New records of pathogenic bacteria in different species of fleas collected from domestic and peridomestic animals in Spain. A potential zoonotic threat?

Climate change is causing many vectors of infectious diseases to expand their geographic distribution as well as the pathogens they transmit are also conditioned by temperature for their multiplication. Within this context, it is worth highlighting the significant role that fleas can play as vectors of important pathogenic bacteria. For this purpose, our efforts focused on detecting and identifying a total of 9 bacterial genera (Rickettsia sp.; Bartonella sp.; Yersinia sp.; Wolbachia sp., Mycobacterium sp., Leishmania sp., Borrelia sp., Francisella sp. and Coxiella sp.) within fleas isolated from domestic and peridomestic animals in the southwestern region of Spain (Andalusia). Over a 19-months period, we obtained flea samples from dogs, cats and hedgehogs. A total of 812 fleas was collected for this study. Five different species were morphologically identified, including C. felis, C. canis, S. cuniculi, P. irritans, and A. erinacei. Wolbachia sp. was detected in all five species identified in our study which a total prevalence of 86%. Within Rickettsia genus, two different species, R. felis and R. asembonensis were mainly identified in C. felis and A. erinacei, respectively. On the other hand, our results revealed a total of 131 fleas testing positive for the presence of Bartonella sp., representing a prevalence rate of 16% for this genus identifying two species B. henselae and B. clarridgeiae. Lastly, both Y. pestis and L. infantum were detected in DNA of P. irritans and C. felis, respectively isolated from dogs. With these data we update the list of bacterial zoonotic agents found in fleas in Spain, emphasizing the need to continue conducting future experimental studies to assess and confirm the potential vectorial role of certain synanthropic fleas.

Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas, Chile.

Seventy-five flea pools (one to ten fleas per pool) from 51 Andean foxes (Lycalopex culpaeus) and five South American grey foxes or chillas (Lycalopex griseus) from the Mediterranean region of Chile were analyzed for the presence of DNA of Bartonella spp. and Rickettsia spp. through quantitative real-time PCR for the nouG and gltA genes, respectively. Positive samples were further characterized by conventional PCR protocols, targeting gltA and ITS genes for Bartonella, and gltA, ompA, and ompB genes for Rickettsia. Bartonella was detected in 48 % of the Pulex irritans pools (B. rochalimae in three pools, B. berkhoffii in two pools, B. henselae in one pool), and 8 % of the Ctenocephalides felis felis pools (B. rochalimae, one pool). Rickettsia was confirmed in 11 % of P. irritans pools and 92 % of the Ct. felis pools. Characterization confirmed R. felis in all sequenced Rickettsia-positive pools. All Ct. canis pools were negative. A Ct. felis pool from a wild-found domestic ferret (Mustela putorius furo) also resulted positive for R. felis. Although opportunistic, this survey provides the first description of zoonotic pathogens naturally circulating in fleas parasitizing Chilean free-living carnivores.

Complete bacterial profile and potential pathogens of cat fleas Ctenocephalides felis.

Fleas are important ectoparasites and vectors associated with a wide range of pathogenic diseases, posing threats to public health concerns, especially cat fleas that spread worldwide. Understanding the microbial components is essential due to cat fleas are capable of transmitting pathogens to humans, causing diseases like plague and murine typhus. In the present study, metagenomic next-generation sequencing was applied to obtain the complete microbiota and related functions in the gut of Ctenocephalides felis. A total of 1,870 species was taxonomically recognized including 1,407 bacteria, 365 eukaryotes, 69 viruses, and 29 archaea. Proteobacteria was the dominant phylum among the six samples. Pathogens Rickettsia felis, Acinetobacter baumannii, Coxiella burnetii, and Anaplasma phagocytophilum were taxonomically identified and had high abundances in all samples. The resistance gene MexD was predominant in microbial communities of all cat fleas. We also performed epidemiological surveys of pathogens R. felis, A. baumannii, C. burnetii, and A. phagocytophilum among 165 cat fleas collected from seven provinces in China, while only the DNAs of R. felis (38/165, 23.03%) and C. burnetii (2/165, 1.21%) were obtained. The data provide new insight and understanding of flea intestinal microbiota and support novel information for preventing and controlling fleas and their transmitted diseases.

