Efficient and unbiased sampling of ecological interactions is essential to our understanding of the functions they mediate. Seed dispersal by frugivorous birds is a key mutualism for plant regeneration and community dynamics. Mist-netting is one of the most widely used methods to sample avian seed dispersal through the identification of seeds in droppings of captured birds kept inside cloth bags. However, birds may drop seeds on the ground before being extracted from the net, leading to a fraction of missing information due to ineffective sampling. Worryingly, this fraction could be unevenly distributed across bird and plant species, leading to sampling biases. Here, we assess the effectiveness of using a 1-m wide mesh below mist nets to sample seeds dropped by entangled birds. We used data from birds mist-netted during one-year-round. We sampled nearly 50% of interaction events and 75% of dispersed seeds on the mesh band below the mist nets (i.e. lost information without this optimization). The proportion of seeds sampled on the mesh bands was not evenly distributed among bird species but strongly related to bird size, ranging from 57-63% in warblers to 84-94% in thrushes. Moreover, the proportion of seeds sampled on the mesh was negatively related to seed size, although this relationship was weaker. We also evaluated accumulation curves of species and pairwise interactions with increasing sampling effort, both with and without using the mesh bands. The number of seed species sampled increased by 21% when using the mesh bands and the number of pairwise interactions by 36%. Our findings provide strong evidence on how inefficient and biased traditional mist-netting can be for sampling community-wide seed-dispersal interactions. We thus urge the use of mesh bands in future studies to increase sampling effectiveness and avoid biases, which will ultimately improve our understanding of the seed dispersal function.
Nosema ceranae is a highly prevalent intracellular parasite of honey bees’ midgut worldwide. This Microsporidium was monitored during a long-term study to evaluate the infection at apiary and intra-colony levels in six apiaries in four Mediterranean countries (France, Israel, Portugal, and Spain). Parameters on colony strength, honey production, beekeeping management, and climate were also recorded. Except for São Miguel (Azores, Portugal), all apiaries were positive for N. ceranae, with the lowest prevalence in mainland France and the highest intra-colony infection in Israel. A negative correlation between intra-colony infection and colony strength was observed in Spain and mainland Portugal. In these two apiaries, the queen replacement also influenced the infection levels. The highest colony losses occurred in mainland France and Spain, although they did not correlate with the Nosema infection levels, as parasitism was low in France and high in Spain. These results suggest that both the effects and the level of N. ceranae infection depends on location and beekeeping conditions. Further studies on host-parasite coevolution, and perhaps the interactions with other pathogens and the role of honey bee genetics, could assist in understanding the difference between nosemosis disease and infection, to develop appropriate strategies for its control.
A total of 106 West African taurine cattle belonging to the Lagunaire breed of Benin (33), the N’Dama population of Burkina Faso (48), and N’Dama cattle sampled in Congo (25) were analyzed for Copy Number Variations (CNVs) using the BovineHDBeadChip of Illumina and two different CNV calling programs: PennCNV and QuantiSNP. Furthermore, 89 West African zebu samples (Bororo cattle of Mali and Zebu Peul sampled in Benin and Burkina Faso) were used as an outgroup to ensure that analyses reflect the taurine cattle genomic background. Analyses identified 307 taurine-specific CNV regions (CNVRs), covering about 56 Mb on all bovine autosomes. Gene annotation enrichment analysis identified a total of 840 candidate genes on 168 taurine-specific CNVRs. Three different statistically significant functional term annotation clusters (from ACt1 to ACt3) involved in the immune function were identified: ACt1 includes genes encoding lipocalins, proteins involved in the modulation of immune response and allergy; ACt2 includes genes encoding coding B-box-type zinc finger proteins and butyrophilins, involved in innate immune processes; and Act3 includes genes encoding lectin receptors, involved in the inflammatory responses to pathogens and B- and T-cell differentiation. The overlap between taurine-specific CNVRs and QTL regions associated with trypanotolerant response and tick-resistance was relatively low, suggesting that the mechanisms underlying such traits may not be determined by CNV alterations. However, four taurine-specific CNVRs overlapped with QTL regions associated with both traits on BTA23, therefore suggesting that CNV alterations in major histocompatibility complex (MHC) genes can partially explain the existence of genetic mechanisms shared between trypanotolerance and tick resistance in cattle. This research contributes to the understanding of the genomic features of West African taurine cattle.
