Studies in Avian Biology No. 22:254-263, 2001.
IMMUNOGENETICS AND RESISTANCE TO AVIAN MALARIA IN HAWAIIAN HONEYCREEPERS (DREPANIDINAE)
SUSAN I. JARVI, CARTER T. ATKINSON, AND ROBERT C. FLEISCHER
Abstract. Although a number of factors have contributed to the decline and extinction of Hawai‘i’s endemic terrestrial avifauna, introduced avian malaria (Plasmodium relicturn)is probably the single most important factor preventing recovery of these birds in low-elevation habitats. Continued decline in numbers, fragmentation of populations, and extinction of species that are still relatively common will likely continue without new, aggressive approaches to managing avian disease. Methods of in- tervention in the disease cycle such as chemotherapy and vaccine development are not feasible because of efficient immune-evasion strategies evolved by the parasite, technical difficulties associated with treating wild avian populations, and increased risk of selection for more virulent strains of the parasite. We are investigating the natural evolution of disease resistance in some low-elevation native bird populations, particularly Hawai‘i ‘Amakihi (Hemignathus virens), to perfect genetic methods for iden- tifying individuals with a greater immunological capacity to survive malarial infection. We are focusing on genetic analyses of the major histocompatibility complex, due to its critical role in both humoral and cell-mediated immune responses. In the parasite, we are evaluating conserved ribosomal genes as well as variable genes encoding cell-surface molecules as a first step in developing a better under- standing of the complex interactions between malarial parasites and the avian immune system. A goal is to provide population managers with new criteria for maintaining long-term population stability for threatened species through the development of methods for evaluating and maintaining genetic diver- sity in small populations at loci important in immunological responsiveness to pathogens. Key Words: avian malaria; Drepanidinae; genetics; Hawai ‘i honeycreeper; Mhc; P lasmodium relic- turn..
The Hawaiian honeycreepers (Drepanidinae) are and Atkinson 1995, Reynolds and Snetsinger a morphologically and ecologically diverse sub- this volume). Although a large number of factors family of cardueline finches that probably contributed to the extinction of Hawaiian hon- evolved from a single finch species that colo- eycreepers (Ralph and van Riper 1985, Scott et nized the Hawaiian Islands an estimated 4-4.5 al. 1988, James and Olson 1991), introduced dis- million years ago (Tarr and Fleischer 1993, ease and disease vectors are likely the greatest 1995; Fleischer et al. 1998). The honeycreepers threat facing them today, particularly at eleva- radiated very rapidly to fill a variety of ecolog- tions lower than 1,500 m (Warner 1968, van ical niches. While the progenitor of the subfam- Riper et al. 1986, Atkinson et al. 1995). ily was presumably a finch-billed, granivorous form, the more than 50 species and subspecies PARASITES IN PARADISE of honeycreepers derived from this ancestor had There are no native mosquitoes in Hawaii,‘ great diversity of morphological types ranging but a bird-biting species (C&x quinquefascia- from nectivores with long, decurved sickle bills tus) was accidentally introduced to Maui in 1826 to seedeaters with massive, powerful beaks (Per- (Hardy 1960). The spread of this mosquito kins 1903, Amadon 1950, Raikow 1977, Freed throughout low- and mid-elevation habitats and et al. 1987a, James and Olson 1991). introduction and release of domestic fowl, game While the Drepanidines have long been con- birds, and cage birds allowed two introduced sidered an exceptional example of adaptive ra- diseases to escape into native populations, avian diation, they, along with many other Hawaiian malaria (Plasmodium relicturn), and avian pox birds, are also an unfortunate paradigm of ex- (Poxvirus avium). Although there is little direct tinction and endangerment (Scott et al. 1986, evidence that diseases caused by these organ- 1988; James and Olson 1991). Of a total of 54 isms were responsible for the major declines and described species, 14 became extinct after Pol- extinctions of honeycreepers during the past 100 ynesian colonization and are known only from years, considerable indirect evidence has accu- subfossil remains. Another 14 became extinct mulated in recent years that supports this hy- following Western contact and are present in pothesis. Anecdotal reports by early naturalists museum collections from the 1800s. Of the re- of sick and dead birds with large pox-like tu- maining 26 species, 18 are currently listed as mors suggests that avian poxvirus was having endangered by the U.S. Fish and Wildlife Ser- major impacts on forest bird populations as early vice and many of these are perched on the brink as the 1890s (Perkins 1903). It is less clear when of extinction, and some may be extinct (Jacobi malaria was first introduced to Hawaii‘ since the
254 MALARIA IN HAWAIIAN BIRDS-larvi et al. 255
TABLE I. MORTALITY IN NONNATIVE FOREST BIRDS IN TABLE 2. MOUALITYINHONEYCREEPERSEXPERIMEN- HAWAI‘I EXPERIMENTALLY INFECTED WITH HAWAI‘I ISO- TALLY INFECTED WITH HAWAI‘I ISOLATES OF P. dictum. I,ATES OF P. rektum (SCIENTIFIC NAMES FOR ALL BIRD SPECIES LISTED BELOW CAN HE FOCJNI) IN TABLE 2 FOLLOWING THE INTRODUC- TION OF THIS VOLUME.)
