Additional Species Research Articles

An updated checklist of the skippers (Lepidoptera: Hesperiidae) of Bhutan

The authors conducted a comprehensive review of Hesperiidae species in Bhutan, scrutinizing three existing checklists and 25 new records presented by Van der Poel et al. (2023). A thorough examination of all available documents and evidence confirmed 145 Hesperiidae species in Bhutan, with four additional species listed as tentative and one as “cf.” (requiring further research). The updated checklist excludes 14 species that were listed in one or more of the three existing checklists and provides justifications for not including these species. Additionally, 11 unverifiable or wrongly identified species presented by non-peer-reviewed sources were not included in the checklist. Sixteen species with no verifiable records in the last 70 years were also identified. This review provides a comprehensive and authoritative checklist of Hesperiidae species in Bhutan.

Haymaking complemented by moderate disturbances can sustain and restore species-rich alpine to subalpine grasslands

AbstractThe high plant diversity in alpine to subalpine grasslands is threatened by the abandonment of land use. In addition, changing environmental conditions might lead to vegetation shifts even when traditional land use is maintained, as observed in grasslands in Switzerland during the last decades. Maintaining and restoring the diversity of such grasslands might therefore require modified management methods. We conducted a six-year experiment to assess the responses of plant species richness, mean ecological indicator values, and vegetation composition to five management treatments, including scraping as additional management measure: haymaking (in autumn), haymaking complemented by scraping (i.e. manual raking) in autumn, haymaking complemented by scraping in spring, only scraping in spring, and abandonment of land use. We hypothesized that haymaking complemented by scraping in either season would remove additional biomass and increase species richness by creating open patches that can reduce inter-specific competition and promote species establishment. We found positive effects of haymaking complemented by scraping on plant species richness and habitat quality, indicated by the increased mean indicator value for light. Abandonment showed the opposite effects and increased mean indicator values for nutrients. Interestingly, haymaking combined with scraping in autumn promoted the development of the vegetation towards the composition similar to the resident vegetation type. Our findings show that extensive land use is essential to maintain species-rich alpine to subalpine grasslands. Further, they imply that modified land use can compensate for the negative developments such as reduced habitat quality and species richness caused by environmental changes and help restore the vegetation.

Distribution, morphological and molecular characterization of root-lesion nematodes (Pratylenchus spp.) (Tylenchida: Pratylenchidae) in maize (Zea mays L.) (Poales: Poaceae) in Türkiye.

Root-lesion nematodes (RLN) are the most economically important pathogenic nematodes attacking maize. Significant economic losses due to lesion nematodes have been reported in maize producing countries in the world. This study was conducted to determine the distribution and identity of root-lesion nematodes (Pratylenchus spp.) (Tylenchida: Hoplolaimidae) in maize (Zea mays L.) (Poales: Poaceae) fields of the Black Sea region of Türkiye. For this purpose, 39 locations were surveyed and soil samples were taken from 17 regional provinces. Nematodes were extracted using the modified Baerman funnel technique. The species were identified based on sequences of the Internal Transcribed Spacer (ITS) region of ribosomal DNA, as well as morphological characters and morphometrics. In addition, species identifications were confirmed using species-specific primers in the D3 expansion region of 26S rDNA. At the end of the study, 51.3% of the maize production areas sampled in the region were infected with root-lesion nematode species. Pratylenchus agilis, P. mediterraneus, P. neglectus, P. penetrans, P. thornei, and P. vulnus were identified, and were present in 25%, 5%, 25%, 10%, 15%, and 20% of samples, respectively. To our knowledge, this is the first report of P. agilis in Türkiye. The present study concluded that the molecular analysis of Pratylenchus sequences based on the ITS and D3 region of ribosomal RNA genes allowed the identification of six root lesion nematode species. This study is of great importance in terms of adding additional species to the root-lesion nematode fauna in Turkey and will provide data for future research on the management of these nematodes.

Genome-Wide Analyses of CCHC Family Genes and Their Expression Profiles under Drought Stress in Rose (Rosa chinensis).

CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses (Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses (Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba, Ostreococcus lucimarinus, Arabidopsis thaliana, and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses.