Revisiting the sialome of the cat flea Ctenocephalides felis.

The hematophagous behaviour emerged independently in several instances during arthropod evolution. Survey of salivary gland and saliva composition and its pharmacological activity led to the conclusion that blood-feeding arthropods evolved a distinct salivary mixture that can interfere with host defensive response, thus facilitating blood acquisition and pathogen transmission. The cat flea, Ctenocephalides felis, is the major vector of several pathogens, including Rickettsia typhi, Rickettsia felis and Bartonella spp. and therefore, represents an important insect species from the medical and veterinary perspectives. Previously, a Sanger-based sialome of adult C. felis female salivary glands was published and reported 1,840 expressing sequence tags (ESTs) which were assembled into 896 contigs. Here, we provide a deeper insight into C. felis salivary gland composition using an Illumina-based sequencing approach. In the current dataset, we report 8,892 coding sequences (CDS) classified into 27 functional classes, which were assembled from 42,754,615 reads. Moreover, we paired our RNAseq data with a mass spectrometry analysis using the translated transcripts as a reference, confirming the presence of several putative secreted protein families in the cat flea salivary gland homogenates. Both transcriptomic and proteomic approaches confirmed that FS-H-like proteins and acid phosphatases lacking their putative catalytic residues are the two most abundant salivary proteins families of C. felis and are potentially related to blood acquisition. We also report several novel sequences similar to apyrases, odorant binding proteins, antigen 5, cholinesterases, proteases, and proteases inhibitors, in addition to putative novel sequences that presented low or no sequence identity to previously deposited sequences. Together, the data represents an extended reference for the identification and characterization of the pharmacological activity present in C. felis salivary glands.

Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection.

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.

Investigation of the genetic diversity and flea-borne pathogens in Ctenocephalides felis samples collected from goats in Izmir and Sanliurfa provinces of Turkey.

The cat flea "Ctenocephalides felis" has veterinary and medical importance since it is a vector for numerous important pathogens. In this study, a total of 249 flea samples were collected from goats bred in eight different farms (located in Izmir and Sanliurfa provinces of Turkey) and morphologically identified under microscopy. Later, the genetic diversity was investigated in 117 of C. felis samples that were morphologically identified by sequencing the mitochondrial cox1 gene, followed by phylogenetic tree, haplotype, genetic differentiation and gene flow analyses. In addition, Rickettsia spp. and Bartonella spp. which are zoonoses were screened in 27 pools comprising 249 flea samples by PCR. The phylogenetic tree showed that 117 flea samples were clustered in Clade 1 together with isolates from Australia, New Zealand, the Czech Republic, and India. Four haplotypes (haplotypes I, II, III and IV) were detected within the C. felis species. The most prevalent haplotype was haplotype I (57/117; 48.7 %). Among the population of flea samples in Izmir and Sanliurfa, the Fst and Nm values were 0.16261 and 2.57, respectively, indicating a moderate genetic differentiation and high gene flow. Rickettsia spp. was detected in four of C. felis pool samples whereas Bartonella spp. was detected in 25 of them. BLAST analysis identified R. raoultii as well as B. henselae and B. elizabethae. In conclusion, the findings showed that C. felis samples collected from goats in Turkey were classified within Clade 1 representing four different haplotypes with a moderate genetic diversity for the first time. Also, R. raoultii, B. henselae and B. elizabethae were demonstrated for the first time in cat flea samples collected in Turkey.

Fleas from the Silk Road in Central Asia: identification of Ctenocephalides canis and Ctenocephalides orientis on owned dogs in Uzbekistan using molecular identification and geometric morphometrics.

