On-host flea phenology and flea-borne pathogen surveillance among mammalian wildlife of the pineywoods of East Texas.

Flea-borne diseases are endemic in Texas, U.S.A., with an increasing incidence of flea-borne typhus and cat scratch disease. Knowledge of flea natural history could provide information to protect public health, yet many knowledge gaps remain outside of plague-endemic regions. Our objective was to characterize seasonal activity patterns of fleas on common mammalian wildlife species and test fleas and wildlife for Rickettsia and Bartonella pathogens. We performed one year of monthly trapping for rodents and medium-sized mammals in a national forest with high recreational use and urban encroachment in East Texas. From 90 mammal captures representing seven species, 101 fleas were collected representing Polygenis spp., Ctenocephalides felis, and Orchopeas species. Virginia opossums (Didelphis virginianus) hosted 99% of the collected fleas (100 fleas) and a single flea was on an eastern woodrat (Neotoma floridana). Flea infestation prevalence of opossums was 79% (23/29). Mean flea abundance was 4.39 fleas, with intensity peaking in spring. One cat flea removed from an opossum was positive for Bartonella henselae. Furthermore, we identified tissue or blood of four raccoons (Procyon lotor) and one golden mouse (Ochrotomys nuttalli) positive for Rickettsia amblyommatis. These findings provide an ecological basis for the maintenance of vectors and pathogens from sylvatic settings.

Comparison of flea diversity in the burrows of Richardson's ground squirrels (Urocitellus richardsonii) in urban and rural sites in central Saskatchewan, Canada.

Spatial and temporal differences in the relative abundance of arthropod vectors are important factors that influence the risk of disease for mammalian hosts. Seasonal changes in the diversity and abundance of fleas (Siphonaptera) in Richardson's ground squirrel (Urocitellus richardsonii) burrows were studied at two sites in central Saskatchewan. A total of 225 fleas (151 at an urban site and 74 at a rural site) were collected. Flea prevalence differed among seasons at the urban site but not at the rural site. Of the nine flea species detected (eight at the urban site and six at the rural site), Oropsylla rupestris, O. bruneri, O. labis, O. tuberculata, and Aetheca wagneri are vectors of Yersinia pestis, the causative agent of plague. The presence and abundance of some fleas differed between sites and seasons. Neopsylla inopina and O. rupestris were the most abundant species at the urban site during the spring and summer, respectively, while O. bruneri was the most abundant species at the rural site. Our findings may have implications for the management of the black-tailed prairie dogs (Cynomys ludovicianus) in southwestern Saskatchewan because they coexist with U. richardsonii, are hosts for Oropsylla, and are at great risk of plague exposure/infection.

The CpxAR signaling system confers a fitness advantage for flea gut colonization by the plague bacillus.

The adaptation of Yersinia pestis, the flea-borne plague agent, to fluctuating environmental conditions is essential for the successful colonization of the flea vector. A previous comparative transcriptomic analysis showed that the Cpx pathway of Y. pestis is up-regulated in infected fleas. The CpxAR two-component system is a component of the envelope stress response and is critical for maintaining the integrity of the cell. Here, a phenotypic screening revealed a survival defect of the cpxAR mutant to oxidative stress and copper. The measured copper concentration in the digestive tract contents of fed fleas increased fourfold during the digestive process. By direct analysis of phosphorylation of CpxR by a Phos-Tag gel approach, we demonstrated that biologically relevant concentrations of copper triggered the system. Then, a competitive challenge highlighted the role of the CpxAR system in bacterial fitness during flea infection. Lastly, an in vitro sequential exposure to copper and then H2O2 to mimic the flea suggests a model in which, within the insect digestive tract, the CpxAR system would be triggered by copper, establishing an oxidative stress response. IMPORTANCE: The bacterium Yersinia pestis is the agent of flea-borne plague. Our knowledge of the mechanisms used by the plague bacillus to infect the flea vector is limited. The up-regulation of the envelope stress response under the control of the Cpx signaling pathway was previously shown in a transcriptomic study. Here, our in vivo and in vitro approaches suggest a model in which Y. pestis uses the CpxAR phosphorelay system to sense and respond to the copper present in the flea gut, thereby optimizing the flea gut colonization. In other words, the system is essential for bacterial fitness in the flea.

Transovarial transmission of Yersinia pestis in its flea vector Xenopsylla cheopis.