The genus Cortinarius should not (yet) be split

The genus Cortinarius (Agaricales, Basidiomycota) is one of the most species-rich fungal genera, with thousands of species reported. Cortinarius species are important ectomycorrhizal fungi and form associations with many vascular plants globally. Until recently Cortinarius was the single genus of the family Cortinariaceae, despite several attempts to provide a workable, lower-rank hierarchical structure based on subgenera and sections. The first phylogenomic study for this group elevated the old genus Cortinarius to family level and the family was split into ten genera, of which seven were described as new. Here, by careful re-examination of the recently published phylogenomic dataset, we detected extensive gene-tree/species-tree conflicts using both concatenation and multispecies coalescent approaches. Our analyses demonstrate that the Cortinarius phylogeny remains unresolved and the resulting phylogenomic hypotheses suffer from very short and unsupported branches in the backbone. We can confirm monophyly of only four out of ten suggested new genera, leaving uncertain the relationships between each other and the general branching order. Thorough exploration of the tree space demonstrated that the topology on which Cortinarius revised classification relies on does not represent the best phylogenetic hypothesis and should not be used as constrained topology to include additional species. For this reason, we argue that based on available evidence the genus Cortinarius should not (yet) be split. Moreover, considering that phylogenetic uncertainty translates to taxonomic uncertainty, we advise for careful evaluation of phylogenomic datasets before proposing radical taxonomic and nomenclatural changes.

Effects of climate and anthropogenic pressures on chemical warfare agent transfer in the Baltic Sea food web

The Baltic Sea is a severely disturbed marine ecosystem previously used as a dumping ground for chemical warfare agents (CWA), which are now known to enter its food web. We have performed a modelling exercise using a calibrated and validated Central Baltic Ecopath with Ecosim (EwE) model to recreate the potential environmental pathways of the infamous Clark I (diphenylchlorarsine). Observations from modelling timestamps covering recent times correspond with in situ detections in sediments and Atlantic cod (Gadus morhua). Under applied modelling conditions and scenarios, there is an active transfer of Clark I from sediments through the Baltic Sea food-web. According to our results, Clark I bioaccumulates within the Baltic Sea food web exclusively throughout the detritus-based food chain. The EwE model for the Central Baltic Sea also allows the simulation of changes in the food web under multiple anthropogenic stressors and management efforts, including recommendations from the Helsinki Commission Baltic Sea Action Plan (HELCOM BSAP). Among all investigated scentarios and factors, the commercial fishing is the most impactful on Clark I accumulation rate and contamination transfer within the Baltic Sea food web. The study indicates the need to extend the existing monitoring approach by adding additional species representing a broader range of ecological niches and tiers within the food chains. From the environmental perspective, the remediation of Chemical Weapons by removal should be considered as part of the integrated management of the Baltic Sea.

Cryptostroma corticale (Ellis & Everh.) P. H. Greg. & S. Waller causing sooty bark disease in British Columbia, Canada

Sooty bark disease is an invasive disease causing significant mortality of sycamore maple (Acer pseudoplatanus L.) in Europe, where it is emerging due to increasing drought and heat events. The causal agent, Cryptostroma corticale (Ellis & Everh.) P. H. Greg. & S. Waller, is a fungus endemic to the Great Lake region in eastern Canada, where it does not appear to cause disease within its natural host range (e.g. sugar maple, Acer saccharum Marshall). Sooty bark disease was reported causing mortality on sycamore maple and additional species within Washington State beginning in 2017. In summer 2022, sooty bark disease was found on a sycamore maple near Vancouver, representing the first report of the disease and causal agent within the province of British Columbia (BC). In this study, we identify the causal agent of sooty bark disease by morphological and molecular methods and confirm its pathogenicity in a controlled growth chamber experiment fulfilling Koch’s postulates on sycamore maple. Cryptostroma corticale has so far been found in BC on sycamore maple, Norway maple (A. platanoides L.) and bigleaf maple (A. macrophyllum Pursh). Sooty bark disease has been previously shown to increase in severity and occurrence under drought and warm conditions; we anticipate rising sooty bark disease cases as BC experiences increasingly frequent and extreme summer droughts and heat events.

Aqueous Two-Phase Extraction of Single-Chirality Carbon Nanotubes with Functionalized ssDNA for Biomarker-Specific Antibody Conjugation

