Susan L. Balenger

Ultrafast Evolution and Loss of CRISPRs Following a Host Shift in a Novel Wildlife Pathogen, Mycoplasma gallisepticum

Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8−1.2×10−5 per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994–95) strains and have lost the CRISPR–associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs.

Rapid evolution of disease resistance is accompanied by functional changes in gene expression in a wild bird

Wild organisms are under increasing pressure to adapt rapidly to environmental changes. Predicting the impact of these changes on natural populations requires an understanding of the speed with which adaptive phenotypes can arise and spread, as well as of the underlying mechanisms. However, our understanding of these parameters is poor in natural populations. Here we use experimental and molecular approaches to investigate the recent emergence of resistance in eastern populations of North American house finches (Carpodacus mexicanus) to Mycoplasma galliseptum (MG), a severe conjunctivitis-causing bacterium. Two weeks following an experimental infection that took place in 2007, finches from eastern US populations with a 12-y history of exposure to MG harbored 33% lower MG loads in their conjunctivae than finches from western US populations with no prior exposure to MG. Using a cDNA microarray, we show that this phenotypic difference in resistance was associated with differences in splenic gene expression, with finches from the exposed populations up-regulating immune genes postinfection and those from the unexposed populations generally down-regulating them. The expression response of western US birds to experimental infection in 2007 was more similar to that of the eastern US birds studied in 2000, 7 y earlier in the epizootic, than to that of eastern birds in 2007. These results support the hypothesis that resistance has evolved by natural selection in the exposed populations over the 12 y of the epizootic. We hypothesize that host resistance arose and spread from standing genetic variation in the eastern US and highlight that natural selection can lead to rapid phenotypic evolution in populations when acting on such variation.

Innate immunity and the evolution of resistance to an emerging infectious disease in a wild bird

Innate immunity is expected to play a primary role in conferring resistance to novel infectious diseases, but few studies have attempted to examine its role in the evolution of resistance to emerging pathogens in wild vertebrate populations. Here, we used experimental infections and cDNA microarrays to examine whether changes in the innate and/or acquired immune responses likely accompanied the emergence of resistance in house finches (Carpodacus mexicanus) in the eastern United States subject to a recent outbreak of conjunctivitis‐causing bacterium (Mycoplasma gallisepticum—MG). Three days following experimental infection with MG, we observed differences in the splenic transcriptional responses between house finches from eastern U.S. populations, with a 12‐year history of MG exposure, versus western U.S. populations, with no history of exposure to MG. In particular, western birds down‐regulated gene expression, while eastern finches showed no expression change relative to controls. Studies involving poultry have shown that MG can manipulate host immunity, and our observations suggest that pathogen manipulation occurred only in finches from the western populations, outside the range of MG. Fourteen days after infection, eastern finches, but not western finches, up‐regulated genes associated with acquired immunity (cell‐mediated immunity) relative to controls. These observations suggest population differences in the temporal course of the response to infection with MG and imply that innate immune processes were targets of selection in response to MG in the eastern U.S. population.

Sexual selection in a socially monogamous bird: Male color predicts paternity success in the mountain bluebird, sialia currucoides

Ornamental traits are thought to evolve because they give individuals an advantage in securing multiple mates. Thus, the presence of ornamentation among males in many monogamous bird species presents something of a conundrum. Under certain conditions, extra-pair paternity can increase the variance in reproductive success among males, thus increasing the potential for sexual selection to act. We addressed this possibility in the mountain bluebird (Sialia currucoides), a socially monogamous songbird in which males possess brilliant ultraviolet (UV)-blue plumage. Specifically, we asked whether a male’s success at siring offspring within his own nest and within the nests of other males was related to his coloration. In pairwise comparisons, males that sired extra-pair offspring were not more colorful than the males that they cuckolded. However, males that sired at least one extra-pair offspring were, on average, brighter and more UV-blue than males that did not sire extra-pair offspring. Brighter, more UV-blue males sired more offspring both with their own mate and tended to sire more offspring with extra-pair mates and thus sired more offspring overall. Our results support the hypothesis that the brilliant UV-blue ornamental plumage of male mountain bluebirds evolved at least in part because it provides males with an advantage in fertilizing the eggs of multiple females.

CHANGES IN EGG SIZE AND CLUTCH SIZE WITH ELEVATION IN A WYOMING POPULATION OF MOUNTAIN BLUEBIRDS

Abstract Few studies have examined how avian life-history traits vary within populations as elevation increases and climate becomes more severe. We compared egg and clutch sizes of Mountain Bluebirds (Sialia currucoides) nesting at two elevations (1500 m and 2500 m above sea level) in the Bighorn Mountains of Wyoming over two years. Eggs laid by females at the high-elevation site were, on average, significantly (6%) smaller in volume than eggs laid by their lower-elevation counterparts. Across elevations, egg size showed a significant positive correlation with female body condition (weight relative to size), and high-elevation females had significantly lower indices of condition than low-elevation females. Temperatures during clutch formation were colder at the high-elevation site, and egg size was negatively related to temperature after controlling for the effects of female condition. Clutches of females at high elevations were, on average, marginally smaller (by 5%, 0.3 eggs) than clutches of low-elevation females. Unlike egg size, clutch size was unrelated to either female condition or temperature during clutch formation. This suggests that, when under energetic or nutritional stress at high elevations, females sacrifice egg size before sacrificing clutch size.