The aim of this work was to study the cooperage potential of the oak species Quercus pyrenaica, which is widespread throughout the Iberian Peninsula. A red wine of 2016 vintage was aged in new barrels made from Quercus pyrenaica, Quercus petraea and Quercus alba for 12 months. This process was repeated with a similar red wine from the subsequent vintage using the same barrels in order to compare the performances of the new Q. pyrenaica barrels and Q. pyrenaica barrels that had been used for one year with equivalent Q. petraea and Q. alba barrels. The results indicate that Q. pyrenaica releases levels of β-methyl-γ-octalactone similar to those released by Q. alba and clearly higher than Q. petraea, whereas it releases levels of ellagitannins similar to Q. petraea and clearly higher than Q. alba. These data indicate that Q. pyrenaica is more similar to American oak from an aromatic point of view, since it provides mainly coconut notes, but is more similar to French oak in terms of wine structuration. Moreover, based on the preferences of a trained panel, the wines aged in the Q. pyrenaica barrels were ranked in second position, just behind the wines aged in the Q. petraea barrels and ahead of those aged in the Q. alba barrels. Consequently, Q. pyrenaica seems to have characteristics midway between the two most commonly used oak species for cooperage, confirming its high potential in this regard.
Pseudomonas viridiflava was originally reported as a bean pathogen, and subsequently as a wide-host range pathogen affecting numerous plants species. In addition, several authors have reported the epiphytic presence of this bacterium in “non-host plants”, which may act as reservoir of P. viridiflava and source of inoculum for crops. A new biotype of this bacterium, showing an atypical LOPAT profile, was found in Asturias, a Northern region of Spain, causing significant damage in beans, kiwifruit, lettuce, and Hebe. In order to investigate the involvement of weeds in bean disease, samples were collected from beans and weeds growing in the same fields. A total of 48 isolates of P. viridiflava were obtained, 39 from weeds and 9 from beans. 48% and 52% of them showed typical (L− O− P+ A− T+) and atypical (L+ O− P v A− T+) LOPAT profiles, and they displayed high biochemical diversity. Regarding virulence factors, the T-PAI and S-PAI pathogenicity islands were found in 29% and 70.8% of the isolates, 81.2% displayed pectinolytic activity on potato slices, and 59% of the weed isolates produced symptoms after inoculation on bean pods. A phylogenetic tree based on concatenated rpoD, gyrB, and gltA sequences separated the strains carrying S-PAI and T-PAI into different clusters, both containing isolates from beans and weeds, and pathogenic as well as non-pathogenic strains. Closely related strains were found in the two hosts, and more than half of the weed isolates proved to be pathogenic in beans. This is consistent with the role of weeds as a reservoir and source of inoculum for bean infection. Detection of P. viridiflava in weeds throughout the year further supports these roles.
