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.     

Detection of Rickettsia spp. and Bartonella spp. in Ctenocephalides felis fleas from free‐ranging crab‐eating foxes (Cerdocyon thous)

Fleas are insects with a worldwide distribution that have been implicated in the transmission of several pathogens. The present study aimed to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Bartonella spp. (Rhizobiales: Bartonellaceae) in fleas from free‐ranging crab‐eating foxes Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from Rio Grande do Sul, southern Brazil. Fleas were collected manually from animals and used for the molecular detection of Rickettsia spp. and Bartonella spp. Twenty‐nine C. thous were sampled in six municipalities. Four foxes were parasitized by 10 fleas, all of which were identified as Ctenocephalides felis (Bouché, 1935) (Siphonaptera: Pulicidae). DNA from Rickettsia felis Bouyer et al., 2001 and Rickettsia asembonensis Maina et al., 2016 were found in three and eight fleas, respectively. In four fleas, DNA of Bartonella sp. was identified. Phylogenetic analysis grouped Bartonella sp. together with other genotypes previously reported in C. felis worldwide. The scenario described in the present study highlights a Neotropical canid parasitized by the invasive cosmopolitan cat flea, which in turn, is carrying potentially invasive vector‐borne microorganisms. These findings suggest that C. felis is adapted to wild hosts in wilderness areas in southern Brazil, hypothetically exposing the Neotropical fauna to unknown ecological and health disturbances.     

Molecular detection of zoonotic bartonellae (B. henselae,B. elizabethae and B. rochalimae) in fleas collected from dogs in Israel

Fleas represent an acknowledged burden on dogs worldwide. The characterization of flea species infesting kennel dogs from two localities in Israel (Rehovot and Jerusalem) and their molecular screening for Bartonella species (Rhizobiales: Bartonellaceae) was investigated. A total of 355 fleas were collected from 107 dogs. The fleas were morphologically classified and molecularly screened targeting the Bartonella 16S–23S internal transcribed spacer (ITS). Of the 107 dogs examined, 80 (74.8%) were infested with Ctenocephalides canis (Siphonaptera: Pulicidae), 68 (63.6%) with Ctenocephalides felis, 15 (14.0%) with Pulex irritans (Siphonaptera: Pulicidae) and one (0.9%) with Xenopsylla cheopis (Siphonaptera: Pulicidae). Fleas were grouped into 166 pools (one to nine fleas per pool) according to species and host. Thirteen of the 166 flea pools (7.8%) were found to be positive for Bartonella DNA. Detected ITS sequences were 99–100% similar to those of four Bartonella species: Bartonella henselae (six pools); Bartonella elizabethae (five pools); Bartonella rochalimae (one pool), and Bartonella bovis (one pool). The present study indicates the occurrence of a variety of flea species in dogs in Israel; these flea species are, in turn, carriers of several zoonotic Bartonella species. Physicians, veterinarians and public health workers should be aware of the presence of these pathogens in dog fleas in Israel and preventive measures should be implemented.     

Prevalence of Rickettsia and Bartonella species in Spanish cats and their fleas

The aim of this study was to determine the prevalence of Bartonella henselae, Rickettsia felis, and Rickettsia typhi in fleas and companion cats (serum and claws) and to assess their presence as a function of host, host habitat, and level of parasitism. Eighty‐nine serum and claw samples and 90 flea pools were collected. Cat sera were assayed by IFA for Bartonella henselae and Rickettssia species IgG antibodies. Conventional PCRs were performed on DNA extracted from nails and fleas collected from cats. A large portion (55.8%) of the feline population sampled was exposed to at least one of the three tested vector‐borne pathogens. Seroreactivity to B. henselae was found in 50% of the feline studied population, and to R. felis in 16.3%. R. typhi antibodies were not found in any cat. No Bartonella sp. DNA was amplified from the claws. Flea samples from 41 cats (46%) showed molecular evidence for at least one pathogen; our study demonstrated a prevalence rate of 43.3 % of Rickettsia sp and 4.4% of Bartonella sp. in the studied flea population. None of the risk factors studied (cat's features, host habitat, and level of parasitation) was associated with either the serology or the PCR results for Bartonella sp. and Rickettsia sp.. Flea‐associated infectious agents are common in cats and fleas and support the recommendation that stringent flea control should be maintained on cats.     

