The fitness of animals in nature is mediated by behavior, through individuals' relative effectiveness in securing food, shelter, and mates. Especially in its ecological context, animal behavior is responsible for the movement of genes among population units and contributes strongly to the relative success and failure of particular lineages. Behavioral ecology, an evolutionary approach to the study of animal behavior that emerged as a discipline in the 1970s, has produced bodies of rigorous theory on reproductive and foraging strategies, social systems, and communication. These theories are increasingly being evaluated within a phylogenetic context.

During the same decades, developing population-genetics methods brought historical perspective with the phylogeographic approach, as well as current estimates of gene flow (even asymmetrical gene flow between population subunits), and accurate measures of individual reproductive success, each with resolution that was not possible through more traditional measures. These approaches mean that we can interpret the behavior of animals in a richer context than was possible before, accounting for the roles that individuals play in social systems, that social systems play in populations, and that populations play in relation to species. As we approached the problem of studying diseases among birds in the Galápagos Islands, we took the perspective of behavioral ecologists, curious about the variable roles among individuals, populations, and species in the dynamics of disease transmission.

Veterinary medicine, likewise, has evolved significantly in the decades since the 1970s, incorporating the latest developments from population biology, human medicine, and the biology and control of pathogenic organisms.

Seeking to understand threats of disease on the Galápagos Islands by joining these perspectives, we have embarked on studies that have implications in both basic and applied realms. At the outset, we anticipated that understanding the population dynamics of Galápagos birds would help us interpret parasite and pathogen distribution and host susceptibility, and that our clinical findings in parasite and pathogen distribution would inform our behavioral ecological studies of Galápagos bird populations.

Historically, the effect of disease on wildlife populations has been understudied, aside from a few cases of conservation interest. Parasite- induced diseases have been underestimated, partly from the erroneous view that parasites that coevolve with their hosts do not have severe negative effects on them and partly from logistical difficulties (Gulland 1995). Disease, however, has been shown to affect survival, reproduction, and movement in host populations, and to affect community structure in turn (reviewed in Scott 1988). At the same time, emergent diseases are more common because of an expanding human population, associated growth of domestic animal populations, increased travel and commerce, human alteration of habitats, and global climate change (Daszak et al. 2000). Outbreaks of “mad cow disease” (bovine spongiform encephalopathy; Brown et al. 2001), foot-and-mouth disease (Picornaviridae: Aphthovirus; Kitching 1999), West Nile virus (Flavivirus; Lanciotti et al. 1999), SARS (Coronavirus), monkeypox (Orthopoxvirus), and avian influenza (Orthomyxoviridae) have demonstrated the virulence of some pathogens and their potential to cross over to new hosts, including humans. Thus, interest grows in the dynamics of infectious diseases in wildlife (Daszak et al. 2000, Friend et al. 2001), especially in threatened species, as we recognize the potential for disease to limit populations (Thorne and Williams 1988, Deem et al. 2001).

Parasites can have especially severe effects when they are transmitted to novel environments, where populations may lack natural resistance. Island populations may be at higher risk, because their parasite diversity is less than that of their continental counterparts (Lewis 1968a, b; Dobson 1988; Fromont et al. 2001; Goüy de Bellocq et al. 2002). Founders likely carry only a subset of the parasites found in the donor population, and virulent pathogens that need large host populations may be lost quickly (Dobson and May 1986, Dobson 1988). Paucity of parasites reduces selection for resistance and enhances host population densities, both of which facilitate the transmission of introduced parasites (Dobson 1988).

The Galápagos Islands are volcanic in origin (Christie et al. 1992, White et al. 1993) and are located on the equator, ∼1,000 km west of South America. Their isolation and relative desolation delayed their permanent colonization by humans until the 1800s, and their biodiversity remains mostly intact, only ∼5% of species having been lost (Gibbs et al. 1999); 26 of 28 breeding landbird species are endemic, and there have been no extinctions of bird species. Currently, humans occupy only 4 of the 11 main islands, and most of those 4 islands and the remainder of the archipelago have been protected as the Galápagos National Park and Marine Reserve since the 1950s. However, the resident human population has grown rapidly and exotic species are continually being introduced despite increasing efforts to exclude them. The Charles Darwin Foundation and the Galápagos National Park fear the introduction of avian diseases that could result in extinctions of Galápagos avifauna, similar to what happened in Hawaii (Warner 1968; van Riper et al. 1986, 2002; Wikelski et al. 2004). The appearance of avianpox-like lesions in domestic chickens (Gallus gallus) on the islands, and then in endemic birds, heightened concerns regarding the possibility of disease transmission from introduced birds to endemics. In fact, it was presumed that pox was brought to the archipelago via chickens in the 20th century (Grant et al. 2000). In the mid-1980s, the mosquito Culex quinquefasciatus, a vector of avian malaria, was reported from Galápagos, and its establishment has recently been confirmed (Whiteman et al. 2005). To date, however, the especially pathogenic strains of avian malaria of the genus Plasmodium have not been found. We have undertaken a large project, in collaboration with the Galápagos National Park and the Charles Darwin Foundation, to monitor disease status of Galápagos avian endemics and introduced birds. Partnering with international experts on major taxonomic groups of parasites, we use morphological and molecular approaches to identify organisms. We pursue phylogeographic studies to understand the history of the parasite lineages on the archipelago, and the relationships among parasite lineages and among parasites and host species. The result is a multilayered program examining the interplay between hosts and parasites and the behavioral ecology of both hosts and pathogens, which provides valuable information to the managers of this World Heritage Site and adds new dimensions to the study of evolutionary ecology of all Galápagos organisms, regardless of their body size.