Fungi in the sooty blotch and flyspeck (SBFS) complex blemish fruit and reduce the market value of fresh-market apples. In 2010, apples were collected from 16 orchards in northern Spain that had received few to no fungicide sprays. SBFS colonies with the subtending cuticle were excised, pressed, and shipped to Ames, Iowa, United States, for isolation. A total of 213 sequences were aligned after a portion of the rRNA was amplified with primer pair VG9/LR5, and two regions were sequenced with primer pairs ITS1/ITS4 and LROR/LR5. Distance and parsimony analyses of the 28S gene sequences were used to compare the collection with previously isolated SBFS species. Most isolates (89%) were within the subclass Dothideomycetes, order Capnodiales. Within this order, the predominant genus was Schizothyrium (anamorph Zygophiala) (86 isolates), including S. pomi, Z. cryptogama, Z. cylindrica, and two previously undescribed putative species. Also widely prevalent were Microcyclosporella mali (45 isolates), four Microcyclospora spp. (36 isolates), and four Stomiopeltis-like putative species (34 isolates). Seven isolates were within the Eurotiomycetes. Twenty-five putative species were delineated using the ITS sequences and morphological characterization. These included 11 species previously named and reported as members of the SBFS complex, two putative SBFS species that were previously reported but have not yet been described, and 12 newly detected putative SBFS species. The findings add substantially to knowledge of the taxonomic diversity of this ectophytic fungal assemblage in Europe. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Introduction: Different gene expression between male and female bovine embryos leads to metabolic differences. Objective: We used UHPLC-MS/MS to identify sex metabolite biomarkers in embryo culture medium (CM). Methods: Embryos were produced in vitro under highly variable conditions, i.e., fertilized with 7 bulls, two breeds, and cultured with BSA or BSA + serum until Day-6. On Day-6, embryos were cultured individually for 24 h. CM of Day-7 embryos (86 female and 81 male) was collected, and Day-6 and Day-7 embryonic stages recorded. Results: A study by sample subsets with fixed factors (culture, bull breed, and Day-6 and Day-7 stages) tentatively identified 31 differentially accumulated metabolites through 182 subsets. Day-6 and Day-7 stage together affected 13 and 11 metabolites respectively, while 19 metabolites were affected by one or another stage and/or day. Culture supplements and individual bull changed 19 and 15 metabolites, respectively. Single bull exerted the highest influence (20 metabolites with the significantly highest p values). Lipid (93 subsets; 11 metabolites) and amino acid (55 subsets; 13 metabolites) were the most relevant classes for sex identification. Conclusions: Single biomarker led to inefficient sex diagnosis, while metabolite combinations accurately identified sex. Our study is a first in non-invasive sex identification in cattle by overcoming factors that induce metabolic variation.
The assessment of feed intake in stabled horses is a difficult task to accomplish. Faecal markers, namely n-alkanes, have been used successfully for the estimation of this important nutritional parameter. This usually involves the dosing of synthetic n-alkanes via different matrices, a laborious task that may also influence the animal normal foraging behaviour. An experiment was conducted to evaluate a relative simple methodology to quantify feed intake in horses, based on the provision of measured amounts of a concentrate supplement labelled with beeswax and the utilisation of n-alkanes as faecal markers. Four Lusitano horses were used in three consecutive experimental periods. Animals were fed on cereal straw and different proportions of a previously prepared beeswax-labelled concentrate supplement (BLCS; 0.05, 0.10 and 0.20, DM basis). Beeswax labelling was performed to provide a distinct n-alkane profile for the concentrate feed. Prior to feed intake calculations, proportions of labelled concentrate supplement in the diets were estimated using n-alkanes C25 to C33 by least-square optimisation procedures. Results showed that the beeswax labelling resulted in high n-alkane concentrations in the concentrate feed, especially for the odd-chain n-alkanes. Estimates of diet composition did not differ from the measured values, except for the diet with highest BLCS incorporation, with an underestimation of 10%. DM intake was accurately estimated by the “labelled supplement method” in all diets. However, for the lowest BLCS incorporation, DM intake was underestimated by 16% whereas for the higher levels of BLCS in the diet, measured and estimated DM intake values were almost identical with a slight overestimation of only 0.7 and 0.2% (10 and 20% of BLCS, respectively). Results indicate that both diet composition and feed intake can be accurately estimated in horses using the “labelled supplement method”, even when very low levels of the labelled concentrate supplement are included in the animals’ diet. This method eliminates the need for daily dosing with external synthetic markers, providing advantages in terms of minimising animal management and interference with their normal foraging behaviour.
