Effects of sex and sampling site on the relative proportion of pesticides in uropygial gland secretions of European Blackbirds (Turdus merula)

ETHICS AND PERMISSIONS This project was undertaken with all the necessary permits issued by the Regional Department of the Environment (Consejería de Medio Ambiente, Junta de Andalucía) and CSIC bio-ethics commit- tee. Ringing licences 66 042 and 660 019. Bio- Ethics permit: 25–05–15-254. Regional Depart- ment of the Environment Permit 2013_21_22- 2011_15 DGGMN 2014_2015.Data Availability Statement All data generated or analysed during this study are included in this published article. Data availability: the sequences from parasite lineages generated dur- ing the current study are available in MALAVI repository (http://130.235.244.92/Malavi/index. html) and GenBank (ncbi.nlm.nih.gov/genbank/). The lineages submitted are from Plasmodium SYAT05 (Plasmodium vaughani) and LINN1 (= pSPHUjJ 2), and three different lineages of Leucocy- tozoon: TUMER01, TUMER02 and NEVE01. The GenBank numbers are as follows: Leucocytozoon: ON730883 – Lineage NEVE01, ON730884 – Line- age TUMER02 and ON730885 – Lineage TUMER01; Plasmodium: ON730886 – Linage LINN1 and ON730887 – Lineage SYAT05.This study was funded by project P11-RNM-7038 from the Junta de Andalucía and projects CGL2015-65055-P and PGC2018-095704-B-I00 from the Spanish Ministry of Science and Innovation and European Regional Development Fund (FEDER). A.D.F. was supported by a Severo- Ochoa grant from the Spanish Ministry of Economy and Competitiveness (SVP-2014-068571). L.G. was supported by a Marie Sklodowska-Curie Fellowship from the European Commission (grant number 747729, EcoEvoClim).Dichlorodiphenyltrichloroethane (DDT) is a pesticide that was commonly used for decades worldwide. The use of DDT was banned in the 1970s and 1980s in Europe because of its high toxicity and persistence in the environment, bioaccumulation in living organisms and biomagnification through food webs. However, monitoring using both invasive and non-invasive methods has routinely reported the occurrence of DDT metabolites such as dichlorodiphenyldichloroethylene (DDE) in wild birds, providing valuable information about the exposure to pesticides and potential differences between species and over time. Here, we analysed the relative proportion of DDE in the uropygial gland secretions of European Blackbirds Turdus merula from two localities in southern Spain. Given the negative effects of this pollutant on animal immunity, we also tested for associations between the prevalence of haemosporidians and the relative proportion of DDE in their secretions. Relative proportions of DDE varied between sampling sites and were higher in females than in males, regardless of their age. In spite of the potential immunosuppressive effect of DDE, haemosporidian infection was not associated with DDE presence.Project P11-RNM-7038 from the Junta de AndalucíaProjects CGL2015-65055-P and PGC2018-095704-B-I00 from the Spanish Ministry of Science and Innovation and European Regional Development Fund (FEDER)Severo Ochoa grant from the Spanish Ministry of Economy and Competitiveness (SVP-2014-068571)Marie Sklodowska-Curie Fellowship from the European Commission (grant number 747729, EcoEvoClim

The role of uropygial secretion and birds body odour on their interaction with mosquitoes and parasites