BACKGROUND: The Silk Road connected the East and West for over 1500 years. Countries in Central Asia are valuable in addressing the hypothesis that parasites on domestic animals were introduced along the Silk Road. Adult fleas are obligate parasites, having worldwide distribution. In dogs, Ctenocephalides canis, C. felis and C. orientis are the most common species identified. The distribution of the Oriental cat flea, C. orientis, is restricted to southeast Asia. The purpose of this study was to determine the diversity of dog fleas from Uzbekistan, a country in Central Asia, with particular reference to C. orientis. METHODS: Fleas were collected from 77 dogs from 5 locations in Uzbekistan. The cox1 gene sequences from Ctenocephalides spp. were compared to global collection of Ctenocephalides cox1 haplotypes. Landmark-based geometric morphometrics have been applied to the head and curvature to compare C. canis and C. canis using canonical variate analysis and discriminant function analysis. RESULTS: Overall, 199 fleas were collected and identified as C. canis (n = 115, 58%), C. orientis (n = 53, 27%) and Pulex irritans (n = 22, 11%). None of the fleas were C. felis. All Ctenocephalides spp. fleas were subject to cox1 amplification and 95% (166/175) yielded DNA sequence. There were 25 cox1 haplotypes; 14 (22/25, 88%) were C. canis cox1 haplotypes and 3 (3/25, 12%) were C. orientis cox1 haplotypes. Molecular analysis confirmed the absence of C. felis. Four (4/22) and one (1/3) cox1 haplotypes were identical to cox1 haplotypes belonging to C. canis and C. orientis cox1 haplotypes identified elsewhere, respectively. Overall morphometric analysis confirmed significant differences between the head shape of C. canis and C. orientis and improved four-fivefold the species identification compared to traditional morphological key. CONCLUSION: We report for the first time the presence of C. orientis in Uzbekistan. Differentiation of C. orientis from C. canis and C. felis remains difficult in regions where these species coexist. Studies in Central and Southeast Asia should confirm species identity using cox1 locus to enable retracing of the distribution of the Ctenocephalides in Asia. The presence of C. orientis suggests that this species may have been introduced from the east along the ancient Silk Road.

Population genetics and genetic variation of Ctenocephalides felis and Pulex irritans in China by analysis of nuclear and mitochondrial genes.

BACKGROUND: Fleas are the most economically significant blood-feeding ectoparasites worldwide. Ctenocephalides felis and Pulex irritans can parasitize various animals closely related to humans and are of high veterinary significance. METHODS: In this study, 82 samples were collected from 7 provinces of China. Through studying the nuclear genes ITS1 and EF-1α and two different mitochondrial genes cox1 and cox2, the population genetics and genetic variation of C. felis and P. irritans in China were further investigated. RESULTS: The intraspecies differences between C. felis and P. irritans ranged from 0 to 3.9%. The interspecific variance in the EF-1α, cox1, and cox2 sequences was 8.2-18.3%, while the ITS1 sequence was 50.1-52.2%. High genetic diversity was observed in both C. felis and P. irritans, and the nucleotide diversity of cox1 was higher than that of cox2. Moderate gene flow was detected in the C. felis and P. irritans populations. Both species possessed many haplotypes, but the haplotype distribution was uneven. Fu's Fs and Tajima's D tests showed that C. felis and P. irritans experienced a bottleneck effect in Guangxi Zhuang Autonomous Region and Henan province. Evolutionary analysis suggested that C. felis may have two geographical lineages in China, while no multiple lineages of P.irritans were found. CONCLUSIONS: Using sequence comparison and the construction of phylogenetic trees, we found a moderate amount of gene flow in the C. felis and P. irritans populations. Both species possessed many haplotypes, but the distribution of haplotypes varied among the provinces. Fu's Fs and Tajima's D tests indicated that both species had experienced a bottleneck effect in Guangxi and Henan provinces. Evolutionary analysis suggested that C. felis may have two geographical lineages in China, while no multiple lineages of P.irritans were found. This study will help better understand fleas' population genetics and evolutionary biology.

Mitochondrial phylogenomics provides insights into the taxonomy and phylogeny of fleas.

BACKGROUND: Fleas (Insecta: Siphonaptera) are obligatory hematophagous ectoparasites of humans and animals and serve as vectors of many disease-causing agents. Despite past and current research efforts on fleas due to their medical and veterinary importance, correct identification and robust phylogenetic analysis of these ectoparasites have often proved challenging. METHODS: We decoded the complete mitochondrial (mt) genome of the human flea Pulex irritans and nearly complete mt genome of the dog flea Ctenocephalides canis, and subsequently used this information to reconstruct the phylogeny of fleas among Endopterygota insects. RESULTS: The complete mt genome of P. irritans was 20,337 bp, whereas the clearly sequenced coding region of the C. canis mt genome was 15,609 bp. Both mt genomes were found to contain 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes. The coding region of the C. canis mt genome was only 93.5% identical to that of the cat flea C. felis, unequivocally confirming that they are distinct species. Our phylogenomic analyses of the mt genomes showed a sister relationship between the order Siphonaptera and orders Diptera + Mecoptera + Megaloptera + Neuroptera and positively support the hypothesis that the fleas in the order Siphonaptera are monophyletic. CONCLUSIONS: Our results demonstrate that the mt genomes of P. irritans and C. canis are different. The phylogenetic tree shows that fleas are monophyletic and strongly support an order-level objective. These mt genomes provide novel molecular markers for studying the taxonomy and phylogeny of fleas in the future.

