Phenology underlies apparent urbanisation effects on avian malaria in juvenile songbirds

Phenology underlies apparent urbanisation effects on avian malaria in juvenile songbirds | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Phenology underlies apparent urbanisation effects on avian malaria in juvenile songbirds View ORCID Profile Davide M Dominoni , View ORCID Profile Bedur Faleh Ali Albalawi , Magdalena Mladenova , View ORCID Profile Barbara Helm , View ORCID Profile Francesco Baldini doi: https://doi.org/10.1101/2025.06.23.661047 Davide M Dominoni 1 School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow , Glasgow, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Davide M Dominoni For correspondence: davide.dominoni{at}glasgow.ac.uk Bedur Faleh Ali Albalawi 2 Department of Biology, University of Tabuk , Tabuk 47512, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bedur Faleh Ali Albalawi Magdalena Mladenova 1 School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow , Glasgow, UK 3 Veeva Systems , Pleasanton, California Find this author on Google Scholar Find this author on PubMed Search for this author on this site Barbara Helm 1 School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow , Glasgow, UK 4 Bird Migration Unit, Swiss Ornithological Institute , Sempach, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Barbara Helm Francesco Baldini 1 School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow , Glasgow, UK 5 Ifakara Health Institute, Environmental Health, and Ecological Sciences Department , Morogoro, United Republic of Tanzania Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Francesco Baldini For correspondence: davide.dominoni{at}glasgow.ac.uk Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Urbanisation can modify species interactions, including those between parasites and their hosts. In birds, urbanisation can either increase or decrease avian malaria infection, depending on host species, parasite or study location. However, temporal coordination between parasites and hosts, which may impact infection outcomes, has not been studied in urban ecology. To fill this gap, we collected blood samples from wild blue tit nestlings (Cyanistes caeruleus) in urban and forest habitats to examine how their hatch dates affected the prevalence and intensity of malaria infection. To separate parasites and quantify parasite load, we newly developed a species-specific qPCR assay. We found that Leucocytozoon prevalence was strongly affected by urbanisation, but effects depended on study year. This was driven by hatch date: nestlings that hatched earlier in the spring had a lower probability of being infected, independent of habitat type. In the few heavily infected nestlings, intensity of infection was associated with low body weight, suggesting fitness effects of infection. These results highlight the importance of breeding early to avoid early-life infection with malaria parasites, and that apparent urbanisation effects on infection arose from phenological differences between urban and forest habitats. Underappreciated phenological changes may underline other ecological effects of urbanisation. Introduction As human populations continue to grow and cities expand into previously undeveloped areas, wildlife encounters with humans and human activities become more frequent [ 1 , 2 ]. This often leads to a restructuring of species composition and biodiversity via changes in several ecological processes, including species interactions [ 3 ]. An example of species interactions that may be affected by urbanisation are those between wildlife and their pathogens [ 4 – 7 ]. Evidence suggests that urban areas act as hotspots for the emergence of zoonotic diseases [ 5 , 8 , 9 ], including vector-borne diseases such as West Nile virus, Dengue fever, and Lyme disease [ 10 – 12 ]. These diseases can also be transmitted from wildlife to humans, thus posing risks to public health [ 13 , 14 ]. The anticipated surge in urbanisation worldwide is posed to further exacerbate disease risks [ 15 , 16 ], requiring a better understanding of host-parasite interactions. Avian malaria parasites have been of central interest in the ecological literature of host-parasite interactions and vector-borne diseases over recent decades [ 17 ]. Identified in bird species around the world, it can pose significant threats to avian biodiversity, particularly for species in isolated island ecosystems that have not previously been exposed to the disease [ 18 ]. Its wide distribution also enables comparative studies across geographical areas and ecological contexts, including urban environments [ 17 , 19 ]. Avian malaria is caused by parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon , which are transmitted through the bite of insect vectors [ 20 ]. These vectors are usually Culicidae mosquitoes in the case of Plasmodium species, Culicoides biting midges for Haemoproteus species and Leucocytozoon caulleryi , and Simulium blackflies for other Leucocytozoon species [ 21 ]. These parasites infect host blood cells (mainly red cells, but in case of Leucocytozoon also the white blood cells), tissues and other major organs such as liver, brain, heart and lungs [ 20 ]. The effects of haemosporidian infections on their host are complex as symptoms in birds can range from undetectable or minor illness to severe disease and death. In many cases, infected birds may show no visible symptoms but can still spread the disease through their vectors [ 20 ], yet infections can also affect various host traits. Examples include parental effort [ 22 , 23 ], body condition and body mass [ 24 , 25 ], global gene expression [ 26 ] as well as specific physiological processes such as iron balance [ 27 ] and telomere loss [ 28 , 29 ]. In turn, these changes have been suggested to negatively affect fitness-related traits, such as reproductive success [ 29 – 31 ] and survival [ 28 , 32 , 33 ], potentially leading to population decline [ 34 ]. Conversely, several studies have pointed to opposite or absent links between malaria infection and fitness [ 35 ]. Effects of malaria often depend on the parasite strain [ 36 ], on the species, population, age, sex or nutritional condition of the host [ 29 , 37 , 38 ] and on environmental conditions (e.g., food availability) during both growth and adulthood [ 39 , 40 ]. Several studies have also suggested that evolution of resistance in response to malaria infection should be more common than currently appreciated [ 18 , 41 ]. Studies of the impact of urbanisation on the prevalence of haemosporidian parasites showed mixed results. The relationship between urbanisation and malaria infection is context-specific and depends on several factors ([ 42 – 47 ], reviewed in [ 48 ]), including effects of year of study, latitude, city, and habitat type of surrounding rural areas (e.g., desert vs tropical rainforest). An overlooked factor that may modulate the impact of urbanisation on malaria parasite prevalence is phenology. Phenology, which affects disease patterns via fluctuations in abundance and condition of hosts, vectors and parasites [ 49 – 51 ], can differ between habitats [ 52 – 54 ]. The emergence and transmission of avian malaria in wild populations is strongly seasonal [ 55 – 58 ], partly due to seasonal changes in composition and abundance of vectors [ 56 , 59 , 60 ] and to seasonal relapse of parasites in birds [ 61 ]; additionally, year-to year variation in malaria prevalence is common [ 62 , 63 ]. Mosquitoes and other malaria vectors, such as midges and black flies, breed in stagnant or flowing water, and their populations can fluctuate depending on seasonal and yearly environmental conditions, such as temperature and rainfall [ 64 , 65 ]. During rainy seasons, when river flows and stagnant water reserves expand, vector populations can increase, thus potentially escalating the transmission of avian malaria. Conversely, in dry seasons or cold winters when vector populations decrease, the transmission of avian malaria is usually low [ 64 ]. In turn, aspects of phenology may differ between urban and non-urban habitats. One example are urban heat islands - areas in the city where temperatures are higher than in surrounding rural areas [ 66 ]. Higher urban temperature may increase the winter survival of vectors and advance both vector and bird phenology [ 67 , 68 ], further extending the transmission season of the disease [ 69 ]. The relative seasonal timing of host life-history stages and vector emergence may be affected by urbanisation, too, for example because species differ in response to ambient temperature [ 70 , 71 ]. Thus, urbanisation can potentially alter the seasonal patterns of avian malaria transmission and modulate its impact on urban bird populations. Importantly, effects of many diseases also differ depending on host condition, physiology, and the timing of host, vector and parasite life-cycle stages in relation to each other [ 50 , 51 ]. For example, reproductive stages can be particularly sensitive to pathogens due to seasonal relapse in adults and developmental processes in offspring [ 72 , 73 ]. Phenological contributions to the prevalence of patent avian malaria infection (when parasites can be detected circulating in the blood rather than dormant in tissues) can be particularly well studied in nestlings, whose time of exposure had only started with hatching. Being naïve to infections, the nestlings’ host response is driven by innate immune responses directly linked to the physiological effects of the parasite (e.g. anaemia, tissue damage), rather than being shaped by acquired immune responses as in