Fleas of wild carnivores in Romania: diversity, distribution, and host-associations.

BACKGROUND: Fleas are important hematophagous insects, infesting mammals and birds with a worldwide distribution. Fleas of medical importance have been reported from various carnivores worldwide, such as felids, canids, or mustelids. Romania hosts a wide carnivore diversity, but very little is known about flea species that parasitize these animals in Romania. This study aimed to provide a better understanding of the fleas' diversity and their distribution in a relatively large and diverse number of wild carnivore hosts from Romania. METHODS: From 2013 to 2021, 282 carcasses of wild carnivores from different locations in Romania were collected and examined for the presence of ectoparasites. All collected fleas were morphologically identified using specific keys and descriptions. An analysis of the co-occurrence networks was performed. RESULTS: A total of 11 flea species were identified: Pulex irritans (41.09%), Paraceras melis (20.11%), Ctenocephalides felis (7.33%), Ctenocephalides canis (7.83%), Monopsyllus sciurorum (11.11%), Chaetopsylla trichosa (21.96%), Chaetopsylla homoea (5.5%), Chaetopsylla tuberculaticeps (100%), Chaetopsylla rothschildi (13.33%), Chaetopsylla sp. (14.34%), Chaetopsylla globiceps (5.12%), Echidnophaga gallinacea (10%). The statistical analyses showed a significant difference between the infestation of Martes foina with females being more frequently infected than males (66% versus 33%). Paraceras melis infesting Meles meles had a significantly higher prevalence in female badgers than in males (× 2 = 7.7977, P < 0.01) and higher intensities of infestations in males than in females (t = 1.871, P < 0.05). CONCLUSIONS: This is the first large-scale study investigating the distribution and diversity of flea species infesting wild carnivores in Romania. Three flea species were identified for the first time in Romania (E. gallinacea, C. homoea, and C. tuberculaticeps).

Simplifying Access to Targeted Protein Degraders via Nickel Electrocatalytic Cross-Coupling.

C-C linked glutarimide-containing structures with direct utility in the preparation of cereblon-based degraders (PROTACs, CELMoDs) can be assessed in a single step from inexpensive, commercial α-bromoglutarimide through a unique Brønsted-acid assisted Ni-electrocatalytic approach. The reaction tolerates a broad array of functional groups that are historically problematic and can be applied to the simplified synthesis of dozens of known compounds that have only been procured through laborious, wasteful, multi-step sequences. The reaction is scalable in both batch and flow and features a trivial procedure wherein the most time-consuming aspect of reaction setup is weighing out the starting materials.

Season and host-community composition inside roosts may affect host-specificity of bat flies.

Bat flies are one of the most abundant ectoparasites of bats, showing remarkable morphological adaptations to the parasitic habit, while the relationship with their hosts is characterized by a high level of specificity. By collecting bat flies from live hosts, our intention was to elucidate the seasonal differences in bat fly occurrence and to describe factors regulating the level of incipient host specificity. Our results indicate that the prevalence and the intensity of infestation is increasing from spring to autumn for most host species, with significant differences among different fly species. Males showed higher infestation levels than females in autumn, suggesting a non-random host choice by flies, targeting the most active host sex. Bat-bat fly host specificity shows seasonal changes and host choice of bat flies are affected by the seasonal differences in hosts' behavior and ecology, the intensity of infestation and the species composition of the local host community. Nycteribiid bat flies showed lower host specificity in the swarming (boreal autumn) period, with higher prevalence recorded on non-primary hosts. Choosing a non-primary bat host may be an adaptive choice for bat flies in the host's mating period, thus increasing their dispersive ability in a high activity phase of their hosts.

Nickel-Electrocatalytic Decarboxylative Arylation to Access Quaternary Centers.