Dynamic gene expression in salivary glands of the cat flea during Rickettsia felis infection.

The cat flea, Ctenocephalides felis, is an arthropod vector capable of transmitting several human pathogens including Rickettsia species. Earlier studies identified Rickettsia felis in the salivary glands of the cat flea and transmission of rickettsiae during arthropod feeding. The saliva of hematophagous insects contains multiple biomolecules with anticlotting, vasodilatory and immunomodulatory activities. Notably, the exact role of salivary factors in the molecular interaction between flea-borne rickettsiae and their insect host is still largely unknown. To determine if R. felis modulates gene expression in the cat flea salivary glands, cat fleas were infected with R. felis and transcription patterns of selected salivary gland-derived factors, including antimicrobial peptides and flea-specific antigens, were assessed. Salivary glands were microdissected from infected and control cat fleas at different time points after exposure and total RNA was extracted and subjected to reverse-transcriptase quantitative PCR for gene expression analysis. During the experimental 10-day feeding period, a dynamic change in gene expression of immunity-related transcripts and salivary antigens between the two experimental groups was detected. The data indicated that defensin-2 (Cf-726), glycine-rich antimicrobial peptide (Cf-83), salivary antigens (Cf-169 and Cf-65) and deorphanized peptide (Cf-75) are flea-derived factors responsive to rickettsial infection.

The complete mitochondrial genome sequences of the cat flea Ctenocephalides felis felis (Siphonaptera: Pulicidae) support the hypothesis that C. felis isolates from China and USA were the same C. f. felis subspecies.

The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the most important ectoparasite in cats and dogs worldwide. Over the years, there has been much dispute regarding the taxonomic and systematic status of C. felis. Mitochondrial (mt) genome sequences are useful genetic markers for the identification and differentiation of ectoparasites, but the mt genome of C. felis and its subspecies has not yet been entirely characterized. In the present study, the entire mt genome of C. f. felis from China was sequenced and compared with that of C. felis from the USA. Both contain 37 genes and a long non-coding region of >6 kbp. The molecular identity between the Chinese and American isolates was 99%, except for the non-coding region. The protein-coding genes showed differences at both the nucleotide (1.2%) and amino acid (1%) levels. Interestingly, the cox1 gene of the Chinese isolate had an unusual putative start codon (TTT). Taken together, our analyses strongly support the hypothesis that C. felis isolates from China and the USA were the same C. f. felis subspecies. The mt genome sequence of the C. f. felis China isolate presented in this study provides useful molecular markers to further address the taxonomy and systematics of C. felis.

Flea-borne pathogens in the cat flea Ctenocephalides felis and their association with mtDNA diversity of the flea host.

Flea-borne pathogens were screened from 100 individual cat fleas using a PCR approach, of which 38 % were infected with at least one bacterium. Overall, 28 % of the flea samples were positive for Bartonella as inferred from ITS DNA region. Of these, 25 % (7/28) were identified as Bartonella clarridgeiae, 42.9 % (12/28) as Bartonella henselae consisted of two different strains, and 32.1 % (9/28) as Bartonella koehlerae, which was detected for the first time in Malaysia. Sequencing of gltA amplicons detected Rickettsia DNA in 14 % of cat flea samples, all of them identified as Rickettsia asembonensis (100 %). None of the flea samples were positive for Mycoplasma DNA in 16S rRNA gene detection. Four fleas were co-infected with Bartonella and Rickettsia DNAs. Statistical analyses reveal no significant association between bacterial infection and mtDNA diversity of the cat flea. Nevertheless, in all types of pathogen infections, infected populations demonstrated lower nucleotide and haplotype diversities compared to uninfected populations. Moreover, lower haplotype numbers were observed in infected populations.