Chemical signals are implied in vital proceses such as food acquisition, reproduction, predation avoidance and interspecific interactions. In blood sucking insects such as mosquitoes, chemical signals play a key role by allowing the detection of their vertebrate hosts. Besides interspecific differences, vertebrate host characteristics such as age, sex, size and infection by blood parasites, may affect the chemical signals emitted by hosts, which may in turn affect vector-host interactions and ultimately, the transmission dynamics of vector-borne diseases. Mosquitoes transmit parasites and other pathogens causing important diseases that affect humans, wildlife, and domestic animals. This is the case of malaria parasites, which may largely affect host population dynamics. However, the mechanisms affecting the interactions between mosquitoes, parasites, and vertebrate hosts are not fully understood, especially in the case of wild nonmodel species. The aim of this thesis is to assess the role of different bird chemical cues (uropygial gland secretions and body odour) in mosquito attraction and the effect of host infection by avian malaria parasites on these interactions. To deal with this important question, I used a multidisciplinary approach that combines techniques from different research areas (e.g. molecular parasitology, entomology, and ornithology), and data from field and laboratory experiments under controlled conditions. As study system, I used i) two wild passerines as vertebrate hosts, the common blackbird (Turdus merula) and the house sparrow (Passer domesticus), ii) two species of mosquitoes, namely the common house mosquito (Culex pipiens) and the marshland mosquito (Aedes caspius), and iii) avian haemospodians, including mosquito-borne parasites of the genus Plasmodium. In order to better understand the dynamics of host-vector-pathogen interactions, I tested the host manipulation hypothesis, which argues that parasites may modify certain characteristics of their hosts to increase their transmission success. Thus, malaria infected birds would be more attractive to mosquitoes than uninfected ones, thereby enhancing the contact rates between parasites and insect vectors. However, the mechanisms underlying this differential attraction are still unknown. I studied the potential effect of parasite infections on the odours emitted by birds and how this may in turn affect the interactions with mosquitoes. Secretions of the uropygial gland are considered as one of the main sources of bird odour, so the effects of parasites on bird-vector interactions could be driven by their effects on the composition of these secretions. I performed different studies to determine the potential factors affecting the composition of birds’ uropygial secretions, including parasite infections, and subsequently, I tested the role of secretions and body odour in mosquito attraction. The overall composition of uropygial secretions of wild birds differed between sexes and age classes, while this was not the case for the infection by avian haemosporidian parasites neither the habitat type (forest vs urban areas). Further analyses revealed the presence of the pollutant DDE in uropygial secretions. DDE is mainly derived from the DDT used decades ago. The relative proportion of DDE was higher in older and forest-dwelling birds, and increased with bird´s body mass. Overall these results support the role of both intrinsic and extrinsic factors in between-individuals variation in the composition of uropygial gland secretions. However, these results do not provide support for the potential effect of parasite infections on the composition of bird uropygial secretions.On the basis of the importance of the uropygial gland secretion as a source of bird odour, I evaluated the role of this secretion in the attraction of two species of mosquitoes with differential feeding patterns, namely the ornithophilic Cx. pipiens and the mammophilic Ae. capius. Both species of mosquitoes were similarly attracted to this stimulus (uropygial gland secretions + CO2) than to the control (only CO2) in a dual choice olfactometer, suggesting that the attraction of mosquitoes to avian hosts is not mediated by this chemical cue. Subsequently, I tested the role of Plasmodium infection in the attraction of Cx. pipiens mosquitoes towards both uropygial secretions and bird´s body odours (headspace). Culex pipiens were more attracted towards the headspace of Plasmodium infected than uninfected birds, while no differences were found in the attraction of mosquitoes when the stimulus tested was the uropygial gland secretion. These results suggest that Plasmodium parasite modify bird body odour increasing the attraction of mosquitoes and therefore, increasing its capacity of transmission to new vertebrate hosts. However, these effects are not driven by changes in the volatile fraction of the uropygial gland secretion. In sum, this thesis provides novel evidence into the complex mechanisms that drive the interactions between parasites, vectors, and vertebrates, and highlights the role of chemical cues such as the odour of birds in the attraction of mosquitoes to individuals infected by haemosporidians. These results may have important implications for the epidemiology of Plasmodium parasites in natural environments and open new questions for future studies on the identification of key components of bird´s odours determining mosquito attraction.Las señales químicas están implicadas en procesos vitales tan importantes como la búsqueda de alimento, reproducción y defensa frente a depredadores, entre otras, desempeñando, por tanto, un importante papel en las interacciones intra e interespecíficas. En insectos que se alimentan de sangre, como los mosquitos, el uso de pistas químicas resulta esencial para detectar a sus hospedadores vertebrados. Además de las diferencias existentes entre especies de hospedadores, ciertas características individuales como la edad, el sexo, el tamaño corporal