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[EN] Targeted molecular methods such as conventional PCR (cPCR) and quantitative PCR (qPCR), combined with species-specific primers and probes, are widely applied for pest species detection. Besides, the potential of qPCR to quantify DNA in samples makes it an invaluable molecular tool to infer the predation levels on specific prey by analysing predators' stools. Nevertheless, studies on the diet of bats failed to find any empirical relationship, and it remains to be evaluated. Thus, we developed and evaluated two species-specific PCR assays to detect and quantify DNA of a major forest pest, the pine processionary, Thaumetopoea pityocampa, in bats' faeces. Further, we empirically compared a range of different known DNA concentrations (input) of the target species mixed with mocks and bat faecal samples against DNA abundances yielded by qPCR (output) for a quantitative assessment. Overall, cPCR showed a lower detection rate than qPCR, but augmenting the replicate effort from one to three replicates led to a greater increase in the detection rate of the cPCR (from 57 to 80%) than the qPCR (from 90 to 99%). The quantitative experiment results showed a highly significant correlation between the input and output DNA concentrations (t = 10.84, p < 0.001) with a mean slope value of 1.05, indicating the accuracy of our qPCR assay to estimate DNA abundance of T. pityocampa in bat faeces. The framework of this study can be taken as a model to design similar assays applicable to other species of interest, such as agricultural pests or insects of public health concern.

There is growing evidence about the role of insectivorous bats against agricultural pests in various crops. Nevertheless, little research addressed the aggregational and functional responses of bat assemblages to changes in pest availability across a spatio-temporal scale. Therefore, we examined the activity and diet habits of different bat species using DNA metabarcoding by simultaneously monitoring the relative abundance of two major pests (the European grapevine moth, Lobesia botrana, and the leaf rolling tortrix, Sparganothis pilleriana) through the grape growing season, in a vineyard region of the Iberian Peninsula. During pest major irruptions, we found the highest bat activity levels and frequencies of grape pests in the diet of bats, although not all bat species contributed equally to pest suppression. Bats of different foraging guilds positively responded to pest abundances, indicating distinct bat species may synergistically play a role at suppressing agricultural pests at broad scales of the aerospace. For instance, narrow space foragers exploiting major irruptions in grape interior, edge space foragers hampering pest dispersion at local scale, and open space foragers preventing infestations of new grapevine patches at broader scales. Yet, our study exposed the current methodological constraints regarding pest dispersion dynamics, acoustic monitoring of bats? foraging activity or the unfeasibility of metabarcoding to reliably quantify prey abundance in bats diet, and thus further improvement in these issues is required in order to gain insight on the agroecological interactions between bats and pests.

197 p., Artropodo fitofagoek nekazal ekoizpenean kalte oso larriak eragiten dituzte urtero eta izurrite hauenkontra modu jasangarrian aurre egiteko bideen artean eragile biologikoak proposatu izan dira. Saguzarintsektiboroak, haien dieta ohiturak direla eta, artropodoen harrapakari amorratuak dira, gau bakar bateanberaien gorputz masa bezainbeste intsektu jan ditzaketelarik. Oraintsu arte ordea, ezinezkoa izan zaigusaguzarren dieta osatzen duten espezieen zerrenda ezagutzea, baina, azken hamarkadan teknikamolekularren aurrerapenei esker, saguzarrek izurriteen dinamikan eduki dezaketen garrantzia ageriangeratu da. Hala ere, badira oraindik ere bete beharreko hutsune ugari.Tesi honetan saguzar eta izurrien arteko elkarrekintzak aztertu dira, horretarako hurbilketa integral batburutu delarik: saguzarren ekologia trofikoa, izurrien sakabanaketa-ereduak, modelizazioespazio-tenporala eta izurrien detekzio eta kuantifikaziorako metodologia molekular ezberdinenkonparaketa burutu da. Testuinguru honetan, tesiaren helburu orokorra da saguzar-intsektiboroekmahastietan eskaintzen dituzten zerbitzu-ekosistemikoak hobeto ulertzea eta teknika molekularezberdinek saguzarren gorotzetan izurrien jarraipenerako duten eginkizuna aztertzea. Horretarako,hurrengo galderei erantzuten saiatu gara: batetik, zein den saguzarrek izurrien erregulatzaile gisa dutenahalmena eta zein izurri-espezie kontsumitzen dituzten; ea saguzarrek mahastietako izurrien populazioakjarraitu eta ustiatzen dituzten; saguzarrek denboran zehar ematen diren izurrien gorabeherekikoerantzuten duten; zein metodo molekular den egokiagoa helburu diren izurrien detekziorako,metabarcoding edo PCR metodoak eta azkenik, qPCR-a metodo aski sendoa eta fidagarria ote denpinu-beldarra T. pityocampa saguzarren gorotzetan kuantifikatzeko. Oro har, emaitzek aditzera ematendute saguzarrek kultibo oso ezberdinetako izurriak kontsumitzeko ahalmena dutela. Horrez gain, izurriengarai oparoenetan saguzarren aktibitatea altuena izateaz gain, izurr

