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Sex and nest type inuence avian blood parasite
prevalence in a high elevation bird community
Marina D. Rodriguez ( mdrod@colostate.edu )
Colorado State University https://orcid.org/0000-0002-7802-8623
Paul F. Doherty
Colorado State University
Antoinette J. Piaggio
USDA National Wildlife Research Center
Kathryn P. Huyvaert
Colorado State University
Research
Keywords: Haemosporidia, Haemoproteus, Plasmodium, occupancy modeling
DOI: https://doi.org/10.21203/rs.3.rs-67068/v3
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Background - Prevalence of avian haemosporidian parasites and the factors inuencing infection in the
Colorado Rocky Mountains are largely unknown. With climate change expected to promote the expansion
of vector and avian blood parasite distributions, baseline knowledge and continued monitoring of the
prevalence and diversity of these parasites is needed.
Methods - Using an occupancy modeling framework, we conducted a survey of haemosporidian parasite
species infecting an avian community in the Colorado Rocky Mountains in order to estimate prevalence
and diversity of blood parasites and to investigate species-level and individual-level characteristics that
may inuence infection.
Results - We estimated prevalence and diversity of avian haemosporidia across 24 bird species, detecting
39 parasite haplotypes. We found that open cup nesters have higher Haemoproteus prevalence than
cavity or ground nesters. Additionally, we found that male Ruby-crowned Kinglets, White-crowned
Sparrows, and Wilson’s Warblers have higher Haemoproteus prevalence compared to other host species.
Conclusions - Our study presents baseline knowledge of haemosporidian parasite presence, prevalence,
and diversity among avian species in the Colorado Rocky Mountains and adds to our knowledge of host-
parasite relationships of blood parasites and their avian hosts.
Background
Parasitism is an important driver of ecological and evolutionary processes [1-2] as parasites may regulate
host population size, e.g.,Hochachka and Dhondt [3], affect species interactions,. e.g., Ricklefs [4], and
create selection pressures in wild populations, e.g., Laine [5]. Compounding effects of parasites with other
factors such as climate change, competition with invasive species, habitat loss, or harsh environmental
conditions can also drive populations to low numbers, predisposing them to local or global extinctions [6-
9].
Haemosporida (Phylum: Apicomplexa) are protozoan parasites that infect the blood cells of vertebrates
and are transmitted by dipteran vectors [10]. These blood parasites – haemosporidian parasites – are
distributed worldwide and infect a number of vertebrates, including mammals [11], reptiles [12], and birds
[10]. Blood parasites go through sexual reproduction in dipteran vectors and are transmitted to vertebrate
hosts during vectors’ blood meals [10]. Once in a competent host, the parasite undergoes asexual
reproduction and the infected host becomes a reservoir, carrying developed gametocytes within its red
blood cells [10].
Among vertebrates, birds are hosts to the highest diversity of haemosporidian parasites, with records of
birds being infected with over 200 morphologically distinct haemosporidian parasite species [10, 13, 14]
and over 3000 unique haplotypes [13]. The three parasite genera that infect birds include
Haemoproteus
,
Plasmodium
, and
Leucocytozoon
[10 and 15]. Negative effects of infection can be due to changes in host
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behavior [16] or to severe physiological responses, resulting in high mortality rates during the acute phase
of infection [17-18]. Avian hosts can also suffer declines in reproductive success [19-20] and reduced
lifespan when enduring chronic infections [21]. Within species, factors such as age, sex, immune status,
and degree of exposure may also contribute to variation in host susceptibility and mortality [15].
Spatial and temporal dynamics of avian haemosporidian parasite occurrence are governed by
environmental, ecological, and demographic characteristics [22-25]. In temperate environments,
seasonality has a strong inuence on survival and development of both parasites and insect vectors, as
mosquitoes emerge during the spring and are active until the end of the summer [26]. This increase in
parasites and vectors coincides with the breeding season for most avian species, when resource
allocation is diverted to reproduction instead of immune function [27, 28]. Our study took place during the
breeding season, allowing us to survey avian blood parasites at a time when infection frequency is
expected to be highest.
The intensity and seasonality of haemosporidian parasite transmission tends to vary by elevation [29].
