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In colony collapse disorder (CCD), honey bee colonies inexplicably lose their workers. CCD has resulted in a loss of 50 to 90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naive bees suggests that infection may contribute to CCD. We used an unbiased metagenomic approach to survey microflora in CCD hives, normal hives, and imported royal jelly. Candidate pathogens were screened for significance of association with CCD by the examination of samples collected from several sites over a period of 3 years. One organism, Israeli acute paralysis virus of bees, was strongly correlated with CCD.

Trypanosomatids are protozoan parasites of the class Kinetoplastida predominately restricted to invertebrate hosts (i.e. possess a monoxenous life-cycle). However, several genera are pathogenic to humans, animals and plants, and have an invertebrate vector that facilitates their transmission (i.e. possess a dixenous life-cycle). Phytomonas is one dixenous genus that includes several plant pathogens transmitted by phytophagous insects. Trypanosoma and Leishmania are dixenous genera that infect vertebrates, including humans, and are transmitted by hematophagous invertebrates. Traditionally, monoxenous trypanosomatids such as Leptomonas were distinguished from morphologically similar dixenous species based on their restriction to an invertebrate host. Nonetheless, this criterion is somewhat flawed as exemplified by Leptomonas seymouri which reportedly infects vertebrates opportunistically. Similarly, Novymonas and Zelonia are presumably monoxenous genera yet sit comfortably in the dixenous clade occupied by Leishmania . The isolation of Leishmania macropodum from a biting midge ( Forcipomyia spp.) rather than a phlebotomine sand fly calls into question the exclusivity of the Leishmania -sand fly relationship, and its suitability for defining the Leishmania genus. It is now accepted that classic genus-defining characteristics based on parasite morphology and host range are insufficient to form the sole basis of trypanosomatid taxonomy as this has led to several instances of paraphyly. While improvements have been made, resolution of evolutionary relationships within the Trypanosomatidae is confounded by our incomplete knowledge of its true diversity. The known trypanosomatids probably represent a fraction of those that exist and isolation of new species will help resolve relationships in this group with greater accuracy. This review incites a dialogue on how our understanding of the relationships between certain trypanosomatids has shifted, and discusses new knowledge that informs the present taxonomy of these important parasites.

Trypanosomatids are obligate flagellated parasites, with the genera Leishmania and Trypanosoma acting as etiological agents of significant diseases such as leishmaniasis and Chagas disease. Although ecological studies have increasingly highlighted the role of bats as potential reservoirs of these parasites, the diversity of trypanosomatids in urban bat populations remains poorly understood. This study investigates the occurrence and diversity of Trypanosomatidae in bats from Mangabeiras Municipal Park (MMP), an urban park in Belo Horizonte, Minas Gerais, Brazil, a region of ecological interest due to the prior detection of Leishmania in sand flies. A total of 56 bats representing seven species were captured, and 149 biological samples (blood, tissues, and organs) were analyzed using NNN/LIT culture medium. Contamination was reported in 32.2% of the samples, while 67.8% yielded negative results with no growth of trypanosomatids. Detection of trypanosomatids was achieved using the V7V8 Nested-PCR technique, revealing positive results in nine bats: Artibeus lituratus ( Leishmania infantum , Trypanosoma sp. Neobat 3), Anoura caudifer ( Trypanosoma sp. Neobat 4), Carollia perspicillata and Glossophaga soricina ( Leishmania infantum ), Sturnira lilium ( Trypanosoma sp. Neobat 3), and Platyrrhinus lineatus (mixed infection with Leishmania infantum and Leishmania braziliensis ). The integrity of the extracted DNA was confirmed through the amplification of cytb and gamma-actin genes. By expanding knowledge of trypanosomatid diversity in urban bats, this study highlights the ecological and epidemiological relevance of bats as hosts and underscores the need for targeted surveillance to assess their role in pathogen transmission dynamics.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts – including four managed species – from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

Common ravens ( Corvus corax ) are intelligent scavengers that adapt to diverse environments, playing a key ecological role, but their health and ecosystem contributions can be affected by parasites. This study investigates the prevalence and diversity of blood parasite infections in common ravens using molecular techniques. Blood samples ( n  = 42) were collected from dead common ravens in Germany and screened for filarioid nematodes, trypanosomatids, and haemosporidian parasites. The results showed that 26.2% of the common ravens were PCR-positive for at least one parasite, with some cases of mixed infections. Filarioid nematodes were found in 16.7%, trypanosomatids in 4.8%, and haemosporidian parasites in 16.7% of the common ravens. Sequencing revealed the presence of four Leucocytozoon CytB lineages and one Haemoproteus lineage. The findings suggest that common ravens in Germany are often infected with diverse avian blood parasites, with a higher prevalence of filarioid nematodes. Further research is needed to confirm the circulation of these parasites in the common raven population and to identify the specific filarioid nematode species present in Germany.