197 p., Artropodo fitofagoek nekazal ekoizpenean kalte oso larriak eragiten dituzte urtero eta izurrite hauenkontra modu jasangarrian aurre egiteko bideen artean eragile biologikoak proposatu izan dira. Saguzarintsektiboroak, haien dieta ohiturak direla eta, artropodoen harrapakari amorratuak dira, gau bakar bateanberaien gorputz masa bezainbeste intsektu jan ditzaketelarik. Oraintsu arte ordea, ezinezkoa izan zaigusaguzarren dieta osatzen duten espezieen zerrenda ezagutzea, baina, azken hamarkadan teknikamolekularren aurrerapenei esker, saguzarrek izurriteen dinamikan eduki dezaketen garrantzia ageriangeratu da. Hala ere, badira oraindik ere bete beharreko hutsune ugari.Tesi honetan saguzar eta izurrien arteko elkarrekintzak aztertu dira, horretarako hurbilketa integral batburutu delarik: saguzarren ekologia trofikoa, izurrien sakabanaketa-ereduak, modelizazioespazio-tenporala eta izurrien detekzio eta kuantifikaziorako metodologia molekular ezberdinenkonparaketa burutu da. Testuinguru honetan, tesiaren helburu orokorra da saguzar-intsektiboroekmahastietan eskaintzen dituzten zerbitzu-ekosistemikoak hobeto ulertzea eta teknika molekularezberdinek saguzarren gorotzetan izurrien jarraipenerako duten eginkizuna aztertzea. Horretarako,hurrengo galderei erantzuten saiatu gara: batetik, zein den saguzarrek izurrien erregulatzaile gisa dutenahalmena eta zein izurri-espezie kontsumitzen dituzten; ea saguzarrek mahastietako izurrien populazioakjarraitu eta ustiatzen dituzten; saguzarrek denboran zehar ematen diren izurrien gorabeherekikoerantzuten duten; zein metodo molekular den egokiagoa helburu diren izurrien detekziorako,metabarcoding edo PCR metodoak eta azkenik, qPCR-a metodo aski sendoa eta fidagarria ote denpinu-beldarra T. pityocampa saguzarren gorotzetan kuantifikatzeko. Oro har, emaitzek aditzera ematendute saguzarrek kultibo oso ezberdinetako izurriak kontsumitzeko ahalmena dutela. Horrez gain, izurriengarai oparoenetan saguzarren aktibitatea altuena izateaz gain, izurr

