# Immunological markers for tolerance to avian malaria in Hawaii Amakihi: new tools for restoring native Hawaiian forest birds?

August 1, 2013

We evaluated three assays for non-specific or innate immune capacity to see if measurements were independent of malarial infection and capable of distinguishing malaria-tolerant, low-elevation Hawaiʽi ʽAmakihi from malaria-susceptible, high-elevation ʽAmakihi. ʽAmakihi were captured at Malama Ki Forest Reserve (20 m), Hakalau Forest National Wildlife Refuge (1800 m), and Upper Waiakea Forest Reserve (1700 m), bled for collection of plasma and packed erythrocytes for malarial diagnostics, and either transported to Kīlauea Field Station Aviary and held in captivity for 48 hours for inoculation of wing webs with phytohemagglutinin A (PHA) or released immediately in the field after collection of a blood sample. All birds were tested by polymerase chain reaction (PCR) and microscopy to determine infection status. We found no significant association between malarial infection status and degree of wing web swelling after inoculation with PHA (T = -0.174, df = 13, P = 0.864) and no association between origin of birds from low- and high-elevation populations and degree of wing web swelling (T = 0.113, df = 52, P = 0.911). Infected ʽAmakihi from low elevation had significantly higher small molecule plasma antioxidant capacity than uninfected individuals from the same population (T = -2.675, df = 21, P = 0.014), so we limited comparisons to uninfected birds. Uninfected ʽAmakihi from low elevations did not differ in small molecule plasma antioxidant capacity from uninfected ʽAmakihi from high elevation (T = -0.260, df = 46, P = 0.796). Compared to high-elevation birds, low-elevation ʽAmakihi had significantly higher titers of natural antibodies (NAb) as measured by complement-mediated lysis of rabbit erythrocytes (Mann-Whitney U = 27, X2= 32.332, df = 1, P < 0.0001). This innate immunological difference may be related to ability to survive malarial infection and may prove to be important for understanding possible mechanisms for the evolution of disease tolerance in Hawaiʽi’s native bird species.