Multiplexed detection of biomarkers for cancer and chronic inflammatory diseases is necessary to improve diagnostic sensitivity and specificity. Our prior studies and that of others has used near-infrared fluorescent single-walled carbon nanotubes (SWCNT) as sensor transducers because of their sensitivity to the local environment, photostability, and emission in the tissue-transparent window. That work engineered the SWCNT surface through non-covalent interactions with short ssDNA oligonucleotides containing a functional group to which a biomarker-specific antibody was conjugated. Further, previous work by other groups has demonstrated the ability to obtain single SWCNT chiralities using the aqueous two-phase extraction (ATPE) technique with non-functionalized ssDNA sequences. Here, we have incorporated primary amine-functionalized ssDNA sequences into the ATPE separation technique to obtain individual SWCNT chiralities with functionalized ssDNA. To those primary amine-functionalized ssDNA-SWCNT complexes, we conjugated antibodies against two inflammatory cytokines. We investigated the sensitivity and specificity of these individual SWCNT chirality sensors specific to two biomarkers together in buffer and serum, finding that each chirality responds specifically only to its cognate biomarker and not the other. This is the first demonstration of single-chirality, molecularly-specific multiplexed SWCNT sensors. It forms the basis of ongoing work to obtain additional chiral species with other functional groups, as well as to develop multiplexed cytokine sensors for in vivo diagnostics.

Few chemoreceptor genes in the ambrosia beetle Trypodendron lineatum may reflect its specialized ecology

BackgroundChemoreception is crucial for insect fitness, underlying for instance food-, host-, and mate finding. Chemicals in the environment are detected by receptors from three divergent gene families: odorant receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs). However, how the chemoreceptor gene families evolve in parallel with ecological specializations remains poorly understood, especially in the order Coleoptera. Hence, we sequenced the genome and annotated the chemoreceptor genes of the specialised ambrosia beetle Trypodendron lineatum (Coleoptera, Curculionidae, Scolytinae) and compared its chemoreceptor gene repertoires with those of other scolytines with different ecological adaptations, as well as a polyphagous cerambycid species.ResultsWe identified 67 ORs, 38 GRs, and 44 IRs in T. lineatum (‘Tlin’). Across gene families, T. lineatum has fewer chemoreceptors compared to related scolytines, the coffee berry borer Hypothenemus hampei and the mountain pine beetle Dendroctonus ponderosae, and clearly fewer receptors than the polyphagous cerambycid Anoplophora glabripennis. The comparatively low number of chemoreceptors is largely explained by the scarcity of large receptor lineage radiations, especially among the bitter taste GRs and the ‘divergent’ IRs, and the absence of alternatively spliced GR genes. Only one non-fructose sugar receptor was found, suggesting several sugar receptors have been lost. Also, we found no orthologue in the ‘GR215 clade’, which is widely conserved across Coleoptera. Two TlinORs are orthologous to ORs that are functionally conserved across curculionids, responding to 2-phenylethanol (2-PE) and green leaf volatiles (GLVs), respectively.ConclusionsTrypodendron lineatum reproduces inside the xylem of decaying conifers where it feeds on its obligate fungal mutualist Phialophoropsis ferruginea. Like previous studies, our results suggest that stenophagy correlates with small chemoreceptor numbers in wood-boring beetles; indeed, the few GRs may be due to its restricted fungal diet. The presence of TlinORs orthologous to those detecting 2-PE and GLVs in other species suggests these compounds are important for T. lineatum. Future functional studies should test this prediction, and chemoreceptor annotations should be conducted on additional ambrosia beetle species to investigate whether few chemoreceptors is a general trait in this specialized group of beetles.

Seek, and you will find: Cryptic diversity of the cardiopulmonary nematode Angiostrongylus vasorum in the Americas

Angiostrongylus vasorum is a metastrongylid parasite infecting wild canids and domestic dogs. Its patchy distribution, high pathogenicity and taxonomical classification makes the evolutionary history of A. vasorum intriguing and important to study. First larval stages of A. vasorum were recovered from feces of two grey foxes, Urocyon cinereoargenteus, from Costa Rica. Sequencing and phylogenetic and haplotypic analyses of the ITS2, 18S and cytochrome oxidase subunit 1 (cox1) fragments were performed. Then p- and Nei´s genetic distance, nucleotide substitution rates and species delimitation analyses were conducted with cox1 data of the specimens collected herein and other Angiostrongylus spp. Cophylogenetic congruence and coevolutionary events of Angiostrongylus spp. and their hosts were evaluated using patristic and phenetic distances and maximum parsimony reconciliations. Specimens from Costa Rica clustered in a separate branch from European and Brazilian A. vasorum sequences in the phylogenetic and haplotype network analyses using the ITS2 and cox1 data. In addition, cox1 p-distance of the sequences derived from Costa Rica were up to 8.6 % different to the ones from Europe and Brazil, a finding mirrored in Nei´s genetic distance PCoA. Species delimitation analysis supported a separate group with the sequences from Costa Rica, suggesting that these worms may represent cryptic variants of A. vasorum, a new undescribed taxon or Angiocaulus raillieti, a synonym species of A. vasorum described in Brazil. Moreover, nucleotide substitution rates in A. vasorum were up to six times higher than in the congener Angiostrongylus cantonensis. This finding and the long time elapsed since the last common ancestor between both species may explain the larger diversity in A. vasorum. Finally, cophylogenetic congruence was observed between Angiostrongylus spp. and their hosts, with cospeciation events occurring at deeper taxonomic branching of host order. Altogether, our data suggest that the diversity of the genus Angiostrongylus is larger than expected, since additional species may be circulating in wild canids from the Americas.