adults. Moreover, parasite intensity in nestlings can serve as a useful proxy for estimating the time since infection, as it reflects the progression of the acute phase [ 74 ]. Immediate, intensity-dependent effects — such as reductions in body mass— can therefore provide a clear window into the health costs of infection. Additionally, nestling body mass is a commonly used predictor for a nestling’s future fitness [ 75 ]. Thus, the aim of the present study is to disentangle effects of phenology and urbanisation on malaria in nestling songbirds. We study prevalence and intensity of avian malaria in early postnatal life of blue tits ( Cyanistes caeruleus ), a species that has served as a major study system in urban and disease ecology [ 76 – 79 ]. The blue tit is found across Europe in a variety of habitats, including urban, suburban, and rural environments, where they showed distinct responses to urbanisation [ 80 , 81 ]. Seasonal patterns of avian malaria have been described in both the blue tit and its arthropod vectors [ 49 , 60 , 81 ]. In Scotland and northern England, malaria vector emergence usually occurs in mid-late May, with a peak abundance in July [ 60 , 82 – 84 ]. Conversely, in the same areas blue tit nestlings hatch on average in early-mid-May. In Scotland, blue tit nestlings have been found to be infected with both Haemoproteus and Leucocytozoon parasites, but not Plasmodium [ 60 , 81 ]. The blue tits’ well-described breeding biology and physiology, including in our study populations, can aid in the interpretation of data on avian malaria and its impact. Thus, we here examine how the prevalence and intensity of infection with malaria parasites varied between forest and urban blue tit nestlings in Scotland, while explicitly considering the birds’ breeding phenology. To disentangle factors that might blur associations, we also introduce methodological improvements. Although detection of haemosporidian parasites in host blood is relatively easy using molecular techniques and/or microscopy [ 85 , 86 ] it imposes challenges. The nested PCR protocol (nPCR) developed by Bensch et al. (2000) [ 87 ], and modified later [ 86 ], has been widely used to detect the three genera of Haemosporidia and to assess the reliability and sensitivity of newly developed protocols [ 68 ]. Nevertheless, the nPCR and other PCR-based protocols often overestimate mixed infection because of cross-reactivity of genus-specific primers [ 88 ]. Indeed, in a previous comparison of urban – non-urban blue tits, in nine out of 12 samples that were positive for both Leucocytozoon and Haemoproteus/Plasmodium by nPCR, sequencing revealed that only one of the two parasites – either Leucocytozoon or Haemoproteus – was present [ 81 ]. Thus, the field is in demand of a new sensitive and accurate molecular technique to quantify intensity of, and more importantly distinguish between, parasite genera in avian blood. Here we will apply a novel quantitative PCR approach that can distinguish and quantify Leucocytozoon and Haemoproteus with high sensitivity and specificity, which is particularly important when assessing parasite prevalence and intensity in nestlings when parasite development in peripheral blood is at its early stages. With this approach, we here compare malaria infection in forest and urban blue tit nestlings across multiple years during which the birds’ breeding phenology varied distinctly. Specifically, we address the following questions: 1. Is the prevalence of malaria parasites affected by urbanisation and spring phenology? We hypothesise that urban and forest blue tit nestlings differ in the prevalence of malaria parasites. We further predict effects of phenology of host reproduction on prevalence. In arthropods, spring emergence typically responds stronger to temperature than vertebrate reproduction, but patterns differ between terrestrial and aquatic species, and can be more variable between years in birds than in insects [ 82 , 89 , 90 ]. We hypothesise that in Scotland, where blue tits can hatch before vector emergence, low overlap between nestling growth and vector presence will reduce the risk of malaria infection. Thus, early-hatched nestlings should show lower prevalence than late hatched nestlings. Whether these effects are similar in urban and rural blue tits depends on how urbanisation affects host and vector phenology. Thus, if blue tits from both habitats breed on average early, we expect little malaria prevalence in both populations. However, if urban and rural birds differ in reproductive timing, inter-annual variation in host phenology could modulate differences in their malaria prevalence, assuming that vector phenology does not vary in synchrony. 2. Does malaria infection intensity impact nestling body mass and fledging success? We hypothesise that malaria infection will impact on nestling condition and survival prospects. Specifically, we predict that malaria infection will reduce body mass before fledging and the probability of a chick’s fledging, independently of hatching habitat, but depending on time since infection as estimated from malaria intensity. Material and Methods Field work The study took place in three forest sites and two urban sites in Scotland where ∼500 nest boxes were monitored for research. The forest sites were: (i) the Scottish Centre for Ecology and the Natural Environment (SCENE) (56° 7’ N, 4° 36’ W), (ii) the Sallochy campsite (56° 7’ N, 4° 36’ W) and (iii) Cashel farm (56° 6’ N, 4° 34’ W). The urban sites were in Glasgow: (i) Kelvingrove Park (55° 52’ N, 4° 17’ W) and (ii) the Garscube Sports Complex (55° 54’ N, 4° 18’ W). Descriptions of study sites and sample sizes for each site can be found in Table S1. We worked at these sites between April and June for four consecutive years, 2016-2019. At the beginning of April boxes were visited once a week to monitor nest building, laying date, clutch size and the start of incubation. After the tenth day of incubation (first day of incubation was defined as when the last egg was laid), nests were visited every other day to estimate hatch date. When nestlings were found in a nest, we estimated whether these had hatched the same day (day 0) or the day before (day 1) based on their appearance, and thus a hatch date was assigned to every nest. On day 13 after the first egg hatched (referred to as sampling date), we randomly selected three to four nestlings per nest, weighed them, and collected a blood sample (30-50 µl) by puncturing their brachial vein. Nest boxes were checked after fledging to search for any dead nestling. Each nestling was assigned a fledging success of 0 if it died before leaving the nest, or 1 if it successfully fledged. qPCR-based quantification of malaria infection DNA extraction DNA was extracted from blood samples using a commercial kit (DNeasy whole-blood extraction kit, Qiagen) and eluted with 80μL of water. Successful DNA extraction was confirmed using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE) and tested against the presence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bird reference gene, following the protocol by [ 91 ], using primer pair: forward (5’-TGTGATTTCAATGGTGACAGC-3’) and reverse (5’AGCTTGACAAAATGGTCGTTC-3’) and the following thermal cycling profile: 95°C for 10 minutes, 40 cycles at 95°C for 1 minute, 1 minute at 60°C and 1 minute at 72°C, and then a final extension of 5 minutes at 72°C. The PCR products were then run out on a 1% agarose gel stained with SYBRTM Safe DNA Gel Stain (ThermoFisher Scientific, California, USA) and visualised under ultraviolet light. Sequencing Leucocytozoon and Haemoproteus mitochondrial genes To develop a qPCR that could quantify either Leucocytozoon or Haemoproteus parasites in bird DNA without cross-reactivity, we designed genus specific primers. First, we detected Leucocytozoon or Haemoproteus infected birds using the nested PCR protocol by [ 92 ], with some minor modifications (30 seconds instead of one minute for the denaturation, annealing and elongation steps). Then we amplified the conserved region of the mitochondrial genome between cytB and cox1 previously used to detect avian haemosporidian infections using the primer set 292F/631R following [ 92 ]. Briefly, PCRs were performed in 20 μL reaction mixture using 1xGoTaq G2 hot start green master mix (Promega, USA), 0.32μM of each primer, and two μL of DNA. Cycling conditions were as follows: 5 min at 94°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 52°C and 30 seconds at 72°C, and a final elongation step at 72°C for 10 minutes. Amplification was evaluated on a 1% agarose gel, purified and sequenced on both strands (Eurofins Genomics). Designing haemosporidia genus-specific qPCR primers Obtained sequences (GenBank accession numbers TBC) were aligned using Clustal Omega [ 93 ] to reference intergenetic space between cytB and cox1 genes from several haemosporidians, including Leucocytozoon fringilinarum (KY653765.1), Leucocytozoon majoris (FJ168563.1), Leucocytozoon dubreuili (KY653795.1), Haemoproteus coatneyi (KT698210.1), Haemoproteus sacharovi (KY653811.1), Haemoproteus tartakovski (KY653810.1), Haemoproteus. sp . (KY653805.1), Haemoproteus belopolskyi (KY653790.1); Plasmodium relictum (KY653773.1), Plasmodium elongatum (KY653801.1), Plasmodium lutzi (KY653816.1), Plasmodium unalis (KY653814.1) and Plasmodium vaughani (KY653792.1). Based on the alignment, we selected primer sequences that were different between genera, but that were conserved within each genus (Figure S1 and Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Haemosporidia genus-specific quantitative PCR primers Testing haemosporidia genus specific qPCR primers