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Background Fleas are major ectoparasites of dogs and cats, with significant veterinary and public health implications. This study aimed to estimate the prevalence of flea infestation in dogs and cats in mainland Portugal, identify associated risk factors and perform morphological and molecular characterisation of flea specimens. Methods A cross-sectional study was conducted from March 2022 to February 2023 in mainland Portugal. Dogs and cats were screened for flea infestations at veterinary clinics and shelters following World Association for the Advancement of Veterinary Parasitology guidelines. Fleas were morphologically identified to species level, and a subset was characterised molecularly via phylogenetic analysis of the cytochrome c oxidase subunit II gene ( cox2 ) sequences. Epidemiological data were analysed through multivariate logistic regression models to identify possible risk factors associated with flea infestation. Results A total of 1052 dogs and 1039 cats were examined, and flea infestation prevalence was determined to be 33.6% and 36.5%, respectively. Ctenocephalides felis was the predominant flea species in both hosts, accounting for 85.7% of fleas in dogs and 98.8% in cats, with molecular data confirming the subspecies C. felis felis . Other species identified included Ctenocephalides canis (9.6% in dogs; 1.8% in cats), Pulex irritans (4.2% in dogs) and Archaeopsylla erinacei maura (0.8% in dogs). The absence of insecticide use was the strongest predictor of flea infestation in both hosts (dogs: adjusted odds ratio [aOR] 4.87; cats: aOR 4.02). In dogs, the risk of infestation was higher in spring, summer and autumn compared to winter (aOR 2.08–3.72), and lower in the Lisbon Metropolitan Area, Alentejo and Algarve compared to the North region (aOR 0.14–0.45). In cats, risk was reduced in non-northern regions (Lisbon, Alentejo, Centro; aOR 0.10–0.45) and in those cats with non-domestic outdoor lifestyles (aOR 0.19). Conclusions Flea infestations are highly prevalent in dogs and cats across mainland Portugal, with C. felis felis as the dominant species. These findings provide valuable insights for the development of integrated, evidence-based strategies for flea control.

Fleas are the most important insect vectors that parasitize warm-blooded animals and are known vectors of zoonotic pathogens. A recent study showed that Stenoponia polyspina parasitizing Eospalax baileyi in Zoige County have carried Bartonella spp. and Spotted fever group Rickettsia (SFGR). Accurate identification and differentiation of fleas are essential for prevention and control of zoonotic pathogens. To understand phylogenetic relationship of the subfamily Stenoponiinae, we described morphological characteristics of S. polyspina and sequenced its mitogenome with 14,933 bp in size and high A + T content (~ 79%). The S. polyspina mitogenome retained the ancestral pattern of mitochondrial gene arrangement of arthropods without rearrangement. The start codons of 13 protein-coding genes (PCGs) are traditional ATN and the stop codons are TAA or TAG. Anticodon loops of all tRNA genes were 7 bp except for trnL 2 and trnD had anticodon loops with 9 bp and the abnormal anticodon loops may be associated with frameshifting mutation. Genetic distance and Ka/Ks ratios indicated that all 13 PCGs of S. polyspina were subjected to purifying selection, with cox1 at the slowest rate and atp8 at the fastest rate. The mitogenomes of 24 species representing 7 families in the order Siphonaptera were selected to reconstruct phylogenetic tree based on concatenated nucleotide sequences of two datasets (PCGRNA matrix and PCG12RNA matrix) using Bayesian inference (BI) and Maximum likelihood (ML) methods. Phylogenetic tree supported that the superfamilies Ceratophylloidea, Vermipsylloidea, Pulicoidea were monophyletic and the superfamily Hystrichopsylloidea was paraphyletic. The family Ctenophthalmidae was monophyletic in PCGRNA-ML (codon partition) and paraphyletic in the remain trees. S. polyspina belongs to the subfamily Stenoponiinae was closely more related to the subfamily Rhadinopsyllinae. This paper explored phylogenetic position of diverse clades within the order Siphonaptera based on morphological and mitogenome data of S. polyspina . Our research enriched NCBI database of the order Siphonaptera.

