general public. I didn’t pre-bait the site but I was able to leave the trap unattended so the birds could explore. I’ve never tried trapping before so I also brought along a net just in case. The trap ended up being a bust but I did catch two hatch-year white-crowned sparrows with target netting. They’re likely the offspring of a bird (or birds) I caught earlier this spring!

I wasn’t able to determine their sex but if they’re still around in the spring I might recapture them in breeding condition. So without further ado, let me introduce two new study birds: 

My Campaign Has Fledged [Successfully!]

My campaign on Instrumentl, a crowdfunding website specific to women in science, is officially fully funded!

You can still check out my project overview and bio here:
http://www.instrumentl.com/campaigns/denaemmerson

And you can watch my video below, which provides an overview of my project and what your funds will go towards:

A HUGE thank you to those who donated over the three-week campaign! You are amazing and generous and l am forever grateful!

Aaron Borovoy

Gauri Manglik

Tom & Karen Emmerson

Jeff Bell

David Emmerson

Sue Engle

Elin Pierce, PhD

Robin Keith

Sunni Robertson

Martha & Ben Weaver

Samantha Young

Hannah McDonald

Emily Moffitt

Norma Emmerson

Wanda Sowa

Laura Emmerson

Ylenia

Lupe McDonald

Krista Parry

John Richards

Mark Jenkins

Byron Ryono

Wanda Sowa - again!

Todd and Sharon Dunn

Elizabeth Davis

Helen Cheng

Hayley Farr

Alexis Weinnig

Carol Clark 

Luis Amaya

Victoria Kentner

Christopher Quock

As well as several anonymous donors - thank you!!

Western Bird Banding Association 2014 Meeting

This past weekend Allison Nelson (remember her thrush project??) and I road tripped up to Humboldt, CA to share our research at the 2014 Meeting of the Western Bird Banding Association (WBBA.)

The conference spanned from September 18-21 and saying it was bird-heavy is an understatement! We were there with representatives from all of the major banding stations up and down the West Coast and even inland into the Southern US a bit (a couple Arizona and New Mexico folks were in attendance.)

Both HBBO and WBBA use a bird in their logo that should be familiar to you at this point!

Most of the conference took place at the Humboldt Bay Bird Observatory (HBBO) on the Humboldt Bay Wildlife Refuge:

Friday

After a nice meet-and-greet dinner Thursday night the conference kicked off bright and early with a morning of banding at HBBO. Even though most of us in attendance were experienced bird banders, it's always interesting to watch how other stations operate. For example, at the station I work at (Coyote Creek) we score fat on a 0-7 scale, 0 being none and 7 being 'very excessive.' HBBO uses a 'none-full' scale - the bird either had no fat, half fat, or full fat.

Even though we have different methodologies we share many of the same species. Below are two yellow warblers - one from HBBO (top) and one from Coyote Creek (bottom):

There's only so many people that can pack into a banding station so some of us departed for a guided walk of the local dunes. The Lanphere Dunes on the Humboldt Bay Wildlife Refuge contain some of the most eerie and beautiful habitat I've ever seen.

Animal tracks were very easy to spot in the wet sand. This skunk left his footprints behind:

And of course:

This was a bird conference after all!

In the afternoon there were a series of workshops, including one led by Allison on bleeding techniques and geolocators.

One helpful workshop was on bird first aid. It is very rare but if we do come across an injured bird – either as a result of netting or if it had a previous injury – it is our responsibility as banders and conservationists to do all that is in our power to help that bird. In this workshop we learned how to splint a broken leg, how to calm a stressed bird, and how to tend to a dislocated limb. Obviously we didn’t have any birds to work with so we splinted broken reeds for practice.

Friday culminated with a social and a dinner followed by a bluegrass concert featuring Arcata locals, the Compost Mountain Boys:

Saturday

Saturday was the presentation and poster day. The day was packed with presentations on everything from nesting habits, molt patterns, and aging techniques of birds from Canada to South America.

Allison gave a great update on her hermit thrush geolocator project. Two years ago she put geolocators on birds in both the South and North Bay. Last year, some of those birds were recaptured and their geolocators removed. Turns out the South Bay birds fly further north and travel faster than the hermit thrushes in the North Bay. It’s right around the time of year that we will be seeing the hermit thrushes migrate back in and we’re hoping to round up the remaining geolocators.

On Saturday evening there was a social and poster session. Only three of us had posters so we certainly got a bit of foot traffic! Though my project is still in the early stage and there aren’t too many conclusions to draw just yet, I had a great time discussing my methodology and initial data analysis.

C.J. Ralph, head of the HBBO and chair of the conference, stopped by for an engaging explanation:

Sunday

Sunday morning Allison went on a birding tour in the Humboldt County mountains while I spent some time with our host's chickens:

Before hitting the road for our 5 hour drive home we stopped by downtown Arcata to check out the North Country Fair:

Arcata is just quirky like that.