To test the efficiency and specificity of the primers, amplicons were cloned (Invitrogen TA cloning kit) and qPCR tested on different numbers of plasmids from each target gene and two negative controls: uninfected bird sample (to ensure that primers are not amplifying bird genes) and distilled nuclease-free water. Samples were run in two technical replicates (accepted only if no more than 0.5 Cycle threshold (Ct) difference between the replicates). Best primer concentration for L146F/L146R was 300nM each with a primer efficiency of 94.1%, and for H160F/H160R it was 600nM each with a primer efficiency of 106.6%. When a primer set was tested against a different genus, only the most concentrated standard (10 7 gene copies) showed cross-reactivity, but as this was observed ∼20 cycles after the expected Ct for the specific target, it suggested high specificity of each primer set (Figure S2). qPCR quantification of parasite infections Leucocytozoon and Haemoproteus infections were quantified by qPCR using our genus-specific primers against Leucocytozoon or Haemoproteus individually ( Table 1 ), and normalised against the bird reference gene using GAPDH-Forward 5’-TGTGATTTCAATGGTGACAGC-3’ and GADPH-Reverse 5’-AGCTTGACAAAATGGTCGTTC-3’ at 100nM final concentration following [ 91 ]. The total volume of each reaction was 15 μl and included 5μL of DNA diluted to 12ng/μl, primers (see Table 1 ), 7.5 μl of SYBR Select Master Mix (Applied Biosystems), and nuclease-free water volumes. Reactions were run in MicroAmp Optical 96-well plates (Applied Biosystems) on Stratagene Mx3000 (ThermoFisher). Cycling conditions were as follows: 15 min at 95°C, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at specific annealing temperature (57°C for Leucocytozoon , 58°C for Haemoproteus , 60°C for GADPH), followed by a melting curve. Quality checks were performed on the data and any duplicates from individual birds whose differences exceeded >0.5 cycle threshold (Ct) value were repeated. Additionally, samples that showed low quantities of the bird GADPH reference gene, i.e. amplified after 29 Ct, were excluded from the analysis as potential false negatives following observations that target parasite genes in low parasitemic samples tend to amplify 10 Ct after the reference. Finally, the relative infection intensity of malaria parasites was calculated for each sample as: x = 2 -(parasite Ct – GAPDH Ct) . Statistical analyses General approach All analyses were conducted in R, version 4.1.2 [ 94 ]. All models were linear or generalized linear mixed models (LMM or GLMM, respectively), which we ran using the R packages lme4 [ 95 ] or glmm . tmb [ 96 ]. Nestbox identity was always included as random factor to account for variation explained by the same nestbox hosting different broods in different years, and to account for family effects (siblings sampled in the same nest). We checked that models adhered to assumptions of normality and homoscedasticity of residuals using the R package performance [ 97 ]. We used the same package to check for collinearity of predictors based on variance inflation factor (VIF). All models adhered to assumptions and collinearity was an issue only for year and hatch date (see below, VIF for all other predictors < 2). Model selection was conducted only when testing the significance of interactions, using likelihood ratio tests. If an interaction was not significant, it was removed from the model, but we left all main effects in the model as they were all crucial to test our hypotheses. All explanatory variables were scaled and mean-centered (across years) to improve the interpretability and comparability of model coefficients [ 98 ]. For example, when hatch date was used as an explanatory covariate, we calculated mean-centered hatch date as the deviation from the grand mean hatch date of all years, which was day 137 (May 17 th ), and then scaled it. We first ran two models to describe variation in malaria parasite prevalence and hatch date, respectively, across years and habitats. These did not directly address our hypotheses but were instrumental to our understanding of the variation in malaria prevalence and timing of reproduction across years. In addition, hatch date and years caused collinearity problems, so they could not be included in the same models as covariates. We analysed the variation in malaria prevalence (0 = uninfected, 1 = infected) by fitting a generalized linear mixed model (GLMM) with a binomial error structure, using the function glmer . Habitat (a categorical factor with two levels: urban or forest), year, brood size and the interaction between habitat and year were included as fixed effects. We used the same approach, but applying a linear mixed model with the function lmer , to analyse variation in raw hatch date (un-centred). We then moved on to build models to answer our specific questions. Prevalence we analysed the variation in malaria prevalence as function of hatch date and urbanisation with a binomial GLMM. Habitat, nestling hatch date (mean-centered across years) and brood size were modelled as fixed effects. We also included the interaction between habitat and hatch date to specifically test the hypothesis that the relationship between urbanisation and infection prevalence might depend on the phenological timing of the birds. Offspring fitness To test for possible effects of malaria infection on nestlings, we first analysed the relationship between infection intensity and nestling weight. We used infection intensity rather than prevalence because higher intensity should be positively correlated to the number of days the animal has been infected for, and thereby the likelihood that it would cause effects on physiological state and condition [ 20 ]. For the nestling weight, we used a linear mixed model (LMM) with a Gaussian error structure, using the function lmer . Nestling weight was modelled as response variable, while infection intensity, nestling hatch date (mean-centered across years) and brood size were included as fixed effects. We also included the two-way interactions between intensity and habitat, intensity and hatch date, and hatch date and habitat. We repeated this same model using only birds that were heavily infected, which we defined as the upper 75% quantile (N = 11, Figure S3). We used a similar approach to analyse the relationship between malaria infection and a chick’s fledging success, but using a binomial GLMM with the probability of fledging (0 = dead, 1 = fledged) as response variable. However, because models that included infection intensity as predictor did not converge, we decided to use infection prevalence instead. Results Is the prevalence of malaria parasites affected by urbanisation and spring phenology? Haemoproteus prevalence was very low in our dataset, as only 6 out of the 320 nestlings screened were infected with this parasite (1.8 %), equally found in urban (N = 3) and forest sites (N= 3). Conversely, Leucocytozoon prevalence was moderately high: 32.5 % of nestlings screened were infected with this parasite. In only two instances we detected co-infection. As Haemoproteus infection was very low and likely not playing a strong role in our system, we decided to focus only on Leucocytozoon for all the following analyses. Leucocytozoon prevalence differed between habitats, but depending on the year of study (Table S2): while prevalence was higher in forest than urban nestlings in 2016 and 2017, no difference between the two habitats was found in 2018 and 2019 ( Figure 1A , Table S3). Similarly, mean hatch date over the four years spanned c. 20 days and differed between the urban and forest habitats ( Fig 1b ). However, the magnitude and direction of habitat differences depended on the year (Table S4). While forest nestlings hatched earlier than urban ones in 2016, 2017 and 2019, no difference was found in 2018 (Table S5). Download figure Open in new tab Figure 1. (A) Variation in Leucocytozoon prevalence in relation to habitat (forest vs urban) and year. (B) Variation in the date of nestlings hatching in relation to habitat (forest vs urban) and year of the study. Figures show predicted means ± confidence intervals. The next step in our analyses was to test whether year-dependent differences in prevalence could be explained by nestling hatch date. As predicted, we found that Leucocytozoon prevalence did not differ significantly between urban and forest sites but was positively associated with hatch date (Table S6, Figure 2 ). All other variables included in our model did not affect Leucocytozoon prevalence (Table S6). Download figure Open in new tab Figure 2. Variation in Leucocytozoon prevalence in nestling blue tits is positively related to the date of hatching. Nestlings hatched later in the spring have a higher probability to be infected with Leucocytozoon . This effect was independent of habitat. Line represents estimated means from Binomial GLMM, shaded area depicts confidence intervals. Points represent individual birds. Does malaria infection intensity impact nestling body mass and fledging success? We found a strong and significant effect of hatch date on body weight, independent of habitat: both forest and urban nestlings were heavier later than earlier in the season ( Figure 3a and Table S7). Moreover, forest nestlings were heavier than urban nestlings, but nestling body mass was unrelated to brood size (Table S7). Across all infected nestlings, body weight was not affected by Leucocytozoon infection intensity (Table S7). However, infection intensity was heavily skewed, with most birds showing intensity close to 0 and few birds with much higher intensity levels, which