Negative correlations between elevation and abundance of mosquitos, the main vectors of avian blood
parasites, have been found in many systems including the mountains in Colorado’s Front Range [30-31],
where our study site is located. Many parasites have elevation limits because of the constraints of lower
ambient temperatures encountered at higher elevations, though distributions are expanding with climate
change as transmission of most vector-borne parasites may be enhanced by higher ambient
temperatures, e.g. Samuel et al. [32], La Pointe et al. [33]. Changes in environmental conditions for
vectors, such as an increase in mean air temperature and declining precipitation, support the expansion
of haemosporidian parasites into habitats where lower temperatures previously limited transmission [34-
35]. For example,
Culex tarsalis
and
C. pipiens
are important vectors of
Plasmodium
parasites at lower
elevations in northeastern Colorado but the low abundance of these species at higher elevations may
mean that
Plasmodium
is not yet established in areas such as Rocky Mountain National Park [36].
However, little research has been done on the distribution of haemosporidian parasites in Colorado,
especially in high elevation communities like those in the Colorado Rockies.
Across species, factors such as nest type and migration strategy may explain variation in host
susceptibility. Although differences in parasite prevalence across nest types is not always found [37-39],
open-cup nesting has been linked to higher blood parasite prevalence in numerous studies due to higher
vector exposure for incubating individuals compared to cavity or ground nesting birds [40-44]. Migration
has important implications for the emergence and spread of infectious disease-causing parasites due to
long-distance movements and exposure to diverse habitats of infected hosts. Establishment of parasites
and expansion of their ranges may take place through migration of host species as parasites are able to
survive at higher elevations as environmental conditions become more suitable for parasites and vectors
[45]. Migratory birds can harbor high intensity infections and are host to biologically diverse
haemosporidian parasite species [4], allowing them to act as a source of infection to non-migratory birds
who may be more susceptible to blood parasites due to lack of previous exposure at higher elevations
[46-47]. With the potential for migratory birds to spread avian blood parasites to new areas, and a
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warming climate allowing for the spread of blood parasites into new environments, parasite surveillance
is needed in bird communities, even those to previously thought to be in areas with low parasite
prevalence.
Parasite surveys can serve as early indicators of disease outbreaks that could affect the health of avian
populations. The study of blood parasites requires knowledge of baseline levels of haemosporidian
parasite infection in target host populations to aid in detection of temporal shifts of parasite diversity to
evaluate changes in prevalence of infection. Using an occupancy approach, we conducted multiple
screenings for blood parasites per host in order to better estimate detection probability of
haemosporidian parasites within host species [48-49]. Occupancy modeling approaches are useful in
wildlife disease ecology because they acknowledge that uncertainty, such as false negative results, exist
when using imperfect diagnostic tests [50-51]. Variation in detection among multiple screenings of the
same blood sample supports the need to use an occupancy modelling framework in order to take
detection probability into account in wildlife disease studies [49].
The objectives of our study were to: 1) obtain baseline prevalence and diversity estimates for an avian
community in the Colorado Front Range Rocky Mountains where avian parasite surveys have not taken
place, and 2) to test differences in prevalence and diversity for various individual and species
characteristics. Our hypotheses related to the second objective regarding specic host and environmental
predictor variables are presented in Table 1.
Methods
Study system
Our study area was located at the Colorado State University Mountain Campus in Larimer County,
Colorado, USA (N40.5611, W105.5978), within a mountain valley at an elevation of 2,750 meters. The
valley is a breeding site for numerous bird species and no prior research on avian blood parasites has
been conducted there to our knowledge.
Data collection
We collected data during the summers of 2017 and 2018. The eld portion of our study began in early
June when birds begin breeding and continued through the end of the breeding season, around mid-
August. We captured birds using mist nets set in sites with high passerine activity. Netting sites were in
riparian, forested, and edge habitats. Song playbacks were used to attract birds to nets, providing larger
sample sizes to facilitate comparisons of parasite prevalence across host species.
Captured birds were identied at the species level and banded. Sex and age were determined when
possible based on guidelines from Pyle [52], morphological measurements were taken (tarsus length
(mm), wing chord (mm), mass (g)), and 10-20 µl of blood were collected by brachial venipuncture and
stored on Nobuto Blood Filter Strips for later DNA extraction. All birds were handled and sampled under a
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Federal Bird Banding permit from the USGS Bird Banding Laboratory and in accordance with approved
guidelines of the Institutional Animal Care and Use Committee of Colorado State University (Protocol 17-
7309A).
