Request for REU Supplement
We will mentor two students via independent field projects that are closely related and complementary to our ongoing research on hummingbird high-altitude adaptation. These students would accompany us on an expedition to Peru during which they would be paired with Peruvian graduate students, introduced to a rich study system, and trained in a set of powerful methods from which they will be expected to design and carry out hypothesis-based independent projects. Projects will be of one of two types: (1) expansion of our current experimental regime with hummingbirds by adding an additional variable or testing a key assumption; or (2) extension of our current methods to non-hummingbirds to draw comparisons across the same elevational gradient. The American undergraduate students and their Peruvian counterparts will work together in choosing the question and approach, and in seeing the project through to completion.

Study system and subjects:
Our study system is an elevational transect spanning 4000m, from the high Andes to the Amazon. In summer of 2007, we will work at field camps at three localities along this transect. We will use mistnets to capture live hummingbirds. Other captured bird species (often 15-20 per day) span the phylogenetic and ecological spectrum, comprising an untapped pool of research subjects. Because of their remote and restricted distributions, there are few published physiology or life history data for these birds. We expect to encounter several species of flowerpiercers (Thraupidae: Diglossa), which are obligate nectarivores, and therefore make an excellent comparison to hummingbirds. Other commonly netted taxa include flying insect specialists (Tyrannidae), substrate-gleaners (Furnariidae), and frugivores (Thraupidae, Cotingidae, and Pipridae). We are certain to catch species across a range of flight abilities, including aerial specialists in the Hirundinidae, and highly sedentary species in the Rhinocryptidae. Thus, ample subjects will be available for comparative hypothesis testing (and at least preliminary molecular phylogenies are available for all of the above mentioned groups), whether our undergraduate researchers choose to focus on hummingbirds or other bird groups that are codistributed along the elevational transect.

Methods and equipment:
For our project, we apply a series of powerful field techniques to understand physiology and biomechanics of hummingbirds across altitudinal gradients. Our undergraduate researchers will be trained on all of our techniques and equipment so that they can use these tools to design independent studies.

Respirometry: Hummingbirds that we train to feed from syringes will be flown in our respirometry trial chamber. We perform open flow respirometry by pulling air through a mask that is attached to a nectar-filled syringe (Bartholomew and Lighton 1986). The change in fraction of O2 coming from the mask is used to quantify the consumption of O2 (VO2) by the hovering hummingbird. As part of our primary project, we are quantifying VO2 for hummingbirds under a standardized set of conditions to understand variation among species and across elevations. This technique can be adapted to examine the effects of temperature, time of day, angle of mask, or other parameters on hovering VO2. 

Flight biomechanics: We use a hi-speed video camera to capture the wing movements of each hummingbird during stationary hovering flight. The camera is mounted on a tripod and trained on a mirror placed at a 45 degree angle directly below the nectar source. This video allows us to quantify wingbeat frequency and amplitude, which are critical parameters governing vertical force generation in hovering flight (Altshuler and Dudley 2002). However, the extent to which these parameters vary under different conditions and within a single hovering bout is unknown.     

Hematology: Our fieldwork involves drawing blood samples from which we can quantify three parameters: (1) total hemoglobin concentration (tHb), measured with a hemoglobinometer; (2) packed cell volume (PCV or hematocrit), measured with microcapillary tubes, digital calipers, and a portable centrifuge; and (3) red blood cell concentration, measured with a hemacytometer and portable compound microscope.  Those three parameters allow us to calculate the mean erythrocyte volume (MCV), the mean erythrocyte hemoglobin content (MCH), and the mean erythrocyte hemoglobin concentration (MCHC), which together allow us to understand total blood oxygen capacity (Cambpell 1995, Simmons and Lill 2006). An undergraduate researcher would be able to extend this study to non-hummingbird species that are captured in our nets along the same transect. There are many unanswered questions with respect to comparative hematology of birds, especially with respect to variation across elevational gradients (Scott and Milsom 2006). The vast majority of Andean species have never been studied at any level.

Specimen preparation: Undergraduate researchers will be trained in museum specimen preparation techniques in advance of our planned summer research trip. Our specimen preparation protocol includes: (1) collection of RNA quality tissue samples; (2) quantification of morphological parameters, including dimensions of wings, bill, legs, and tail; (3) description of molt status; (4) autopsy data, including skull condition, stomach contents, gonad, and bursa measurements. These data, recorded in their field catalogues using the Grinnellian method (Herman 1989), will be available to augment independent projects and expand the range of hypotheses that can be tested and variables that can be controlled.

Hypotheses:    
We will work closely with the undergraduate researchers to guide them towards hypotheses that are approachable within the constraints of our study system and summer field season. We will insure that the students take the lead on hypothesis testing and study design.
Examples of potential questions that are both novel and tractable:

  1. Is hummingbird VO2 affected by boundary effects related to height above ground?
  2. How do wingbeat frequency and amplitude change during stationary hovering inside and outside of the respirometry mask, or with different mask sizes or positions?
  3. How does blood oxygen carrying capacity (O2cap) vary with altitude at different phylogenetic levels (e.g. within a species, a genus, or a family)?
  4. How does life history (e.g. flight ability or foraging mode) effect O2cap in a phylogenetically-controlled comparison?
  5. Do independent bird lineages increase O2cap at higher elevations by different means (e.g. increase in cell size vs. increase cell number or hemoglobin concentration)?
  6. Does red blood cell volume scale with body mass?