The Thaumapsylla breviceps orientalis is endemic to China and exhibits extreme host specificity (monoxenous). This study reports the first mitogenome sequenced for the family Ischnopsyllidae and provides comprehensive analysis of both the morphological characteristics and mitogenome of T. b. orientalis . The assembled mitogenome is 15,631 bp in length with a high AT content (78.5%), and its codon usage bias is predominantly shaped by natural selection. Evolutionary analysis based on evolutionary rates and nucleotide diversity across different families within Siphonaptera revealed that Pulicidae has the fastest evolutionary rate and the highest nucleotide diversity, a pattern likely driven by differences in their hosts and habitats. We observed that early-diverging flea lineages are predominantly polyxenous, whereas later-diverging lineages are primarily pleioxenous or monoxenous. Phylogenetic results indicate that the taxonomic status of the families Ctenophthalmidae, Vermipsyllidae, and Hystrichopsyllidae requires further study and revision. This research addresses a key knowledge gap in Ischnopsyllidae mitogenomics and clarifies the phylogenetic relationships within the order Siphonaptera.

Accurate differentiation and identification of flea species are essential for both basic and applied research on fleas, as well as for the diagnosis of flea-borne diseases. However, distinguishing between flea species can be challenging, especially among those with minimal morphological differences. Therefore, some scholars have suggested the necessity of comprehensive revisions to the classification of fleas, incorporating morphological, molecular, and phylogenetic data. In this study, we focused on classifying the rodents’ parasitic fleas in southeastern China and provided molecular and phylogenetic data. We also described a new subspecies Ctenophthalmus breviprojiciens fujiansis n. ssp. A total of 392 fleas were collected from 8 species of rodents in 10 counties. Morphologically, they belonged to 10 species, 9 genera and 5 families. Barcode identification based on COI gene and phylogenetic analysis based on five genetic markers ( 18S rDNA , 28S rDNA , EF-1a , COI , COII ) revealed that the molecular and morphological identification of Xenopsylla cheopis , Aviostivalius klossi bispiniformis , Leptopsylla segnis , Monopsyllus anisus and Ctenocephalides felis felis were consistent. The taxonomic status of Neopsylla specialis minpiensis and Peromyscopsylla himalaica sinica as subspecies is questionable due to significant intraspecific genetic distance, and further morphological and molecular data are required to determine if they should be elevated to species level. The molecular identification of C. breviprojiciens n. ssp., N. dispar fukienensis , and Nosopsyllus nicanus could not be completed at this time due to a lack of sequences for related species in existing GenBank databases. Additionally, phylogenetic relationships of 31 species from 9 genera and 5 families of Siphonaptera were inferred based on five molecular markers ( 18S rDNA , 28S rDNA , EF-1a , COI and COII ) using Maximum Likelihood analyses. The analyses revealed that various taxa of Siphonaptera are monophyletic at the subspecies, species, and genus levels. However, at the family level, Leptopsyllidae, Ceratophyllidae, Pulicidae, and Pygiopsyllidae are all monophyletic, while Ctenophthalmidae is paraphyletic. we support the view of some authors that revising the catchall group Ctenophthalmidae and elevating each of its constituent subfamilies to family status.

Fleas, which are ubiquitous small wingless parasitic insects, have a significant impact on human and animal health globally. In this study, we sequenced and analyzed the complete mitochondrial genomes of Paradoxopsyllus custodis and Stenischia montanis yunlongensis . The lengths of these genomes were 15,375 bp and 15,651 bp respectively, encompassing a total of 37 genes. Notably, all nucleotide combinations displayed a marked AT preference, with ATN as start codon for all 13 protein-coding genes in both species. Furthermore, only two genes in Paradoxopsyllus custodis were terminated with an incomplete stop codon T(AA). The five most frequently utilized codons among the 13 PCGs in both species ended with A / U, and their relative synonymous codon usage values surpassed 2. Phylogenetic relationships among fleas were assessed using maximum likelihood (ML) and Bayesian inference (BI), providing support for the paraphyletic of Leptopsyllidae. This study not only enhances our understanding of the mitochondrial genome within the genera Paradoxopsyllus and Stenischia , but also offers valuable genetic markers for the taxonomic identification and phylogenetic evolution within the order Siphonaptera.