DNA extraction, PCR amplication, and sequencing
We assessed Haemosporidian parasite infection prevalence and parasite diversity using molecular
techniques. We took a 2 mm hole punch of blood-soaked Nobuto Blood Filter Strip (with ~15 µL of blood)
for each bird and extracted DNA using the Qiaquick DNeasy 96 Blood and Tissue kit (Qiagen, Valencia,
CA), following the manufacturer’s dried blood spot protocol. We stored extracted DNA at -20 °C prior to
screening. We screened an aliquot of DNA for parasite presence using a nested polymerase chain
reaction (PCR) protocol to amplify a segment of mitochondrial DNA (mtDNA) from the cytochrome
b
gene as outlined in Hellgren et al. [53]. Primers HaemNF1 and HaemNR3 were used to amplify an initial
617-bp segment of mtDNA from species of haemosporidian parasites. The conditions for this PCR were
as follows: 30 seconds at 94°C, 30 seconds at 50°C, and 45 seconds at 72°C for 20 cycles. The samples
were incubated before the cyclic reaction at 94°C for 3 minutes and after the cyclic reaction at 72°C for 10
minutes. An aliquot of the product (1 µL) from the rst PCR reaction was used in a second reaction
amplifying a 479-bp segment of
Haemoproteus
and
Plasmodium
lineages using primers HaemF and
HaemR2 [53]. The conditions of the second round of PCRs are as follows: 30 seconds at 50°C, and 45
seconds at 72°C for 35 cycles. PCR screening was repeated three times for each sample, and each plate
(96 samples) included two positive controls (one for
Haemoproteus
and one for
Plasmodium
) and one
negative control. All PCR reactions were performed at a nal volume of 25 µl using illustra PureTaq
Ready-To-Go™ beads (GE Healthcare) with freeze-dried, pre-formulated reagents. We ran 5 µl of the nal
product on a 2% agarose gel to screen for parasite presence. For host individuals infected with
haemosporidian parasites, the nal PCR product was cleaned with ExoSAP (ThermoFisher Scientic)
prior to sequencing.
The sequencing reaction of 10 μl contained 0.25 μl BigDye™ (Thermo Fisher Scientic), 2.275 μl BigDye™,
1 µl of each nested secondary PCR primer (HaemF and HaemR2), 1 μl of PCR product, and 5.475 μl
molecular grade ddH2O. Cycle sequencing was conducted at 94 C for 2 min; 40 cycles of amplication
at 85 C for 10 s; 53 C for 10 s and 60 C for 2.5 min. The sequencing reactions were cleaned-up using
600 μl of Sephadex® G-50 solution per sample prior to analysis on an automated ABI 3500 Genetic
Analyzer. Forward and reverse reads were assembled and edited using Geneious Prime 2019.0.4
(https://www.geneious.com). Mixed sequences, as indicated by double peaks in a chromatogram, were
considered co-infections [44] We identied all sequences at the genus level using the Basic Local
Alignment Search Tool (BLAST) feature in the MalAvi database [13]( http://mbio-
serv2.mbioekol.lu.se/Malavi/), a database for avian blood parasites. Mitochondrial haplotypes, that is,
sequences differing by one or more bases (<100% identity) from known parasite lineages, were
considered unique lineages [53].
Statistical Analyses
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We repeated each PCR assay three times for each DNA sample in order to obtain a parasite detection
history composed of 1s and 0s for each individual with 1 signifying at least one detected parasite and 0
indicating no parasite detected. With this detection history, we estimated the probability of parasite
detection along with the proportion of individuals infected with blood parasites, corrected for detection
probability. We analyzed detection histories for avian haemosporidian parasites using the single-season
occupancy model in Program MARK [54] to estimate prevalence (i.e., occupancy) of each genus of
parasite for each bird species as well as across species [55 and 49]. In a typical occupancy framework,
randomly selected “sites” are surveyed on multiple occasions within a period where occupancy state is
assumed not to change. Repeated survey occasions at each site allow estimation of two parameters:
occupancy (ψ), the probability that a site is occupied by the species of interest, and detection probability
(p), the probability that the species is detected during a given occasion if the site is occupied [56]. In our
study, each blood sample from an individual bird is analogous to a site, the species of interest are
Haemoproteus
and
Plasmodium
parasites, and the repeated survey occasions are multiple replicates of
PCR assays for each DNA sample. Reinterpreting the model parameters for parasite detection gives ψi as
the prevalence of a parasite infection, and
p
i as the probability of detecting a parasite(s) in site
i
, given
the presence of the parasite(s) in the host.