Characterizing Parasites and Pathogens on the Galápagos Islands

In 2001 and 2002, animal health professionals from the Saint Louis Zoo collaborated with biologists from the University of Missouri at St. Louis to offer Avian Health Workshops to personnel of the Charles Darwin Research Station, the Galápagos National Park, the customs and immigration office of Galápagos, and local veterinary professionals. Lectures covered the biology of avian pathogens and diagnostic symptoms of major avian diseases; hands-on workshops trained participants in phlebotomy for disease testing, smears for blood parasites, and necropsy protocol. Subjects were domestic chickens from local farms and Rock Pigeons (Columba livia) from extermination programs, on the human-inhabited islands of Santa Cruz and San Cristobal. In addition, we began the field surveys of natural populations of endemic birds. During the first two years, plasma and serum samples, blood smears, and cloacal swabs were sampled from Waved Albatrosses (Phoebastria irrorata), endemic to the island of Española (Padilla et al. 2003); Galápagos Hawks (Buteo galapagoensis) from eight islands; Galápagos Doves (Zenaida galapagoensis) from five islands (three uninhabited by humans and two inhabited; Padilla et al. 2004); and from Galápagos Penguins (Spheniscus mendiculus) and Galápagos Flightless Cormorants (Phalacrocorax harrisi) across their narrow ranges within the archipelago (Travis et al. 2006a, b). Sampling was later extended to other species (see below). These endemic species were chosen because we were already involved in studies of their population biology and genetics. They represented two terrestrial species (one of which is the apex predator on the islands) and three marine species (one of which is pelagic and covers huge distances, the other two of which are flightless), and so could be seen as sentinel species for a large proportion of the ecological zones inhabited by birds in the Galápagos Islands. In addition, all are relatively large- bodied, so sufficient samples could be taken to test for several pathogens. These ongoing population studies provided a background against which data on disease could be interpreted more fully. For example, our population-genetics studies show that the Galápagos Hawk is the most recent arrival of the endemic vertebrates on the archipelago (Bollmer et al. 2006), that since its arrival it has diversified rapidly among island populations, and that individuals very rarely move among islands (Bollmer et al. 2005). By contrast, the genes of Galápagos Doves move freely among the islands of the main archipelago (Santiago-Alarcón et al. 2006). Similar studies are underway for the penguins and cormorants. These data contribute to understanding the timing of arrival of hosts and their host-specific pathogens, their historical interisland movements, and the current dynamics of transmission of pathogens among islands and species. For example, comparison of hawks and doves suggests that the doves are far more likely to distribute pathogens among islands.

Veterinary and Evolutionary Approaches

Since 2002, the Saint Louis Zoo has funded a veterinary pathologist to reside on the islands as part of our research program. This person is present in the event of outbreaks and receives carcasses reported to the station, and is involved in a year-round training program for Ecuadorian veterinarians and veterinary pathologists on the islands. We broadened the field survey to include four seabird species on Genovesa and other island populations of landbirds. Upon capture, birds are handled briefly while morphological measures are made and ocular, choanal, and cloacal swabs are taken. Blood samples (≤1% body weight) are taken, and two smears are made on the spot. One drop of blood is preserved in lysis buffer for genetic analyses, whereas the remainder is spun in the field and plasma or serum frozen in liquid nitrogen. Later, samples and tissues are examined by serological tests, polymerase chain reaction (PCR), microscopy, and histopathology (on chickens and Rock Pigeons euthanized humanely as part of the extermination program, or on tissues from animals found dead) for complete blood counts, serum chemistry panel, and tests for enteric pathogens (Salmonella, Shigella, Campylobacter spp.), Chlamydophila, as well as for avian cholera, avian influenza, Newcastle disease, Paramyxovirus 2 and 3, West Nile Virus, infectious bronchitis, avian adenovirus, avian encephalomyelitis virus, avian reovirus, Marek's disease, and infectious bursal disease. Blood smears were examined for hemoparasites in all species, and positives were further examined by PCR for identification (Escalante et al. 1998, Ricklefs and Fallon 2002, Fallon et al. 2003). Introduced and endemic doves were tested for Trichomonas gallinae.

