Testing the resource trade-off hypothesis for carotenoid-based signal honesty using genetic variants of the domestic canary.

Carotenoid-based coloration in birds is widely considered an honest signal of individual condition, but the mechanisms responsible for condition dependency in such ornaments remain debated. Currently, the most common explanation for how carotenoid coloration serves as a reliable signal of condition is the resource trade-off hypothesis, which proposes that use of carotenoids for ornaments reduces their availability for use by the immune system or for protection from oxidative damage. However, two main assumptions of the hypothesis remain in question: whether carotenoids boost the performance of internal processes such as immune and antioxidant defenses, and whether allocating carotenoids to ornaments imposes a trade-off with such benefits. In this study, we tested these two fundamental assumptions using types of domestic canary (Serinus canaria domestica) that enable experiments in which carotenoid availability and allocation can be tightly controlled. Specifically, we assessed metrics of immune and antioxidant performance in three genetic variants of the color-bred canary that differ only in carotenoid phenotype: ornamented, carotenoid-rich yellow canaries; unornamented, carotenoid-rich 'white dominant' canaries; and unornamented, carotenoid-deficient 'white recessive' canaries. The resource trade-off hypothesis predicts that carotenoid-rich individuals should outperform carotenoid-deficient individuals and that birds that allocate carotenoids to feathers should pay a cost in the form of reduced immune function or greater oxidative stress compared with unornamented birds. We found no evidence to support either prediction; all three canary types performed equally across measures. We suggest that testing alternative mechanisms for the honesty of carotenoid-based coloration should be a key focus of future studies of carotenoid-based signaling in birds.

Bacterial Pathogen Emergence Requires More than Direct Contact with a Novel Passerine Host.

While direct contact may sometimes be sufficient to allow a pathogen to jump into a new host species, in other cases, fortuitously adaptive mutations that arise in the original donor host are also necessary. Viruses have been the focus of most host shift studies, so less is known about the importance of ecological versus evolutionary processes to successful bacterial host shifts. Here we tested whether direct contact with the novel host was sufficient to enable the mid-1990s jump of the bacterium Mycoplasma gallisepticum from domestic poultry to house finches (Haemorhous mexicanus). We experimentally inoculated house finches with two genetically distinct M. gallisepticum strains obtained either from poultry (Rlow) or from house finches (HF1995) during an epizootic outbreak. All 15 house finches inoculated with HF1995 became infected, whereas Rlow successfully infected 12 of 15 (80%) inoculated house finches. Comparisons among infected birds showed that, relative to HF1995, Rlow achieved substantially lower bacterial loads in the host respiratory mucosa and was cleared faster. Furthermore, Rlow-infected finches were less likely to develop clinical symptoms than HF1995-infected birds and, when they did, displayed milder conjunctivitis. The lower infection success of Rlow relative to HF1995 was not, however, due to a heightened host antibody response to Rlow. Taken together, our results indicate that contact between infected poultry and house finches was not, by itself, sufficient to explain the jump of M. gallisepticum to house finches. Instead, mutations arising in the original poultry host would have been necessary for successful pathogen emergence in the novel finch host.

Linking stress and immunity: Immunoglobulin A as a non-invasive physiological biomarker in animal welfare studies.

As the animal welfare community strives to empirically assess how care and management practices can help maintain or even enhance welfare, the development of tools for non-invasively measuring physiological biomarkers is essential. Of the suite of physiological biomarkers, Immunoglobulin A (IgA), particularly the secretory form (Secretory IgA or SIgA), is at the forefront because of its crucial role in mucosal immunity and links to physical health, stress, and overall psychological well-being. While interpretation of changes in SIgA concentrations on short time scales is complex, long-term SIgA patterns are consistent: conditions that create chronic stress lead to suppression of SIgA. In contrast, when welfare is enhanced, SIgA is predicted to stabilize at higher concentrations. In this review, we examine how SIgA concentrations are reflective of both physiological stress and immune function. We then review the literature associating SIgA concentrations with various metrics of animal welfare and provide detailed methodological considerations for SIgA monitoring. Overall, our aim is to provide an in-depth discussion regarding the value of SIgA as physiological biomarker to studies aiming to understand the links between stress and immunity.

Ketocarotenoid circulation, but not retinal carotenoid accumulation, is linked to eye disease status in a wild songbird.