We carried out analyses for
Haemoproteus
and
Plasmodium
parasite prevalence separately. We
constructed a candidate model set for an all-species analysis that included all birds captured and
sampled, as well as a species-specic candidate model set for each host species with at least 20 DNA
samples (ten species). In order to address any individual heterogeneity that may exist, we used the
random effects model in Program MARK to incorporate any heterogeneity beyond our predictions in each
model. Our model set consisted of all possible combinations of predictor variables (Table 1) and we used
an information-theoretic approach for model ranking and selection [57]. We calculated Akaike weights (wi;
the weight of evidence in favor of each model being the best model compared to the rest of the models in
the set) and considered the variables with a cumulative weight greater than 0.5 to be the most important
[58].
Results
In 2017, we captured 232 birds, and in 2018, we captured 206 birds. Of the 438 birds captured, 180 were
males, 206 were females, and for the remainder sex could not be determined. We captured 24 hatch-year
birds, 135 second-year birds, 241 after second-year birds, and we could not determine age in the
remaining 38 birds. Body condition indices (mass:tarsus) ranged from 0.20 to 25.71 g/mm. We collected
molecular data from a total of 437 birds belonging to 24 species over the two years of the study (Table
2).
Haemosporidian Parasite Diversity
In total, we detected 10
Plasmodium
and 29
Haemoproteus
cytochrome b haplotypes. Thirty-three
haplotypes had a 100% match to current sequences deposited in the Malavi database (Table 2) [13], and
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the other 6 sequences were considered novel haplotypes (GenBank accession numbers MW147676,
MW147677, MW147678, MW147679, MW147680, MW147681). The most common
Haemoproteus
lineages were TURDUS2 and SISKIN1, which were detected in 15 and 13 individuals, respectively. The
most common
Plasmodium
lineage was PADOM11, which was detected in 6 individuals. Only 1 Warbling
Vireo (
Vireo gilvus
) and 2 Lincoln’s Sparrows (
Melospiza lincolnii
) were found to be infected with both
Plasmodium
and
Haemoproteus
. The greatest parasite diversity was obtained from the Wilson’s Warbler
(
Cardellina pusilla
; 13), the Warbling Vireo (12), and the White-crowned Sparrow (
Zonotrichia leucophrys
;
11), which also had some of the largest sample sizes.
Haemoproteus
We detected
Haemoproteus
parasites in 109 out of 437 birds, a naïve (without taking detection
probability or covariates into account)
Haemoproteus
prevalence of nearly 25% (109/437). Nest type and
year were considered important variables associated with
Haemoproteus
prevalence in the all-species
analysis, with variable weights of 0.54 and 0.85 (Table 3). Open-cup nesters in 2018 had the highest
Haemoproteus
overall prevalence, estimated at 38%, 95% CI: 26.24%- 49.76% (Figure 1). Prevalence was
similar for cavity and ground nesters both years and was higher in 2018. In the all-species analysis, we
found no evidence of unmodeled heterogeneity using a random effects model (Additional File 1:Table
S1). PCR replicate was an important variable when considering detection probability, with a variable
weight of 0.99. Detection probability was estimated at 0.62 (±0.07 SE) for the rst PCR run, 0.38 (±0.07
SE) for the second PCR run, and 0.50 (±0.07 SE) for the third PCR run (Figure 2).