y la infección por parásitos sanguíneos podrían afectar a las señales químicas que emiten, lo que a su vez podría afectar a las interacciones vector-hospedador y, en última instancia, a la dinámica de transmisión de enfermedades transmitidas por vectores. Los mosquitos transmiten parásitos y otros patógenos causantes de importantes enfermedades que afectan a humanos y animales, tanto silvestres como domésticos. Este es el caso de los parásitos de la malaria, que pueden influir en gran medida en la dinámica de las poblaciones de sus hospedadores. Sin embargo, los mecanismos que afectan a las interacciones entre mosquitos, parásitos y hospedadores vertebrados siguen sin estar claros, especialmente en el caso de especies silvestres-no modelo. El objetivo de esta tesis es entender el papel de las diferentes pistas químicas que generan las aves (secreciones de la glándula uropigial y olor corporal) en la atracción de mosquitos, así como el efecto de la infección por parásitos de la malaria aviar del hospedador sobre estas interacciones. Para abordar esta importante pregunta, utilicé un enfoque multidisciplinar que combina diferentes áreas de investigación (p.e., parasitología molecular, entomología y ornitología), datos de campo y experimentos de laboratorio bajo condiciones controladas. Mi sistema de estudio incluye i) dos aves paseriformes silvestres como hospedadores vertebrados, el mirlo común (Turdus merula) y el gorrión común (Passer domesticus), ii) dos especies de mosquitos, el mosquito común (Culex pipiens) y el mosquito de marismas (Aedes caspius), y iii) hemosporidios presentes en aves, incluidos los parásitos del género Plasmodium transmitidos por mosquitos. Para comprender mejor la dinámica de las interacciones entre hospedador-vector-patógeno, evalué la hipótesis de la manipulación del hospedador, la cual propone que los parásitos modifican ciertas características de sus hospedadores para aumentar su éxito de transmisión. Por lo tanto, las aves infectadas con malaria resultarían más atractivas para los mosquitos que aquellas no infectadas, aumentando así las tasas de contacto entre parásitos e insectos vectores. Sin embargo, aún se desconocen los mecanismos que subyacen a esta atracción diferencial. Estudié el efecto potencial de la infección por parásitos sobre los olores emitidos por las aves y como esto a su vez, podría afectar a las interacciones con los mosquitos. Las secreciones de la glándula uropigial son consideradas como una de las principales fuentes de olor de las aves, por lo que los efectos de los parásitos en las interacciones entre las aves y los vectores podrían estar mediados a través de cambios en la composición de estas secreciones. Realicé diferentes estudios para determinar los factores potenciales que afectan a la composición de las secreciones uropigiales de las aves, incluida la infección por parásitos y, posteriormente, evalué el papel de las secreciones y el olor corporal en la atracción de mosquitos. La composición de las secreciones uropigiales de las aves silvestres difirió entre clases de edad y sexo, mientras que no hubo diferencias asociadas a la infección por parásitos hemosporidios ni al tipo de hábitat (bosque vs áreas urbanas). Análisis adicionales revelaron la presencia del contaminante DDE en las secreciones uropigiales de las aves. La degradación del DDT usado hace décadas origina DDE. La proporción relativa de DDE detectada fue mayor en aves adultas y en aquellas capturadas en bosques frente a las de ciudad, siendo superior también en aves con un mayor peso corporal. En general, estos resultados apoyan el papel de factores intrínsecos y extrínsecos en las variaciones interindividuales en la composición de las secreciones de la glándula uropigial. Sin embargo, estos resultados no respaldan un efecto de la infección por parásitos de la malaria aviar en la composición de las secreciones de estas aves. Teniendo en cuenta la importancia de la secreción de la glándula uropigial como fuente principal del olor en las aves, evalué el papel de esta secreción en la atracción de dos especies de mosquito con patrones de alimentación diferentes, el mosquito Cx. pipiens que se alimenta principalmente de aves y el mosquito Ae. Capius que se alimenta de mamíferos. Ambas especies de mosquitos mostraron una atracción similar hacia el estímulo (secreciones de la glándula uropigial + CO2) y el control (solo CO2) en un olfatómetro de doble elección, lo que sugiere que la atracción de los mosquitos hacia las aves no está mediada por la fracción volátil de esta secreción. Posteriormente, evalué el papel de la infección por Plasmodium en la atracción de los mosquitos Cx. pipiens hacia las secreciones uropigiales y olor corporal de las aves. Culex pipiens mostró mayor atracción hacia el olor corporal de las aves infectadas por Plasmodium que al de aves no infectadas, pero no se encontraron diferencias cuando el estímulo utilizado fue la secreción de la glándula uropigial. Estos resultados sugieren que los parásitos de la malaria aviar modifican el olor corporal de las aves, aumentando así la atracción de los mosquitos hacia aves infectadas y, por tanto, su capacidad de transmisión a nuevos hospedadores. No obstante, estos efectos no son producidos por cambios en la composición de la fracción volátil de la secreción de la glandula uropigial. En resumen, esta tesis proporciona nuevas evidencia sobre los complejos mecanismos que rigen las interacciones entre parásitos, vectores y vertebrados, y destaca el papel de los compuestos químicos, como los que generan el olor de las aves, en la atracción de mosquitos hacia individuos infectados por hemosporidios. Estos resultados tienen también implicaciones importantes para la comprensión de la epidemiología de los parásitos del género Plasmodium en ambientes naturales, y generan nuevas preguntas para futuras investigaciones dirigidas a la identificación de compuestos clave que conforman los olores de las aves y determinan la atracción de los mosquitos

