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:
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!