Of all species, the Warbling Vireo, American Robin (
Turdus migratorius
), and Wilson’s Warbler had the
highest naïve
Haemoproteu
s prevalences, at 59%, 35%, and 32%, respectively. In the species-specic
analyses (Additional les: Tables S2-S11), all species had an estimated individual heterogeneity of nearly
zero. In terms of prevalence (ψ), sex was considered an important variable for the Ruby-crowned Kinglet
(
Regulus calendula
), the White-crowned Sparrow, and the Wilson’s Warbler (Figure 3), with variable
weights of 0.57, 0.54, and 0.51, respectively (Table 4). BCI was also considered an important covariate of
prevalence for the Red-breasted Nuthatch (
Sitta canadensis
) and the Ruby-crowned Kinglet (Figure 4),
with variable weights of 0.69 and 0.84, respectively (Table 5). No species had variable weights above 0.5
for age or year. For detection probability (p), PCR run was an important variable for the Lincoln’s Sparrow
and the White-crowned Sparrow, with variable weights of 0.96 and 0.50, respectively (Figure 5).
Plasmodium
We detected
Plasmodium
parasites in at least one PCR replicate in 23 out of 437 birds, which is a total
naïve
Plasmodium
prevalence of 5.3%. When analyzing all species together, we detected no heterogeneity
using a random effects model. We found no predictor variable with a cumulative variable AICc weight of
at least 0.5, and therefore none of our hypothesized variables were considered important in predicting
Plasmodium
infection in the all-species analysis.
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The Wilson’s Warbler and Lincoln’s Sparrow had the most infected individuals per species, with three birds
positive for
Plasmodium
in each. Because of the low number of positives per species, species-specic
analyses could not be carried out for
Plasmodium
prevalence.
Discussion
In this study, we present baseline knowledge of haemosporidian parasite presence, prevalence, and
diversity across a suite of avian species in the Colorado Rocky Mountains. Among the 438 birds of 24
species sampled, thirty-nine unique haemosporidian parasite haplotypes were detected, 21 host species
had at least 1 infected individual, and
Haemoproteus
parasites had a larger host-breadth and occurred at
much higher prevalence compared to
Plasmodium
. In addition, 6 novel haplotypes were detected among
3 different species. Using an occupancy-modelling framework to account for imperfect detection of avian
blood parasites, we found that nest type is an important species-level factor inuencing
Haemoproteus
parasitism at our study site, with open cup nesters having a higher prevalence compared to cavity and
ground nesters. We also found that sex and BCI are important individual-level factors associated with
Haemoproteus
parasitism in some species, with males and birds with higher BCI having a higher blood
parasite prevalence. In addition, we found that out of the 24 hatch-year birds that were sampled during
the study, 7 of them were found to be positive for
Haemoproteus
blood-parasites, meaning that local
transmission was taking place at our sample site.
Haemosporidian lineage diversity
Diversity of haemosporidian parasites in wild birds was high, with a total of 39 lineages of
Haemoproteus
and
Plasmodium
from 21 of the 24 avian species that were sampled (Table 2).
Plasmodium
parasites are
considered generalists in terms of host breadth, while
Haemoproteus
are generally considered more host-
specic [53]. However, we identied
Haemoproteus
in a wider range of bird species than
Plasmodium
and
found that
Haemoproteus
parasites were more prevalent overall.
Despite the small sample sizes for some host species, some patterns were still apparent largely in the
family Turdidae. One lineage, TURDUS2, infects many families of birds throughout Europe [60], Asia [61],
and the United States [62], yet most detections have occurred in the Turdidae, including robins and other
thrushes. Accordingly, American Robins, at our study site had the highest proportion of TURDUS2
detections compared to other species and may act as a reservoir for this parasite lineage. The lineage
TUMIG07, has only been detected in the American Robin and Hermit Thrush (
Catharus guttatus
) in Alaska
[62]. In our study, TUMIG07 was present in these species and six other hosts at our site. The VIGIL07
lineage has only been detected in the Vireonidae family in California [63], New Mexico (Marroquin-Flores
unpublished data), and Michigan [42], and, in our study, the Warbling Vireo had the highest proportion of
positive individuals . Of the 7 detections of the TUMIG08 lineage in the MalAvi database, 4 have been
from the American Robin [62]. Accordingly, 2 of the 4 detections of this lineage at our study site were
from American Robins, with one detection in a Lincoln Sparrow, and the other in a Warbling Vireo. The
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POETR01 lineage has been mainly detected in the Thrush family as well [61], and our one detection of
this lineage was in an American Robin in agreement with previous detections.