Herbivorous arthropods cause immense damage in crop production annually. Consumption of these pests by insectivorous animals is of significant importance to counteract their adverse effects. Insectivorous bats are considered amongst the most voracious predators of arthropods, some of which are known crop pests. In vineyard-dominated Mediterranean agroecosystems, several crops are damaged by the attack of insect pests. In this study we aimed 1) to explore the diet and pest consumption of the lesser horseshoe bat Rhinolophus hipposideros and 2) analyse whether the composition of pest species in its diet changes throughout the season. We employed a dual-primer DNA metabarcoding analysis of DNA extracted from faeces collected in three bat colonies of a wine region in Southwestern Europe during the whole active period of most pest species. Overall, 395 arthropod prey species belonging to 11 orders were detected; lepidopterans and dipterans were the most diverse orders in terms of species. Altogether, 55 pest species were identified, 25 of which are known to cause significant agricultural damage and 8 are regarded as pests affecting grapevines. The composition of pest species in faeces changed significantly with the season, thus suggesting several periods should be sampled to assess the pest consumption by bats. As a whole, the results imply that R. hipposideros acts as a suppressor of a wide array of agricultural pests in Mediterranean agroecosystems. Therefore, management measures favouring the growth of R. hipposideros populations should be considered.

Outbreaks of the processionary moth Thaumetopoea pityocampa (Denis & Schiffer-mailer, 1775), a forest pest from the Palearctic, are thought to induce a behavioral response of bats, but up to now the moth has been seldom identified as bats' prey. Studies on bat diets suggest moths with cyclical outbreaks attract a wide array of bat species from different foraging guilds. We test whether bats feed upon T. pityocampa in the Iberian Peninsula irrespective of the predator's ecological and morphological features. We found that seven out of ten bat species belonging to different foraging guilds contained T. pityocampa DNA in their faeces and no difference was found in the foraging frequency among foraging guilds. A different size of the typical prey or the lack of fondness for moths can explain the absence of the pest in some bat species. Moreover, the intraspecific foraging frequency of T. pityocampa also changed with the sampling site likely representing differential availability of the moth. Lack of information on flight and dispersal behavior or the tympanate nature of the adult moth complicates understanding how different foraging guilds of bats prey upon the same prey. Our data suggests that T. pityocampa is a remarkable food source for many thousands of individual bats in the study area and we anticipate that more bats besides the species studied here are consuming this moth.

Knowledge on the trophic interactions among predators and their prey is important in order to understand ecology and behaviour of animals. Traditionally studies on the diet composition of insectivorous bats have been based on the morphological identification of prey remains, but the accuracy of the results has been hampered due to methodological limitations. Lately, the DNA metabarcoding and High Throughput Sequencing (HTS) techniques have changed the scene since they allows prey identification to the species level, ultimately giving more precision to the results. Nevertheless, the use of one single primer set to amplify faecal DNA produces biases in the assessed dietary composition. Three horseshoe bats overlap extensively in their distribution range in Europe: Rhinolophus euryale, R. hipposideros and R. ferrumequinum. In order to achieve the deepest insight on their prey list we combined two different primers. Results showed that the used primers were complementary at the order and species levels, only 22 out of 135 prey species being amplified by both. The most frequent prey of R. hipposideros belonged to Diptera and Lepidoptera, to Lepidoptera in R. euryale, and Lepidoptera, Diptera and Coleoptera in R. ferrumequinum. The three bats show significant resource partitioning, since their trophic niche overlap is not higher than 34%. Our results confirm the importance of combining complementary primers to describe the diet of generalist insectivorous bats with amplicon metabarcoding techniques. Overall, each primer set showed a subset of the prey composition, with a small portion of the total prey being identified by both of them. Therefore, each primer presented a different picture of the niche overlap among the three horseshoe bats due to their taxonomic affinity.