There is a pressing need, particularly in the field of drug discovery, for general methods that will enable direct coupling of tertiary alkyl fragments to (hetero)aryl halides. Herein a uniquely powerful and simple set of conditions for achieving this transformation with unparalleled generality and chemoselectivity is disclosed. This new protocol is placed in context with other recently reported methods, applied to simplify the routes of known bioactive building blocks molecules, and scaled up in both batch and flow. The role of pyridine additive as well as the mechanism of this reaction are interrogated through Cyclic Voltammetry studies, titration experiments, control reactions with Ni(0) and Ni(II)-complexes, and ligand optimization data. Those studies indicate that the formation of a BINAPNi(0) is minimized and the formation of an active pyridine-stabilized Ni(I) species is sustained during the reaction. Our preliminary mechanistic studies ruled out the involvement of Ni(0) species in this electrochemical cross-coupling, which is mediated by Ni(I) species via a Ni(I)-Ni(II)-Ni(III)-Ni(I) catalytic cycle.

Detection of bacterial and protozoan pathogens in individual bats and their ectoparasites using high-throughput microfluidic real-time PCR.

Among the most studied mammals in terms of their role in the spread of various pathogens with possible zoonotic effects are bats. These are animals with a very complex lifestyle, diet, and behavior. They are able to fly long distances, thus maintaining and spreading the pathogens they may be carrying. These pathogens also include vector-borne parasites and bacteria that can be spread by ectoparasites such as ticks and bat flies. In the present study, high-throughput screening was performed and we detected three bacterial pathogens: Bartonella spp., Neoehrlichia mikurensis and Mycoplasma spp., and a protozoan parasite: Theileria spp. in paired samples from bats (blood and ectoparasites). In the samples from the bat-arthropod pairs, we were able to detect Bartonella spp. and Mycoplasma spp. which also showed a high phylogenetic diversity, demonstrating the importance of these mammals and the arthropods associated with them in maintaining the spread of pathogens. Previous studies have also reported the presence of these pathogens, with one exception, Neoehrlichia mikurensis, for which phylogenetic analysis revealed less genetic divergence. High-throughput screening can detect more bacteria and parasites at once, reduce screening costs, and improve knowledge of bats as reservoirs of vector-borne pathogens. IMPORTANCE The increasing number of zoonotic pathogens is evident through extensive studies and expanded animal research. Bats, known for their role as reservoirs for various viruses, continue to be significant. However, new findings highlight the emergence of Bartonella spp., such as the human-infecting B. mayotimonensis from bats. Other pathogens like N. mikurensis, Mycoplasma spp., and Theileria spp. found in bat blood and ectoparasites raise concerns, as their impact remains uncertain. These discoveries underscore the urgency for heightened vigilance and proactive measures to understand and monitor zoonotic pathogens. By deepening our knowledge and collaboration, we can mitigate these risks, safeguarding human and animal well-being.

Diastereoselective Radical 1,4-Ester Migration: Radical Cyclizations of Acyclic Esters with SmI2.

Reductive cyclizations of carbonyl compounds, mediated by samarium(II) diiodide (SmI2, Kagan's reagent), represent an invaluable platform to generate molecular complexity in a stereocontrolled manner. In addition to classical ketone and aldehyde substrates, recent advances in radical chemistry allow the cyclization of lactone and lactam-type substrates using SmI2. In contrast, acyclic esters are considered to be unreactive to SmI2 and their participation in reductive cyclizations is unprecedented. Here, we report a diastereoselective radical 1,4-ester migration process, mediated by SmI2, that delivers stereodefined alkene hydrocarboxylation products via radical cyclization of acyclic ester groups in α-carbomethoxy δ-lactones. Isotopic labeling experiments and computational studies have been used to probe the mechanism of the migration. We propose that a switch in conformation redirects single electron transfer from SmI2 to the acyclic ester group, rather than the "more reactive" lactone carbonyl. Our study paves the way for the use of elusive ketyl radicals, derived from acyclic esters, in SmI2-mediated reductive cyclizations.

Asymmetric Total Synthesis of (-)-Phaeocaulisin A.