Yersinia pestis, the causative agent of plague, is endemic in certain regions due to a stable transmission cycle between rodents and their associated fleas. In addition, fleas are believed to serve as reservoirs that can occasionally cause enzootic plague cycles and explosive epizootic outbreaks that increase human exposure. However, transmission by fleas is inefficient and associated with a shortened lifespan of the flea and rodent hosts, indicating that there remain significant gaps in our understanding of the vector-animal cycle of Y. pestis. Here, we show that laboratory-reared, infected fleas (Xenopsylla cheopis) can transmit viable Y. pestis from adults to eggs, and the bacteria can be passed through all subsequent life stages of the flea. Thus, our data raise the possibility that transovarial transmission in fleas might contribute to the persistence of Y. pestis in the environment without detectable plague activity in mammals.

Attempted Transmission of Marburg Virus by Bat-Associated Fleas Thaumapsylla breviceps breviceps (Ischnopsyllidae: Thaumapsyllinae) to the Egyptian Rousette Bat (Rousettus aegyptiacus).

Egyptian rousette bats (ERBs) are implicated as reservoir hosts for Marburg virus (MARV), but natural mechanisms involved in maintenance of MARV in ERB populations remain undefined. A number of hematophagous ectoparasites, including fleas, parasitize bats. Subcutaneous (SC) inoculation of ERBs with MARV consistently results in viremia, suggesting that infectious MARV could be ingested by blood-sucking ectoparasites during feeding. In our study, MARV RNA was detected in fleas that took a blood meal during feeding on viremic bats on days 3, 7, and 11 after SC inoculation. Virus concentration in individual ectoparasites was consistent with detectable levels of viremia in the blood of infected host bats. There was neither seroconversion nor viremia in control bats kept in close contact with MARV-infected bats infested with fleas for up to 40 days post-exposure. In fleas inoculated intracoelomically, MARV was detected up to 14 days after intracoelomic (IC) inoculation, but the virus concentration was lower than that delivered in the inoculum. All bats that had been infested with inoculated, viremic fleas remained virologically and serologically negative up to 38 days after infestation. Of 493 fleas collected from a wild ERB colony in Matlapitsi Cave, South Africa, where the enzootic transmission of MARV occurs, all tested negative for MARV RNA. While our findings seem to demonstrate that bat fleas lack vectorial capacity to transmit MARV biologically, their role in mechanical transmission should not be discounted. Regular blood-feeds, intra- and interhost mobility, direct feeding on blood vessels resulting in venous damage, and roosting behaviour of ERBs provide a potential physical bridge for MARV dissemination in densely populated cave-dwelling bats by fleas. The virus transfer might take place through inoculation of skin, mucosal membranes, and wounds when contaminated fleas are squashed during auto- and allogrooming, eating, biting, or fighting.

The first mitogenome of the subfamily Stenoponiinae (Siphonaptera: Ctenophthalmidae) and implications for its phylogenetic position.

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 trnL2 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.

ABIOTIC AND DEMOGRAPHIC DRIVERS OF FLEA PARASITISM ON DEER MICE IN A RECOVERING MIXED-CONIFER FOREST A DECADE POSTFIRE.

With the intensity and frequency of wildfires increasing rapidly, the need to study the ecological effects of these wildfires is also growing. An understudied aspect of fire ecology is the effect fires have on parasite-host interactions, including ectoparasites that might be pathogen vectors. Although some studies have examined the impacts of fire on ticks, studies on other ectoparasites, including pathogen vectors, are rare. To help address this knowledge gap, we examined the abiotic and biotic factors that predict the likelihood and extent of parasitism of deer mice (Peromyscus maniculatus) by fleas within a landscape of unburned and recovering burned (>9 yr postfire) mixed conifer forests. We sampled 227 individual deer mice across 27 sites within the Jemez Mountains of northern New Mexico in 2022 and quantified measures of parasitism by fleas (primarily Aetheca wagneri). These sites were distributed in both unburned areas (n = 15) and recovering burned areas (n = 12), with the latter derived from 2 large fires, the Las Conchas fire (2011) and the Thompson Ridge fire (2013). Using these data, we tested for differences in prevalence, mean abundance, and mean intensity of fleas on deer mice, focusing on the predictive importance of host sex and fire history. We also created generalized linear mixed-effects models to investigate the best host and environmental predictors of parasitism by fleas. Approximately a decade postfire, we found minimal evidence to suggest that fire history influenced either the presence or intensity of fleas on deer mice. Rather, at the current forest-regeneration stage, the extent of parasitism by fleas was best predicted by measures of host sex, body condition, and the trapline's ability to accumulate water, as measured through topography. As host body condition increased, the probability of males being parasitized increased, whereas the opposite pattern was seen for females. Male mice also had significantly greater flea loads. Among potential abiotic predictors, the topographic wetness index or compound topographic index (a proxy for soil moisture) was positively related to flea intensity, suggesting larger flea populations in burrows with higher relative humidity. In summary, although fire may potentially have short-term impacts on the likelihood and extent of host parasitism by fleas, in this recovering study system, host characteristics and topographic wetness index are the primary predictors of parasitism by fleas.