Ectoparasites are collected using a modified dust-ruffling technique (Walther and Clayton 1997; Whiteman and Parker 2004a, b) or, in the case of fresh carcasses, are removed directly from the hosts. Although dust-ruffling does not remove all ectoparasites (e.g., many mites), it is relatively noninvasive and allows a good estimation of ectoparasite infection intensity and prevalence (Walther and Clayton 1997). Ectoparasites are placed in 95–100% ethanol and stored at −20°C to ensure DNA preservation. DNA is extracted from exemplars from each host species or population, depending on the particular requirements of the study. A voucher method, in which an exoskeleton is retained as a voucher specimen, is used (Cruickshank et al. 2001, Whiteman et al. 2004). In the case of hippoboscid flies or other relatively large arthropods, a single leg is removed. DNA is extracted and used as a source of template DNA for PCRs. Arthropod-specific PCR primers are then used to amplify and sequence loci to be used in population or phylogenetic studies (e.g., the 5′ end of the cytochrome-c oxidase subunit I [COI] is used as a DNA barcode; Hebert et al. 2003). Thus, identifications of ectoparasites are made using a combination of morphological and molecular characters.

For many parasites and pathogens, DNA sequence data offer a larger set of characters than morphological data alone, for identification purposes. For example, with our collaborator, Kevin P. Johnson, we genotyped Galápagos Dove lice (Columbicola macrourae and Physconelloides galapagensis) that moved horizontally to Galápagos Hawks after the hawks fed upon the doves and showed that the dove lice were indeed derived from the local Galápagos Dove and were not a typical part of the hawk's ectoparasite community (Whiteman et al. 2004). Genotyping was necessary because female lice of these species are indistinguishable from some congeners. In the same study, we genotyped a louse nymph (Bovicola sp.) collected from a Galápagos Hawk that presumably moved from a goat carcass on which the hawks were feeding. In addition to providing additional characters for identification, DNA sequence data also allow estimation of relationships among Galápagoan taxa and their mainland relatives and estimation of gene flow of parasites among islands, a distinctly evolutionary approach that can build from and then inform the veterinary findings.

Endoparasites are identified using traditional taxonomy from blood smears and formalin- preserved specimens (when hosts are necropsied) and using PCR-based identification (Padilla et al. 2004). Moreover, microparasites (e.g., Haemoproteus, microfilariae, Trypanosoma, avipoxvirus) obtained from avian blood, swabs, or biopsy are identified by extracting DNA (host and parasite) from host tissue samples (e.g., blood or skin biopsies) and PCR- amplifying loci used to test for presence or absence (and then to calculate prevalence). Additionally, the amplicons from a subset of positive samples from each avian population are sequenced using standard techniques (e.g., cytochrome-b oxidase mitochondrial DNA [mtDNA] for haemoproteids; Fallon et al. 2003). Macroendoparasites are extracted using a method similar to the above when possible (preserving a part of the parasite as a voucher). All specimens are labeled fully and deposited in the appropriate institutions. Representatives of all lineages are deposited into the invertebrate collection at the Charles Darwin Research Station after all data have been collected (e.g., DNA sequencing, morphometrics, photographs).

Public databases, such as GenBank, have proved highly useful for obtaining outgroups for phylogenetic analyses, and we have relied on the use of the BLAST search option on the National Center for Biotechnology Information (NCBI) server to determine quickly and roughly the identities of the parasite lineages recovered within Galápagos taxa (e.g., trypanosomes, haemoproteids, avian skin mites, Phthiraptera). Eventually, our goal is to create a public database of all DNA sequences obtained from exemplars of all parasite taxa studied within the Galápagos Islands, with all available data on host, habitat, and collection information linked to the sequences, which will allow rapid determination of whether a given parasite has been recovered previously from a given host and locality. Thus, we can quantify the large amount of parasite diversity endemic to the Galápagos avifauna, which hopefully will encourage studies of other vertebrate groups that harbor their own unique parasite lineages (e.g., reptiles: Ayala and Hutchings 1974).

Preliminary Survey Results