Pathogenic or parasitic infections pose numerous physiological challenges to organisms. Carotenoid pigments have often been used as biomarkers of disease state and impact because they integrate multiple aspects of an individual's condition and nutritional and health state. Some diseases are known to influence carotenoid uptake from food (e.g. coccidiosis) and carotenoid use (e.g. as antioxidants/immunostimulants in the body, or for sexually attractive coloration), but there is relatively little information in animals about how different types of carotenoids from different tissue sources may be affected by disease. Here we tracked carotenoid accumulation in two body pools (retina and plasma) as a function of disease state in free-ranging house finches (Haemorhous mexicanus). House finches in eastern North America can contract mycoplasmal conjunctivitis (Mycoplasma gallisepticum, or MG), which can progress from eye swelling to eye closure and death. Previous work showed that systemic immune challenges in house finches lower carotenoid levels in retina, where they act as photoprotectors and visual filters. We assessed carotenoid levels during the molt period, a time of year when finches uniquely metabolize ketocarotenoids (e.g. 3-hydroxy-echinenone) for acquisition of sexually selected red plumage coloration, and found that males infected with MG circulated significantly lower levels of 3-hydroxy-echinenone, but no other plasma carotenoid types, than birds exhibiting no MG symptoms. This result uncovers a key biochemical mechanism for the documented detrimental effect of MG on plumage redness in H. mexicanus. In contrast, we failed to find a relationship between MG infection status and retinal carotenoid concentrations. Thus, we reveal differential effects of an infectious eye disease on carotenoid types and tissue pools in a wild songbird. At least compared to retinal sources (which appear somewhat more temporally stable than other body carotenoid pools, even to diseases of the eye evidently), our results point to either a high physiological cost of ketocarotenoid synthesis (as is argued in models of sexually selected carotenoid coloration) or high benefit of using this ketocarotenoid to combat infection.

Identification of a novel subtype of South River virus (family Bunyaviridae).

We previously reported the isolation of South River virus (SORV) from a pool of mosquitoes collected in the Yucatan Peninsula of Mexico (Farfan-Ale et al. in Vector Borne Zoonotic Dis 10:777-783, 5). The isolate (designated SORV-252) was identified as SORV after a 197-nucleotide region of its small RNA genome segment was sequenced. In the present study, the complete small and medium RNA genome segments and part of the large RNA genome segment of SORV-252 were sequenced and shown to have 92%, 85% and 90% nucleotide sequence identity, respectively, to the homologous regions of the prototype SORV isolate (NJO-94F). To determine the antigenic relationship between SORV-252 and NJO-94F, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice inoculated with these viruses. SORV-252 and NJO-94F were distinguishable in the cross-neutralization assays; there was a twofold difference in the PRNT titers in one direction and a fourfold difference in the other direction, suggesting that SORV-252 represents a novel subtype of SORV. Additionally, SORV-252 and NJO-94F have distinct plaque morphologies in African green monkey kidney (Vero) cells. In conclusion, we provide evidence that a novel subtype of SORV is present in the Yucatan Peninsula of Mexico.

Levels of pathogen virulence and host resistance both shape the antibody response to an emerging bacterial disease.

Quantifying variation in the ability to fight infection among free-living hosts is challenging and often constrained to one or a few measures of immune activity. While such measures are typically taken to reflect host resistance, they can also be shaped by pathogen effects, for example, if more virulent strains trigger more robust immune responses. Here, we test the extent to which pathogen-specific antibody levels, a commonly used measure of immunocompetence, reflect variation in host resistance versus pathogen virulence, and whether these antibodies effectively clear infection. House finches (Haemorhous mexicanus) from resistant and susceptible populations were inoculated with > 50 isolates of their novel Mycoplasma gallisepticum pathogen collected over a 20-year period during which virulence increased. Serum antibody levels were higher in finches from resistant populations and increased with year of pathogen sampling. Higher antibody levels, however, did not subsequently give rise to greater reductions in pathogen load. Our results show that antibody responses can be shaped by levels of host resistance and pathogen virulence, and do not necessarily signal immune clearance ability. While the generality of this novel finding remains unclear, particularly outside of mycoplasmas, it cautions against using antibody levels as implicit proxies for immunocompetence and/or host resistance.

Universal primers for the amplification and sequence analysis of actin-1 from diverse mosquito species.

We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were designed after actin cDNA sequences of Anopheles gambiae, Bombyx mori, Drosophila melanogaster, and Caenorhabditis elegans. The efficacy of these primers was determined by RT-PCR with the use of total RNA from mosquitoes belonging to 30 species and 8 genera (Aedes, Anopheles, Culex, Deinocerites, Mansonia, Psorophora, Toxorhynchites, and Wyeomyia). The RT-PCR products were sequenced, and sequence data were used to design additional primers. One primer pair, denoted as Act-2F (5'-ATGGTCGGYATGGGNCAGAAGGACTC-3') and Act-8R (5'-GATTCCATACCCAGGAAGGADGG-3'), successfully amplified an RT-PCR product of the expected size (683-nt) in all mosquito spp. tested. We propose that this primer pair can be used as an internal control to test the quality of RNA from mosquitoes collected in vector surveillance studies. These primers can also be used in molecular experiments in which the detection, amplification or silencing of a ubiquitously expressed mosquito housekeeping gene is necessary. Sequence and phylogenetic data are also presented in this report.

Genomic sequence and phylogenetic analysis of Culex flavivirus, an insect-specific flavivirus, isolated from Culex pipiens (Diptera: Culicidae) in Iowa.