were all forest birds (Figure S3). As these low-intensity nestlings were likely only recently infected, we ran a subsequent model using only highly infected individuals to predict nestling weight. We defined highly infected individuals as those with relative level of infection above the 75% quantile of the data. Using this subset of 11 birds, our model showed that nestling weight was significantly negatively related to infection intensity: as infection intensity increases, nestling weight decreases ( Figure 3b and Table S8). Download figure Open in new tab Figure 3. (a) Nestling weight is affected by season but not habitat. In both environments, nestling weight increases over the spring. (b) In the 11 highly infected individuals (which were all forest birds), nestling body weight is negatively related to Leucocytozoon infection intensity (parasite gene quantity relative to bird housekeeping gene). In both panels, lines are estimated means from Gaussian LMM, while shaded areas represent confidence intervals and dots represent raw data. Across all nestlings, fledging success was significantly lower in the city than in the forest ( Figure 4 and Table S9), and positively associated to nestling body mass (Table S9). Leucocytozoon prevalence, brood size and hatch date were not related to fledging success (Table S9). Download figure Open in new tab Figure 4. Urban nestlings have a lower probability of successfully fledging than forest nestlings. Points are estimated means over the four study years from a binomial GLMM, while error bars depict confidence intervals. Discussion This is the first study, to our knowledge, that has explicitly investigated the interplay between phenology and urbanisation in determining avian malaria infection status. Our results provide evidence that the timing of hatching, rather than urbanisation, can explain variation in prevalence of a malaria parasite, Leucocytozoo n, in blue tit nestlings. In both urban and forest habitats, infection prevalence varied between years, but in no consistent manner. On average, nestlings that hatched earlier in the spring had a lower probability of being infected. This effect was quite strong: early in spring the probability of infection was as low as 20%, but later in the spring it rose to 60%. The phenology of hatching also influenced nestlings’ body weight, independently of habitat type. Nestlings born earlier in the spring were lighter than those that hatched later in the season, in contrast to previous studies in woodland passerines [ 99 , 100 ] and the widespread evidence that being early has fitness advantages [ 101 ]. Heavier nestlings were also much more likely to fledge. Low malaria infection intensity had no detectable effects on body mass of 13-day old nestlings. In the few nestlings that were found to be heavily infected, which were all forest birds, the intensity of infection was negatively related to body weight, suggesting that Leucocytozoon infection could potentially affect the health and fitness of blue tit nestlings. However, infection prevalence did not explain variation in fledging success across urban and forest nestlings. Among the concise literature on avian malaria in the context of urbanisation, studies published to date have provided mixed results [ 48 ]. While generally avian malaria prevalence has been found to be lower in urban than non-urban birds of the same species, this pattern can vary depending on the species, life stage and local environmental conditions. For example, in house sparrows ( Passer domesticus ), studies conducted in Spain and USA found higher prevalence of Leucocytozoon in populations inhabiting rural habitats compared to urbanised areas [ 102 , 103 ], but no urban-rural differences were found in France [ 45 ]. In France, malaria infection in adult and nestling great tits ( Parus major ) was higher in urban than rural populations in Montpellier, but lower in urban than rural adults of the same species in Besancon and Dijon [ 46 , 104 ]. A study on five songbird species in an arid region of the southwest USA showed that adult birds generally exhibited lower Haemoproteus parasite prevalence in urban compared to rural populations, but species and sex-specific differences were found [ 47 ]. Our study confirms the mixed evidence. In some years malaria prevalence was higher in forest than urban nestlings, and in other years the pattern was reversed. Our analysis suggests that this pattern is likely explained by the fact that the time of hatching of nestlings in the two populations differed between the years, and that hatching time, rather than habitat type, drives differences in malaria prevalence between urban and forest birds. We thus assume that early hatching nestlings do not overlap much with the emergence of malaria vectors, thereby escaping infection. Passerine bird phenology in temperate areas can vary considerably in time and space [ 105 ] and can be governed by both large-scale environmental variables, such as average spring temperatures [ 106 , 107 ], as well as by microenvironmental factors, such as the availability of insect prey [ 108 ]. Similarly, the phenology of the vectors that transmit malaria parasites can change across different years and over relatively small spatial scales. Thus, the mixed relationships observed between malaria prevalence and urbanisation may reflect variation in both reproductive phenology and vector emergence. Like bird reproduction, vector emergence is influenced by ambient temperature, but organisms vary in response to spring temperature. Thus, while terrestrial invertebrates generally advance phenology faster than vertebrates in warmer springs, these relationships are reversed in freshwater habitats, where the dipteran vectors develop [ 70 , 71 ]. Furthermore, different vector species might respond in distinct ways [ 65 ]. For instance, in our system, avian malaria is transmitted by blackflies, which breed in fast-flowing streams—habitats that may be less sensitive to temperature fluctuations and urbanisation than the small, temporary water bodies used by mosquito vectors. Such different developmental habitats can influence phenological patterns of vectors in an urbanisation context. Urban environments often provide artificial water reservoirs such as ornamental pond, fountains, gutters, and containers, which can serve as potential mosquito vector breeding grounds [ 69 ]. These microhabitats can sustain vector populations beyond the normal breeding season, potentially leading to more persistent transmission of avian malaria throughout the year in urban areas [ 69 , 109 , 110 ]. However, pollution and vector control activities in urban areas can considerably reduce larval numbers [ 111 ], and flowing waters – crucial for blackfly larval development - can be scarce in urban areas but more abundant in rural and agricultural areas. Future studies should investigate how urbanisation and seasonality may affect the abundance and phenology of different vectors. In addition to hatching and vector phenology, seasonal changes of haemosporidian parasitaemia within blue tit hosts could also contribute to phenological effects on nestling malaria prevalence. After infection, birds enter the acute phase after which parasitaemia decreases drastically, known as the chronic phase (or latent stage). The chronic phase is no longer infectious, as parasites only persist in internal organs and not in peripheral blood [ 112 ]. However, when birds in temperate regions start breeding, an increase in parasitaemia is commonly observed [ 112 ]. This relapse is induced by changes in corticosterone and gonadal hormones, that are believed to directly trigger parasites in tissues [ 113 , 114 ]. Evidence of parasite relapse in Haemoproteus and Trypanosoma in blackcaps following increases in day lengths further suggest a suppressive role of melatonin, reducing relapses when nights are long [ 114 ]. Urban-related environmental factors known to affect phenology, such as artificial light at night, may thus also affect seasonal relapse of malaria parasites, e.g., via effects on melatonin or on the immune system [ 115 ]. In a system where most of the hosts have chronic infections, seasonal relapses that are timed with the vector’s peak in abundance are considered an adaptive strategy of the parasite to ensure transmission to the new-born susceptible birds [ 116 ]. Thus, synchrony between vector emergence and avian reproduction is key for parasite persistence across seasons [ 50 ], and these interactions could be facilitated, or reduced, by combinations of environmental change in urban habitats. We could not measure vector abundance and phenology at our sites, nor could we disentangle the effects of ALAN and other urban factors, which could have helped to explain our results. Future studies should integrate bird, vector and environmental data to gain a holistic understanding of how avian malaria infection varies in space and time with respect to urbanisation. Our analyses also revealed a negative relationship between the intensity of Leucocytozoon infection and the nestlings’ body mass before fledging (i.e. when they were two weeks old), at least in those individuals that were heavily infected. High infection levels should reflect the time from first infection, and given that Leucocytozoon parasite life cycle is less than one week, it is likely that these nestlings were infected soon after hatching. However, malaria infection did not affect fledging success, which is more likely to be a function of poor diet leading to early nest mortality, especially in urban environments [ 117 , 118 ]. Nevertheless, since body mass before fledging is