Bartonella quintana detection among arthropods and their hosts: a systematic review and meta-analysis.

BACKGROUND: Bartonella quintana is a body louse-borne bacterium causing bacteremia and infective endocarditis. We aimed to describe B. quintana detection among arthropods and their hosts. METHODS: We searched databases in PubMed Central/MEDLINE, Scopus, Embase, and Web of Science from January 1, 1915 (the year of B. quintana discovery) to January 1, 2024, to identify publications containing specific search terms relating to B. quintana detection among arthropods. Descriptive statistics and meta-analysis of pooled prevalence using random-effects models were performed for all arthropods and body and head lice. RESULTS: Of 1265 records, 62 articles were included, describing 8839 body lice, 4962 head lice, and 1692 other arthropods, such as different species of fleas, bedbugs, mites, and ticks. Arthropods were collected from 37 countries, of which 28 had arthropods with B. quintana DNA. Among articles that reported B. quintana detection among individual arthropods, 1445 of 14,088 (0.1026, 95% CI [0.0976; 0.1077]) arthropods tested positive for B. quintana DNA, generating a random-effects model global prevalence of 0.0666 (95% CI [0.0426; 0.1026]). Fifty-six studies tested 8839 body lice, of which 1679 had B. quintana DNA (0.1899, 95% CI [0.1818; 0.1983]), generating a random-effects model pooled prevalence of 0.2312 (95% CI [0.1784; 0.2843]). Forty-two studies tested 4962 head lice, of which 390 head lice from 20 studies originating from 11 different countries had B. quintana DNA (0.0786, 95% CI [0.0713; 0.0864]). Eight studies detected B. quintana DNA exclusively on head lice. Five studies reported greater B. quintana detection on head lice than body lice; all originated from low-resource environments. CONCLUSIONS: Bartonella quintana is a vector-borne bacterium with a global distribution, disproportionately affecting marginalized populations. Bartonella quintana DNA has been detected in many different arthropod species, though not all of these arthropods meet criteria to be considered vectors for B. quintana transmission. Body lice have long been known to transmit B. quintana. A limited number of studies suggest that head lice may also act as possible vectors for B. quintana in specific low-resource contexts.

Ecologic, Geoclimatic, and Genomic Factors Modulating Plague Epidemics in Primary Natural Focus, Brazil.

Plague is a deadly zoonosis that still poses a threat in many regions of the world. We combined epidemiologic, host, and vector surveillance data collected during 1961-1980 from the Araripe Plateau focus in northeastern Brazil with ecologic, geoclimatic, and Yersinia pestis genomic information to elucidate how these factors interplay in plague activity. We identified well-delimited plague hotspots showing elevated plague risk in low-altitude areas near the foothills of the plateau's concave sectors. Those locations exhibited distinct precipitation and vegetation coverage patterns compared with the surrounding areas. We noted a seasonal effect on plague activity, and human cases linearly correlated with precipitation and rodent and flea Y. pestis positivity rates. Genomic characterization of Y. pestis strains revealed a foundational strain capable of evolving into distinct genetic variants, each linked to temporally and spatially constrained plague outbreaks. These data could identify risk areas and improve surveillance in other plague foci within the Caatinga biome.

Acute and chronic effects of the antifouling booster biocide Irgarol 1051 on the water flea Moina macrocopa revealed by multi-biomarker determination.