Sam- ple SlWXb six Route of inoculation % Morbhty (Leothrix lutea) 5 Blood inoculation 0 (O/5) JapaneseWhite Eye” Laysan Finch” 5 Blood inoculation 100 (5/5) ‘I‘iwi” (Zoster0p.s japoni- 5 Blood inoculation 0 (O/5) 5 Blood inoculation 60 (3/5) CUS) ‘I‘iwih 10 Mosquito bite 90 (9/10) Nutmeg Mannikinh Maui ‘Alauahioc 4 Mosquito bite 75 (3/4) (Lonchum punctu- 7 Mosquito bite 0 (O/7) ‘Apapane” 5 Blood inoculation 40 (2/5) lam) ‘Apapanec 8 Mosquito bite 63 (5/S) Hawai ‘i ‘Ama- LIvan Riper et al. (1986). h Atkinson ct al. (1995). kihi” (high elevation) 6 Blood inoculation 66 (4/6) Hawai‘i ‘Ama- earliest blood smears from native birds date only kihi” to the 1940s and it is unlikely that early natu- (low elevation) 5 Blood inoculation 20 (l/S) ralists would have recognized signs and lesions Hawai‘i ‘Ama- of the disease. van Riper et al. (1986) has hy- kihid pothesized that the introduction occurred in the (high elevation) 20 Mosquito bite 65 (I 3/20) 1920s since this corresponds to a time when Nntc: Mosquito hitc method of inoculalion duplicale
Elevation (m) FIGURE 1. Differences in density of three species of honeycreeperson the northeasternslope of Haleakala Volcano, island of Maui. Data are extracted from density maps published in Scott et al. (1986) to illustrate the lower elevational limits of species that are likely to be highly susceptible to avian malaria (i.e., Maui ‘Alauahio and ‘Akohekohe, Palmeria dole-i) and those more resistant to this disease (i.e., Hawai‘i ‘Amakihi). endangered species, but it is likely that they This putative genetic basis for resistance to share a similar high susceptibility to infection. malaria in low-elevation honeycreeper popula- Interestingly, several of the more abundant tions could involve only a single gene of the honeycreeper species, i.e. Hawai‘i ‘Amakihi, immune system, such as a locus of the major O‘ahu ‘Amakihi (Hemignathus $avus), and histocompatibility complex. Or, like genetically ‘Apapane, appear to have fragmented but appar- based resistance to malaria in humans, there ently stable populations in low- and mid-eleva- could be many loci and genetic systems in- tion habitats where pox and malaria transmission volved to varying degrees, with epistasis also a occurs. Based on differential mortality between possible factor (Nagel and Roth 1989, McGuire mid- and high-elevation Hawai‘i ‘Amakihi that et al. 1994, Hill 1996, Riley 1996, Weatherall were infected experimentally with P. relicturn 1996, Hill and Weatherall 1998). If there is a (Table 2), van Riper et al. (1986) hypothesized genetic basis to resistance to malaria, then in- that these low-elevation populations were evolv- dividuals who are resident at higher elevations ing “immunogenetic” resistance to malaria from represent a population that has not been tested continual exposure to the parasite. van Riper significantly or subjected to natural selection by (1991) also found some evidence that Hawaiian malarial infection. These individuals act as a isolates of P. relicturn are less virulent than control for the experimental or selected popu- those found on mainland North America, sug- lation that has lived with malaria at low eleva- gesting that both native hosts and introduced tions for many generations. Thus by comparing malaria are exerting strong selective pressures allele frequencies or heterozygosities of different on each other and actively coevolving (Atkinson genetic markers between these two populations, et al. 1995). There is some evidence that selec- we may find evidence for the previous (and like- tion for less virulent strains of pox may also be ly continuing) action of natural selection. This occurring, since the massive, debilitating lesions linkage disequilibrium caused by selection that were described by workers in the early (Ghosh and Collins 1996) might also allow us 1900s are found rarely today in naturally infect- ultimately to identify the specific target(s) of se- ed honeycreepers (C. T Atkinson, unpubl. data). lection. Evidence for this is still limited but consistent To understand why and how some species and with current evolutionary theory that predicts individuals within species appear to be able to selection for intermediate levels of virulence and survive disease epidemics, while others suc- host susceptibility after a pathogen is introduced cumb, requires knowledge of the complex inter- into a highly susceptible host population (An- actions between malarial parasites and the avian derson and May 1982, Ewald 1994). immune system and how these interactions may MALARIA IN HAWAIIAN BIRDS-lurvi et al. 257 place selective pressures on both parasite and immune system. Mhc molecules function to dis- host. We have initiated research on genes of the tinguish foreign invaders by presenting a peptide immune system in Hawaiian honeycreepers and fragment of the invader (i.e., a parasite of any ribosomal genes and selected cell-surface mol- kind, or peptides from a foreign graft) in the ecules of Hawaiian isolates of P. relicturn as a antigen-binding region (ABR) of the molecule first step toward developing a better understand- to T-cell receptors. This initiates a cascade of ing of these complex relationships. events that leads to production of lymphocytes, cytokines, and