Patterns across host species
When analyzing
Haemoproteus
prevalence across host species, nest type and year were important
variables associated with infection (Figure 1). Although some studies have found contradicting results
[37, 38, and 64], open-cup nesting has been linked to higher
Haemoproteus
prevalence in many studies,
e.g., Gonzales et al. [40], Smith et al. [42], Fechio [43]. Higher prevalence in open-cup nesters may indicate
that
Haemoproteus
vectors – biting midges – are more likely to come in contact with species that have
open-cup nests than with ground or cavity nesters, perhaps because open-cup nesters are more
vulnerable to exposure to blood feeding by the vectors.
Overall
Haemoproteus
prevalence also displayed marked variation between years in our study, with 2018
higher than 2017 (Figure 3). Interannual variation in avian blood parasite prevalence is common and has
been found in many studies, e.g., Bensch et al. [64], Wood et al. [23], Lachish et al. [24], Podmokła et al.
[65]. One potential explanation is that the vectors responsible for
Haemoproteus
transmission uctuate in
abundance in response to weather variation (e.g., temperature and rainfall), which alters the habitat and
microclimate they require for breeding to conditions that are more favorable for vectors. Alternatively,
annual variation in host demography and population dynamics could also play a role in driving this
annual variation in prevalence [66-67].
Age, sex, BCI, and migration were not considered important variables across species in our study
although they have been linked to higher haemosporidian parasite prevalence in other studies, e.g.,
Hatchwell et al. [68], Deviche et al. [69], Garvin et al. [70], and Calero-Riestra and Garcia [71]. Further
studies are needed to address the inuence of host traits on patterns of avian haemosporidian parasite
infection and to determine whether such patterns exist and persist at larger spatial scales and across a
wider host-parasite community.
When analyzing
Plasmodium
prevalence among host species, no associations were found between
prevalence and species-level traits, likely due to the low number of individuals that were positive for the
parasite. Elevation governs the distribution of parasites belonging to different genera, with
Plasmodium
parasites being more prevalent at lower altitudes and
Haemoproteus
parasite prevalence increasing with
elevation [25] and our site is very high which may expalin the lack of Plasmodium detections.
Accordingly, Eisen et al. [36] found that
Culex
spp. mosquitoes, the main vectors of
Plasmodium
parasites, had not yet established in areas in and around Rocky Mountain National Park, at similar
elevations as our study site. Associations between exposure to mosquitoes and
Plasmodium
prevalence
across host species has been demonstrated [28], supporting the idea that
Plasmodium
vectors may be
absent, or in low numbers at our study site.
Patterns of Haemoproteus infection within individual species
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Sex was associated with
Haemoproteus
infection in the Ruby-crowned Kinglet, White-crowned Sparrow,
and Wilson’s Warbler (Figure 4). Sex-related differences in haemosporidian parasite prevalence are often
observed in nature, however, sex-bias in parasitism often varies between and within host-parasite
systems [72]. Contrary to our prediction, our study demonstrated a strong male-biased parasite
prevalence in the three species mentioned above, with Ruby-crowned Kinglet having the largest difference
between sexes (53% in males vs. 1% in females). Although the greater stress of reproduction in females
might translate to weakened immune responses and higher prevalence [73], there is overwhelming
evidence that sex-associated hormones can directly inuence the differential susceptibility of each sex to
infections [74]. For example, testosterone has immunosuppressive effects in many species, leading to
higher susceptibility of males to parasite infections (75-77]. This is not the case for every host-parasite
relationship, as was illustrated by the lack of an association between parasite prevalence and sex in the
other seven species that we analyzed.