New taxonomic and evolutionary insights relevant to the cat flea, Ctenocephalides felis: A geographic perspective.

The cat flea, Ctenocephalides felis, is an obligate haematophagous ectoparasite of wildlife and domestic cats and dogs worldwide. Since cat fleas can affect the health of humans and their pets, an uncertain taxonomy of this taxon can greatly inhibit pest and disease management. To address the evolution and taxonomy of the cat flea, we set out to determine 1) how many genetically distinct taxa exist, 2) whether there is morphological support for the genetically distinct taxa, and 3) the role of host range and paleoclimatic events in speciation. We collected a total of 3352 fleas sampled from 576 domestic cats and dogs as well as 10 wildlife species across 30 localities in South Africa. A total of three flea genera, five species, and three of the currently recognized cat flea subspecies, C. f. damarensis, C. f. strongylus and C. f. felis were obtained. Geometric morphometric analyses on head shape were performed on 68 female and 107 male cat flea individuals. Principal component analysis demonstrated large overlap in head shape variation between C. f. strongylus and C. f. felis, rendering this character not useful for phylogenetic inferences. DNA was extracted from 188 Ctenocephalides spp. and mitochondrial COII and nuclear EF1-α sequences were generated. Bayesian and Maximum Likelihood analyses as well as a TCS parsimony haplotype network of the mitochondrial DNA confirmed the presence of three well supported monophyletic clades. These assemblages did not fully corroborate the existence of the three C. felis subspecies. A single well-supported molecular clade included only C. f. damarensis morphotypes that were mostly collected from wildlife. The recognition of this subspecies as a distinct taxon was further corroborated by sequence distances and also the number of plantar spiniform bristles on fore-tarsi V in males. Despite the overall lack of support for the recognition of C. f. damarensis and C. f. strongylus, a geographic trend was visible whereby one genetic lineage corresponded to the western dryer hot subregion, whereas the other was found throughout the region. Bayesian dating suggested that these two clades diverged during the early Pliocene (4.18 mya), a date that corresponds well with the establishment of a dry hot climate in the west of southern Africa. If so, the off-host environment, particularly temperature and humidity, are important factors to consider in the evolution of the cat flea. The present study rejects recent assertions that the three cat flea subspecies are valid entities and rather point to a situation where more sampling is required before the taxonomic status of C. f. damarensis can be resolved.

Investigation of Ctenocephalides felis on domestic dogs and Rickettsia felis infection in the Democratic Republic of Sao Tome and Principe.

Rickettsia felis is an obligate intracellular Gram-negative bacterium which causes flea-borne spotted fever in humans. In the past decades, R. felis has been detected worldwide in Ctenocephalides felis fleas and various other arthropods. However, due to its shared symptoms with other common vector-borne diseases, human infection is prone to be underestimated or misdiagnosed, especially in the malaria-endemic areas including sub-Saharan Africa, where confirmatory laboratory diagnoses are not usually available. In this study, a 'One Health' approach was adopted to explore potential vector-borne and zoonotic pathogens in the Democratic Republic of Sao Tome and Principe (DRSTP), an island nation in the Gulf of Guinea. By collaborating with local veterinarians, 1,187 fleas were collected from 95 domestic dogs across the country and later identified as Ct. felis using taxonomic keys. A cytochrome oxidase gene-based phylogenetic analysis revealed that all collected fleas belonged to a single haplotype and were identical to isolates from Ivory Coast and Brazil that clustered into a clade of tropical distribution. Additional samples of 14 chigoe fleas (Tunga penetrans) were collected from the surrounding environment of the dogs' resting spots. Rickettsia felis infection in fleas was examined by molecular methods targeting the citrate synthase (gltA)- and outer membrane protein A (ompA)-coding genes as well as the R. felis-specific pRF plasmid. The bacterial DNA was detected in 21.01% (146/695) of cat fleas but none of the chigoe fleas. Microimmunofluorescence assay was then performed to assess pathogen exposure of the residents. Of 240 dried blood spots from participants with dog contacts, 8 (3.33%) exhibited R. felis antibodies. Our findings demonstrated the presence of R. felis in DRSTP. Further extensive epidemiological studies regarding its prevalence and its role in causing febrile illness while the nation is entering pre-elimination stage of malaria will be carried out.