one of the most important predictor of future recruitment probability in small woodland passerines [ 75 , 119 ], our results point to potential long-term fitness consequences of early life infection with malaria parasites in these species, as suggested for great reed warblers ( Acrocephalus arundinaceus ) [ 28 ]. Molecular tools have significantly advanced avian malaria research by offering greater sensitivity and specificity than microscopy. Nested PCR (nPCR) has been instrumental in detecting Plasmodium, Haemoproteus , and Leucocytozoon [ 86 , 87 ], but it has limitations—namely, overestimating mixed infections due to primer cross-reactivity [ 88 ] and its inability to quantify parasite load. To address these issues, we developed a SYBR-based qPCR assay using species-specific primers for Leucocytozoon and Haemoproteus . This method requires two separate reactions, avoids the need for probes, and enables sensitive, cost-effective quantification. While validated only in Scottish samples, in silico analyses suggest broad applicability due to conserved primer regions across lineages. Compared to nPCR, our assay improves specificity by distinguishing single from mixed infections, detects down to 10 −5 parasite gene copies per blood cell, and allows quantification. Other recent methods have tackled similar challenges: a genus-specific nPCR improves detection of mixed infections and phylogenetic resolution [ 120 ], and a multiplex PCR enables simultaneous detection of all three genera [ 121 ], though neither quantifies parasite load. Quantitative methods like a universal qPCR [ 122 ] and digital droplet PCR (ddPCR) [ 123 ] offer absolute quantification but cannot distinguish genera or detect mixed infections. A comparative study highlighted differences in detection rates across microscopy, multiplex, and nPCR, suggesting that combining methods may be optimal [ 124 ]. Future work should compare our qPCR with these approaches to evaluate its strengths and best-use scenarios. Our study has some limitations. First, we sampled five populations in a small geographical area in and around Glasgow. Thus, it is unclear if our results can be generalized elsewhere. Second, as mentioned above, we do not have information on vector abundance and phenology in the different years and habitats, which does not allow us to verify our hypothesis that nestlings avoid overlapping with malaria vectors in years when they hatch earlier than average. This also prevents us from understanding how environmental conditions during spring, particularly temperature, can influence vector phenology, which might be particularly relevant in the context of climate warming, and how this might affect host-parasite interactions. Third, even though we sampled hundreds of birds over four years, Leucocytozoon prevalence was relatively low, especially in some years, which resulted in a small sample size for the fitness analyses. Moreover, Haemoproteus prevalence was extremely low and Plasmodium infection was never detected. The exact reasons still need to be clarified. For example, low or absent Plasmodium transmission might be a consequence of low abundance of Culex mosquito vectors; however this is less plausible for Haemoproteus , whose Culicoides biting midge vectors are highly abundant in the study sites. Alternatively, as the latent period of Leucocytozoon is much shorter (∼3 days) than in Plasmodium and Haemoproteus (∼10-14 days) [ 112 ], early sampling of nestlings at day 13 after hatching might have selectively favored the detection of the fastest parasite genus. In summary, our study revealed previously unappreciated relationships between passerine reproductive timing, malaria infection and urbanisation. Our results highlight the importance of breeding early to avoid early-life infection with malaria parasites, at least in blue tits, and supports recent findings that infection can have a detectable impact on fitness-related traits. This has potential implications for climate warming, which is already leading many passerines to breed earlier. Whether insect vectors are also responding in the same direction, and to the same extent, is largely unknown, as long-term time series of vector phenology in relation to local environmental conditions are scarce. There is a clear research gap at the interface between bird and vector phenology, especially in an urbanisation context. Thus, we anticipate future studies to answer exciting questions on this topic. Acknowledgements We thank Robyn Womack, Luke Woodford, Georgia Kirby, Heather Ferguson, and many master and undergraduate students who were involved with field sampling for this project. We thank the staff of SCENE, particularly Matt Newton and Hannele Honkanen, for hosting us and supporting our work. We thank Glasgow City Council and the Loch Lomond and the Trossachs National Park for giving us access to the urban and forest field sites, respectively. This research was funded by the University of Glasgow and by Funding through a Marie-Curie Career Integration Grant to BH [EC CIG (618578) Wildclocks]. BAA was funded by the Saudi Ministry of Education and by the University of Tabuk. FB was funded by AXA RF fellowship (14-AXA-PDOC-130), EMBO LT fellowship (43-2014), the Medical Research Council GCRF Infections Foundation Awards (MR/P025501/1) and the Academy Medical Sciences Springboard Award (ref:SBF007\100094). Footnotes https://github.com/dmdominoni/urban_malaria/tree/main References 1. ↵ Gaynor KM , Hojnowski CE , Carter NH , Brashares JS . 2018 The influence of human disturbance on wildlife nocturnality . Science 360 , 1232 – 1235 . ( doi: 10.1126/science.aar7121 ) OpenUrl Abstract / FREE Full Text 2. ↵ Soulsbury CD , White PCL , Soulsbury CD , White PCL . 2015 Human–wildlife interactions in urban areas: a review of conflicts, benefits and opportunities . Wildl. Res . 42 , 541 – 553 . ( doi: 10.1071/WR14229 ) OpenUrl CrossRef 3. ↵ Shochat E , Lerman SB , Anderies JM , Warren PS , Faeth SH , Nilon CH . 2010 Invasion, Competition, and Biodiversity Loss in Urban Ecosystems . BioScience 60 , 199 – 208 . ( doi: 10.1525/bio.2010.60.3.6 ) OpenUrl CrossRef Web of Science 4. ↵ Delgado-V . C a. , French K. 2012 Parasite-bird interactions in urban areas: Current evidence and emerging questions . Landscape and Urban Planning 105 , 5 – 14 . ( doi: 10.1016/j.landurbplan.2011.12.019 ) OpenUrl CrossRef 5. ↵ Hassell JM , Begon M , Ward MJ , Fèvre EM . 2017 Urbanization and Disease Emergence: Dynamics at the Wildlife–Livestock–Human Interface . Trends in Ecology and Evolution 32 , 55 – 67 . ( doi: 10.1016/j.tree.2016.09.012 ) OpenUrl CrossRef PubMed 6. Becker DJ , Streicker DG , Altizer S. 2015 Linking anthropogenic resources to wildlife-pathogen dynamics: a review and meta-analysis . Ecology Letters , n/a-n/a. ( doi: 10.1111/ele.12428 ) OpenUrl CrossRef PubMed 7. ↵ Bradley C a. , Altizer S. 2007 Urbanization and the ecology of wildlife diseases . Trends in Ecology and Evolution 22 , 95 – 102 . ( doi: 10.1016/j.tree.2006.11.001 ) OpenUrl CrossRef PubMed Web of Science 8. ↵ D Gil , H Brumm Martin LB , Boruta M. 2013 The impact of urbanization on avian disease transmission and emergence . In Avian Urban Ecology: Behavioural and Physiological Adaptations (eds D Gil , H Brumm ), pp. 116 – 128 . Oxford : Oxford University Press . 9. ↵ Allen T , Murray KA , Zambrana-Torrelio C , Morse SS , Rondinini C , Di Marco M , Breit N , Olival KJ , Daszak P. 2017 Global hotspots and correlates of emerging zoonotic diseases . Nat Commun 8 , 1124 . ( doi: 10.1038/s41467-017-00923-8 ) OpenUrl CrossRef PubMed 10. ↵ Lines J , Harpham T , Leake C , Schofield C. 1994 Trends, priorities and policy directions in the control of vector-borne diseases in urban environments . Health policy and planning 9 , 113 – 129 . ( doi: 10.1093/heapol/9.2.113 ) OpenUrl CrossRef PubMed Web of Science 11. Millán J , Proboste T , Fernández de Mera IG , Chirife AD , de la Fuente J, Altet L. 2016 Molecular detection of vector-borne pathogens in wild and domestic carnivores and their ticks at the human–wildlife interface . Ticks and Tick-borne Diseases 7 , 284 – 290 . ( doi: 10.1016/j.ttbdis.2015.11.003 ) OpenUrl CrossRef 12. ↵ LaDeau SL , Allan BF , Leisnham PT , Levy MZ . 2015 The ecological foundations of transmission potential and vector-borne disease in urban landscapes . Functional Ecology , n/a-n/a. ( doi: 10.1111/1365-2435.12487 ) OpenUrl CrossRef 13. ↵ Worsley-Tonks KEL , Gehrt SD , Anchor C , Escobar LE , Craft ME . 2021 Infection risk varies within urbanized landscapes: the case of coyotes and heartworm . Parasites & Vectors 14 , 464 . ( doi: 10.1186/s13071-021-04958-1 ) OpenUrl CrossRef PubMed 14. ↵ In press. Parasitic infections of brushtail possums Trichosurus vulpecula in urbanised environments and bushland in the greater Perth region, Western Australia - Hillman - 2018 - Wildlife Biology - Wiley Online Library . See https://onlinelibrary.wiley.com/doi/full/10.2981/wlb.00442 (accessed on 27 September 2023 ). 15. ↵ Colón-González FJ , Sewe MO , Tompkins AM , Sjödin H , Casallas A , Rocklöv J , Caminade C , Lowe R. 2021 Projecting the risk of mosquito-borne diseases in a warmer and more populated world: a multi-model, multi-scenario intercomparison modelling study . The Lancet Planetary Health 5 , e404 – e414 . ( doi: 10.1016/S2542-5196(21)00132-7 ) OpenUrl CrossRef 16. ↵ Seto KC , Guneralp B , Hutyra LR . 