Direct Hydrogenation of Sterically Hindered, Unactivated Alkenes Catalyzed by Phosphino(silyl)‐Nickel Complexes

AbstractReadily accessible (PSi)Ni(II)‐benzyl complexes supported by bidentate phosphino(silyl) ligation were found to be effective pre‐catalysts for the direct hydrogenation of a variety of highly sterically hindered, unfunctionalized alkenes under relatively mild conditions (2.5‐5 mol% Ni, 10 atm H2, 50 °C) and without the need for additional additives or activator species. A range of substrates, including di‐, tri‐, and tetra‐substituted alkenes were evaluated in this regard and afforded good to moderate yields. Substrates featuring α,β‐unsaturated carbonyl functionality were also readily hydrogenated with no evidence for reduction of carbonyl or ester C−O functionalities. Deuteration experiments highlight the occurrence of chain walking which occurs in the background of the reported catalytic chemistry.

Risk assessment of the laboratory host range and a molecular characterisation determining the field host range of Lixus aemulus, for the biological control of Chromolaena odorata in South Africa

Chromolaena odorata (Asteraceae: Eupatorieae) is a sprawling shrub native to the Americas, and a destructive invader of much of the humid tropics and subtropics of the Old World. Opportunistic native-range exploration in 1995 identified a stem-boring weevil, Lixus aemulus, as a promising biological control candidate agent. Host-specificity testing was conducted on L. aemulus in South Africa using laboratory no-choice and paired-choice tests. Three invasive alien plants closely related to C. odorata may be utilized by L. aemulus but no indigenous, ornamental or crop species in South Africa was or is expected to be attacked by the weevil. A native-range field survey was conducted in Brazil to determine the exact identity of the host plant L. aemulus had been collected in 1995, and to identify additional host-plant species. Genetic assessments of the Lixus sp.(p.). adults collected on the three host plants (C. odorata, Chromolaena laevigata and Heterocondylus vitalbae) reveal these individuals are L. aemulus and the weevil can be classed as an oligophage in its native range. Over 5,500 adults were released in South Africa, but overall establishment has been poor. The most likely explanation appears to be a climate mismatch between the region of South Africa invaded by C. odorata and the collection locality in Rio Branco, Acre state, Brazil. Additionally, because the full extent of the native range of L. aemulus is unknown, it is uncertain whether individuals can be sourced from an area whose climate resembles that of South Africa. Furthermore, despite being oligophagous, L. aemulus may perform sub-optimally on the southern African C. odorata biotype.

Additional new species of Xenodidymella from pasture-medicinal plants in Iran.

Xenodidymella species have a wide range of hosts and can be found as pathogens and saprobes. In this study, two new species of Xenodidymella were found from leaf diseases of three pasture-medicinal plants in Ilam Province, in the west of Iran, and proposed here as X. ilamica and X. scandicis spp. nov. These species were identified based on morphological features and phylogenetic analyses of the internal transcribed spacer regions 1 & 2 and 5.8S nrDNA (ITS), partial beta-tubulin gene (tub2), and partial RNA polymerase II second largest subunit (rpb2) gene. The four Xenodidymella strains isolated in this study were delimited into two sister clades, with the two isolates of X. ilamica from the leaf spot of Colchicum speciosum and Ficaria kochii and two isolates of X. scandicis from leaf blight of Scandix pecten-veneris. Morphologically, X. scandicis produces larger, ostiolate or poroid pycnidia in vitro, while pycnidia in the cultures of X. ilamica are non-ostiolate and smaller. Some pycnidia in old cultures of X. scandicis produce a neck, but a distinct neck in X. ilamica has not been observed. Moreover, three plants under study are new hosts for the genus Xenodidymella.