antibodies, and eventual elimi- AVIAN IMMUNE SYSTEMS AND THE nation of the parasite. MHC The Mhc is polymorphic (multiallelic) and Many of the genes and molecules involved in multigenic in most species investigated to date. immune system processes that are described in The human Mhc is known to contain over 200 mammals are also known in birds and many genes, many of which are directly involved in were, in fact, initially discovered in birds. The the adaptive immune response. The chicken Mhc domestic chicken (Gallus domesticus) has long (B system; Briles et al. 1948) is the smallest served as the “laboratory mouse” of avian re- known Mhc, containing only 19 known genes search, largely because of its agricultural impor- coupled to a large family of B-G genes (Kauf- tance and domestication. Thus, much of the cur- man et al. 1999). Studies of the avian Mhc have rent knowledge of avian immune systems comes recently been extended to galliformes other than from studies completed in chickens. Avian im- chickens and include pheasants (Jarvi and Briles mune system cells appear to function in a way 1992, Jarvi et al. 1996; Wittzell et al. 1994, similar to those in mammals, but obvious dis- 1995), turkeys (Emara et al. 1993), and quail tinctions in structure and distribution of lym- (Shiina et al. 1995) as well as passeriformes phoid tissue exist in birds. Birds lack the lymph (Edwards et al. 1995, Vincek et al. 1995; S. I. nodes that are so common in mammals, but they Jarvi et al., unpubl. data) and cranes (Jarvi et al. have unique avian lymphatic tissues such as the 1995, 1999). B-G molecules are expressed on a oculo-nasal Harderian gland and the bursa of Fa- variety of tissues in chickens (Miller et al. 1990, bricious (Eerola et al. 1987). Early studies illu- Salomonsen et al. 1991), but their function is minating the role of the bursa of Fabricious in still unknown. B-G genes have been shown to antibody production established the foundation exist in pheasants (Jarvi and Briles 1992, Jarvi for the T-cell, B-cell concept (Click et al. 1956). et al. 1996) and cranes (Jarvi et al. 1995, 1999). The B-cell system involves production of spe- Recently a second cluster of at least two Mhc cific antibody (humoral immunity) and the T-cell class I and two class II genes (called R&-Y) has system involves cell-mediated immunity. This been identified in chickens (Briles et al. 1993; duality of the immune system is now known to Miller et al. 1994a,b) and pheasants (Wittzell et be universal among all vertebrates. In mammals al. 1995, Jarvi et al. 1996). The function and and birds, both humoral and cell-mediated re- expression of these genes is currently under in- sponses are involved in the immune response vestigation. The simplicity of Mhc structure and against malaria. Among the many cells and mol- function in birds as compared to mammals has ecules with important roles in immunity (e.g., been thought to account for the more obvious antibodies, macrophages, neutrophils, natural associations of Mhc genotype and susceptibility killer cells, and a variety of cell communication to infectious disease (for review see Kaufman molecules, such as interleukins and cytokines), and Wallney 1996). molecules encoded by genes within the major One of the earliest reported Mhc associations histocompatibility complex (Mhc) are of special with infectious disease was in chickens with significance due to their critical role in both hu- Mareks’ disease (Hansen et al. 1967, Briles and moral and cell-mediated immunological re- Oleson 1971). Mareks’ disease is a naturally oc- sponses. curring, herpes-virus induced lymphoma of Mhc class I and class II genes have been chickens. The virus initially infects B-cells and found in all well-characterized vertebrates and macrophages, and eventually T-cells, which re- date as far back as cartilaginous fish. The Mhc sult in lethal lymphomas. Birds possessing the was first identified as the genetic locus respon- B21 Mhc haplotype show strong resistance (as sible for allograft (tissue) rejection, but it is now much as 95% survival; Pazderka et al. 1975, known to be responsible for determining what is Longenecker et al. 1976; Briles et al. 1977, viewed as “self” versus “non-self” by the im- 1980, 1983; Bacon and Whitter 1980). The B21 mune system. Class I molecules are present on haplotype occurs frequently in many apparently essentially all nucleated cells in the body (in- unrelated populations of chickens, including Red cluding erythrocytes in birds), whereas class II Jungle Fowl (Gallus gallus; the hypothesized molecules are present on only certain cells of the species progenitor), indicating that it may have 258 STUDIES IN AVIAN BIOLOGY NO. 22 special survival value for the species (Longe- species with some evidence of natural resistance necker and Mossman I98 I). to malaria, and also the I‘ iwi,‘ a highly suscep- Rous Sarcoma virus (RSV) is a retrovirus tible species with declining numbers that is cur- which causes fatal tumors in some chickens but rently limited to high-elevation habitats. Be- not others. When studied in congenic strains of cause of the extreme variability and putative dis- chickens, Mhc haplotype B12 is involved with ease and fitness associations of the