The present study investigated the presence of endo- and ecto-parasites, and vector-borne pathogens, in dogs from four islands of Greece. A total of 200 (123 owned and 77 sheltered) dogs were examined with different microscopic, serological and molecular methods. Of the examined dogs, 130 (65%) were positive for one or more parasites and/or vector-borne pathogens. The most common zoonotic intestinal helminths recorded were Ancylostomatidae (12.5%) and Toxocara canis (3.5%). Ninety-three dogs (46.5%) seroreacted to Rickettsia conorii. Twenty-two (11%) of them were also PCR positive and 7 (3.5%) showed corpuscles suggestive of Rickettsia spp. on the blood smears. Nineteen dogs (9.5%) were seropositive for Ehrlichia canis, three of them being also PCR positive. Dogs positive for Anaplasma phagocytophilum-Anaplasma platys (1%), Dirofilaria immitis (0.5%) and Babesia canis (0.5%) were also found. Fleas and ticks were recorded in 53 (26.5%) and 50 (25%) dogs, respectively, and all specimens were identified as Ctenocephalides felis felis and Rhipicephalus sanguineus sensu lato. Binary multiple univariate Generalized Linear Models were used to investigate factors and clinical signs related to the recorded positivity, while the association of specific signs with the pathogens was evaluated using tests of independence. Knowledge of occurrence and impact of zoonotic parasites and vector-borne pathogens in dog populations is crucial to prevent the infection in animals and people, and to control the risk of spreading of these pathogens in endemic and non-endemic areas.

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis , has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis , is a well-described biological vector of R . felis . Unique to insect-borne rickettsiae, R . felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R . felis . While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner -based transposon systems in Rickettsiales. Thus, this study aimed to assess R . felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R . felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3’ end of sca1 . Alterations in sca1 expression resulted in unique infection phenotypes. While the R . felis sca1 :: tn mutant portrayed enhanced growth kinetics compared to R . felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R . felis sca1 :: tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R . felis sca1 :: tn during flea infection.

We studied patterns of compositional, functional, and phylogenetic α- and β-diversity in flea and gamasid mite infracommunities of small Siberian mammals, taking into account host-associated (species) and environmental (biome or sampling period) factors. We asked: (a) How do these factors and their interactions affect infracommunity diversity? (b) Does infracommunity composition, in terms of species, traits, and phylogenetic lineages, deviate from random? (c) Are species, traits, and phylogenetic lineages in infracommunities clustered or overdispersed?, and (d) Do patterns of diversity differ between the three diversity facets and/or the two ectoparasite taxa? We found that the α-diversity of infracommunities was strongly affected by host species, biome, and sampling period. The highest proportion of infracommunity diversity in both taxa was associated with the interaction between either host species and biome or host species and sampling period. Infracommunities of both taxa within, as well as between, host species, biomes, and sampling periods were characterized by the clustering of species, traits and lineages. The patterns of the effects of host species, biome, and sampling period on infracommunity diversity were congruent among the three diversity facets in both fleas and mites. We conclude that the assembly patterns in ectoparasite infracommunities mirror those characteristics of component and compound communities.