All in all it was a successful and informative trip. Allison was even elected and unanimously approved to the HBBO board! Next year's WBBA meeting will be hosted by the Vancouver Avian Research Centre so we'll be heading north of the border! 

Avian Malaria (& Relatives) Workshop - Part 3 - Indentifying Blood Parasites

This post brings me to my third and concluding update on the Third Annual International Workshop on Malaria and Related Haemosporidian Parasites of Wildlife (whew - that title is epic!) [Here's Part 1 and Part 2]

The first two days of the four day workshop focused on blood smears and identifying parasites by their physical characteristics. The second two days focused on vectors and genetic techniques.

Vectors

Malaria, as we all know, is a vector-borne disease transmitted by mosquitoes. Insect vectors are the sites of sexual reproduction for haemosporidian parasites; without these vectors the parasites would not be able to complete their life cycle.

Therefore, as avian parasitologists, it is important for us to know how to identify vectors that could potentially spreading infectious agents to our birds. It's not just mosquitoes we need to be concerned about either. Each of the three parasite genera, Plasmodium, Haemoproteus, and Leucocytozoon, are transmitted by different types of insects.

The following vector images are from Avian Malaria Parasites and Other Haemosporidia by Gediminas Valkiūnas. 

Plasmodium is transmitted by mosquitoes:

Haemoproteus is transmitted by biting midges and hippoboscid flies:

And Leucocytozoon is transmitted by black flies:

Each vector has its own unique set of habitat requirements. Combined with the habitat requirements of the bird hosts and the parasites, understanding these insect-vectored diseases quickly becomes quite complex.

For the purposes of this workshop we focused on mosquito vectors. We learned how to trap, identify, and dissect mosquitoes. This last skill in particular was difficult to master. Mosquitoes seem pretty big when you're swatting them on your arm but when you're actually trying to keep parts of them intact as you sever them with a small knife, believe me they seem very small.

Mosquito Traps

Photo by Francisco Ferreira

This trap is called a gravid trap. It attracts gravid ("pregnant") female mosquitoes looking for somewhere to lay their eggs. In the bottom bucket there is a putrid liquid called (very technically) "smelly water" - a lovely mixture of cow feces, water, and yeast left to rot in the hot sun for a couple of months. The vertical contraption contains a fan at the top to suck up female mosquitoes as they come to lay their eggs.

Ellen Martinsen explaining the inner mechanism of the gravid trap - Photo by Francisco Ferreira

Photo by Francisco Ferreira

This trap is a CDC Light Trap. Developed by the Centers for Disease Control, this trap uses both carbon dioxide and light to attract in mosquitoes. You may know this already but it is only female mosquitoes that bite you. Ingeniously but frustratingly, females use your exhaled breath (CO2, carbon dioxide) to find you. This trap works in much the same way - in the dark thermos there is dry ice that sublimates to release CO2 gas. Mosquitoes fly towards it and are sucked into the mesh bag by a fan at the top of the trap. Unlike the gravid trap the CDC light trap is also equipped with a flashing light that draws in both males and females. 

Identification and Dissection

One thing that is important to note at this stage is the importance of keeping your mosquito alive prior to dissection. A dead mosquito is a squishy mosquito and impossible to dissect properly. For this reason the traps are set out at night and checked the following day. Any trapped mosquitoes are brought back to the lab and kept alive in a mini habitat, such as this bug tent:

Just like parasite identification, mosquito ID was difficult at first but once we knew what to look for it became much easier. We used various physical characteristics including wings and palps to tell species apart:

From our Malaria Workshop handout materials (unknown source beyond that)

Once we knew our species and whether it was a male or a female (no use dissecting the males - they don't bite birds and therefore wont carry parasites), we moved on to dissection. We targeted both the salivary glands and the midgut of our insect vectors.

Presence of parasites in the salivary glands indicates the mosquito is a suitable vector - when mosquitoes bite they also inject an anticoagulant to keep the blood flowing. Parasites in the salivary glands will be injected into the host along with the anticoagulant. 

The midgut is the site of parasite reproduction. The "male" and "female" gametes fuse to form a zygote, which then implants in the gut lining of the mosquito and proceeds to divide and divide and divide. Eventually these divisions burst out and make their way to the salivary glands.

Carter Atkinson demonstrates mosquito dissection.

Below is a video from the 1980's that we watched in the workshop. Despite its age, it is an incredibly helpful video to reference technique.

I really cannot reiterate this enough - mosquitoes are small. It was absolutely essential to use a dissecting microscope just to see what we were doing. These were the tiny tools we used:

Initially we chopped off a lot of mosquito heads and lost the glands and guts into a boogery mess of mashed mosquito. But with some fine tuning of our large (comparatively) human hands, we were eventually able to isolate our organs of interest. 

Genetic Techniques

The last half of the last day focused on the use of genetics to determine the species and lineage of parasite as well as their phylogenetic relationships. We looked at several genetic sequences and became familiar with a couple different computer programs used to manipulate this kind of data. 