Mhc, a num- tumor regression (more resistant to RSV) where- ber of authors have suggested that selection for as B4 is a progressor (more susceptible to RSV; increased Mhc diversity occurs via interactions reviewed in Plachy et al. 1992). Further studies of the molecule with diverse antigen types, but show the existence of 17 virally derived peptides the type of selection has been open to debate that are capable of binding the major B12 class (e.g., Hughes and Nei 1988, Hedrick et al. I molecule, whereas only two virally derived 1991). Selection may be directional in situations peptides were identified with the motif of the where a particular Mhc variant provides immu- major B4 class I molecule (Kaufman and Wall- nity to a particular disease (Hedrick et al. 1991). ney 1996). This may partially explain the resis- If a number of different diseases infect a popu- tance (or enhanced immune response) that oc- lation over time, selection could balance the fre- curs in B12 chickens. Other strong disease as- quencies, resulting in a high level of variability sociations with different Mhc alleles in chickens (i.e., frequency dependent selection; Hedrick et include coccidiosis (Clare et al. 1989) and fowl al. 1991). If a population, such as low-elevation cholera (Lamont et al. 1987). Association be- Amakihi,‘ experiences a devastating epidemic of tween specific Mhc alleles and resistance to dis- a single disease, we may see the “signature” of ease in humans exists, but it is not as apparent. the selection as a greatly increased or modified Substantial evidence exists for protective alleles frequency of a particular haplotype in the pop- against severe malaria in some human popula- ulation of survivors. Alternatively, selection tions but not in others (Hill et al. 1991, 1994; may favor heterozygous individuals (heterozy- Riley 1996, Hill and Weatherall 1998, Gilbert et gote advantage), because the Mhc products of al. l998), and that particular class I1 haplotypes two alleles can recognize more, different anti- affect the probability that a hepatitis B infection gens (i.e., polypeptide products) of a particular will become persistent (Thursz et al. 1995). disease than one allele. It has been shown that Mhc heterozygotes What sample sizes are needed to have suffi- have higher hatchability and viability than ho- cient power to detect this selection? We are cur- mozygotes (Schultz and Briles 1953), and that rently comparing samples from low- and high- individual Mhc haplotypes are associated with elevation Hawaii‘ Amakihi.‘ Most of the low- level of hatchability and viability in chickens elevation populations have been in contact with (Briles and Allen 196 I ). Evidence for overdom- mosquitoes and malaria for 50-100 yrs. Given inance of Mhc alleles (heterozygous advantage) mortality rates of 50-70% in malaria-challenged was demonstrated very early in chickens for high-elevation birds, there is potential for very traits such as viability, hatchability, body strong selection with coefficients of 0.5 or high- weight, and survivor egg production (Briles er. Using equations from Hart1 and Clark (1989), 1954, Briles et al. 1957, Abplanalp et al. 1992, we have modeled the potential impacts of direc- Sato et al. 1992) and in humans for susceptibil- tional selection by malaria on single-locus allele ity to hepatitis B infection (Thursz et al. 1997) frequencies and on the power to detect signifi- and survivorship to HIV-1 infections (Carring- cant differences (Cohen 1988) given sample ton et al. 1999). Retention of polymorphism sizes of 50 low- and 200 high-elevation birds (multiple alleles) as well as heterozygosity for (Fig. 2). Whether we use 50 or 100 generations genes in the Mhc is likely important for long- of selection, or have an initial allele frequency term population stability. The Mhc, therefore, of the positively selected allele as 5% (based on appears to be an excellent candidate gene region a “typical” Mhc haplotype frequency in chick- in which to search for disease relationships in ens), or 30% (based on the survival rate in high- Hawaiian honeycreepers. elevation challenges), we generally have suffi- cient power (>80%) to detect selection coeffi- GENETIC ANALYSES OF HOSTS cients as low as 0.02 with our sample. Satta et We know that susceptibility to malaria differs al. (1995) recently estimated overdominant se- among and within honeycreeper species and that lection coeflicients on mammalian Mhc loci to relative susceptibility or resistance can explain range from about 0.002 to 0.05; thus, this type their current elevational distribution to this dis- of comparison should be able to detect at least ease. To investigate the natural evolution of dis- the higher range of their values. ease resistance in honeycreepers, we have initi- Our model does not include the ameliorating ated studies of the Mhc in Hawaii‘ Amakihi,‘ a effects of migration. However, the Amakihi‘ is MALARIA IN HAWAIIAN BIRDS-Jur-vi et al. 259