2012 Global forecasts of urban expansion to 2030 and direct impacts on biodiversity and carbon pools . PNAS 109 , 16083 – 16088 . ( doi: 10.1073/pnas.1211658109 ) OpenUrl Abstract / FREE Full Text 17. ↵ Bensch S , Hellgren O , Pérez-Tris J. 2009 MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages . Molecular Ecology Resources 9 , 1353 – 1358 . ( doi: 10.1111/j.1755-0998.2009.02692.x ) OpenUrl CrossRef PubMed 18. ↵ Woodworth BL et al. 2005 Host population persistence in the face of introduced vector-borne diseases: Hawaii amakihi and avian malaria . Proceedings of the National Academy of Sciences of the United States of America 102 , 1531 – 1536 . ( doi: 10.1073/pnas.0409454102 ) OpenUrl Abstract / FREE Full Text 19. ↵ Beadell JS et al. 2006 Global phylogeographic limits of Hawaii’s avian malaria . Proceedings of the Royal Society B: Biological Sciences 273 , 2935 – 2944 . ( doi: 10.1098/rspb.2006.3671 ) OpenUrl CrossRef PubMed Web of Science 20. ↵ Valkiunas G. 2004 Avian Malaria Parasites and other Haemosporidia . CRC Press . 21. ↵ Valkiūnas G , Atkinson CT . 2020 Introduction to Life Cycles, Taxonomy, Distribution, and Basic Research Techniques . In Avian Malaria and Related Parasites in the Tropics: Ecology, Evolution and Systematics (eds dD Santiago-Alarcon, A Marzal), pp. 45 – 80 . Cham : Springer International Publishing . ( doi: 10.1007/978-3-030-51633-8_2 ) OpenUrl CrossRef 22. ↵ Mukhin A , Palinauskas V , Platonova E , Kobylkov D , Vakoliuk I , Valkiūnas G. 2016 The Strategy to Survive Primary Malaria Infection: An Experimental Study on Behavioural Changes in Parasitized Birds . PLOS ONE 11 , e0159216 . ( doi: 10.1371/journal.pone.0159216 ) OpenUrl CrossRef PubMed 23. ↵ Knowles SCL , Palinauskas V , Sheldon BC . 2010 Chronic malaria infections increase family inequalities and reduce parental fitness: Experimental evidence from a wild bird population . Journal of Evolutionary Biology 23 , 557 – 569 . ( doi: 10.1111/j.1420-9101.2009.01920.x ) OpenUrl CrossRef PubMed Web of Science 24. ↵ Merino S , Moreno J , José Sanz J , Arriero E. 2000 Are avian blood parasites pathogenic in the wild? A medication experiment in blue tits (Parus caeruleus) . Proceedings of the Royal Society of London. Series B: Biological Sciences 267 , 2507 – 2510 . ( doi: 10.1098/rspb.2000.1312 ) OpenUrl CrossRef PubMed Web of Science 25. ↵ Valkiūnas G , Zičkus T , Shapoval AP , Iezhova TA . 2006 Effect of Haemoproteus belopolskyi (Haemosporida: Haemoproteidae) on Body Mass of the Blackcap Sylvia atricapilla . Journal of Parasitology 92 , 1123 – 1125 . ( doi: 10.1645/GE-3564-RN.1 ) OpenUrl CrossRef PubMed 26. ↵ Videvall E , Cornwallis CK , Palinauskas V , Authors C. 2015 The Avian Transcriptome Response to Malaria Infection . 27. ↵ Palinauskas V , Valkiūnas G , Bolshakov CV , Bensch S. 2008 Plasmodium relictum (lineage P-SGS1): Effects on experimentally infected passerine birds . Experimental Parasitology 120 , 372 – 380 . ( doi: 10.1016/j.exppara.2008.09.001 ) OpenUrl CrossRef PubMed Web of Science 28. ↵ Asghar M , Hasselquist D , Hansson B , Zehtindjiev P , Westerdahl H , Bensch S. 2015 Hidden costs of infection: Chronic malaria accelerates telomere degradation and senescence in wild birds . Science 347 , 436 – 438 . ( doi: 10.1126/science.1261121 ) OpenUrl Abstract / FREE Full Text 29. ↵ Sudyka J , Podmokła E , Drobniak SM , Dubiec A , Arct A , Gustafsson L , Cichoń M. 2019 Sex-specific effects of parasites on telomere dynamics in a short-lived passerine—the blue tit . Sci Nat 106 , 6 . ( doi: 10.1007/s00114-019-1601-5 ) OpenUrl CrossRef 30. Ots I , Hõrak P. 1998 Health impact of blood parasites in breeding great tits . Oecologia 116 , 441 – 448 . ( doi: 10.1007/s004420050608 ) OpenUrl CrossRef PubMed Web of Science 31. ↵ Marzal A , De Lope F , Navarro C , M??ller AP . 2005 Malarial parasites decrease reproductive success: An experimental study in a passerine bird . Oecologia 142 , 541 – 545 . ( doi: 10.1007/s00442-004-1757-2 ) OpenUrl CrossRef PubMed Web of Science 32. ↵ Ilgūnas M , Bukauskaitė D , Palinauskas V , Iezhova TA , Dinhopl N , Nedorost N , Weissenbacher-Lang C , Weissenböck H , Valkiūnas G. 2016 Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites . Malaria Journal 15 , 256 . ( doi: 10.1186/s12936-016-1310-x ) OpenUrl CrossRef PubMed 33. ↵ Dawson RD , Bortolotti GR . 2000 Effects of Hematozoan Parasites on Condition and Return Rates of American Kestrels . The Auk 117 , 373 – 380 . ( doi: 10.1093/auk/117.2.373 ) OpenUrl CrossRef 34. ↵ Dadam D , Robinson RA , Clements A , Peach WJ , Bennett M , Rowcliffe JM , Cunningham AA . 2019 Avian malaria-mediated population decline of a widespread iconic bird species . Royal Society Open Science 6 , 182197 . ( doi: 10.1098/rsos.182197 ) OpenUrl CrossRef PubMed 35. ↵ Marzal A , Bensch S , Reviriego M , Balbontin J , De Lope F. 2008 Effects of malaria double infection in birds: One plus one is not two . Journal of Evolutionary Biology 21 , 979 – 987 . ( doi: 10.1111/j.1420-9101.2008.01545.x ) OpenUrl CrossRef PubMed Web of Science 36. ↵ Lachish S , Knowles SCL , Alves R , Wood MJ , Sheldon BC . 2011 Fitness effects of endemic malaria infections in a wild bird population: The importance of ecological structure . Journal of Animal Ecology 80 , 1196 – 1206 . ( doi: 10.1111/j.1365-2656.2011.01836.x ) OpenUrl CrossRef PubMed 37. ↵ Emmenegger T , Riello S , Schmid R , Serra L , Spina F , Hahn S. 2023 Avian Haemosporidians Infecting Short- and Long-Distance Migratory Old World Flycatcher Species and the Variation in Parasitaemia After Endurance Flights . Acta Parasit . ( doi: 10.1007/s11686-023-00710-0 ) OpenUrl CrossRef 38. ↵ Emmenegger T , Alves JA , Rocha AD , Costa JS , Schmid R , Schulze M , Hahn S. 2020 Population- and age-specific patterns of haemosporidian assemblages and infection levels in European bee-eaters (Merops apiaster) . International Journal for Parasitology 50 , 1125 – 1131 . ( doi: 10.1016/j.ijpara.2020.07.005 ) OpenUrl CrossRef PubMed 39. ↵ Lachish S , Knowles SCL , Alves R , Sepil I , Davies A , Lee S , Wood MJ , Sheldon BC . 2013 Spatial determinants of infection risk in a multi-species avian malaria system . Ecography 36 , 587 – 598 . ( doi: 10.1111/j.1600-0587.2012.07801.x ) OpenUrl CrossRef 40. ↵ Isaksson C , Sepil I , Baramidze V , Sheldon BC . 2013 Explaining variance of avian malaria infection in the wild: The importance of host density, habitat, individual life-history and oxidative stress . BMC Ecology 13 . ( doi: 10.1186/1472-6785-13-15 ) OpenUrl CrossRef PubMed 41. ↵ Kilpatrick AM . 2006 Facilitating the evolution of resistance to avian malaria in Hawaiian birds . Biological Conservation 128 , 475 – 485 . ( doi: 10.1016/j.biocon.2005.10.014 ) OpenUrl CrossRef Web of Science 42. ↵ Caizergues AE , Robira B , Perrier C , Jeanneau M , Berthomieu A , Perret S , Gandon S , Charmantier A. 2023 Cities as parasitic amplifiers? Malaria prevalence and diversity along an urbanization gradient in great tits ., 2023.05.03.539263. ( doi: 10.1101/2023.05.03.539263 ) OpenUrl Abstract / FREE Full Text 43. Jiménez-Peñuela J , Ferraguti M , Martínez-de la Puente J, Soriguer RC , Figuerola J. 2021 Urbanization effects on temporal variations of avian haemosporidian infections . Environmental Research 199 , 111234 . ( doi: 10.1016/j.envres.2021.111234 ) OpenUrl CrossRef 44. Santiago-Alarcon D , Havelka P , Pineda E , Segelbacher G , Schaefer HM . 2013 Urban forests as hubs for novel zoonosis: blood meal analysis, seasonal variation in Culicoides (Diptera: Ceratopogonidae) vectors, and avian haemosporidians . Parasitology , 1 – 12 . ( doi: 10.1017/S0031182013001285 ) OpenUrl CrossRef PubMed 45. ↵ Bichet C , Brischoux F , Ribout C , Parenteau C , Meillère A , Angelier F. 2020 Physiological and morphological correlates of blood parasite infection in urban and non-urban house sparrow populations . PLOS ONE 15 , e0237170 . ( doi: 10.1371/journal.pone.0237170 ) OpenUrl CrossRef PubMed 46. ↵ Bailly J et al. 2016 Negative impact of urban habitat on immunity in the great tit Parus major . Oecologia 182 , 1053 – 1062 . ( doi: 10.1007/s00442-016-3730-2 ) OpenUrl CrossRef PubMed 47. ↵ Bobby Fokidis H , Greiner EC , Deviche P. 2008 Interspecific variation in avian blood parasites and haematology associated with urbanization in a desert habitat . Journal of Avian Biology 39 , 300 – 310 . ( doi: 10.1111/j.0908-8857.2008.04248.x ) OpenUrl CrossRef 48. ↵ Sehgal RNM . 2015 Manifold habitat effects on the prevalence and diversity of avian blood parasites . International Journal for Parasitology: Parasites and Wildlife 4 , 421 – 430 . ( doi: 10.1016/j.ijppaw.2015.09.001 ) OpenUrl CrossRef PubMed 49. ↵ Cosgrove CL , Wood MJ , Day KP , Sheldon BC . 2008 Seasonal variation in Plasmodium prevalence in a population of blue tits Cyanistes caeruleus . Journal of Animal Ecology 77 , 540 – 548 . ( doi: 10.1111/j.1365-2656.2008.01370.x ) OpenUrl CrossRef PubMed Web of Science 50. ↵ Martinez-Bakker M , Helm B. 2015 The influence of biological rhythms on host–parasite interactions . Trends in Ecology & Evolution 30 , 314 – 326 . ( doi: 10.1016/j.tree.2015.03.012 ) OpenUrl CrossRef PubMed 51. ↵ Stevenson TJJ et al. 2015 Disrupted seasonal biology impacts health, food security and ecosystems . Proceedings of the Royal Society B: Biological Sciences 282 , 20151453 . ( doi: 10.1098/rspb.2015.1453 ) OpenUrl CrossRef PubMed 52. ↵ Dominoni DM , Kjellberg Jensen J , de Jong M , Visser ME , Spoelstra K. 2020 Artificial light at night, in interaction with spring temperature, modulates timing of reproduction in a passerine bird . Ecological Applications 30 , e2062 . ( doi: 10.1002/eap.2062 ) OpenUrl CrossRef 53. Hajdasz AC , Otter KA , Baldwin LK , Reudink MW . 2019 Caterpillar phenology predicts differences in timing of mountain chickadee breeding in urban and rural habitats . Urban Ecosystems 22 , 1113 – 1122 . ( doi: 10.1007/s11252-019-00884-4 ) OpenUrl CrossRef 54. ↵ Ffrench-Constant RH , Somers-Yeates R , Bennie J , Economou T , Hodgson D , Spalding A , McGregor PK . 