Irgarol 1051 is an herbicide extensively utilized in antifouling paint due to its ability to inhibit photosynthesis. Irgarol and its photodegradation products are highly persistent in waters and sediments, although they are present in low concentrations. However, our understanding of the harmful effects of Irgarol on non-target organisms remains limited. In this study, we assessed the effects of acute (24 h) and chronic (14 days across three generations) exposure to different concentrations (including the 1/10 NOEC, NOEC, and 1/10 LC50 calculated from the 24-h acute toxicity test) of Irgarol using the water flea Moina macrocopa. Acute exposure to 1/10 LC50 significantly decreased survival, feeding rate, thoracic limb activity, heart rate, and acetylcholinesterase activity. Elevated levels of intracellular reactive oxygen species and malondialdehyde, along with a significant increase in catalase and superoxide dismutase activity, suggested the induction of oxidative stress in response to 1/10 LC50. An initial boost in glutathione level and the enzymatic activities of glutathione peroxidase and glutathione reductase, followed by a plunge, implies some compromise in the antioxidant defense system. Upon chronic exposure to the NOEC value, both generations F1 and F2 displayed a significant decrease in survival rate, body length, number of neonates per brood, and delayed sexual maturation, suggesting maternal transfer of potential damage through generations. Taken together, Irgarol induced acute toxicity through physiological and cholinergic damage, accompanied by the induction of oxidative stress, in the water flea. Even its sub-lethal concentrations can induce detrimental effects across generations when consistently exposed.

Duplex Reverse-Transcription Real-Time Polymerase Chain Reaction Assay Targeting 23S rRNA Single Nucleotide Polymorphisms for the Detection of Flea-Borne Rickettsioses.

Flea-borne spotted fever and flea-borne (murine) typhus are rickettsioses caused by Rickettsia felis and Rickettsia typhi, respectively, and typically present as undifferentiated febrile illnesses. The relative contribution of these agents to flea-borne rickettsioses in California is unclear. We have developed a duplex reverse transcription real-time polymerase chain reaction (RT-rtPCR) assay targeting R. felis- and R. typhi-specific 23S ribosomal RNA single nucleotide polymorphisms to better understand the respective roles of these agents in causing flea-borne rickettsioses in California. This assay was compared with an established duplex R. felis- and R. typhi-ompB rt-PCR assay and was shown to have 1,000-fold and 10-fold greater analytical sensitivity for the detection of R. felis and R. typhi, respectively. Retrospective testing of clinical specimens with both assays established R. typhi as the major etiologic agent of flea-borne rickettsioses in California.

Epidemiological exploration of fleas and molecular identification of flea-borne viruses in Egyptian small ruminants.

The study aimed to investigate molecularly the presence of flea-borne viruses in infested small ruminants with fleas. It was carried out in Egypt's Northern West Coast (NWC) and South Sinai Governorate (SSG). Three specific primers were used targeting genes, ORF103 (for Capripoxvirus and Lumpy skin disease virus), NS3 (for Bluetongue virus), and Rdrp (for Coronavirus), followed by gene sequencing and phylogenetic analyses. The results revealed that 78.94% of sheep and 65.63% of goats were infested in the NWC area, whereas 49.76% of sheep and 77.8% of goats were infested in the SSG region. Sheep were preferable hosts for flea infestations (58.9%) to goats (41.1%) in the two studied areas. Sex and age of the animals had no effects on the infestation rate (p > 0.05). The season and site of infestation on animals were significantly different between the two areas (p < 0.05). Ctenocephalides felis predominated in NWC and Ctenocephalides canis in SSG, and males of both flea species were more prevalent than females. Molecular analysis of flea DNA revealed the presence of Capripoxvirus in all tested samples, while other viral infections were absent. Gene sequencing identified three isolates as sheeppox viruses, and one as goatpox virus. The findings suggest that Capripoxvirus is adapted to fleas and may be transmitted to animals through infestation. This underscores the need for ongoing surveillance of other pathogens in different regions of Egypt.

Unique microbial communities of parasitic fleas on wild animals from the Qinghai-Tibet Plateau.