Mechanisms of evolutionary change in structural plumage coloration among bluebirds (Sialia spp.)

Combinations of microstructural and pigmentary components of barbs create the colour displays of feathers. It follows that evolutionary changes in colour displays must reflect changes in the underlying production mechanisms, but rarely have the mechanisms of feather colour evolution been studied. Among bluebirds in the genus Sialia, male rump colour varies among species from dark blue to light blue while breast colour varies from blue to rusty. We use spectrometry, transmission electron microscopy and Fourier analysis to identify the morphology responsible for these divergent colour displays. The morphology of blue rump barbs is similar among the three species, with an outer keratin cortex layer surrounding a medullary ‘spongy layer’ and a basal row of melanin granules. A spongy layer is also present in blue breast barbs of mountain bluebirds Sialia currucoides and in rusty breast barbs of western Sialia mexicana and eastern bluebirds Sialia sialis. In blue barbs melanin is basal to the spongy layer, but is not present in the outer cortex or spongy layer, while in rusty barbs, melanin is present only in the cortex. The placement of melanin in the cortex masks expression of structural blue, creating a rusty display. Such shifts in microstructures and pigments may be widespread mechanisms for the evolutionary changes in the colours of feathers and other reflective structures across colourful organisms.

Experimental evidence for distinct costs of pathogenesis and immunity against a natural pathogen in a wild bird

Protective immunity is expected to evolve when the costs of mounting an immune response are less than those of harbouring pathogens. Estimating the costs of immunity vs. pathogenesis in natural systems is challenging, however, because they are typically closely linked. Here we attempt to disentangle the relative cost of each using experimental infections in a natural host–parasite system in which hosts (house finches, Carpodacus mexicanus) differ in resistance to a bacterium (Mycoplasma gallisepticum, MG), depending on whether they originate from co‐evolved or unexposed populations. Experimental infection with a 2007‐strain of MG caused finches from co‐evolved populations to lose significantly more mass relative to controls, than those from unexposed populations. In addition, infected co‐evolved finches that lost the most mass harboured the least amounts of MG, whereas the reverse was true in finches from unexposed populations. Finally, within co‐evolved populations, individuals that displayed transcriptional evidence of higher protective immune activity, as indicated by changes in the expression of candidate immune and immune‐related genes in a direction consistent with increased resistance to MG, showed greater mass loss and lower MG load. Thus, mass loss appeared to reflect the costs of immunity vs. pathogenesis in co‐evolved and unexposed populations, respectively. Our results suggest that resistance can evolve even when the short‐term energetic costs of protective immunity exceed those of pathogenesis, providing the longer‐term fitness costs of infection are sufficiently high.

Roaming Romeos: male crickets evolving in silence show increased locomotor behaviours

Loss of sexual signals should be strongly selected against when these signals are necessary for mate attraction or acquisition. Male Teleogryllus oceanicus field crickets produce a long-distance calling song to attract females. Separate genetic mutations recently evolved on the Hawaiian Islands of Kauai and Oahu, rendering approximately 90% and 50% of males, respectively, incapable of calling. We examined whether males from three populations, each with a distinct prevalence of this silent ‘flatwing’ phenotype, show behavioural plasticity in response to being reared in a call-less environment. Crickets from Kauai, Oahu and Mangaia, a Cook Islands population without the flatwing phenotype, either were or were not exposed to calling song during late juvenile and early adult development. Movement assays showed that when males originating from Kauai were reared in silence, they moved sooner, moved more and spent more time walking during silent behaviour trials than did their counterparts reared with calling song. Males from Oahu and Mangaia, however, showed no such effect of acoustic rearing environment on these behaviours. This suggests that there has been directional selection on Kauai for males to respond to a silent environment by increasing their mobility, thus compensating for their lack of song and increasing their chance of encountering receptive females.

Primer design and transcript quantification of a highly multiplexed RT-PCR for a nonmodel avian species

Multiplexed qRT‐PCR assays are currently lacking for nearly all species without genome or transcriptome resources. Here, we present a strategy for primer design of highly multiplexed qRT‐PCR assays, evaluate Beckman Coulter’s Quant Tool gene expression quantification software and provide details of our assay for the North American songbird Carpodacus mexicanus (house finch), for which only small sections of genome sequence are available. We combined Beckman Coulter’s eXpress Designer module for creating custom multiplex primers with the free, online program Amplify 3 to design and evaluate primers computationally before testing them empirically. We also generated a standard curve for each gene included in the final multiplex. We compared models using cubic and quadratic polynomial estimators that did and did not force the intercept through zero. Ultimately, we used the sequences available for 316 clones differentially expressed in cDNA macroarray and microarray comparisons, and from these sequences, we were able to generate a set of transcript‐specific primers for use with the GeXP analyser for 20 house finch genes.