2016 Light pollution is associated with earlier tree budburst across the United Kingdom . Proceedings of the Royal Society B: Biological Sciences 283 , 20160813 . ( doi: 10.1098/rspb.2016.0813 ) OpenUrl CrossRef PubMed 55. ↵ García JT , Viñuela J , Calero-Riestra M , Sánchez-Barbudo IS , Villanúa D , Casas F. 2021 Risk of Infection, Local Prevalence and Seasonal Changes in an Avian Malaria Community Associated with Game Bird Releases . Diversity 13 , 657 . ( doi: 10.3390/d13120657 ) OpenUrl CrossRef 56. ↵ Han Y , Hellgren O , Wu Q , Liu J , Jin T , Bensch S , Ding P. 2023 Seasonal variations of intensity of avian malaria infection in the Thousand Island Lake System, China . Parasites & Vectors 16 , 218 . ( doi: 10.1186/s13071-023-05848-4 ) OpenUrl CrossRef PubMed 57. Hernández-Lara C , González-García F , Santiago-Alarcon D. 2017 Spatial and seasonal variation of avian malaria infections in five different land use types within a Neotropical montane forest matrix . Landscape and Urban Planning 157 , 151 – 160 . ( doi: 10.1016/j.landurbplan.2016.05.025 ) OpenUrl CrossRef 58. ↵ Kim KS , Tsuda Y. 2010 Seasonal changes in the feeding pattern of Culex pipiens pallens govern the transmission dynamics of multiple lineages of avian malaria parasites in Japanese wild bird community . Molecular Ecology 19 , 5545 – 5554 . ( doi: 10.1111/j.1365-294X.2010.04897.x ) OpenUrl CrossRef PubMed Web of Science 59. ↵ Cornet S , Nicot A , Rivero A , Gandon S. 2014 Evolution of Plastic Transmission Strategies in Avian Malaria . PLOS Pathogens 10 , e1004308 . ( doi: 10.1371/journal.ppat.1004308 ) OpenUrl CrossRef PubMed 60. ↵ Woodford L et al. 2018 Vector species-specific association between natural Wolbachia infections and avian malaria in black fly populations . Sci Rep 8 , 4188 . ( doi: 10.1038/s41598-018-22550-z ) OpenUrl CrossRef PubMed 61. ↵ Deviche P , Greiner EC , Manteca X. 2001 Seasonal and age-related changes in blood parasite prevalence in dark-eyed juncos (Junco hyemalis, Aves, Passeriformes) . Journal of Experimental Zoology 289 , 456 – 466 . ( doi: 10.1002/jez.1027 ) OpenUrl CrossRef PubMed 62. ↵ Lachish S , Knowles SCL , Alves R , Wood MJ , Sheldon BC . 2011 Infection dynamics of endemic malaria in a wild bird population: parasite species-dependent drivers of spatial and temporal variation in transmission rates . Journal of Animal Ecology 80 , 1207 – 1216 . ( doi: 10.1111/j.1365-2656.2011.01893.x ) OpenUrl CrossRef PubMed 63. ↵ Bensch S , Waldenstr??m J , Jonz??n N , Westerdahl H , Hansson B , Sejberg D , Hasselquist D. 2007 Temporal dynamics and diversity of avian malaria parasites in a single host species . Journal of Animal Ecology 76 , 112 – 122 . ( doi: 10.1111/j.1365-2656.2006.01176.x ) OpenUrl CrossRef PubMed Web of Science 64. ↵ Montarsi F , Mazzon L , Cazzin S , Ciocchetta S , Capelli G. 2015 Seasonal and daily activity patterns of mosquito (Diptera: Culicidae) vectors of pathogens in Northeastern Italy . Journal of Medical Entomology 52 , 56 – 62 . ( doi: 10.1093/jme/tju002 ) OpenUrl CrossRef PubMed 65. ↵ Ferraguti M , Martínez-de la Puente J, Ruiz S , Soriguer RC , Figuerola J. 2024 Landscape and mosquito community impact the avian Plasmodium infection in Culex pipiens . iScience 27 , 109194 . ( doi: 10.1016/j.isci.2024.109194 ) OpenUrl CrossRef PubMed 66. ↵ Kolokotroni M , Giridharan R. 2008 Urban heat island intensity in London: An investigation of the impact of physical characteristics on changes in outdoor air temperature during summer . Solar Energy 82 , 986 – 998 . ( doi: 10.1016/j.solener.2008.05.004 ) OpenUrl CrossRef Web of Science 67. ↵ Capilla-Lasheras P , Thompson MJ , Sánchez-Tójar A , Haddou Y , Branston CJ , Réale D , Charmantier A , Dominoni DM . In press. A global meta-analysis reveals higher variation in breeding phenology in urban birds than in their non-urban neighbours . Ecology Letters n/a. ( doi: 10.1111/ele.14099 ) OpenUrl CrossRef PubMed 68. ↵ Ishtiaq F , Bowden CGR , Jhala YV . 2017 Seasonal dynamics in mosquito abundance and temperature do not influence avian malaria prevalence in the Himalayan foothills . Ecology and Evolution 7 , 8040 – 8057 . ( doi: 10.1002/ece3.3319 ) OpenUrl CrossRef 69. ↵ Townroe S , Callaghan A. 2014 British container breeding mosquitoes: The impact of urbanisation and climate change on community composition and phenology . PLoS ONE 9 . ( doi: 10.1371/journal.pone.0095325 ) OpenUrl CrossRef PubMed 70. ↵ Thackeray SJ et al. 2010 Trophic level asynchrony in rates of phenological change for marine, freshwater and terrestrial environments . Global Change Biology 16 , 3304 – 3313 . ( doi: 10.1111/j.1365-2486.2010.02165.x ) OpenUrl CrossRef Web of Science 71. ↵ Thackeray SJ et al. 2016 Phenological sensitivity to climate across taxa and trophic levels . Nature 535 , 241 – 245 . ( doi: 10.1038/nature18608 ) OpenUrl CrossRef PubMed 72. ↵ McDevitt-Galles T , Moss WE , Calhoun DM , Johnson PTJ . 2020 Phenological synchrony shapes pathology in host–parasite systems . Proceedings of the Royal Society B: Biological Sciences 287 , 20192597 . ( doi: 10.1098/rspb.2019.2597 ) OpenUrl CrossRef PubMed 73. ↵ Farzan S , Yang LH . 2018 Experimental shifts in phenology affect fitness, foraging, and parasitism in a native solitary bee . Ecology 99 , 2187 – 2195 . ( doi: 10.1002/ecy.2475 ) OpenUrl CrossRef PubMed 74. ↵ Khan RA , Fallis AM . 1970 Life Cycles of Leucocytozoon dubreuili Mathis and Leger, 1911 and L. fringillinarum Woodcock, 1910 (Haemosporidia: Leucocytozoidae)* . The Journal of Protozoology 17 , 642 – 658 . ( doi: 10.1111/j.1550-7408.1970.tb04742.x ) OpenUrl CrossRef Web of Science 75. ↵ Both C , Visser ME , Verboven N. 1999 Density–dependent recruitment rates in great tits: the importance of being heavier . Proceedings of the Royal Society of London. Series B: Biological Sciences 266 , 465 – 469 . ( doi: 10.1098/rspb.1999.0660 ) OpenUrl CrossRef Web of Science 76. ↵ Branston CJ , Capilla-Lasheras P , Pollock CJ , Griffiths K , White S , Dominoni DM . 2021 Urbanisation weakens selection on the timing of breeding and clutch size in blue tits but not in great tits . Behavioral Ecology and Sociobiology 75 , 1 – 12 . ( doi: 10.1007/s00265-021-03096-z ) OpenUrl CrossRef 77. Bambini G , Schlicht E , Kempenaers B. 2019 Patterns of female nest attendance and male feeding throughout the incubation period in Blue Tits Cyanistes caeruleus . Ibis 161 , 50 – 65 . ( doi: 10.1111/ibi.12614 ) OpenUrl CrossRef 78. Jarrett C , Powell LL , McDevitt H , Helm B , Welch AJ . 2020 Bitter fruits of hard labour: diet metabarcoding and telemetry reveal that urban songbirds travel further for lower-quality food . Oecologia 193 , 377 – 388 . ( doi: 10.1007/s00442-020-04678-w ) OpenUrl CrossRef 79. ↵ Chatelain M , Massemin S , Zahn S , Kurek E , Bulska E , Szulkin M. 2021 Urban metal pollution explains variation in reproductive outputs in great tits and blue tits . Science of The Total Environment 776 , 145966 . ( doi: 10.1016/j.scitotenv.2021.145966 ) OpenUrl CrossRef 80. ↵ McGlade CLO , Capilla-Lasheras P , Womack RJ , Helm B , Dominoni DM . 2023 Experimental light at night explains differences in activity onset between urban and forest Great tits . ( doi: 10.5281/zenodo.8021624 ) OpenUrl CrossRef 81. ↵ Capilla-Lasheras P et al. 2017 Elevated Immune Gene Expression Is Associated with Poor Reproductive Success of Urban Blue Tits . Frontiers in Ecology and Evolution 5 , 64 . ( doi: 10.3389/fevo.2017.00064 ) OpenUrl CrossRef 82. ↵ Sanders CJ , Shortall CR , England M , Harrington R , Purse B , Burgin L , Carpenter S , Gubbins S. 2019 Long-term shifts in the seasonal abundance of adult Culicoides biting midges and their impact on potential arbovirus outbreaks . Journal of Applied Ecology 56 , 1649 – 1660 . ( doi: 10.1111/1365-2664.13415 ) OpenUrl CrossRef PubMed 83. White SM , Sanders CJ , Shortall CR , Purse BV . 2017 Mechanistic model for predicting the seasonal abundance of Culicoides biting midges and the impacts of insecticide control . Parasites Vectors 10 , 162 . ( doi: 10.1186/s13071-017-2097-5 ) OpenUrl CrossRef PubMed 84. ↵ Searle KR , Blackwell A , Falconer D , Sullivan M , Butler A , Purse BV . 2013 Identifying environmental drivers of insect phenology across space and time: Culicoides in Scotland as a case study . Bulletin of Entomological Research 103 , 155 – 170 . ( doi: 10.1017/S0007485312000466 ) OpenUrl CrossRef PubMed 85. ↵ Godfrey RD Jr , Fedynich AM , Pence DB . 1987 QUANTIFICATION OF HEMATOZOA IN BLOOD SMEARS . Journal of Wildlife Diseases 23 , 558 – 565 . ( doi: 10.7589/0090-3558-23.4.558 ) OpenUrl CrossRef PubMed 86. ↵ Hellgren O , Waldenström J , Bensch S. 2004 A NEW PCR ASSAY FOR SIMULTANEOUS STUDIES OF LEUCOCYTOZOON, PLASMODIUM, AND HAEMOPROTEUS FROM AVIAN BLOOD . Journal of Parasitology 90 , 797 – 802 . ( doi: 10.1645/GE-184R1 ) OpenUrl CrossRef PubMed Web of Science 87. ↵ Bensch S , Stjernman M , Hasselquist D , Örjan Ö , Hannson B , Westerdahl H , Pinheiro RT . 2000 Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds . Proceedings of the Royal Society of London. Series B: Biological Sciences 267 , 1583 – 1589 . ( doi: 10.1098/rspb.2000.1181 ) OpenUrl CrossRef PubMed Web of Science 88. ↵ Valkiūnas G , Bensch S , Iezhova TA , Križanauskienė A , Hellgren O , Bolshakov CV . 2006 Nested Cytochrome B Polymerase Chain Reaction Diagnostics Underestimate Mixed Infections of Avian Blood Haemosporidian Parasites: Microscopy is Still Essential . Journal of Parasitology 92 , 418 – 422 . ( doi: 10.1645/GE-3547RN.1 ) OpenUrl CrossRef PubMed 89. ↵ Both C , van Asch M , Bijlsma RG , van den Burg AB , Visser ME . 