A chromosome-level assembly of the cat flea genome uncovers rampant gene duplication and genome size plasticity.

BACKGROUND: Fleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e., the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea, Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe. RESULTS: By combining Illumina and PacBio sequencing of DNA derived from multiple inbred female fleas with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly for C. felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ~ 38% of protein-coding genes with two or more copies and over 4000 tRNA genes. A broad range of genome size determinations (433-551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) in C. felis. Similarly, broad genome sizes were also calculated for individuals of Xenopsylla cheopis (Oriental rat flea), indicating that this remarkable "genome-in-flux" phenomenon could be a siphonapteran-wide trait. Finally, from the C. felis sequence reads, we also generated closed genomes for two novel strains of Wolbachia, one parasitic and one symbiotic, found to co-infect individual fleas. CONCLUSION: Rampant CNV in C. felis has dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novel Wolbachia endosymbionts-potential tools for blocking pathogen transmission-these oddities highlight a unique and underappreciated disease vector.

High frequency of knockdown resistance mutations in the para gene of cat flea (Ctenocephalides felis) samples collected from goats.

Fleas are ectoparasites of mammals and birds. In livestock such as sheep and goat, flea bites cause many clinical signs. Several types of insecticides including pyrethroids are used to struggle against fleas. The widespread use of these insecticides causes an increase in the number of resistant individuals in flea populations. T929V and L1014F mutations corresponding to pyrethroid resistance have been found in the para gene of cat fleas. We aimed to investigate T929V and L1014F mutations in flea samples (n:162) collected from goats in seven different farms where cypermethrin, a synthetic pyrethroid, had been used intensively. To achieve this aim, collected flea samples were morphologically identified under a stereo microscope and DNA isolation was conducted by HotSHOT method. Later, a bi-PASA targeting the para gene was applied to identify both mutations in corresponding samples. According to the results obtained, all fleas were Ctenocephalides felis. Frequencies of T929V and L1014F mutations in fleas were 92.6% (150/162) and 95.7% (155/162), respectively. In conclusion, the frequency of mutations related to pyrethroid resistance was very high in the fleas collected from all the farms and it was thought that the high frequency of these mutations can be attributed to intensive use of pyrethroids.

New insights into the haplotype diversity of the cosmopolitan cat flea Ctenocephalides felis (Siphonaptera: Pulicidae).

The present study investigated the genetic profile of the cosmopolitan cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) from Malaysia and the reference data available in the National Center for Biotechnology Information (NCBI) GenBank. A set of sequences of 100 Malaysian samples aligned as 550 characters of the cytochrome c oxidase subunit I (cox1) and 706 characters of the II (cox2) genes revealed ten haplotypes (A1-A10) and eight haplotypes (B1-B8), respectively. The concatenated sequences of cox1 and cox2 genes with a total of 1256 characters revealed 15 haplotypes (AB1-AB15). Analyses indicated that haplotype AB1 was the most frequent and the most widespread haplotype in Malaysia. Overall haplotype and nucleotide diversities of the concatenated sequences were 0.52909 and 0.00424, respectively, with moderate genetic differentiation (FST = 0.17522) and high gene flow (Nm = 1.18). The western population presented the highest genetic diversity (Hd = 0.78333, Pi = 0.01269, Nh = 9), whereas the southern population demonstrated the lowest diversity (Hd = 0.15667, Pi = 0.00019, Nh = 3). The concatenated sequences showed genetic distances ranged from 0.08 % to 4.39 %. There were three aberrant haplotypes in cox2 sequences that highly divergent, suggesting the presence of cryptic species or occurrence of introgression. In the global point of view, the aligned sequences of C. felis revealed 65 haplotypes (AA1-AA65) by the cox1 gene (n = 586), and 27 haplotypes (BB1-BB27) by the cox2 gene (n = 204). Mapping of the haplotype network showed that Malaysian C. felis possesses seven unique haplotypes in both genes with the common haplotypes demonstrated genetic affinity with C. felis from Southeast Asia for cox1 and South America for cox2. The topologies of cox1 and cox2 phylogenetic trees were concordant with relevant grouping pattern of haplotypes in the network but revealed two major lineages by which Malaysian haplotypes were closely related with haplotypes from the tropical region.