Biodiversity surveys remain a critical tool for characterizing the global species richness of parasites. In high-latitude regions of the world, characterizing parasite biodiversity is of particular importance due to the rapid rate at which the climate is changing and potentially shifting parasite distributions and abundances. We sampled a bird community on Prince of Wales Island in southern Alaska, United States, to test for the abundance and richness of haemosporidian and trypanosomatid parasites in this understudied region. We tested for parasites in 67 archived tissue samples of 18 bird species, of which five had not been previously tested for haemosporidians and 11 had not been sampled for trypanosomatids using molecular methods. We recovered two novel mitochondrial genetic lineages of haemosporidians (genera Haemoproteus and Leucocytozoon ), and three novel 18S rRNA genotypes belonging to Trypanosoma of uncertain species-level affinities. Surprisingly, we also identified a trypanosomatid from the genus Zelonia , a group of monoxenous parasites of insects, from an avian tissue. While this anomalous record may have been the result of environmental contamination, it nonetheless reflects the first record of Zelonia in the entire Nearctic region.

Little is known about the role of horse flies in potential pathogen transmission in Chile. This study provides evidence of the molecular detection of microorganisms in southern Chile. In the present study, adult Osca lata horse flies were trapped from Punucapa (39°45’06”S/73°16’08”W, Región de Los Ríos) and Puyehue (40°39’10”S/72°10’57”W, Región de Los Lagos), Chile. Among the 95 samples analyzed by PCR using specific primers, microorganisms were detected in 23.2% (n = 22) of the samples. Rickettsia spp. DNA was detected in 15.8% (n = 15) of the samples, Trypanosomatidae DNA in 5.3% (n = 5) of the samples, and filarial DNA in 2.1% (n = 2) of the samples. This study found that horse flies in the region are capable of carrying a variety of both parasites and endosymbionts. Further research is needed to understand the specific impact of horse flies as mechanical or biological vectors and develop effective control measures to prevent the spread of any microorganisms associated with disease.

Background Biting midges of the genus Culicoides play an important role in the transmission of pathogenic arboviruses and parasites. Thailand has documented more than 100 species of Culicoides ; however, several cryptic species complexes remain to be clarified. Recent studies in areas with leishmaniasis indicate that several species of Culicoides might be potential vectors of Leishmania in the subgenus Mundinia , but evidence supporting the hypothesis is still lacking. Therefore, the diversity of Culicoides biting midges and their potential role as vectors of leishmaniasis in southern Thailand remains uncertain. Methods Female Culicoides biting midges were collected using Centers for Disease Control and Prevention (CDC) ultraviolet (UV) light traps from four locations within leishmaniasis-affected areas in three provinces of southern Thailand, including Nakhon Si Thammarat, Krabi, and Surat Thani. Culicoides species were identified based on the morphology of wing spot patterns and subsequently confirmed by cytochrome c oxidase subunit I ( COI ) Sanger sequencing. A potential cryptic species was classified using an integrative taxonomic approach associated with DNA barcoding identification by Barcode of Life Database (BOLD) and Basic Local Alignment Search Tool (BLAST) searches. Furthermore, three different methods of species delimitation, namely ASAP [Assemble Species by Automatic Partitioning], TCS [Templeton, Crandall, and Sing], and PTP [Poisson Tree Processes], were employed to verify the sequences into the molecular operational taxonomic unit (MOTU). Detection of Leishmania and other trypanosomatid parasites was performed by polymerase chain reaction (PCR) based on the ITS1 region and small subunit SSU ribosomal RNA (rRNA) gene, followed by Sanger sequencing and haplotype diversity analysis. The identification of host blood sources was carried out using host-specific multiplex PCR. Results A total of 716 unfed midges and 159 blood-fed specimens were morphologically identified into 25 species belonging to five subgenera ( Avaritia , Hoffmania , Meijerehelea , Remmia , and Trithecoides ) and four species groups ( Clavipalpis , Ornatus , Shermani , and Shortti ). Two unidentified specimens were classified into two subgenera ( Trithecoides and Avaritia ). The DNA barcoding identification exhibited an 82.20% success rate. Species delimitation analyses demonstrated the presence of cryptic species complexes, categorized into six species: Culicoides actoni , C. orientalis , C. huffi , C. palpifer , C. clavipalpis , and C. jacobsoni . Furthermore, 6.42% of the Culicoides biting midges tested positive for Leishmania DNA in three sampling sites in Nakhon Si Thammarat and Surat Thani provinces (with no positive results in Krabi province). Furthermore, the sympatric infection of Leishmania martiniquensis and Leishmania orientalis was identified in several Culicoides species in Ron Phibun and Phunphin districts in Nakhon Si Thammarat and Surat Thani, respectively. In contrast, L. orientalis was detected in Sichon district, Nakhon Si Thammarat province. A genetic diversity analysis revealed high haplotype diversity and relatively low nucleotide diversity in both parasite populations. Additionally, Crithidia sp. and Crithidia brevicula were detected in Culicoides peregrinus and Culicoides subgenus Trithecoides . The analysis of the host blood meal from Ron Phibun also demonstrated that Culicoides had fed on cows, dogs, and chickens, and mixed blood preferences for humans and cows or chickens and cows were detected. Conclusions The findings of the present study demonstrate the presence of mixed blood hosts and co-circulation of L. martiniquensis and L. orientalis in Culicoides in areas of leishmaniasis, as well as cryptic species of Culicoides biting midges, through an integrative taxonomic approach. These findings support the hypothesis that Culicoides biting midges may serve as potential vectors in southern Thailand, and vector diversity is a contributing factor to the risk of zoonotic transmission. Graphical Abstract

Background Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse. Results Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis , Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly. Conclusion The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.