The therapeutic properties of Curcuma (ginger and turmeric's family) have long been known in traditional medicine. However, only recently have guaiane-type sesquiterpenes extracted from Curcuma phaeocaulis been submitted to biological testing, and their enhanced bioactivity was highlighted. Among these compounds, phaeocaulisin A has shown remarkable anti-inflammatory and anticancer activity, which appears to be tied to the unique bridged acetal moiety embedded in its tetracyclic framework. Prompted by the promising biological profile of phaeocaulisin A and by the absence of a synthetic route for its provision, we have implemented the first enantioselective total synthesis of phaeocaulisin A in 17 steps with 2% overall yield. Our route design builds on the identification of an enantioenriched lactone intermediate, tailored with both a ketone moiety and a conjugated alkene system. Taking advantage of the umpolung carbonyl-olefin coupling reactivity enabled by the archetypal single-electron transfer (SET) reductant samarium diiodide (SmI2), the lactone intermediate was submitted to two sequential SmI2-mediated cyclizations to stereoselectively construct the polycyclic core of the natural product. Crucially, by exploiting the innate inner-sphere nature of carbonyl reduction using SmI2, we have used a steric blocking strategy to render sites SET-unreceptive and thus achieve chemoselective reduction in a complex substrate. Our asymmetric route enabled elucidation of the naturally occurring isomer of phaeocaulisin A and provides a synthetic platform to access other guaiane-type sesquiterpenes from C. phaeocaulis─as well as their synthetic derivatives─for medicinal chemistry and drug design.

Molecular epidemiological study of Trichomonas gallinae focusing on central and southeastern Europe.

Trichomonas gallinae is a geographically widespread protozoan parasite of birds. In this study, oropharyngeal swab samples were collected in Hungary and Romania from 99 columbiform birds, including 76 feral pigeons (Columba livia domestica: 42 kept for racing, 32 with urban and two with rural habitat), four common wood pigeons (C. palumbus), 16 ring doves (Streptopelia risoria) and three Eurasian collared doves (S. decaocto). These samples were analyzed for the presence of T. gallinae using molecular methods. Racing feral pigeons had significantly higher prevalence of T. gallinae infection than urban feral pigeons. The rate of PCR-positivity was the highest among wood pigeons and ring doves. Based on 18S rRNA gene, T. gallinae was the most heterogenous among racing feral pigeons sampled in a trading-breeding place. Clinical signs were associated with only one 18S rRNA gene subtype. The most divergent 18S rRNA gene subtype, Trichomonas sp. Hu-TG37 clustered with T. canistomae and T. tenax and represents probably a new species. To our knowledge, this is the first report on the genetic diversity of T. gallinae in the southeastern European region. The results suggest that most detected T. gallinae 18S rRNA gene subtypes are not host-specific and do not cause clinical signs. The highest number of 18S rRNA gene subtypes was demonstrated among racing feral pigeons. Significantly more captive than free-living columbiform birds had T. gallinae infection. These data highlight the importance of epizootic monitoring of the genetic diversity and presence of T. gallinae in trading-breeding places of pigeons and doves.

Argasid Ticks of Palearctic Bats: Distribution, Host Selection, and Zoonotic Importance.

The soft ticks (Ixodida: Argasidae) are ectoparasites of terrestrial vertebrates with a wide geographic distribution, occurring on all continents. These ticks are obligate blood-feeders, most of them show high degrees of host-specialization and several species in arid and tropical regions are important parasites of livestock and men. Species commonly occurring on domestic animals and man are generally well-known, with many studies focusing on their ecology, distribution or vectorial role. However, wildlife-specialist soft ticks are less studied. Nearly half of all soft tick species are bat specialists, with five species (Carios vespertilionis, Chiropterargas boueti, Chiropterargas confusus, Reticulinasus salahi, and Secretargas transgariepinus) occurring in the Western Palearctic. There is no comprehensive study on the distribution, hosts or pathogens in these soft ticks, although most species were shown to carry several viral, bacterial, or protozoan pathogens and also to occasionally infest humans. Based on a literature survey and 1,120 distinct georeferenced records, we present here the geographical range, host selection and vectorial potential for bat-specialist soft ticks occurring in the Western Palearctic (chiefly Europe, North Africa, and the Middle East). Carios vespertilionis shows the largest distribution range and was found on most host species, being ubiquitous wherever crevice-roosting bats occur. All the other species were located only in areas with Mediterranean climate, with Ch. boueti, Chiropteraragas confusus, and R. salahi are missing entirely from Europe. These three species have a host spectrum of bats roosting primarily in caves, while S. transgariepinus and Ca. vespertilionis is feeding primarily on crevice-roosting bat species. All but one of these soft tick species are known to feed on humans and may be vectors of important disease agents (Rickettsia spp., Borrelia spp., Bartonella spp., Ehrlichia spp., Babesia spp., several nairo-, and flaviviruses). As several crevice-roosting bat species show a continuous adaptation to human-altered areas, with certain species becoming common city-dwellers in the Western Palearctic, the study of bat specialist soft ticks is also important from an epidemiologic point of view.