Feasibility
We have all necessary permits in place for proposed work in Peru. We have taken two expeditions to Peru under our current grant (July-August and November-December, 2006) during which we collected preliminary data on each of these topics suggesting that each would be feasible and fruitful.

Candidates for REU support:
We have pre-selected two undergraduate students to participate in this project. Our selection criteria were that the students already have some experience in museum curatorial techniques, but have not had experience doing independent research beyond the classroom.  We found two students that fit these criteria well. Both would be excellent candidates for going on to graduate school in biology.
Jessica Castillo:  Jessica is currently a junior at UC-Berkeley, majoring in Conservation and Resource Studies. She is a second-generation Hispanic-American, her father’s family is from Mexico and her mother’s family is from Colombia. She has done excellent work as a volunteer at the Museum of Vertebrate Zoology (MVZ), including specimen preparation and public outreach.
Zachary Hanna:  Zach is currently a junior at UC-Berkeley, majoring in Molecular Environmental Biology.  Zach has a strong background in natural history. Since arriving at UC-Berkeley, Zach has worked in several different labs in biology and related fields. Most recently, Zach has enjoyed working in the specimen preparation lab at the MVZ, where he has become a valued team member.

International student partners:
Each supported undergraduate would work in partnership with a Peruvian student who is at a similar level of training. We have pre-selected two Peruvian students, each of whom will be paid as field assistants via our existing budget. All students will prepare specimens and tissue samples to be deposited in the collection of the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru.
Dora Susanibar:  Dora recently finished her coursework for her master’s degree in Biology from the Universidad Nacional Mayor de San Marcos, Lima, Peru. She is currently working on final revisions to her master’s thesis which is a meticulous study of the diet of the Black-chested Buzzard-Eagle (Geranoaetus melanoleucus) at Lomas de Lachay, Lima, Peru.  Dora already has a tremendous amount of field experience, but very little independent research experience.
Miriam Torres:  Miriam is currently a master’s degree student at Universidad Nacional Mayor de San Marcos, Lima, Peru.  Her nearly completed thesis is a study of the status and habitat requirements of resident and migratory waterbird populations in coastal estuaries near Lima, Peru. Miriam has already prepared over 100 bird specimens, and she is eager for opportunities to do independent research.

Proposed 10-week curriculum:
Pre-training in museum curatorial methods: Ongoing, January-May 2007.

  1. Practice specimen preparation, tissue and data collection, catalog and field journal use.
  2. Note: Peruvian students have already been trained in these methods on our previous trips.

Fieldwork in Peru: June 5 – August 5:

  1. Week 1: Meet Peruvian counterparts, learn field methods and equipment.
  2. Week 2: Hypothesis formation and study design.
  3. Week 3-8: Data collection.

Data analysis and report preparation: August 6-August 20

  1. Students compile data from field catalogs and prepare technical lab reports, and individually meet with PI’s to discuss: (1) the future of the student-led project (including co-authored publication); and (2) the student’s future in research, including continuation of student-PI mentorship and the possibility of applying to grad school in biology.

Prior experience in mentoring undergraduates:
PI McGuire’s has mentored undergraduates through an NSF-funded BSI program for the island of Sulawesi, Indonesia, providing training in field techniques for 19 Indonesian students and junior research scientists, and seven American student researchers (four of whom were undergrads). Of those students, two of the Indonesians are now in Ph.D. programs in the U.S. and Canada, three of the American undergrads are now in grad school, and the fourth undergraduate is now applying to graduate schools. McGuire has trained ~20 undergrads in molecular lab techniques. Senior collaborator Witt also has trained six undergrads in molecular lab methods, four of whom went on to graduate school. Witt provided field-based training for four Peruvian and two Bolivian students and during international fieldwork.

REFERENCES:
Altshuler, D. L., and R. Dudley. 2002. The ecological and evolutionary interface of hummingbird flight physiology. Journal of Experimental Biology 205:2325-2336.
Bartholomew, G. A., and J. R. B. Lighton. 1986. Oxygen-consumption during hover-feeding in free-ranging Anna Hummingbirds. Journal of Experimental Biology 123:191-199.
Campbell, T. W. 1995. Avian hematology and cytology. Iowa State University Press, Ames, IA.
Herman, S.G. 1989. The naturalist field journal, based on the method by J. Grinnell. Vermillion, SD: Buteo Books.
Scott GR, Milsom WK. 2006. Flying high: A theoretical analysis of the factors limiting exercise performance in birds at altitude. Respiratory Physiology & Neurobiology 154: 284-301.

Simmons P., and A. Lill. 2006. Development of parameters influencing blood oxygen carrying capacity in the welcome swallow and fairy martin. Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology 143: 459-468.