Climate change models predict southerly shift of the cat flea (Ctenocephalides felis) distribution in Australia.

BACKGROUND: Bioclimatic variables play an integral part in the life-cycle of Ctenocephalides felis, the most common flea found on companion animals. It is essential that we understand the effects of climate on C. felis distribution as fleas are a major veterinary and public health concern. This study investigated the current distribution of C. felis in Australia and future projections based on climate modelling. RESULTS: Typing of C. felis was undertaken using the cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA (mtDNA) region and current distribution of haplotypes was mapped by Maximum Entropy (Maxent) niche modelling. All C. felis haplotypes have been predicted to persist in environments along the eastern and southern coastlines of Australia and distinct ecological niches were observed for two C. felis haplogroups. Clade 'Cairns' haplogroup thrives under the northern coastal tropical conditions whilst Clade 'Sydney' haplogroup persists in temperate climates along the eastern and southern coasts. The model was then used to predict areas that are projected to have suitable climatic conditions for these haplogroups in 2050 and 2070 under the Intergovernmental Panel on Climate Change (IPCC) climate change scenarios. Under all IPCC Representative Concentration Pathways (RCP) climate change scenarios, the geographical range of all haplotypes was reduced by 5.59-42.21% in 2050 and 27.08-58.82% by 2070. The ranges of all clades were predicted to shift south along the eastern coastline. CONCLUSIONS: As future temperatures exceed critical threshold temperatures for C. felis development in the northern tropical areas, Clade 'Cairns' haplogroup is predicted to shift south along the coastline and possibly outcompete the temperate haplogroup in these areas. If C. felis haplogroups possess distinct climatic niches it suggests a potential for these to be biologically distinct and have differing developmental rates and vector capabilities.

Out-of-Africa, human-mediated dispersal of the common cat flea, Ctenocephalides felis: The hitchhiker's guide to world domination.

The cat flea (Ctenocephalides felis) is the most common parasite of domestic cats and dogs worldwide. Due to the morphological ambiguity of C. felis and a lack of - particularly largescale - phylogenetic data, we do not know whether global C. felis populations are morphologically and genetically conserved, or whether human-mediated migration of domestic cats and dogs has resulted in homogenous global populations. To determine the ancestral origin of the species and to understand the level of global pervasion of the cat flea and related taxa, our study aimed to document the distribution and phylogenetic relationships of Ctenocephalides fleas found on cats and dogs worldwide. We investigated the potential drivers behind the establishment of regional cat flea populations using a global collection of fleas from cats and dogs across six continents. We morphologically and molecularly evaluated six out of the 14 known taxa comprising genus Ctenocephalides, including the four original C. felis subspecies (Ctenocephalides felis felis, Ctenocephalides felis strongylus, Ctenocephalides felis orientis and Ctenocephalides felis damarensis), the cosmopolitan species Ctenocephalides canis and the African species Ctenocephalides connatus. We confirm the ubiquity of the cat flea, representing 85% of all fleas collected (4357/5123). Using a multigene approach combining two mitochondrial (cox1 and cox2) and two nuclear (Histone H3 and EF-1α) gene markers, as well as a cox1 survey of 516 fleas across 56 countries, we demonstrate out-of-Africa origins for the genus Ctenocephalides and high levels of genetic diversity within C. felis. We define four bioclimatically limited C. felis clusters (Temperate, Tropical I, Tropical II and African) using maximum entropy modelling. This study defines the global distribution, African origin and phylogenetic relationships of global Ctenocephalides fleas, whilst resolving the taxonomy of the C. felis subspecies and related taxa. We show that humans have inadvertently precipitated the expansion of C. felis throughout the world, promoting diverse population structure and bioclimatic plasticity. By demonstrating the link between the global cat flea communities and their affinity for specific bioclimatic niches, we reveal the drivers behind the establishment and success of the cat flea as a global parasite.

RNA interference in the cat flea, Ctenocephalides felis: Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2.

Effective RNA interference (RNAi) methods have been developed in many pest species, enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of GSTσ was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% of Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administration of dsRNA in whole blood via a membrane feeding system, resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 h after exposure to exogenous dsRNA, implicating the short-interfering RNA pathway. To our knowledge this study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight into how the gene knockdown response progresses.