Aerospace-foraging bats eat seasonably across varying habitats.

Recent research has confirmed the efficiency of insectivorous bats as pest suppressors, underlining the ecological services they offer in agroecosystems. Therefore, some efforts try to enhance bat foraging in agricultural landscapes by acting upon environmental factors favouring them. In this study, we monitored a Miniopterus schreibersii colony, in the southern Iberian Peninsula. We intensively sampled their faeces and analysed them by metabarcoding to describe how the bent-winged bat diet would change with time, and to test whether their most-consumed prey would seasonally depend on different landscapes or habitats. Our results confirm that M. schreibersii are selective opportunist predators of moths, dipterans, mayflies, and other fluttering insects, shifting their diet to temporary peaks of prey availability in their foraging range, including both pest and non-pest insects. Supporting our hypothesis, throughout the year, M. schreibersii consume insects linked to diverse open habitats, including wetlands, grassland, diverse croplands, and woodland. The importance of each prey habitat varies seasonally, depending on their insect phenology, making bats indirectly dependent on a diverse landscape as their primary prey source. Bats' predation upon pest insects is quantitatively high, consuming around 1610 kg in 5 months, of which 1467 kg correspond to ten species. So, their suppression effect may be relevant, mainly in patchy heterogeneous landscapes, where bats' foraging may concentrate in successive outbursts of pests, affecting different crops or woodlands. Our results stress that to take advantage of the ecosystem services of bats or other generalist insectivores, keeping the environmental conditions they require to thrive, particularly a heterogeneous landscape within the colony's foraging area, is crucial.

Molecular assays to reliably detect and quantify predation on a forest pest in bats faeces.

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.

Bats from different foraging guilds prey upon the pine processionary moth.

Outbreaks of the processionary moth Thaumetopoea pityocampa (Denis & Schiffermüller, 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.

Pest consumption in a vineyard system by the lesser horseshoe bat (Rhinolophus hipposideros).

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.

Trait-based functional dietary analysis provides a better insight into the foraging ecology of bats.

The degree of trophic specialization determines the ability of predators to cope with changing foraging conditions, but in predators that prey on hundreds of species it is challenging to assess, especially when prey identity varies among predator individuals and across space and time. Here, we test the hypothesis that a bat species foraging on flying insects like moths will show ample flexibility in trophic niche, and this irrespective of phylogenetic relationships among moths, so as to cope with a high diversity of prey types that vary across seasons. We predict that individual bats will show functional dietary differences consistent with energetic requirements and hunting skills. We used DNA metabarcoding to determine the diet of 126 Mediterranean horseshoe bats (Rhinolophus euryale) from two different sites during three seasons. Simultaneously, we measured moth availability and characterized the traits of 290 moth taxa. Next, we explored the relationship between phylogeny and traits of all consumed and available moth taxa. Finally, we assessed the relationship between individual traits of bats and traits related to prey profitability, for which we used the RLQ and fourth-corner statistical techniques. Seasonality was the main factor explaining the functional dietary variation in adult bats, with moths consumed irrespective of their phylogenetic relationships. While adults consumed moths with a broad range in wing loading, body mass and echolocation detection ability, juveniles consumed slower, smaller and lighter moths, which suggests that young individuals may undergo some fitness gain and/or psychomotor learning process during which they would acquire more effective foraging skills. Our approach revealed a degree of functional flexibility in the trophic niche previously unknown for an insectivorous bat. Rhinolophus euryale consumed a wide variety of moth taxa differing in profitability throughout seasons and between ontogenetic stages. We showed the validity of trait-based approaches to gain new insights in the trophic specialization of predators consuming hundreds of species of prey.

Gaining ecological insight on dietary allocation among horseshoe bats through molecular primer combination.

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.

Assessing niche partitioning of co-occurring sibling bat species by DNA metabarcoding.

Niche partitioning through foraging is a mechanism likely involved in facilitating the coexistence of ecologically similar and co-occurring animal species by separating their use of resources. Yet, this mechanism is not well understood in flying insectivorous animals. This is particularly true of bats, where many ecologically similar or cryptic species coexist. The detailed analysis of the foraging niche in sympatric, cryptic sibling species provides an excellent framework to disentangle the role of specific niche factors likely involved in facilitating coexistence. We used DNA metabarcoding to determine the prey species consumed by a population of sympatric sibling Rhinolophus euryale and Rhinolophus mehelyi whose use of habitat in both sympatric and allopatric ranges has been well established through radio tracking. Although some subtle dietary differences exist in prey species composition, the diet of both bats greatly overlapped (Ojk  = 0.83) due to the consumption of the same common and widespread moths. Those dietary differences we did detect might be related to divergences in prey availabilities among foraging habitats, which prior radio tracking on the same population showed are differentially used and selected when both species co-occur. This minor dietary segregation in sympatry may be the result of foraging on the same prey-types and could contribute to reduce potential competitive interactions (e.g., for prey, acoustic space). Our results highlight the need to evaluate the spatial niche dimension in mediating the co-occurrence of similar insectivorous bat species, a niche factor likely involved in processes of bat species coexistence.

What mechanism of niche segregation allows the coexistence of sympatric sibling rhinolophid bats?

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.