The Varroa destructor mite is a serious worldwide pest of honeybees that is usually controlled with pyrethroid-based acaricides. However, the intensive use of these substances over the past decades has led to the development of resistance in these mites. Here, Varroa samples collected between 2006 and 2021 from apiaries across Spain were studied to evaluate the presence of mutations producing pyrethroid resistance, particularly those in the gene encoding the voltage-gated sodium channel (VGSC). Genotyping of the IIS4-IIS5 region of this gene detected the L925V (Leucine ‘CTG’ to valine ‘GTG’) mutation at position 925 and confirmed the presence of the M918L (Methionine ‘ATG’ to Leucine ‘TTG’) mutation at position 918 in these Spanish Varroa mites. Interestingly, the M918L mutation was always found in combination with L925V, both of which were always homozygous. Over and above the high frequency of pyrethroid-resistant mutations in Spanish Varroa populations, this apparently recent association of the M918L and L925V point mutations is a combination that appears to trigger greater resistance than that produced by L925V alone.
Beekeepers have various options to control the parasitic mite Varroa destructor in honey bee colonies, but no empirical data are available on the methods they apply in practice. We surveyed 28,409 beekeepers maintaining 507,641 colonies in 30 European countries concerning Varroa control methods. The set of 19 different Varroa diagnosis and control measures was taken from the annual COLOSS questionnaire on honey bee colony losses. The most frequent activities were monitoring of Varroa infestations, drone brood removal, various oxalic acid applications and formic acid applications. Correspondence analysis and hierarchical clustering on principal components showed that six Varroa control options (not necessarily the most used ones) significantly contribute to defining three distinctive clusters of countries in terms of Varroa control in Europe. Cluster I (eight Western European countries) is characterized by use of amitraz strips. Cluster II comprises 15 countries from Scandinavia, the Baltics, and Central-Southern Europe. This cluster is characterized by long-term formic acid treatments. Cluster III is characterized by dominant usage of amitraz fumigation and formed by seven Eastern European countries. The median number of different treatments applied per beekeeper was lowest in cluster III. Based on estimation of colony numbers in included countries, we extrapolated the proportions of colonies treated with different methods in Europe. This suggests that circa 62% of colonies in Europe are treated with amitraz, followed by oxalic acid for the next largest percentage of colonies. We discuss possible factors determining the choice of Varroa control measures in the different clusters.
There is mounting evidence that acaricides are among the most prevalent medicinal compounds in honey bee hive matrices worldwide. According to OCDE guideline No. 245 chronic lethal concentration of tau-fluvalinate (at concentrations ranging from 77.5 to 523.18 ppm), coumaphos (59.8 ppm) and dimethoate (0.7 ppm) were determined. The activity of the biomarkers acetylcholinesterase (AChE), carboxylesterase (CbE), glutathione S-transferase (GST), catalase (CAT) and malondialdehyde (MDA) was analysed and as they are implicated in neurotoxicity, biotransformation and antioxidant defences, these values were combined into an integrated biomarker response (IBR). There was enhanced AChE, CAT and GST activity in honey bees exposed to tau-fluvalinate, while dimethoate inhibited AChE activity. Both dimethoate and coumaphos inhibited CbE activity but they enhanced CAT activity and MDA formation. Our results highlight how these biomarkers may serve to reveal honey bee exposure to commonly used acaricides.
Nosema ceranae is a highly prevalent pathogen of Apis mellifera, which is distributed worldwide. However, there may still exist isolated areas that remain free of N. ceranae. Herein, we used molecular tools to survey the Azores to detect N. ceranae and unravel its colonisation patterns. To that end, we sampled 474 colonies from eight islands in 2014/2015 and 91 from four islands in 2020. The findings revealed that N. ceranae was not only present but also the dominant species in the Azores. In 2014/2015, N. apis was rare and N. ceranae prevalence varied between 2.7% in São Jorge and 50.7% in Pico. In 2020, N. ceranae prevalence increased significantly (p < 0.001) in Terceira and São Jorge also showing higher infection levels. The spatiotemporal patterns suggest that N. ceranae colonised the archipelago recently, and it rapidly spread across other islands, where at least two independent introductions might have occurred. Flores and Santa Maria have escaped the N. ceranae invasion, and it is remarkable that Santa Maria is also free of Varroa destructor, which makes it one of the last places in Europe where the honey bee remains naive to these two major biotic stressors.