TABLE 3. POWER TO DETECT FREQUENCY DIFFERENCES BETWEEN SURVIVORS AND NONSURVIVORS AFTER DIREC- 0.8 TIONAL MALARIA SELECTION AT THE GIVEN SELECTION CO- p gem EFFICIENT (S) WITH THE GIVEN SAMPLE SIZE (N) iii 0.6 ..a.. 0.05 100 iz Selection coefficient -*- 0.30 100 ~ 2 0.4 N 0.05 0.10 0.25 0.50 -9-0.05 50 25 0.061 0.077 0.153 0.440 0.2 -o- 0.30 50 100 0.077 0.120 0.354 0.915 250 0.097 0.184 0.655 0.999 0 0 0.02 0.04 0.06 0.08 s (selection coefficient) I‘ iwi‘ class II genes were carried out (Figs. 3a and 3b, respectively). All blots were produced FIGURE 2. Power (Cohen 1988) to detect frequency differences between low- and high-elevation popula- by digestion of approximately 15 kg of genomic tions after 50 or 100 generations of malaria selection DNA with either PvuIl, Pstl, or HindIII. Blots (Hard and Clark 1989) in low-elevationpopulations. were then hybridized with a mixture of cloned, sequenced, 2P-labeled‘ Mhc class II antigen- binding region from Hawaii‘ Amakihi‘ and known to have high breeding and natal philo- I‘ iwi‘ at 50°C in a rotating hybridization oven. patry (van Riper 1984; U.S. Geological Survey, Blots were washed in 1 X SSC for at least one unpubl. data), and it is our view that migration hour and autoradiograms were produced. Band is not sufficient to counter the expected selection sharing coefficients (s) were calculated (using intensities. We also assessedthe power to detect methods described in Lynch 1988) from banding significant differences in allele frequencies be- patterns derived from individuals on four Ama-‘ tween malaria challenge survivors, fatalities, and kihi blots and four I‘ iwi‘ blots. Data are pre- controls (Table 3). In this case, selection must sented as composite histograms in Figures 3a be very strong (>0.25) in order for us to detect and 3b. The Mhc class II banding patterns of a significant difference given sample sizes from Amakihi‘ and I‘ iwi‘ are markedly distinct, as is recent challenge experiments (Atkinson et al. reflected by a mean band sharing coefficient of 1995). 0.617 over four Amakihi‘ blots (Figure 3a) and Our goals are to perfect genetic methods for 0.883 over four I‘ iwi‘ blots (Figure 3b). The ac- evaluating Mhc diversity among and within spe- tual number of bands varies depending on re- cies to look for elevation-dependent allele fre- striction enzyme used (either HindUI, PvuII, or quency distributions. Birds involved in experi- PstI). Digestion of genomic Amakihi‘ DNA re- mental malaria challenges are also included in sults in a range of four to nine bands/lane where- this ongoing study with the hopes of developing as I‘ iwi‘ generally have from one to four bands/ methods for identifying individuals with a great- lane. One would expect these two species to er ability to survive malarial infection. General possess a similar number of class II genes since methodology includes the use of the polymerase they are thought to be monophyletic, and also chain reaction (PCR) to amplify the Mhc anti- since some I‘ iwi‘ were found with six or more gen-binding region (ABR) of class II (beta) bands/lane. Therefore, the observed decrease in genes from the genomic DNA from several in- bands/lane among I‘ iwi‘ as compared to Ama-‘ dividuals (S. I. Jarvi et al., unpubl. data). This kihi (particularly a decrease in polymorphic targets the gene region in which much of the bands, i.e., an increase in band-sharing coeffi- variability of Mhc molecules is concentrated. cients) likely represents a decrease in class II Products from PCR were cloned using methods Mhc diversity. The limited Mhc diversity found that allow specific cloning of homoduplex PCR in I‘ iwi‘ could play a role in the high mortality products by either PCR+l (Borriello and Krau- observed in this species. That is, if Mhc class I ter 1991) or single-stranded conformational and class II antigen-binding variability is low, it polymorphism (SSCP) isolation (Oto et al. is less likely that malarial-encoded peptides will 1993), PCR reamplification, and direct cloning. be presented to or be recognized by the hosts’ Both strands of the cloned products were se- immune system. Insufficient stimulus to the im- quenced using a 373 ABI automated sequencing mune system could explain the prolonged par- system, and sequences verified as originating asitemias that are common in honeycreepers from the class II ABR by comparison with nu- with acute infections, where the parasite repro- merous known class II sequences available duces unchecked in the bloodstream. This is in through Genbank.@ Restriction fragment-length stark contrast to nonnative species where pat- polymorphism (RFLP) analyses of Amakihi‘ and asitemias peak sharply and immediately decline 260 STUDIES IN AVIAN BIOLOGY NO. 22
70 cr 60 x=0.617
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Amakihi‘ Bandsharing Coefficient
250 x=0.883
200
150
100
50
0 I I I *,,~I I I II! 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 I‘ iwi’ Bandsharing Coefficient
FIGURE 3. Summary of RFLP (Southern blot) analyses of Hawaiian honeycreeper Mhc class II genes. Data are compiled as the percentage of shared bands between individuals (i.e., the bandsharing coefficient, computed according to methods of Lynch 1988) versus the number of individual comparisons. A. Four Hawai‘i ‘Amakihi Southern blots include a total of 58 different individuals originating from several elevations on the island of Hawai‘i. B. Four ‘I‘iwi blots include a total of 41 different individuals originating from high elevations on the island of Hawai‘i.