This picture is a little difficult to see but this is what a genetic sequence looks like read out on a computer screen. Peaks of different colors correspond to specific nucleotide bases - A, G, T, or C. Mutations unique to different parasites help to determine the specific infection present in a bird. These can then be pooled to create a phylogenetic tree, which helps to visualize relationships and evolutionary patterns. 

Saying Goodbye

It was so tough to say goodbye to everybody the last day. An early morning shuttle sent some students to bed early but not before a final round of "selfies" from the 2014 Malaria RCN Student Cohort:

Photos by Adam Krupski

Thank you to the Malaria RCN for this amazing opportunity. I am so grateful to have participated in this workshop and meet all these amazing biologists from around the world! 

Avian Malaria (& Relatives) Workshop - Part 2 - Indentifying Blood Parasites

Here I am one year into my Master's program at San Francisco State University. Time has flown by so fast it's hard to believe a whole year has gone by already. But at the same time when I look at where I was at this time in 2013 I realize how much my skills and experience have progressed.

One year ago I didn't know how to handle a bird or set up a mist net - now I can handle and take a blood sample from a bird. I have a formal training course under my belt and work at a bird banding station.

One year ago I didn't have a single wildlife permit - now I have three (and IACUC approval from my school.)

One year ago I barely knew how to run a gel or do nested PCR or sequence a DNA segment - now these are integral pieces of my research project.

And one year ago I didn't know a thing about avian blood parasites. Now it's the focus of my project and I've participated in the International Workshop on Malaria and Related Haemosporidian Parasites of Wildlife.

Meet this year's "Haemosporidian Workshop" participants! pic.twitter.com/ui2YKOn5Nk

— Malaria RCN (@MalariaRCN) August 1, 2014

In a graduate program you pick up a lot of your knowledge and tips from your advisor and fellow lab mates. That being said, nothing beats an intensive, concentrated training experience. That's what the Malaria Workshop was - after four packed days of content, I felt that my knowledge about blood parasites had taken a giant leap forward. I even gained skills that I never dreamed I would have... believe it or not, I can now dissect out the salivary glands of a mosquito.

Blood Sampling and Parasite ID

The first two days of the workshop emphasized identifying parasites in blood smears under a microscope. In a past post about Avian Malaria I went into a little detail about the causative agent of malaria, Plasmodium (different species within this genus cause malaria in humans, birds, reptiles, etc.) In addition to Plasmodium there are two other genera of blood parasites within the order Haemosporidia we are concerned with: Haemoproteus and Leucocytozoon.

These three genera each have unique methods of reproduction and are spread by different insect vectors. Once you know what you're looking for, they're quite easy to tell apart under a microscope. Here's what the first two days of the workshop looked like:

First we caught some local birds:

Gray Catbird - Photo by Elin Videvall

And took some blood samples:

Strahil taking a blood sample from the brachial vein of a flycatcher - Photo by Strahil Peev

And made some blood smears - sometimes using the "Polaroid method":

Dovile blotting and drying a smear

But preferably with a fan:

Gediminas Valkiūnas fan drying blood smears - It is important to dry the slides right away because soon as they are exposed to air the parasites begin to change form. Fan drying suspends that transformation and preserves their current form.

Then we fixed the slides in methanol: 

Photo by Elin Videvall

Then we stained the slides with Giemsa stain: 

This process stains the cells purple - the nuclei (bird red blood cells have nuclei) and parasites are stained dark purple while the cytoplasm stays clear - Photo by Elin Videvall

Then we looked under our microscopes!:

Photo by Elin Videvall

We did this for several afternoons, looking at both our local bird slides and some "known positive" slides to practice our parasite ID. Our course instructors were very helpful when we inevitably got stuck:

Photo by Elin Videvall

This is what we saw:

Most of what you see here are avian red blood cells, which are nucleated. Right at the tip of the pointer is a Haemoproteus parasite. There are many many species of Haemoproteus that infect birds but they can be differentiated if you know what to look for. 

We would look for defining morphological characteristics such as:

• Location of the pigment granules (in this picture the small dark dots scattered throughout the cell)
• Location of the parasite nucleus (light pink spot in the top right corner)
• How much of the host cell the parasite takes up and what membranes it touches (in this case the whole cell and both the nucleus and outer cell membrane - this isn't always the case!)
• General size and shape 
• And more

Gediminas Valkiūnas' book has a very helpful key - we used it (as well as his direct instruction) extensively:

And this is Leucocytozoon:

Leucocytozoon parasites are much larger than the other two genera. They're the Jabba the Hutt of the parasite world:

To the untrained eye (us before this workshop) Leucocytozoon looks suspiciously like white blood cells so we had a special lecture on that as well:

Interspersed with all these parasites we also each gave a 5 minute presentation of our research projects. It was so interesting to see the different pieces of the blood parasite world that people had carved out internationally. 

Paulo gives his 5 minute presentation - Photo by Strahil Peev

And this was just the first two days! Stay tuned for the third and final installment - Vectors and Summary