Fleas, one of the most significant ectoparasites, play a crucial role as vectors in spreading zoonotic diseases globally. The Qinghai Province, as part of the Qinghai-Tibet Plateau, is one of the provinces in China with the largest number of flea species. In this study, we characterized the microbial communities of eighty-five adult fleas, belonging to nineteen species within four families (Ceratophyllidae, Ctenophthalmidae, Leptopsyllidae, and Pulicidae). We identified a total of 1162 unique operational taxonomic units at the genus level, with flea-borne pathogens such as Wolbachia, Bartonella, Rickettsia being the members of top abundant taxa. Except for comparison between Ctenophthalmidae and Leptopsyllidae families, the analyses of both alpha- and beta- diversity indicators suggested that bacterial diversity varied among flea families. This could be attributed to flea phylogeny, which also influenced by their geographical sites and animal hosts. Results of Linear discriminant analysis effect size (LEfSe) indicated that 29 genera in Ceratophylloidea, 11 genera in Ctenophthalmidae, 15 genera in Leptopsyllidae, and 22 genera in Pulicidae were significantly responsible for explaining the differences among the four flea families (linear discriminant analysis score > 2, P < 0.05). Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) analyses showed that the functional pathways varied significantly across flea families, which was supported by the significant correlation between the functional pathways and the microbial communities.

Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Epidemiological survey and genetic diversity of Bartonella in fleas collected from rodents in Fujian Province, Southeast China.

BACKGROUND: Fleas, considered to be the main transmission vectors of Bartonella, are highly prevalent and show great diversity. To date, no investigations have focused on Bartonella vectors in Southeast China. The aim of this study was to investigate the epidemiological and molecular characteristics of Bartonella in fleas in Southeast China. METHODS: From 2016 to 2022, flea samples (n = 1119) were collected from 863 rodent individuals in seven inland and coastal cities in Southeast China. Flea species, region, gender, host species and habitat were recorded. The DNA samples from each individual flea were screened by real-time PCR for the Bartonella ssrA gene. All positive samples were confirmed by PCR based on the presence of the gltA gene and sequenced. The factors associated with Bartonella infection were analyzed by the Chi-square test and Fisher's exact test. ANOVA and the t-test were used to compare Bartonella DNA load. RESULTS: Bartonella DNA was detected in 26.2% (293/1119) of the flea samples, including in 27.1% (284/1047) of Xenopsylla cheopis samples, 13.2% (5/38) of Monopsyllus anisus samples, 8.3% (2/24) of Leptopsylla segnis samples and 20.0% (2/10) of other fleas (Nosopsyllus nicanus, Ctenocephalides felis, Stivalius klossi bispiniformis and Neopsylla dispar fukienensis). There was a significant difference in the prevalence of Bartonella among flea species, sex, hosts, regions and habitats. Five species of Bartonella fleas were identified based on sequencing and phylogenetic analyses targeting the gltA gene: B. tribocorum, B. queenslandensis, B. elizabethae, B. rochalimae and B. coopersplainsensis. CONCLUSIONS: There is a high prevalence and diversity of Bartonella infection in the seven species of fleas collected in Southeast China. The detection of zoonotic Bartonella species in this study, including B. tribocorum, B. elizabethae and B. rochalimae, raises public health concerns.

Dissimilarity in flea and host assemblages and their interaction networks along a spatial distance gradient: different patterns revealed by different network dissimilarity metrics.

We investigated the distance-decay pattern (an increase in dissimilarity with increasing geographic distance) in regional assemblages of fleas and their small mammalian hosts, as well as their interaction networks, in four biogeographic realms. Dissimilarity of assemblages (ßtotal) was partitioned into species richness differences (ßrich) and species replacement (ßrepl) components. Dissimilarity of networks was assessed using two metrics: (a) whole network dissimilarity (ßWN) partitioned into species replacement (ßST) and interaction rewiring (ßOS) components and (b) D statistics, measuring dissimilarity in the pure structure of the networks, without using information on species identities and calculated for hosts-shared-by-fleas networks (Dh) and fleas-shared-by-hosts networks (Df). We asked whether the distance-decay pattern (a) occurs among interactor assemblages or their interaction networks; (b) depends on the network dissimilarity metric used; and (c) differs between realms. The ßtotal and ßrepl of flea and host assemblages increased with distance in all realms except for host assemblages in the Afrotropics. ßrich for flea and host assemblages increased with distance in the Nearctic only. In networks, ßWN and ßST demonstrated a distance-decay pattern, whereas ßOS was mainly spatially invariant except in the Neotropics. Correlations of Dh or Df and geographic distance were mostly non-significant. We conclude that investigations of dissimilarity in interaction networks should include both types of dissimilarity metrics (those that consider partner identities and those that consider the pure structure of networks). This will allow elucidating the predictability of some facets of network dissimilarity and the unpredictability of other facets.

The complete mitochondrial genome of the rodent flea Nosopsyllus laeviceps: genome description, comparative analysis, and phylogenetic implications.