Recent advances in the chemistry of ketyl radicals.

Ketyl radicals are valuable reactive intermediates for synthesis and are used extensively to construct complex, functionalized products from carbonyl substrates. Single electron transfer (SET) reduction of the C[double bond, length as m-dash]O bond of aldehydes and ketones is the classical approach for the formation of ketyl radicals and metal reductants are the archetypal reagents employed. The past decade has, however, witnessed significant advances in the generation and harnessing of ketyl radicals. This tutorial review highlights recent, exciting developments in the chemistry of ketyl radicals by comparing the varied contemporary - for example, using photoredox catalysts - and more classical approaches for the generation and use of ketyl radicals. The review will focus on different strategies for ketyl radical generation, their creative use in new synthetic protocols, strategies for the control of enantioselectivity, and detailed mechanisms where appropriate.

First record of Ixodes simplex found on a human host, with a review of cases of human infestation by bat tick species occurring in Europe.

Ixodes simplex is a bat tick species, a common parasite of the Schreibers' bent-winged bat, Miniopterus schreibersii. Its distribution is linked to the range of its host, free stages occurring exclusively inside the underground bat shelters. Here we present the first case of human infestation with I. simplex. An adult female tick was found attached to the upper limb after a visit to the underground shelter of a large bat colony. This unusual host selection is a likely consequence of the reduction of suitable hosts, as the number of bats was much lower at the time of the visit than in previous years. Bat ticks rarely feed on humans, with soft ticks (Argasidae) being more commonly involved. In the light of the potential vectorial capacity of I. simplex, the incidence and potential future risks are discussed.

First report of the bat fly species Basilia italica in Romania.

Bat flies are haematophagous ectoparasites, highly specialised to bats and are also considered to have vectorial potential for several pathogens like Bartonella spp. or Polychromophilus spp. In Romania, past studies mostly focused on the ectoparasitic fauna of cave-dwelling bats, listing the occurrence of 10 bat fly species in the country, with only scarce information on bat flies infesting crevice-roosting bat species. Here we report the occurrence of Basilia italica, a rare nycteribiid species infesting primarily forest-dwelling bats. This is the first country-record for Romania and also represents the easternmost occurrence of this species. Further studies are needed to evaluate the vectorial potential of B. italica.

Wide Distribution and Diversity of Malaria-Related Haemosporidian Parasites (Polychromophilus spp.) in Bats and Their Ectoparasites in Eastern Europe.

Malaria is responsible for major diseases of humans, while associated haemosporidians are important factors in regulating wildlife populations. Polychromophilus, a haemosporidian parasite of bats, is phylogenetically close to human-pathogenic Plasmodium species, and their study may provide further clues for understanding the evolutionary relationships between vertebrates and malarial parasites. Our aim was to investigate the distribution of Polychromophilus spp. in Eastern Europe and test the importance of host ecology and roost site on haemosporidian parasite infection of bats. We sampled bats and their ectoparasites at eight locations in Romania and Bulgaria. DNA was extracted from blood samples and ectoparasites and tested individually for the presence of DNA of Polychromophilus spp. using a nested PCR targeting a 705 bp fragment of cytB. Two species of Polychromophilus were identified: Po. melanipherus in Miniopterus schreibersii and associated ectoparasites and Po. murinus in rhinolophid and vespertilionid bats (6 species) and their ticks and nycteribiid flies. Only cave-dwelling bat species (and their ectoparasites) showed infections, and we found a strong correlation between infections with Polychromophilus parasites and Nycteribiidae prevalence. We report the high genetic diversity of Polychromophilus spp. in Eastern Europe, suggesting that the simultaneous presence of varied host and vector assemblages enhances bat haemosporidian parasite diversity.