2009 Climate change and unequal phenological changes across four trophic levels: constraints or adaptations? Journal of Animal Ecology 78 , 73 – 83 . ( doi: 10.1111/j.1365-2656.2008.01458.x ) OpenUrl CrossRef PubMed Web of Science 90. ↵ Burgess MD et al. 2018 Tritrophic phenological match–mismatch in space and time . Nat Ecol Evol 2 , 970 – 975 . ( doi: 10.1038/s41559-018-0543-1 ) OpenUrl CrossRef 91. ↵ Atema E , Oers KV , Verhulst S. 2013 GAPDH as a Control Gene to Estimate Genome Copy Number in Great Tits, with Cross-Amplification in Blue Tits GAPDH as a control gene to estimate genome copy number in Great Tits, with cross-amplification in Blue Tits . 101 , 49 – 54 . OpenUrl 92. ↵ Fallon SM , Ricklefs RE , Swanson BL , Bermingham E. 2003 DETECTING AVIAN MALARIA: AN IMPROVED POLYMERASE CHAIN REACTION DIAGNOSTIC . para 89 , 1044 – 1047 . ( doi: 10.1645/GE-3157 ) OpenUrl CrossRef PubMed Web of Science 93. ↵ Sievers F et al. 2011 Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega . Molecular Systems Biology 7 , 539 . ( doi: 10.1038/msb.2011.75 ) OpenUrl CrossRef PubMed Web of Science 94. ↵ R Development Core Team . 2015 R: A language and environment for statistical computing . URL http://www.R-project.org . 95. ↵ Bates D , Mächler M , Bolker BM , Walker SC . 2015 Fitting linear mixed-effects models using lme4 . Journal of Statistical Software 67 , 1 – 113 . ( doi: 10.18637/jss.v067.i01 ) OpenUrl CrossRef PubMed 96. ↵ Brooks ME , Kristensen K , Benthem KJ van , Magnusson A , Berg CW , Nielsen A , Skaug HJ , Mächler M , Bolker BM . 2017 glmmTMB Balances Speed and Flexibility Among Packages for Zero-inflated Generalized Linear Mixed Modeling . The R Journal 9 , 378 – 400 . OpenUrl 97. ↵ Lüdecke (@strengejacke) D et al. 2023 performance: Assessment of Regression Models Performance . 98. ↵ Schielzeth H. 2010 Simple means to improve the interpretability of regression coefficients . Methods in Ecology and Evolution 1 , 103 – 113 . ( doi: 10.1111/j.2041-210X.2010.00012.x ) OpenUrl CrossRef 99. ↵ Askenmo C. 1973 Nestling Weight and Its Relation to Season and Brood-Size in the Pied Flycatcher Ficedula hypoleuca (Pallas) . Ornis Scandinavica (Scandinavian Journal of Ornithology) 4 , 25 – 31 . ( doi: 10.2307/3676287 ) OpenUrl CrossRef 100. ↵ Womack RJ , Capilla-Lasheras P , McGlade CLO , Dominoni DM , Helm B. 2022 Reproductive fitness is associated with female chronotype in a songbird ., 2022.07.01.498449. ( doi: 10.1101/2022.07.01.498449 ) OpenUrl Abstract / FREE Full Text 101. ↵ Verboven N , Visser ME . 1998 Seasonal Variation in Local Recruitment of Great Tits: The Importance of Being Early . Oikos 81 , 511 – 524 . ( doi: 10.2307/3546771 ) OpenUrl CrossRef Web of Science 102. ↵ Jiménez-Peñuela J , Ferraguti M , Martínez-de la Puente J, Soriguer R , Figuerola J. 2019 Urbanization and blood parasite infections affect the body condition of wild birds . Science of The Total Environment 651 , 3015 – 3022 . ( doi: 10.1016/j.scitotenv.2018.10.203 ) OpenUrl CrossRef PubMed 103. ↵ Santiago-Alarcon D , Carbó-Ramírez P , Macgregor-Fors I , Chávez-Zichinelli CA , Yeh PJ . 2020 The prevalence of avian haemosporidian parasites in an invasive bird is lower in urban than in non-urban environments . Ibis 162 , 201 – 214 . ( doi: 10.1111/ibi.12699 ) OpenUrl CrossRef 104. ↵ Caizergues AE , Robira B , Perrier C , Jeanneau M , Berthomieu A , Perret S , Gandon S , Charmantier A. 2024 Cities as parasitic amplifiers? Malaria prevalence and diversity in great tits along an urbanization gradient . Peer Community Journal 4 . ( doi: 10.24072/pcjournal.405 ) OpenUrl CrossRef 105. ↵ In press . Tritrophic phenological match–mismatch in space and time | Nature Ecology & Evolution . See https://www.nature.com/articles/s41559-018-0543-1 (accessed on 1 February 2024 ). 106. ↵ Visser ME , Holleman LJM . 2001 Warmer springs disrupt the synchrony of oak and winter moth phenology . Proceedings of the Royal Society B: Biological Sciences 268 , 289 – 294 . ( doi: 10.1098/rspb.2000.1363 ) OpenUrl CrossRef PubMed Web of Science 107. ↵ Phillimore AB , Leech DI , Pearce-Higgins JW , Hadfield JD . 2016 Passerines may be sufficiently plastic to track temperature-mediated shifts in optimum lay date . Global Change Biology , 1 – 14 . ( doi: 10.1111/gcb.13302 ) OpenUrl CrossRef PubMed 108. ↵ Noordwijk AJVJV , McCleery RHH , Perrins CMM . 1995 Selection for the Timing of Great Tit Breeding in Relation to Caterpillar Growth and Temperature . Journal of Animal Ecology 64 , 451 – 458 . ( doi: 10.2307/5648 ) OpenUrl CrossRef Web of Science 109. ↵ Kolimenakis A , Heinz S , Wilson ML , Winkler V , Yakob L , Michaelakis A , Papachristos D , Richardson C , Horstick O. 2021 The role of urbanisation in the spread of Aedes mosquitoes and the diseases they transmit—A systematic review . PLOS Neglected Tropical Diseases 15 , e0009631 . ( doi: 10.1371/journal.pntd.0009631 ) OpenUrl CrossRef PubMed 110. ↵ Snow K , Medlock J. 2006 The potential impact of climate change on the distribution and prevalence of mosquitoes in Britain . European Mosquito Bulletin , 1 – 10 . 111. ↵ Stockwell PJ , Wessell N , Reed DR , Kronenwetter-Koepel TA , Reed KD , Turchi TR , Meece JK . 2006 A FIELD EVALUATION OF FOUR LARVAL MOSQUITO CONTROL METHODS IN URBAN CATCH BASINS . moco 22 , 666 – 671 . ( doi: 10.2987/8756-971X(2006)22[666:AFEOFL]2.0.CO;2 ) OpenUrl CrossRef 112. ↵ Valkiunas G. 2014 Avian Malaria Parasites and other Haemosporidia . Boca Raton : CRC Press . ( doi: 10.1201/9780203643792 ) OpenUrl CrossRef 113. ↵ Applegate JE . 1970 Population Changes in Latent Avian Malaria Infections Associated with Season and Corticosterone Treatment . The Journal of Parasitology 56 , 439 – 443 . ( doi: 10.2307/3277599 ) OpenUrl CrossRef PubMed Web of Science 114. ↵ Valkiūnas G , Bairlein F , Iezhova TA , Dolnik OV . 2004 Factors affecting the relapse of Haemoproteus belopolskyi infections and the parasitaemia of Trypanosoma spp. in a naturally infected European songbird, the blackcap, Sylvia atricapilla . Parasitol Res 93 , 218 – 222 . ( doi: 10.1007/s00436-004-1071-2 ) OpenUrl CrossRef PubMed 115. ↵ Becker DJ , Singh D , Pan Q , Montoure JD , Talbott KM , Wanamaker SM , Ketterson ED. 2020 Artificial light at night amplifies seasonal relapse of haemosporidian parasites in a widespread songbird . Proceedings of the Royal Society B: Biological Sciences 287 , 20201831 . ( doi: 10.1098/rspb.2020.1831 ) OpenUrl CrossRef 116. ↵ Allan RA , Mahrt JL . 1989 Influence of Transmission Period on Primary and Relapse Patterns of Infection of Leucocytozoon spp . and Haemoproteus mansoni. The American Midland Naturalist 121 , 341 – 349 . ( doi: 10.2307/2426038 ) OpenUrl CrossRef 117. ↵ Pollock CJ , Capilla-Lasheras P , McGill RAR , Helm B , Dominoni DM . 2017 Integrated behavioural and stable isotope data reveal altered diet linked to low breeding success in urban-dwelling blue tits (Cyanistes caeruleus) . Scientific Reports 7 , 5014 . ( doi: 10.1038/s41598-017-04575-y ) OpenUrl CrossRef 118. ↵ Seress G , Sándor K , Evans KL , Liker A. 2020 Food availability limits avian reproduction in the city: An experimental study on great tits Parus major . Journal of Animal Ecology 89 , 1570 – 1580 . ( doi: 10.1111/1365-2656.13211 ) OpenUrl CrossRef PubMed 119. ↵ Linden M , Gustafsson L , Part T. 1992 Selection on Fledging Mass in the Collared Flycatcher and the Great Tit . Ecology 73 , 336 – 343 . ( doi: 10.2307/1938745 ) OpenUrl CrossRef Web of Science 120. ↵ Musa S , Hemberle T , Bensch S , Palinauskas V , Baltrūnaitė L , Woog F , Mackenstedt U. 2024 Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites . Front. Cell. Infect. Microbiol . 14 . ( doi: 10.3389/fcimb.2024.1385599 ) OpenUrl CrossRef 121. ↵ Ciloglu A , Ellis VA , Bernotienė R , Valkiūnas G , Bensch S. 2019 A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites . Parasitol Res 118 , 191 – 201 . ( doi: 10.1007/s00436-018-6153-7 ) OpenUrl CrossRef PubMed 122. ↵ Bell JA , Weckstein JD , Fecchio A , Tkach VV . 2015 A new real-time PCR protocol for detection of avian haemosporidians . Parasites & Vectors 8 , 383 . ( doi: 10.1186/s13071-015-0993-0 ) OpenUrl CrossRef PubMed 123. ↵ Huang X , Huang D , Liang Y , Zhang L , Yang G , Liu B , Peng Y , Deng W , Dong L. 2020 A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays . Parasites & Vectors 13 , 354 . ( doi: 10.1186/s13071-020-04195-y ) OpenUrl CrossRef PubMed 124. ↵ Musa S , Mackenstedt U , Woog F , Dinkel A. 2022 Untangling the actual infection status: detection of avian haemosporidian parasites of three Malagasy bird species using microscopy, multiplex PCR, and nested PCR methods . Parasitol Res 121 , 2817 – 2829 . ( doi: 10.1007/s00436-022-07606-4 ) OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted June 27, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Phenology underlies apparent urbanisation effects on avian malaria in juvenile songbirds Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Phenology underlies apparent urbanisation effects on avian malaria in juvenile songbirds Davide M Dominoni , Bedur Faleh Ali Albalawi , Magdalena Mladenova , Barbara Helm , Francesco Baldini bioRxiv 2025.06.23.661047; doi: https://doi.org/10.1101/2025.06.23.661047 Share This Article: Copy Citation Tools Phenology underlies apparent urbanisation effects on avian malaria in juvenile songbirds Davide M Dominoni , Bedur Faleh Ali Albalawi , Magdalena Mladenova , Barbara Helm , Francesco Baldini bioRxiv 2025.06.23.661047; doi: https://doi.org/10.1101/2025.06.23.661047 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Ecology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41936) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15153) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)