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.

Role of the Yersinia pestis phospholipase D (Ymt) in the initial aggregation step of biofilm formation in the flea.

Transmission of Yersinia pestis by fleas depends on the formation of condensed bacterial aggregates embedded within a gel-like matrix that localizes to the proventricular valve in the flea foregut and interferes with normal blood feeding. This is essentially a bacterial biofilm phenomenon, which at its end stage requires the production of a Y. pestis exopolysaccharide that bridges the bacteria together in a cohesive, dense biofilm that completely blocks the proventriculus. However, bacterial aggregates are evident within an hour after a flea ingests Y. pestis, and the bacterial exopolysaccharide is not required for this process. In this study, we characterized the biochemical composition of the initial aggregates and demonstrated that the yersinia murine toxin (Ymt), a Y. pestis phospholipase D, greatly enhances rapid aggregation following infected mouse blood meals. The matrix of the bacterial aggregates is complex, containing large amounts of protein and lipid (particularly cholesterol) derived from the flea's blood meal. A similar incidence of proventricular aggregation occurred after fleas ingested whole blood or serum containing Y. pestis, and intact, viable bacteria were not required. The initial aggregation of Y. pestis in the flea gut is likely due to a spontaneous physical process termed depletion aggregation that occurs commonly in environments with high concentrations of polymers or other macromolecules and particles such as bacteria. The initial aggregation sets up subsequent binding aggregation mediated by the bacterially produced exopolysaccharide and mature biofilm that results in proventricular blockage and efficient flea-borne transmission. IMPORTANCE: Yersinia pestis, the bacterial agent of plague, is maintained in nature in mammal-flea-mammal transmission cycles. After a flea feeds on a mammal with septicemic plague, the bacteria rapidly coalesce in the flea's digestive tract to form dense aggregates enveloped in a viscous matrix that often localizes to the foregut. This represents the initial stage of biofilm development that potentiates transmission of Y. pestis when the flea later bites a new host. The rapid aggregation likely occurs via a depletion-aggregation mechanism, a non-canonical first step of bacterial biofilm development. We found that the biofilm matrix is largely composed of host blood proteins and lipids, particularly cholesterol, and that the enzymatic activity of a Y. pestis phospholipase D (Ymt) enhances the initial aggregation. Y. pestis transmitted by flea bite is likely associated with this host-derived matrix, which may initially shield the bacteria from recognition by the host's intradermal innate immune response.

Records of fleas (Siphonaptera) from Delaware.

We present an annotated checklist of fleas (Siphonaptera) known to occur in the state of Delaware based on an examination of Siphonaptera collections at the University of Delaware and the Carnegie Museum of Natural History, as well as new specimens of fleas we collected from wildlife, other hosts, and tick flags. We review published records and compile them herein with our new records, which include 3 species previously unreported from Delaware. With these additions, there are now 18 flea species from 19 avian and mammalian hosts documented from Delaware.

Survey of Rickettsia species in hematophagous arthropods from endemic areas for Japanese spotted fever in China.

Japanese spotted fever (JSF) is caused by Rickettsia japonica, mainly vectored by hard ticks. However, whether R. japonica can be transmitted by other arthropods remains unknown. Moreover, it is of interest to investigate whether other Rickettsia species cause spotted fever in endemic areas. In this study, a survey of Rickettsia species was performed in hematophagous arthropods (mosquitoes, tabanids, and ticks) from endemic areas for JSF in Hubei Province, central China. The results showed that the diversity and prevalence of Rickettsia species in mosquitoes are low, suggesting that mosquitoes may not be the vector of zoonotic Rickettsia species. A novel Rickettsia species showed a high prevalence (16.31%, 23/141) in tabanids and was named "Candidatus Rickettsia tabanidii." It is closely related to Rickettsia from fleas and mosquitoes; however, its pathogenicity in humans needs further investigation. Five Rickettsia species were identified in ticks. Rickettsia japonica, the agent of JSF, was detected only in Haemaphysalis longicornis and Haemaphysalis hystricis, suggesting that they may be the major vectors of R. japonica. Notably, two novel species were identified in H. hystricis ticks, one belonging to the spotted fever group and the other potentially belonging to the ancestral group. The latter one named "Candidatus Rickettsia hubeiensis" may provide valuable insight into the evolutionary history of Rickettsia.