(van Riper et al. 1986; C. T. Atkinson, unpubl. tion at the Mhc for its possible relationship to data). disease resistance, other genetic markers can be Comparisons of mitochondrial DNA gene se- used to identify different genetic systems that quences derived from I‘ iwi‘ and Amakihi‘ re- may also confer resistance. These markers can veal predictable levels of nucleotide diversity include putatively random ones (such as micro- among Amakihi,‘ but all sequences derived from satellites, minisatellites, Randomly Amplified I‘ iwi‘ are invariant (Feldman 1994; C. Tarr, pers. Polymorphic DNA (RAPDs), or Amplified Frag- comm.). Further studies are needed to clarify ment Length Polymorphisms (AFLPs)), or gene any potential selective role of the parasite. systems known to play a role in malaria resis- In addition to systematically analyzing varia- tance in humans (e.g., G6PDH, TNF-a, etc.; MALARIA IN HAWAIIAN BIRDS-Jurvi et al. 261
Riley 1996, Weatherall 1996). Microsatellites proximately 129 million years ago (Escalante have been developed for drepanidines (Tarr and Ayala 1994), possibly explaining why more 1995, Tarr et al. 1998), and panels of chicken species are found in reptiles and birds (1 lo+) cDNA probes are available (Bumstead et al. than in mammals (40+; Levine 1988). Most of 1995) for screening immune system and other what we know about the life cycles, pathogenic- genes. These markers may prove useful for three ity, and immunology of the avian parasites was primary purposes: (1) An alternative measure of established during the first half of the 20” ’ cen- variability will be available for comparisons tury when several species that readily infect do- among elevations and susceptibility classes in mestic birds (e.g., P. gallinaceum and P. lophu- Amakihi,‘ and among different honeycreeper rue) were used as primary laboratory models for species. This tests whether a correlation between studies of human malaria. With the development Mhc gene diversity (heterozygosity) and resis- of rodent, primate, and in vitro models, research tance is due to the variation within the Mhc itself shifted away from avian parasites in the 1950s (and/or its linked genes) or to genomic variabil- and we consequently know relatively little about ity in general. (2) Markers can be assayed to how unique avian immune system molecules determine if particular allelic variants or heter- and processes might influence parasite interac- ozygotes show strong associations with malaria tions with the avian host. resistance (i.e., differ between low and high el- Having successfully persisted in a variety of evation or challenge survivors and fatalities). vertebrate species over such a long period of This approach is standard in medical genetics time, Plasmodium spp. have necessarily evolved and is outlined by Ghosh and Collins (1996) as effective mechanisms for survival. Immune-eva- the “linkage disequilibrium” approach, as it re- sion strategies and the processes involved in nat- quires that the actual disease resistance muta- ural immunity to malaria are complex and poor- tions be in linkage disequilibrium with particular ly understood, even in mammalian hosts where alleles. (3) Microsatellite and AFLP markers are most research has focused in recent years. Much excellent for the construction of linkage maps. of this complexity is due to multiple stages of Such a map would be important for locating the the parasite life cycle that alternate between the relative position of the Mhcand’ other immune vertebrate host and the mosquito vector. In system genes, or any other markers that show a mammals, transmission occurs to a new host relationship with disease resistance. when an infected anopheline mosquito inocu- While mapping disease resistance to the Mhc lates sporozoites into the bloodstream during a is strong evidence favoring that Mhc itself is re- blood meal. These invade hepatocytes, undergo sponsible, it does not rule out other genes linked one generation of asexual reproduction, and re- within the region. In fact a number of other lease merozoites into the bloodstream, which in- genes coding for immune system molecules vade circulating erythrocytes. Multiple cycles of have been localized to the Mhc region in mam- asexual reproduction occur in the circulating mals and birds (Bumstead et al. 1995), including blood cells, during which some merozoites are tumor necrosis factors (Hedrick et al. 1991), produced that invade erythrocytes and develop complement proteins (Hedrick et al. 1991), pro- into gametocytes. These circulating gametocytes teasomes for antigen degradation (Fehling et al. complete the vertebrate phase of the life cycle 1994), and transporter-associated antigen pro- and are capable of infecting new mosquito hosts. cessing proteins (de la Salle et al. 1994, Suh et The complex interactions that occur between de- al. 1994, Bumstead et al. 1995). Some of these veloping parasites, host cells, and the host im- may be involved in disease resistance and serve mune system in mammals results in production as the targets of selection. of antibodies, activation of a variety of different effector cells, production of lymphokines, and a GENETIC ANALYSIS OF PARASITES cascade of events that control parasite numbers A second key to understanding disease resis- without