Disease is an integral part of any organisms' life, and bees have evolved immune responses and a suite of hygienic behaviours to keep them at bay in the nest. It is now evident that flowers are another transmission hub for pathogens and parasites, raising questions about adaptations that help pollinating insects stay healthy while visiting hundreds of plants over their lifetime. Drawing on recent advances in our understanding of how bees of varying size, dietary specialization and sociality differ in their foraging ranges, navigational strategies and floral resource preferences, we explore the behavioural mechanisms and strategies that may enable foraging bees to reduce disease exposure and transmission risks at flowers by partitioning overlapping resources in space and in time. By taking a novel behavioural perspective, we highlight the missing links between disease biology and the ecology of plant–pollinator relationships, critical for improving the understanding of disease transmission risks and the better design and management of habitat for pollinator conservation. This article is part of the theme issue ‘Natural processes influencing pollinator health: from chemistry to landscapes’.
Crithidia acanthocephali is a trypanosomatid species that was initially described in the digestive tract of Hemiptera. However, this parasite was recently detected in honey bee colonies in Spain, raising the question as to whether bees can act as true hosts for this species. To address this issue, worker bees were experimentally infected with choanomastigotes from the early stationary growth phase and after 12 days, their hindgut was extracted for analysis by light microscopy and TEM. Although no cellular lesions were observed in the honey bee’s tissue, trypanosomatids had differentiated and adopted a haptomonad morphology, transforming their flagella into an attachment pad. This structure allows the protozoa to remain attached to the gut walls via hemidesmosomes-such as junctions. The impact of this species on honey bee health, as well as the pathogenic mechanisms involved, remains unknown. Nevertheless, these results suggest that insect trypanosomatids may have a broader range of hosts than initially thought.
Liquid chromatography high-resolution mass spectrometry fingerprinting together with pattern recognition techniques was used to determine the metabolites involved in the susceptibility of apple cultivars to rosy apple aphid (RAA). Preprocessing of ultra-high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry raw data of resistant and susceptible apple cultivars was carried out with XCMS and CAMERA packages. Univariate statistical tools and multivariate data analysis highlighted significant different profiles of the apple metabolomes according to their tolerance to RAA. Optimized and cross-validated Partial least squares discriminant analysis and orthogonal projections to latent structures discriminant analysis models confirmed trans-4-caffeoylquinic acid and 4-p-coumaroylquinic acid as biomarkers for the identification of resistant and susceptible apple cultivars to RAA and disclosed that only hydroxycinnamic acids are involved in the disease susceptibility of cultivars. In this sense, the final steps of the biosynthesis of caffeoylquinic acid (CQA) and p-coumaroylquinic acid (p-CoQA) become decisive because the isomerization of 5-CQA to 4-CQA is favored in resistant cultivars, whereas the isomerization of 5-p-CoQA to 4-p-CoQA is favored in susceptible cultivars.
Nosema ceranae is an intracellular parasite that infects honeybees’ gut altering the digestive functions; therefore, it has the potential of affecting the composition of the gut microbiome. In this work, individual bees of known age were sampled both in spring and autumn, and their digestive tracts were assessed for N. ceranae infection. Intestinal microbiome was assessed by sequencing the bacterial 16S rRNA gene in two different gut sections, the anterior section (AS; midgut and a half of ileum) and the posterior section (PS; second half of ileum and rectum). A preliminary analysis with a first batch of samples (n = 42) showed that AS samples had a higher potential to discriminate between infected and non-infected bees than PS samples. As a consequence, AS samples were selected for subsequent analyses. When analyzing the whole set of AS samples (n = 158) no changes in α- or β-diversity were observed between infected and non-infected bees. However, significant changes in the relative abundance of Proteobacteria and Firmicutes appeared when a subgroup of highly infected bees was compared to the group of non-infected bees. Seasonality and bees’ age had a significant impact in shaping the bacteriome structure and composition of the bees’ gut. Further research is needed to elucidate possible associations between the microbiome and N. ceranae infection in order to find efficient strategies for prevention of infections through modulation of bees’ microbiome.