Revision of the genus Kunungua (Hemiptera, Heteroptera, Miridae) with descriptions of three new species and new generic synonymy

This study provides a taxonomic revision of the genus Kunungua Carvalho, 1951 (Hemiptera, Heteroptera, Miridae, Bryocorinae, Eccritotarsini) with the description of three new species, K. atramentomaculatasp. nov., K. geminasp. nov., and K. ornatasp. nov. Revised diagnoses for the genus and three additional species are given, along with a key to the species. Habitus photographs, illustrations of male genitalic structures, and distributional information are provided for each species. The placement of Kunungua within the Prodromus group of genera is discussed. The genus Duducoris Odhiambo, 1962 is recognised as a new junior subjective synonym of Prodromus Distant, 1904, resulting in new combinations for the five species contained in the subsumed genus. Kunungua pallida Linnavuori, 1975 is excluded from the genus Kunungua and treated as a species of uncertain generic placement.

NEW SPECIES OF FROGS FROM LOW ANO MODERATE ELEVATIONS FROM THE CALDAS TRANSECT OF THE EASTERN FLANK OF THE CORDILLERA CENTRAL

Four additional species of Eleutlzerodactylus are reported from eastern Caldas, Colombia, bring to 28 the numher of species known along a transect between 600 and 2650 m. Eleutherodactylus orpacobates is reported for the first time away from the Cordillera Occidental and three species are named as new.

THE RELATIONSHIPS OF AN ENSEMBLE OF GUATEMALAN AND MEXICAN FROGS (ELEUTHERODACTYLUS: LEPTODACTYLIDAE: AMPHIBIA)

Eleutherodactylus daryi and E. greggi are shown to be species of the Eleutherodactylus gollmeri group which is defined on the basis of having a synapomorphy (fusion of sacrum and presacral vertebrae). Eleutherodactylus omiltemanus is a species of the rhodopis Series rather than thefitzingeri Series. The Eleutherodactylus omiltemanus group disappears because ali of its contained members are placed in other species groups. Eleutherodactylus saltator is a synonym of E. mexicanus. Eleutherodactylus rhodopis sensu lato contains two species, a larger upland E. rhodopis and the more widely distibuted lowland E. loki. The rhodopis Series, containing the rhodopis group, is not definable because no synapomorphies are apparent. Eleutherodactylus occidentalis and E. omiltemanus are assigned to this "group. " Within this "group " there is a cluster of species having black mesorchia (E. hobartsmithi, E. mexicanus, E. pygmaeus, and E. sarton), one having a shortened inner toe (E. jota and E. podiciferus), and one having conical subarticular tubercles (E. bransfordii, E. lauraster, E. loki, E. occidentalis, E. omiltemanus, E. rhodopis, and E. stejnegerianus). Sixty of the 84 essentially Mesoamerican species of the subgenus Craugastor are placed in a monophyletic subunit based on the common presence of marked sexual dimorphism in tympanum size. The subunit includes the nine species ofthe gollmeri group, the thirteen species of the rhodopis group, and 38 additional species (the three species of the biporcatus group, the eleven species of the milesi group, eight species of thefitzingeri group, 15 species of the rugulosus group, and E. uno). The 24 species of Craugastor not included in that clade are the 13 species of the alfredi group (including E. bocourti), the two species of the augusti group, the two species of the bufoniformis group, three species removed from thefitzingeri group (E. andi, E. cuaquero, and E. emcelae), and four species removed from the rugulosus group (E. anatipes, E. anomalus, E. cheiroplethus, and E. zygodactylus).

ADDITIONAL NEW SPECIES OF FROGS (GENUS ELEUTHERODACTYLUS) FROM CLOUD FORESTS OF EASTERN DEPARTAMENTO DE CALDAS, COLOMBIA

Four additional species of the genus Eleutherodactylus are described from the high cloud forests of the municipalities of Pensilvania and Samaná, Caldas. With these descriptions, 18 species of the genus have been recorded from this small region lying between 1800 and 2150 meters.