Impact of abiotic factors, habitat type and urban wildlife on the ecology of hard ticks (Acari: Ixodidae) in urban and peri-urban habitats.

BACKGROUND: Ticks are increasingly acknowledged as significant vectors for a wide array of pathogens in urban environments with reports of abundant tick populations in recreational areas. The study aims to contribute to a better knowledge of the abiotic and biotic factors which impact the ecology of hard ticks in urban and peri-urban habitats in Romania. METHODS: Questing ticks were collected by flagging in seven recreational locations, from four types of habitats in Cluj-Napoca, Romania: parks; gardens; a cemetery; and peri-urban forests. Hedgehogs, birds and micromammals were also sampled and searched for ticks, using standard methods (i.e. torch-based searches, ornithological mist nets, snap-traps, etc.), while vegetation was evaluated on surveyed areas. Data on questing ticks were converted to abundance indices. Mood's median tests were used to assess the relationship between the abiotic and biotic factors and the abundance of questing ticks. RESULTS: Two species of questing ticks were found: Ixodes ricinus (96.8%) and Haemaphysalis punctata (3.2%). Ixodes ricinus was also the predominant engorged tick collected from urban wildlife. For I. ricinus the highest mean total abundance index/location (total no. of ticks/100 m2) was recorded in the urban gardens (3.79, 95% CI: ± 1.59) and parks (2.68, 95% CI: ± 0.75), whereas the lowest mean total abundance index was noted in the peri-urban forests (0.06, 95% CI: ± 0.03) and the urban cemetery (0.04, 95% CI: ± 0.02). The adults and nymphs of I. ricinus displayed a bimodal activity pattern, while the larvae showed a unimodal questing behaviour with an autumnal peak. Positive correlations were found between the mean total abundance index and the rise in the daily mean temperature and relative humidity, and between the global abundance of questing ticks and the presence of hedgehogs in the respective locations (P < 0.01). CONCLUSIONS: Ticks were collected in all the recreational sites surveyed in Cluj-Napoca. Ixodes ricinus was the dominant questing and engorged tick species. Several abiotic and biotic factors shape the ecology of ticks in Cluj-Napoca city, with climate and the local presence of suitable hosts being the most important.

Biotic and abiotic factors influencing the prevalence, intensity and distribution of Eucoleus aerophilus and Crenosoma vulpis in red foxes, Vulpes vulpes from Romania.

To understand the importance of host' habitat choice in determining parasite burden, we studied the distribution of two helminth parasites of the red fox (Vulpes vulpes) in south-eastern Europe (Romania): Crenosoma vulpis and Eucoleus aerophilus, both widely distributed respiratory nematodes parasitic in various carnivores. Even though the life cycle and biology of the two nematodes follow a different pattern, both parasites appear to be co-distributed and often co-infect foxes with variable prevalences across their range. Between July 2016 and August 2018, 550 red foxes, V. vulpes were collected by hunters in different localities from 22 counties of Romania and examined by necropsy. All parasites found in the trachea and bronchial system were collected and preserved in 70% ethanol. We characterised red fox/parasite habitats using seven predictors (fragmentation, altitude, presence/absence of water surface, per cent cover of arable land/grassland/urbanized areas/forest cover/wetlands). Prevalence, abundance, intensity, and sex ratio were calculated and statistically analysed using the R software. Out of the 550 examined foxes, 76.2% were infected with lungworms. The overall prevalence was 32.0% for C. vulpis and 72.5% for E. aerophilus. The mean intensity of infection was 13.70 for C. vulpis 6.15 for E. aerophilus. For both nematodes, the prevalence was significantly higher in males than in females, and there was no influence of hosts' age. No statistical differences were found for intensity and mean intensity in the case of infection with C. vulpis and E. aerophilus between age and sex categories. The abundance of C. vulpis showed a strong positive relationship with the presence of wetlands and habitat fragmentation. We found a significant correlation between the abundance of E. aerophilus and altitude, with foxes from higher elevations showing higher prevalences.