Rosmarinus officinalis L. (synonym Salvia rosmarinus Schleid) grows in the Mediterranean basin and is known to be a source of natural bioactive compounds and one of the most important aromatic species in terms of the marketing of the essential oil. However, wild collection and the lack of selection lead to the absence of standardized material that ensures the homogeneity and quality of the essential oils over time. In the present work, thirteen wild Spanish populations of rosemary were cultivated in two experimental fields and their essential oil composition monitored during two years. The main compounds present in the essential oils were camphor (21.9%), α-pinene (14.8%), 1,8-cineole (11.6%), β-pinene + myrcene (11.3%) and camphene (8.3%), although their proportions differ greatly among populations. Other terpenes as limonene had a significant presence in some populations, up to 10.2%. The results showed that the variability in the composition of essential oil was mainly controlled by genetics and little affected by soil and climate conditions. Statistical processing allowed to group populations into three different groups based on the geographical origin of the populations. In conclusion, the characterization of essential oils of these populations is a starting point for the development of breeding programmes aimed to commercialize standardized plants (varieties).
The diversity of traits within animal assemblages has been shown to affect the magnitude of animal‐provided ecological functions. However, little is known about how consistent trait diversity effects are across ecological functions and ecosystems. More importantly, the importance of trait diversity in driving ecosystem functioning, relative to other components of biodiversity, has rarely been assessed. It also remains unclear how environmental gradients filter trait diversity and, ultimately, modulate ecological functions. Here we test how different biodiversity components (i.e., trait diversity, phylogenetic diversity and abundance) affect the magnitude of avian seed dispersal and insect predation along large environmental gradients. We sampled frugivorous and insectivorous birds and their ecological functions across gradients of forest cover and fruit and insect abundances in woodland pastures and apple orchards in Northern Spain. We measured 6 morphological traits and compiled phylogenetic information on 43 bird species. We used Structural Equation Models to disentangle the effects of environmental gradients and biodiversity components on ecological functions. We found that different avian functions in the same agroecosystem were controlled by different biodiversity components. While seed dispersal was positively driven by bird abundance in woodland pastures, insect predation responded positively to trait and phylogenetic diversity. The positive effects of trait diversity on insect predation were, on the other hand, consistent across woodland pastures and apple orchards. Our results also pinpointed forest cover and resource availability as filters of the different components of avian diversity, suggesting that environmental gradients condition the effects of biodiversity on avian ecological functions. Our findings reveal variable effects of trait diversity on two different avian ecological functions, but consistent effects on the same function across agroecosystems. Consolidating the generalities of trait diversity effects will require further multi‐function studies, as well as a unifying framework for animal‐driven functions that integrates the causal links between environmental gradients, the different biodiversity components, and ecological functions.
Trained immunity (TRAIM) may be defined as a form of memory where innate immune cells such as monocytes, macrophages, dendritic and natural killer (NK) cells undergo an epigenetic reprogramming that enhances their primary defensive capabilities. Cross‐pathogen protective TRAIM can be triggered in different hosts by exposure to live microbes or microbe‐derived products such as heat‐inactivated Mycobacterium bovis or with the glycan α‐Gal to elicit protective responses against several pathogens. We review the TRAIM paradigm using two models representing distinct scales of immune sensitization: the whole bacterial cell and one of its building blocks, the polysaccharides or glycans. Observations point out to macrophage lytic capabilities and cytokine regulation as two key components in nonspecific innate immune responses against infections. The study of the TRAIM response deserves attention to better characterize the evolution of host‐pathogen cooperation both for identifying the etiology of some diseases and for finding new therapeutic strategies. In this field, the zebrafish provides a convenient and complete biological system that could help to deepen in the knowledge of TRAIM‐mediated mechanisms in pathogen‐host interactions. This article is protected by copyright. All rights reserved.
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