completely eliminating the infection. tance in Hawaiian honeycreepers concerns ge- Production of nonsterilizing immunity is char- netic diversity of malarial parasites themselves acteristic of Plasmodium in its various vertebrate and how they may exert selective pressure on hosts, including birds, and has been termed con- the host. The malarial parasites of vertebrates comitant immunity. (Plasmodium spp.) are a closely related group of A number of key differences exist between Apicomplexan parasites that share common the life cycles of avian and mammalian malarial morphological and developmental characteris- parasites that may be important in how parasites tics in all of the reptilian, avian, and mammalian interact with the immune system. The pre-eryth- hosts in which they occur (Garnham 1966). Spe- rocytic stages of avian parasites (i.e., those that cies of Plasmodium are thought to have diverged develop from sporozoites) invade and develop from other members of the Apicomplexa ap- in blood forming cell types, such as hemocytob- 262 STUDIES IN AVIAN BIOLOGY NO. 22 lasts, and cells of the lymphoid-macrophage sys- fers to the expression of different alleles of a tem rather than hepatocytes (Huff 1969). These gene in different populations, whereas antigenic cell types include macrophages, stem cells, and variation is the process by which a clonal para- endothelial cells that line blood capillaries. Avi- site population can switch its antigenic pheno- an parasites undergo several cycles of reproduc- type (reviewed in Reeder and Brown 1996). In tion in these cell types before invading eryth- fact, polymorphic regions of the CSP have been rocytes and, unlike most mammalian parasites shown to serve as T-cell epitopes (Good et al. that have a self-limiting cycle in the host liver, 1988). persist in cells of the lymphoid-macrophage sys- We are using a variety of molecular tech- tem for the duration of the infection and most niques to evaluate these genes or portions of likely for the life of the host. These persistent these genes in P. relictum as a means of iden- tissue stages provide a source of parasites for tifying variation sufficient to warrant strain di- relapsing erythrocytic infections and, more im- vergence. We are also using these techniques to portantly, stimulate concomitant immunity in the develop a PCR-based diagnostic test for P. re- host, providing protection from reinfection with lictum that will supplement both the PCR test homologous strains of the parasite. described by Feldman et al. (1995) and serolog- We have initiated genetic studies of Hawaiian ical tests for antibodies to the parasite that we isolates of P. relictum to determine if multiple are currently using (Atkinson et al. 2001). strains that differ in pathogenicity are present in We began analyses of the 18s ribosomal Hawaii‘ and whether they are responsible for pe- genes using highly conserved PCR primers spe- riodic epidemic outbreaks that occur in mid-el- cific for an approximately 580 base pairs (bp) evation habitats (C. T. Atkinson, unpubl. data). segment of 18s ribosomal genes (Feldman et al. We are also interested in developing reliable 1995, Shehata et al. this volume). We selected PCR-based methods for diagnostic purposes. To individuals that had been previously screened accomplish this, we are evaluating diversity of for the presence or absence of Plasmodium by regions of several genes including the more con- blood smear and immunoblot methods (C. T. At- served 18s ribosomal genes (Waters and Mc- kinson, unpubl. data) to provide a basis for com- Cutchan 1989, Feldman et al. 1995), and several parison. PCR-based techniques for studies of hu- variable genes encoding cell-surface proteins in- man Plasmodium spp. are generally used in cluding thrombospondin-related analogous pro- combination with serological or other immuno- tein (TRAP), circumsporozoite protein (CSP), logical methods due to the high percentage of and merozoite surface antigen 2 (MSA-2). The false negatives (0.05) and false positives (0.16) genes encoding cell-surface proteins were ini- in PCR-based diagnostic tests (reviewed in tially characterized in P. falciparum, and we are Weiss 1995). These ribosomal primers are also currently developing PCR primers specific for highly conserved. This means that they would the homologous gene regions in P. relictum likely anneal to ribosomal regions of DNA of (Felger et al. 1993, 1994; McCutchan et al. any number of organisms under the appropriate 1996, Templeton and Kaslow 1997). The 185 conditions, as has been demonstrated in other ribosomal genes have a low mutation rate of ap- species (Perkins and Martin 1999). In our hands proximately 2% per 110 million years (Ochman we have found that these primers amplify mul- and Wilson 1987, Wilson et al. 1987) and are tiple fragments from individuals which makes it especially useful for phylogenetic analyses (e.g., difficult to distinguish the (theoretically) Plas- Escalante and Ayala 1994). Sporozoite and modium-specific 580 bp band from other similar- merozoite cell-surface proteins are all quite vari- sized bands that may also be amplified from able and are thought to be under positive Dar- whole blood. Upon cloning and sequencing am- winian selection by the host immune system, plified DNA from 20 