A preliminary overview on the influence of rehabilitation on blood parasite infection in wild owls Strigidae

Trabajo presentado en La 10ª edición de la conferencia de la EOU ( Unión Europea Ornitólogos/as). Badajoz (España) del 24 al 28 de agosto de 2015.The haemosporidian parasites of birds usually infect wild birds in natural condi'ons and could modulate their health status. The rehabilita'on centres have a remarkable role in avian conserva'on. This study is focused on the influence of rehabilita'on period over the presence of haemosporidian infec'on in the recovered individuals in a rehabilitation centre in Madrid (Central Spain).The haemosporidian parasites of birds have captured the interest of avian researchers in the last century. They usually infect wild birds in natural conditions, and the parasite presence (or absence) is often used as a general health index of the avian host. Nowadays, the rehabilitation centres have a remarkable role in avian conservation. Among others activities these centres heal a vast number of individuals suffering from different kinds of accidents/problems. More of them are released once they get recovered. This study is focused on the influence of rehabilitation period over the presence of haemosporidian infection in the recovered individuals. We carried out haemosporidian diagnosis on owl in a rehabilitation centre to measure if any parasitemia variation happens during the recovery of birds. We assessed the parasitemia at the beginning and at the end of the rehabilitation stint. Our preliminary results show that infection levels by these blood parasites decrease during the period spent in the rehabilitation centre. A non-specific treatment against haemosporidians would be enough to decrease the parasitemia furtherPeer reviewe

The prevalence and molecular characterisation of blood parasites infecting the vulnerable Tamarugo Conebill (Conirostrum tamarugense) and other birds in the Pampa del Tamarugal, Chile

Blood parasites exert significant selective pressure, which can alter population dynamics, and the introduction of new parasite lineages in a region could lead to the extinction of endemic avian species that have not coevolved with them. The avifauna of the Pampa del Tamarugal in the desert of Atacama, Chile, is highly depauperate, with two species accounting for the greatest numbers: the widespread Rufous-collared Sparrow (Zonotrichia capensis) and the endemic Tamarugo Conebill (Conirostrum tamarugense). We used molecular and microscopic methods to estimate the prevalence of haemoparasites in birds inhabiting the Pampa del Tamarugal. The molecular screening of the samples from the most common species indicated that the Tamarugo Conebill was only infected by parasites of the genus Leucocytozoon (prevalence 3.5%) and the Rufous-collared Sparrow was uninfected. Only two other bird species (of a total of seven) were infected, the House Sparrow (Passer domesticus), by Plasmodium relictum (prevalence 30%), and the Eared Dove (Zenaida auriculata), by Haemoproteus multipigmentatus (prevalence 100%). The occurrence of House Sparrows infected with the Plasmodium haplotype GRW4 (involved in Hawaiian bird extinctions) may entail the risk of potential host-switching, something particularly relevant for the conservation of the endemic and vulnerable Tamarugo Conebill.Peer reviewe

Strangers spreading diseases: the role of nonindigenous species as vectos of blood parasites for endemic threatened forest bird species in Chile

Trabajo presentado en 32nd IUBS General Assembly and Conference »IUBS 2015 – Frontiers in Unified Biology« 14–16 December 2015 Berlin .Peer reviewe

Effects of Mosquito Microbiota on the Survival Cost and Development Success of Avian Plasmodium

Both intrinsic and extrinsic factors affect the capacity of mosquitoes for the transmission of vector-borne pathogens. Among them, mosquito microbiota may play a key role determining the development of pathogens in mosquitoes and the cost of infections. Here, we used a wild avian malaria-mosquito assemblage model to experimentally test the role of vector microbiota on the cost of infection and their consequences for parasite development. To do so, a cohort of Culex pipiens mosquitoes were treated with antibiotics, including gentamicin sulfate and penicillin-streptomycin, to alter their microbiota, and other cohort was treated with sterilized water as controls. Subsequently, both cohorts were allowed to feed on Plasmodium infected or uninfected house sparrows (Passer domesticus). The antibiotic treatment significantly increased the survival rate of mosquitoes fed on infected birds while this was not the case of mosquitoes fed on uninfected birds. Additionally, a higher prevalence of Plasmodium in the saliva of mosquitoes was found in antibiotic treated mosquitoes than in mosquitoes of the control group at 20 days post exposure (dpe). Analyses of the microbiota of a subsample of mosquitoes at 20 dpe suggest that although the microbiota diversity did not differ between individuals of the two treatments, microbiota in control mosquitoes had a higher number of unique features and enriched in biochemical pathways related to the immune system than antibiotic treated ones. In sum, this study provides support for the role of mosquito microbiota on mosquito survival and the presence of parasite DNA in their saliva

Urbanization modifies plumage and beak colour of common blackbirds in Europe

Trabajo presentado en BOU Annual Conference, University of Leicester, Uk. 5 to 7 April. (2016)Peer reviewe

The expansion of monk parakeet in mediterranean urban areas: implications for the transmission of parasites by native and invasive vectors

Trabajo presentado en XIV MEDECOS & XIII AEET Meeting. Seville, 31st January - 4th February 2017Peer reviewe

Correction to: Aedes vittatus in Spain: current distribution, barcoding characterization and potential role as vectors of human diseases

Unfortunately, the original version of this article [1] contained an error. In the distribution map in Fig. 3, the presence of the mosquito Aedes vittatus was incorrectly indicated for Libya and Egypt.Peer reviewe