Body condition index (BCI) was positively associated with
Haemoproteus
infection in the Red-breasted
Nuthatch and the Ruby-crowned Kinglet when analyzed separately (Figure 5). Similar results have been
found in the American Kestrel (
Falco sparverius
), the Yellow-rumped Warbler (
Setophaga coronate
), and
the Great Tit (
Parus majo
r) [78-79]. Although the reason for this positive correlation is unknown, it may be
due to infected individuals having lower capture probability. If infected individuals with low body
condition are less active and are less likely to y into mist nets, that leaves only infected individuals with
greater body condition to be caught. Similarly, if individuals with low body condition are unable to survive
the acute stage of
Haemoproteus
infection, then this may leave more infected individuals with higher
body condition. The eight other species in our study did not show an apparent relationship between
prevalence and BCI, which is a common result in wildlife studies given that host condition and its
responsiveness to infection could change in response to foraging resources that uctuate in space and
time [80-81]. Some parasites cause minimal or no effects on condition in certain host taxa [81] and some
infections might only exert negative tness effects during stressful periods or under resource limitation
[82-83].
We found no relationship between
Haemoproteus
prevalence and individual-level traits (sex, age, BCI) in
the American Robin, Mountain Chickadee (
Poecile gambeli
), Pine Siskin (
Carduelis pinus
), or Dark-eyed
Junco (
Junco hyemalis
), contrary to our hypotheses. Our results suggest that the individual-level traits
examined in this study may not be important predictors of
Haemoproteus
infection for all species.
Detection probability
PCR replicate was an important variable associated with detection probability for
Haemoproteus
infection for three species (Lincoln Sparrow, Warbling Vireo, and White-Crowned Sparrow) as well as for
the all-species analysis, with PCR results varying among the three PCR runs (Figures 2 and 5). Nested
PCR assays for haemosporidian parasites are known to be vulnerable to false negative results for
samples with low parasite intensities [84] and is most likely responsible for the variation we found
between PCR replicates.
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Detection probability for
Plasmodium
parasites was lower than for
Haemoproteus
consistent with the
idea of lower detection in blood samples [85] because
Plasmodium
enters latent, exoerythrocytic phases
during chronic infection and may even be absent in the blood stream [10]. Thus, sampling peripheral
blood may not allow for detection of all true infections with
Plasmodium
, leading to underestimates of
prevalence.
Conclusion
Our results suggest that open cup nesting birds in the Colorado Rocky Mountains are commonly infected
with avian blood parasites and that male Ruby-crowned Kinglets, White-crowned Sparrows, and Wilson’s
Warblers have higher prevalence compared to females of these species. We present baseline knowledge
of blood parasite presence, prevalence, and diversity across avian species in the Colroado Rocky
Mountains, providing a strong foundation for subsequent studies. With climate change expected to
support the expansion of avian blood parasite distributions, such that monitoring avian haemosporidian
parasites should continue in order to detect potential climate-related changes in prevalence and diversity
over time. Our study is the only avian blood parasite survey conducted in the Colorado Rocky Mountains,
to date; additional research in this area examining host-parasite relationships would help to determine
whether anthropogenic changes – such as climate change – leading to potential changes in vector
communities or parasite distributions may pose a threat to resident avian populations.
Declarations
Ethics approval and consent to participate – Permission for capturing and taking blood samples from
birds was approved by the Colorado State University Institution for Animal Care and Use Committee
(Protocol 17-7309A).
Consent for publication – Not applicable.
Availability of data and materials - The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Competing interests - The authors declare that they have no competing interests.
Funding – This study was funded in part by the Colorado Chapter of The Wildlife Society’s Small Grants
Program and the National Science Foundation’s Graduate Research Fellowship Program.
Authors' contributions – MR and KH conceived the idea. MR, KH, and PD designed the eld study. MR
carried out the laboratory work with the instruction and laboratory equipment from KH and AP. MR
conducted the analysis with guidance from PD. MR wrote the paper and KH, PD, and AP reviewed the
manuscript.
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Acknowledgements – We would like to thank the Colorado Chapter of the Wildlife Society for funding
through their Small Grants Program. Our sincere thanks to Seth Webb and all of the staff Colorado State
University Mountain Campus for approval and support in collecting avian blood samples on the
premises. We thank eld assistants Arlene Cortez and Martin Rodriguez, as well as laboratory assistants
Jarred Bland and Valeria Aspinall. Thanks to Doreen Grin for sequence training and guidance.
Permission for capturing birds and taking blood samples from birds in Colorado was approved by the
Colorado State University Institution for Animal Care and Use Committee (Protocol 17-7309A).
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Tables
Due to technical limitations, table 1,2,3,4 is only available as a download in the Supplemental Files
section.