Adult mosquitoes (Diptera: Culicidae) were collected in 2007 and tested for specific viruses, including West Nile virus, as part of the ongoing arbovirus surveillance efforts in the state of Iowa. A subset of these mosquitoes (6,061 individuals in 340 pools) was further tested by reverse transcription-polymerase chain reaction (RT-PCR) using flavivirus universal primers. Of the 211 pools of Culex pipiens (L.) tested, 50 were positive. One of 51 pools of Culex tarsalis Coquillet was also positive. The flavivirus minimum infection rates (expressed as the number of positive mosquito pools per 1,000 mosquitoes tested) for Cx. pipiens and Cx. tarsalis were 10.3 and 1.2, respectively. Flavivirus RNA was not detected in Aedes triseriatus (Say) (52 pools), Culex erraticus (Dyar & Knab) (25 pools), or Culex territans Walker (one pool). Sequence analysis of all RT-PCR products revealed that the mosquitoes had been infected with Culex flavivirus (CxFV), an insect-specific virus previously isolated in Japan, Indonesia, Texas, Mexico, Guatemala and Trinidad. The complete genome of one isolate was sequenced, as were the envelope protein genes of eight other isolates. Phylogenetic analysis revealed that CxFV isolates from the United States (Iowa and Texas) are more closely related to CxFV isolates from Asia than those from Mexico, Guatemala, and Trinidad.

Detection of Mycoplasma gallisepticum in House Finches ( Haemorhous mexicanus) from Arizona.

In 1994, an endemic poultry pathogen, Mycoplasma gallisepticum (MG), was identified as the causative agent of a novel disease in house finches ( Haemorhous mexicanus). After an initial outbreak in Maryland, MG spread rapidly throughout eastern North American populations of house finches. Subsequently, MG spread slowly through the northern interior of North America and then into the Pacific Northwest, finally reaching California in 2006. Until 2009, there were no reports of MG in the southwestern United States east of California. In August 2011, after reports of house finches displaying conjunctivitis characteristic of MG infection in Arizona, we trapped house finches at bird feeders in central Arizona (Tempe) and southern Arizona (Tucson and Green Valley) to assay for MG infection. Upon capture, we noted whether birds exhibited conjunctivitis, and we collected choanal swabs to test for the presence of MG DNA using PCR. We detected MG in finches captured from Green Valley (in ∼12% of birds captured), but not in finches from Tucson or Tempe. Based on resampling of house finches at these sites in July 2014, we suggest that central Arizona finches likely remain unexposed to MG. We also suggest that low urban connectivity between arid habitats of southern and central Arizona or a reduction in the prevalence of MG after its initial arrival in Arizona may be limiting the spread of MG from south to north in Arizona. In addition, the observed conjunctivitis-like signs in house finches that were negative for MG by PCR may be caused primarily by avian pox virus.

Rapid Antagonistic Coevolution in an Emerging Pathogen and Its Vertebrate Host.

Host-pathogen coevolution is assumed to play a key role in eco-evolutionary processes, including epidemiological dynamics and the evolution of sexual reproduction [1-4]. Despite this, direct evidence for host-pathogen coevolution is exceptional [5-7], particularly in vertebrate hosts. Indeed, although vertebrate hosts have been shown to evolve in response to pathogens or vice versa [8-12], there is little evidence for the necessary reciprocal changes in the success of both antagonists over time [13]. Here, we generate a time-shift experiment to demonstrate adaptive, reciprocal changes in North American house finches (Haemorhous mexicanus) and their emerging bacterial pathogen, Mycoplasma gallisepticum [14-16]. Our experimental design is made possible by the existence of disease-exposed and unexposed finch populations, which were known to exhibit equivalent responses to experimental inoculation until the recent spread of genetic resistance in the former [14, 17]. Whereas inoculations with pathogen isolates from epidemic outbreak caused comparable sub-lethal eye swelling in hosts from exposed (hereafter adapted) and unexposed (hereafter ancestral) populations, inoculations with isolates sampled after the spread of resistance were threefold more likely to cause lethal symptoms in hosts from ancestral populations. Similarly, the probability that pathogens successfully established an infection in the primary host and, before inducing death, transmitted to an uninfected sentinel was highest when recent isolates were inoculated in hosts from ancestral populations and lowest when early isolates were inoculated in hosts from adapted populations. Our results demonstrate antagonistic host-pathogen coevolution, with hosts and pathogens displaying increased resistance and virulence in response to each other over time.

Detection of flaviviruses and orthobunyaviruses in mosquitoes in the Yucatan Peninsula of Mexico in 2008.

A total of 191,244 mosquitoes from 24 species were collected in the Yucatan Peninsula of Mexico from January to December 2008, and tested for the presence of cytopathic virus by virus isolation in Vero cells. Eighteen virus isolates were obtained, all of which were orthobunyaviruses. These were identified by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing as Cache Valley virus (n=17) and South River virus (n=1). A subset (n=20,124) of Culex quinquefasciatus collected throughout the year was further tested by RT-PCR using flavivirus-specific primers. Flavivirus RNA was present in this mosquito species year-round. The overall flavivirus minimal infection rate, expressed as the number of positive mosquito pools per 1000 mosquitoes tested, was 7.7 and the monthly flavivirus minimal infection rates ranged from 4.3 to 16.6. Approximately one-third of the RT-PCR products were sequenced and all corresponded to Culex flavivirus, a recently discovered insect-specific flavivirus.