Abstract Molecular Biology has proven to be an essential tool in public health laboratories, as it provides significant advances in diagnosing and treating diseases and plays a crucial role in preventing and controlling epidemics. Considering the need to detect etiological agents from various vectors in the health service to improve knowledge of the regional transmission cycles of diseases involving arthropods, molecular analysis is an alternative for rapidly diagnosing natural infection by these organisms. This study aims to standardize commercial automated DNA extraction kits to obtain the DNA of pathogens from different vectors. Specimens of sandflies, ticks, fleas and kissing bugs received at LACEN-RO were screened and used in the tests. Three kits were evaluated: (i) Extracta Kit DNA and RNA from Pathogens (MPTA-PU16-B); (ii) Extracta Kit DNA from Bacteria (MBXD-PU16-B); and (iii) Extracta Kit DNA and RNA from Pathogens GOLD (MVXA-PU96 B/W Gold C). The specific protocol was used for each kit, and the EXTRACTA® 32 device was used to perform the automated extraction, following the manufacturer’s instructions. PCRs were performed with universal primers for the mitochondrial DNA cytochrome c oxidase subunit I (COI) region and specific primers for Rickettsia spp., Leishmania spp. and Trypanosoma cruzi. The PCR products were detected using agarose gel electrophoresis, which enabled observing the amplified DNA, demonstrating the success of automated vector DNA extraction technique. These findings will enable future epidemiological studies to more quickly and effectively identify vectors and their parasites involved in the epidemiological cycles of diseases which occur in Rondônia state (Northern Brazil). Resumo A Biologia Molecular tem se mostrado uma ferramenta essencial em laboratórios de saúde pública, pois proporciona avanços significativos no diagnóstico e tratamento de doenças e desempenha um papel crucial na prevenção e controle de epidemias. Considerando a necessidade de detectar agentes etiológicos de diversos vetores no serviço de saúde para aprimorar o conhecimento dos ciclos de transmissão de doenças que envolvem artrópodes, a análise molecular é uma alternativa para o diagnóstico rápido da infecção natural por esses organismos. Este estudo tem como objetivo padronizar kits comerciais automatizados de extração de DNA para obtenção do DNA de patógenos de diferentes vetores. Espécimes de flebotomíneos, carrapatos, pulgas e barbeiros recebidos no LACEN-RO foram triados e utilizados nos testes. Três kits foram avaliados: (i) Extracta Kit DNA and RNA from Pathogens (MPTA-PU16-B); (ii) Extracta Kit DNA from Bacteria (MBXD-PU16-B); e (iii) Extracta Kit DNA and RNA from Pathogens GOLD (MVXA-PU96 B/W Gold C). O protocolo específico foi utilizado para cada kit, e o dispositivo EXTRACTA® 32 foi utilizado para realizar a extração automatizada, seguindo as instruções do fabricante. As PCRs foram realizadas com primers para a região citocromo c oxidase subunidade I do DNA mitocondrial (COI) e primers específicos para Rickettsia spp., Leishmania spp. e Trypanosoma cruzi. Os produtos da PCR foram detectados por eletroforese em gel de agarose, o que permitiu a observação do DNA amplificado, demonstrando o sucesso da técnica automatizada de extração de DNA vetorial. Esses resultados permitirão que futuros estudos epidemiológicos identifiquem de forma mais rápida e eficaz os vetores e seus parasitas envolvidos nos ciclos epidemiológicos de doenças que ocorrem no estado de Rondônia.

Background Fleas are one of the most common and pervasive ectoparasites worldwide, comprising at least 2500 valid species. They are vectors of several disease-causing agents, such as Yersinia pestis . Despite their significance, however, the molecular genetics, biology, and phylogenetics of fleas remain poorly understood. Methods We sequenced, assembled, and annotated the complete mitochondrial (mt) genome of the rodent flea Nosopsyllus laeviceps using next-generation sequencing technology. Then we combined the new mitogenome generated here with mt genomic data available for 23 other flea species to perform comparative mitogenomics, nucleotide diversity, and evolutionary rate analysis. Subsequently, the phylogenetic relationship within the order Siphonaptera was explored using the Bayesian inference (BI) and maximum likelihood (ML) methods based on concentrated data for 13 mt protein-coding genes. Results The complete mt genome of the rodent flea N. laeviceps was 16,533 base pairs (bp) in a circular DNA molecule, containing 37 typical genes (13 protein-coding genes, 22 transfer RNA [tRNA] genes, and two ribosomal RNA [rRNA] genes) with one large non-coding region (NCR). Comparative analysis among the order Siphonaptera showed a stable gene order with no gene arrangement, and high AT content (76.71–83.21%) with an apparent negative AT and GC skew except in three fleas Aviostivalius klossi bispiniformis , Leptopsylla segnis , and Neopsylla specialis . Moreover, we found robust evidence that the cytochrome c oxidase subunit 1 ( cox1 ) gene was the most conserved protein-coding gene (Pi = 0.15, non-synonymous/synonymous [Ka/Ks] ratio = 0.13) of fleas. Phylogenomic analysis conducted using two methods revealed different topologies, but both results strongly indicated that (i) the families Ceratophyllidae and Leptopsyllidae were paraphyletic and were the closest to each other, and (ii) the family Ctenophthalmidae was paraphyletic. Conclusions In this study, we obtained a high-quality mt genome of the rodent flea N. laeviceps and performed comparative mitogenomics and phylogeny of the order Siphonaptera using the mt database. The results will enrich the mt genome data for fleas, lay a foundation for the phylogenetic analysis of fleas, and promote the evolutionary analysis of Siphonaptera. Graphical Abstract