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.

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.

Myotis emarginatus seems fond of spiders and flies, a unique feeding style among European bats. The importance of each prey type varies among studies, so this paper aims to expand on the knowledge to unveil the trophic niche of M. emarginatus by studying its diet changes throughout the maternity season. We sampled five maternity colonies in the Basque Country every 2 weeks for the whole duration of the maternity season and studied their diet using DNA metabarcoding methods. We observed significant changes in diet diversity and composition. At the beginning of the season, M. emarginatus consumed a variety of prey orders, Diptera being the most abundant; but as the season progressed, the relative consumption of Araneae increased. By August, 80% of the weighted percentage of occurrences (wPOO) corresponded to the orb-web-building spider Araneus diadematus. Orb-web-building spiders need habitats of high vertical complexity to grow in abundance. Therefore, conserving them is essential to preserve attractive hunting grounds for colonies of M. emarginatus. Besides, diurnal flies, mainly Stomoxys calcitrans, were also considerably consumed in the colonies studied, which can become detrimental to the well-being of cattle, and can act as a vector of many diseases.