Cascades, Catalysis and Chiral Ligand Control with SmI2; The Rebirth of a Reagent.

This review focuses on recent developments from our laboratory in the field of radical reactions mediated by the archetypal reductive single electron transfer (SET) reagent, SmI2. Namely, we have expanded the scope of reducible carbonyl moieties to esters and amides and have exploited the resultant ketyl radicals in radical cascade reactions that generate unprecedented scaffolds. Moreover, we have taken the first steps to address the long-standing challenges of catalysis and chiral ligand control associated with the reagent.

Bats and ticks: host selection and seasonality of bat-specialist ticks in eastern Europe.

BACKGROUND: Parasites may actively seek for hosts and may use a number of adaptive strategies to promote their reproductive success and host colonization. These strategies will necessarily influence their host specificity and seasonality. Ticks are important ectoparasites of vertebrates, which (in addition to directly affecting their hosts) may transmit a number of pathogens. In Europe, three hard tick species (Ixodidae: Ixodes ariadnae, I. simplex and I. vespertilionis) and at least two soft tick species (Argasidae: Argas transgariepinus and A. vespertilionis) are specialized for bats. METHODS: Here we report data on the host range of these ticks and the seasonality of tick infestation on wild caught bats in south-east Europe. We collected 1803 ticks from 30 species of bats living in underground shelters (caves and mines) from Romania and Bulgaria. On the basis of tick-host associations, we tested several hypotheses on host-parasite evolutionary adaptations regulating host specificity, seasonality and sympatric speciation. RESULTS: We observed significant differences in host specificity and seasonality of abundance between the morphologically different bat specialist ticks (I. simplex and I. vespertilionis) likely caused by their host choice and their respective host-seeking behavior. The two highly generalist, but morphologically similar tick species (I. ariadnae and I. vespertilionis) showed temporal differences in occurrence and activity, thus exploiting significantly different host communities while occurring in geographical sympatry. CONCLUSIONS: We conclude that bat-specialist ticks show a wide range of adaptations to their hosts, with differences in specificity, seasonality of occurrence, the prevalence and intensity of infestation and all these contribute to a successful division of temporal niches of ticks sharing morphologically similar hosts occurring in geographical sympatry.

Illustrated redescription of the male of Ixodes simplex Neumann, 1906.

Ixodes simplex is a tick species of bats, mainly reported from Schreibers' Bent-winged bat, Miniopterus schreibersii, all over its host's range. While being a common parasite of cave-dwelling bat species, the initial description of males is poor (based on a sole damaged specimen), lacking distinctive features. This study describes the male of I. simplex also providing measurements for a number of characters, using a series 10 of specimens. Males of I. simplex show the following characteristics distinct from males of I. ariadnae and I. vespertilionis: idiosoma is shorter (2.6 mm vs. 3.4-4.7 mm); the palps of I. simplex males are laterally curved (bent) (while straight in I. ariadnae and gradually curving in I. vespertilionis), the genital opening is anterior in position between coxae II (whereas posterior to second intercoxal space in I. vespertilionis); coxae II are to some extent rectangular (vs. rounded in I. ariadnae); coxae IV have a prominent tuft of long hairs (missing in the other two species). Most importantly, the legs of I. simplex males are considerably shorter than in males of the other two species (2.7-3.3 mm vs. 7-10 mm). The description is completed with high-quality pictures highlighting all the mentioned features.

Gold-Catalyzed Direct Alkynylation of Azulenes.

A novel catalytic method for the direct C-H alkynylation of azulenes is developed. The gold catalyzed functionalization of this special carbacycle is achieved with hypervalent iodonium reagent TIPS-EBX under mild reaction conditions. With the aid of the developed procedure, several TIPS alkynylated azulene derivatives were synthesized bearing important functional groups for further functionalization.