individuals, we have found i.e., amino acid variability is higher, especially that the length of this region varies, ranging within certain gene regions of the molecules, from approximately 570 to 600 bp in length. than would be expected under circumstances of From initial nucleic acid comparisons (MEGA neutrality (Hughes and Hughes 1995). Balanced and PC Gene), no distinct groupings were seen host-parasite interactions may likely involve se- based on geographic origin of the samples. lective pressure by the parasite on molecules of The actual number of ribosomal genes in P. the immune system (e.g., Mhc molecules) as relictum is unknown but ranges from 4-10 well as selection on variable parasite molecules rDNA units in other Plasmodium species (e.g., TRAP, CSP MSA-2) by the hosts’ immune (McCutchan 1986). For understanding more system. These highly variable parasite molecules completely the diversity levels in this gene re- are important in fundamental primary mecha- gion, we are using an SSCP-based approach. nisms of immune evasion, antigenic diversity The patterns produced by SSCP reveal the pres- and antigenic variation. Antigenic diversity re- ence of as many as 10 bands, suggesting that P. MALARIA IN HAWAIIAN BIRDS-an/i‘ et al. 263 relictum, if haploid, contains multiple rDNA alleles, especially at loci important in immuno- gene units with a minimum copy number of five. logical responsiveness to pathogens. At the same We have cloned and sequenced a nearly full time, detailed information about genetic diver- length TRAP gene from P. relictum in order to sity in parasite populations can have important obtain DNA sequence for designing strain-spe- applications in monitoring epidemics and devel- cific primers (S. I. Jarvi, unpubl. data); these are oping quarantine protocols for preventing intro- for use in diagnostic tests to supplement the cur- ductions of new strains of the parasite. Recent rently available PCR test that is based on ribo- data indicates that the dispersal and flight range somal genes (Feldman et al. 1995, Shehata et al. of Culex quinquefasciatus in densely forested this volume), as well as for use in evaluating habitats may be much greater than initially an- diversity at a variable and likely selected gene. ticipated, making it less likely that vector-con- Because the bionomics of the mosquito vec- trol techniques based on elimination of breeding tors of avian malaria can affect evolution of the sites or application of environmentally compat- bird-parasite interactions, research on dispersal, ible larvicides will be effective unless applied host preferences, behavior, susceptibility, and over large geographic areas (D. A. LaPointe, C. genetics of C. quinquefasciatus is needed to help T. Atkinson, unpubl. data). Other approaches for interpret findings from research being conducted breaking the disease cycle, such as chemother- on both honeycreepers and malarial parasites. It apy or vaccine development, are even less fea- is beyond the scope of this paper to report in sible because of efficient immune-evasion strat- detail on projects in progress, but field and lab- egies evolved by the parasite, technical difficul- oratory studies currently underway are using mi- ties associated with treating wild avian popula- tochondrial DNA and a number of microsatellite tions, and the increased risk of selecting for markers as well as other techniques for exam- more virulent strains of the parasite. Until we ining geographic diversity, patterns of introduc- know more about genetic diversity and its rela- tion, dispersal rates, and vectorial capacity of tionship to disease susceptibility in remaining Culex populations in Hawaii‘ (Fonseca et al. threatened and endangered forest bird popula- 1998; D. A. Fonseca, D. A. LaPointe, C. T At- tions, protection of high-elevation habitats, pre- kinson, and R. C. Fleischer, unpubl. data). vention of new introductions of pathogens, and intensive management of adjacent mid-elevation CONCLUSIONS forests to reduce oviposition sites for Culex Genetic studies that help to clarify the com- mosquitoes may be the best short-term approach plex interactions between host and parasite can for preventing further extinctions. provide information critical for the survival and ACKNOWLEDGMENTS management of native forest birds. Immunoge- We thank J. Ballou, S. Banner, D. LaPointe, J. netic studies of honeycreepers will provide nat- Lease, C. McIntosh, J. Schultz, C. Tan; and J. Wilcox ural resource managers new criteria for main- for technical assistance and helpful discussions. The taining and increasing genetic diversity in frag- Mhc studies of honeycreepers were supported by a mented populations of threatened or endangered Smithson grant (to RCF), a Smithsonian Molecular species. Because of dynamic coevolutionary in- Evolution Postdoctoral Fellowship (to SIJ), and a Smithsonian Institution Scholarly Studies grant to teractions among hosts, parasites, and vectors, (RCE SIJ, and J. Ballou). This work was also sup- the best overall strategy may be to aggressively ported by the U.S. Geological Survey-Biological Re- use translocations and captive propagation to sources Division, Pacific Island Ecosystems Research maximize heterozygosity to prevent loss of rare Center (parasite and pathogenicity studies).