187 p., Saguzarren ekologia eta jokamoldea ulertzeko ezinbestekoa da beraien eta harrapakinen arteko elkarrekintza trofikoak identifikatzea. Nahiz eta saguzarren oinarrizko bazka-ekologia ezaguna izan (adb.: non eta zertaz bazkatzen diran), beraien bazka-nitxoaren inguruko zehaztasunak aztertzea metodologikoki guztiz mugatuta egon da. Testuinguru honetan, DNA metabarcoding delako teknika molekularrak saguzarren ekologian sakontzeko aukera berri bat eskaintzen du. Tesi honetan, Rhinolophus euryale eta R. mehelyi ferra-saguzarrak eredu hartuta, saguzarren bazka-ekologiaren inguruko hainbat galdera ekologiko ebatzi dira DNA metabarcodingaren bidez. Tesiaren lehenengo ikerketa-atalean paradigma ekologiko berri bat aurkezten da: R. euryale espeziearentzako harrapakin-eskuragarritasuna ez da bere ehiza-habitat egokien presentziarekin eta hauen kontserbazioarekin soilik bermatzen, hau da, hesi-bizi eta hostozabalen basoekin. Harrapakinek beraien bizi-fase desberdinak garatzeko beharrezkoak dituzten habitatak ere ezinbestekoak dira saguzarrentzat, nahiz eta habitat hauek ez dituzten bazkatzeko erabiltzen. Paradigma berri hau testatzeko, 56 R. euryaleren dieta espezie mailan zehaztu eta berau osatzen duten sits espezieen beldarren beharrizan ekologikoak bilatu dira bibliografian. Jandako espezie askoren larbek saguzarren ehiza-habitatak ez diren bestelako habitat batzuk behar dituzte beraien larba-fasea burutzeko, larreak eta belardiak esaterako. Gainera, larben beharrizan ekologikoak esangarriki aldatzen dira sasoitik sasoira. Beraz, R. euryaleren bazka-beharrizanak asetzeko ez dira nahikoa ehiza-habitat egokiak kontserbatzea paisai jakinean. Ehiza-habitatak bazkaz hornitzeko beharrezkoak diren gainerako eremuak ere guztiz beharrezkoak dira. Azken hauetan egindako edozein eraldaketak eragin zuzena izan dezake R. euryaleren bazka-ekologian. Gure emaitzen arabera, saguzarrak kontserbatzeko beharrezko neurriek ehiza habitatak barneratzeaz gain harrapakinen fase desberdinak garatzeko b

Introduction: Our purpose was to assess how pairs of sibling horseshoe bats coexists when their morphology and echolocation are almost identical. We collected data on echolocation, wing morphology, diet, and habitat use of sympatric Rhinolophus mehelyi and R. euryale. We compared our results with literature data collected in allopatry with similar protocols and at the same time of the year (breeding season). Results:Echolocation frequencies recorded in sympatry for R. mehelyi (mean = 106.8 kHz) and R. euryale (105.1 kHz) were similar to those reported in allopatry (R. mehelyi 105–111 kHz; R. euryale 101–109 kHz). Wing parameters were larger in R. mehelyi than R. euryale for both sympatric and allopatric conditions. Moths constitute the bulk of the diet of both species in sympatry and allopatry, with minor variation in the amounts of other prey. There were no inter-specific differences in the use of foraging habitats in allopatry in terms of structural complexity, however we found inter-specific differences between sympatric populations: R. mehelyi foraged in less complex habitats. The subtle inter-specific differences in echolocation frequency seems to be unlikely to facilitate dietary niche partitioning; overall divergences observed in diet may be explained as a consequence of differential prey availability among foraging habitats. Inter-specific differences in the use of foraging habitats in sympatry seems to be the main dimension for niche partitioning between R. mehelyi and R. euryale, probably due to letter differences in wing morphology. Conclusions: Coexistence between sympatric sibling horseshoe bats is likely allowed by a displacement in spatial niche dimension, presumably due to the wing morphology of each species, and shifts the niche domains that minimise competition. Effective measures for conservation of sibling/similar horseshoe bats should guarantee structural diversity of foraging habitats.