Tannin-on-protein adlayers: Secondary adsorption quantified by quartz crystal microbalance with dissipation monitoring (QCM-D)

The quartz crystal microbalance with dissipation monitoring (QCM-D) technique allows to detect adsorption and mechanical characteristics of protein adsorption layers at solid surfaces, including subsequent secondary adsorption of additional species interacting with the proteins. Here, we study the secondary adsorption of two different classes of tannins - hydrolysable vs. condensed - onto a β-lactoglobulin base layer using QCM-D in a protein/tannin adlayer configuration. Sequential flow experiments of protein solutions, tannin solutions and neat solvent or buffer are used to distinguish reversible from desorption-resistant layer formation, evaluate the viscoelastic characteristics the layers, and map protein/tannin interactions in view of their structural properties and origin. We find that for the majority of tannins, secondary layer structural changes (detected via a change in the dissipation factor) occur before significant mass adsorption (detected via the resonance frequency shift). In contrast, simultaneous onset of structural change and adsorbed mass within an observation window of 100 ms was only found for certain hydrolysable tannins. We use these data to build adsorption maps and group the tannins according to their adlayer properties, providing a physical basis for an improved understanding of the organoleptic perception of tannins and polyphenols, in particular with respect to attributes such as ‘astringency’ and ‘lingering’. Moreover, these maps provide insight into the capacity of different tannins to modify the mechanical properties of protein adsorption layers.

Sporocysts of Sarcocystis bertrami (syn. Sarcocystis fayeri) shed by dogs: Molecular analysis, morphometry and pattern of excretion

Sarcocystis bertrami (synonym: Sarcocystis fayeri) is a coccidian parasite that infects horses and donkeys in several countries. Dogs are known as definitive hosts of the parasite, however, the patent period is not well defined, and S. bertrami shed by dogs has never been confirmed by molecular methods. Here we investigated the shedding of S. bertrami by experimentally infected dogs and examined the excreted parasites by morphological and molecular tools. Three dogs of small breeds (one Yorkshire terrier and two miniature Pinschers) were acquired with ages of 30 and 60 days and were exclusively fed commercial dog food. Two dogs consumed equine muscle tissues containing cysts of S. bertrami. The third dog served as negative control and was simultaneously fed commercial dog food. The two animals that received equine tissues shed sporocysts and/or oocysts in their feces after prepatent periods of 13 and 23 days. The patent periods were 47 and 14 days. Sporocysts showed average dimensions of 14.19 µm (± 0.53) x 10.06 µm (± 0.44). The control dog did not shed sporocysts or oocysts of the parasite. Interestingly, patent periods had never been reported, and for one dog, the patent period (47 days) was longer than that reported for other Sarcocystidae parasites. PCRs to the gene 18S and to the internal transcribed spacer 1 (ITS1) of the rDNA were successfully performed with DNA extracted from sporocysts. ITS1 sequences were also obtained from the equine tissue cysts used to infect the dogs. Nucleotide sequences of cloned fragments of 18S from sporocysts, and ITS1 from both stages (tissue cysts and sporocysts) matched with S. bertrami (18S: 97.50–99.88 %; ITS1: 88.76–95.21 %), although high molecular diversity was observed with data from these loci. PCR to cox1 using sporocysts’ DNA failed to amplify any product. The possibility of the existence of an additional and undescribed Sarcocystis species in the excreted sporocysts, besides S. bertrami, cannot be excluded from this experiment. To our knowledge, this is the first molecular confirmation of S. bertrami in canine feces. Sporocyst dimensions and prepatent periods observed in this study were similar to those previously described for S. bertrami and S. fayeri. In conclusion, the molecular, morphological and biological data generated here fit in previous descriptions for both S. bertrami and S. fayeri.

Farm management practices and host species richness associated with higher likelihood of tuberculosis positive farms in Western Spain

Despite enormous efforts, complete animal tuberculosis (TB) eradication has only been achieved in few regions. Epidemiological analyses are key to identify TB risk factors and set up targeted biosecurity measures. Here, we conducted an in-depth characterization of 84 extensive beef cattle farms from a high TB prevalence region in Western Spain, and assessed how farm management and wildlife presence on farms contribute to cattle TB risk. Twenty-six out of 84 variables were associated with cattle farm TB positivity. Farm management variables associated with TB positivity included older cattle, larger herd size, highly fragmented farm structure and greater connectivity between farms. TB-positive farms provided supplemental feed over a higher number of months, used calf feeders, and had higher number of waterholes. Detecting Eurasian wild boar (Sus scrofa), red fox (Vulpes vulpes), European badger (Meles meles), roe deer (Capreolus capreolus), or Egyptian mongoose (Herpestes ichneumon) on cattle farms was also associated with farm TB positivity. The best ordinal regression model indicated that in farms with a large herd size (> 167 animals) the odds of being positive or recurrently positive (versus negative) was 7.34 (95% CI = 2.43–23.51) times higher that of farms with small herd size. Further, for every additional host species detected in the farm premises, the odds of being TB-positive increased 56%. We conclude that both cattle management and wildlife need to be targeted for successful TB control in grazing-based farming systems.