High phylogenetic diversity of the cat flea (Ctenocephalides felis) at two mitochondrial DNA markers

The cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) (Bouché), is the most common flea species found on cats and dogs worldwide. We investigated the genetic identity of the cosmopolitan subspecies C. felis felis and evaluated diversity of cat fleas from Australia, Fiji, Thailand and Seychelles using mtDNA sequences from cytochrome c oxidase subunit I (cox1) and II (cox2) genes. Both cox1 and cox2 confirmed the high phylogenetic diversity and paraphyletic origin of C. felis felis. The African subspecies C. felis strongylus (Jordan) is nested within the paraphyletic C. felis felis. The south East Asian subspecies C. felis orientis (Jordan) is monophyletic and is supported by morphology. We confirm that Australian cat fleas belong to C. felis felis and show that in Australia they form two distinct phylogenetic clades, one common with fleas from Fiji. Using a barcoding approach, we recognize two putative species within C. felis (C. felis and C. orientis). Nucleotide diversity was higher in cox1 but COX2 outperformed COX1 in amino acid diversity. COX2 amino acid sequences resolve all phylogenetic clades and provide an additional phylogenetic signal. Both cox1 and cox2 resolved identical phylogeny and are suitable for population structure studies of Ctenocephalides species.     

Fleas from domestic dogs and rodents in Rwanda carry Rickettsia asembonensis and Bartonella tribocorum

Fleas (Siphonaptera) are ubiquitous blood‐sucking parasites that transmit a range of vector‐borne pathogens. The present study examined rodents (n = 29) and domestic dogs (n = 7) living in the vicinity of the Volcanoes National Park, Rwanda, for fleas, identified flea species from these hosts, and detected Bartonella (Rhizobiales: Bartonellaceae) and Rickettsia (Rickettsiales: Rickettsiaceae) DNA. The most frequently encountered flea on rodents was Xenopsylla brasiliensis (Siphonaptera: Pulicidae). In addition, Ctenophthalmus (Ethioctenophthalmus) calceatus cabirus (Siphonaptera: Hystrichopsyllidae) and Ctenocephalides felis strongylus (Siphonaptera: Pulicidae) were determined using morphology and sequencing of the cytochrome c oxidase subunit I and cytochrome c oxidase subunit II genes (cox1 and cox2, respectively). Bartonella tribocorum DNA was detected in X. brasiliensis and Rickettsia asembonensis DNA (a Rickettsia felis‐like organism) was detected in C. felis strongylus. The present work complements studies that clarify the distributions of flea‐borne pathogens and potential role of fleas in disease transmission in sub‐Saharan Africa. In the context of high‐density housing in central sub‐Saharan Africa, the detection of B. tribocorum and R. asembonensis highlights the need for surveillance in both rural and urban areas to identify likely reservoirs.     

Assessment of Persistence of Bartonella henselae in Ctenocephalides felis

Bartonella henselae (Rhizobiales: Bartonellaceae) is a Gram-negative fastidious bacterium of veterinary and zoonotic importance. The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the main recognized vector of B. henselae, and transmission among cats and humans occurs mainly through infected flea feces. The present study documents the use of a quantitative molecular approach to follow the daily kinetics of B. henselae within the cat flea and its excreted feces after exposure to infected blood for 48 h in an artificial membrane system. B. henselae DNA was detected in both fleas and feces for the entire life span of the fleas (i.e., 12 days) starting from 24 h after initiation of the blood meal.     

Relationship between the Presence of Bartonella Species and Bacterial Loads in Cats and Cat Fleas (Ctenocephalides felis) under Natural Conditions

Cats are considered the main reservoir of three zoonotic Bartonella species: Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae. Cat fleas (Ctenocephalides felis) have been experimentally demonstrated to be a competent vector of B. henselae and have been proposed as the potential vector of the two other Bartonella species. Previous studies have reported a lack of association between the Bartonella species infection status (infected or uninfected) and/or bacteremia levels of cats and the infection status of the fleas they host. Nevertheless, to date, no study has compared the quantitative distributions of these bacteria in both cats and their fleas under natural conditions. Thus, the present study explored these relationships by identifying and quantifying the different Bartonella species in both cats and their fleas. Therefore, EDTA-blood samples and fleas collected from stray cats were screened for Bartonella bacteria. Bacterial loads were quantified by high-resolution melt real-time quantitative PCR assays. The results indicated a moderate correlation between the Bartonella bacterial loads in the cats and their fleas when both were infected with the same Bartonella species. Moreover, a positive effect of the host infection status on the Bartonella bacterial loads of the fleas was observed. Conversely, the cat bacterial loads were not affected by the infection status of their fleas. Our results suggest that the Bartonella bacterial loads of fleas are positively affected by the presence of the bacteria in their feline host, probably by multiple acquisitions/accumulation and/or multiplication events.     

Presence of Bartonella and Rickettsia spp. in cat fleas and brown dog ticks collected from dogs in American Samoa

Surveys for ectoparasites of dogs on the American Samoa islands of Aunu'u and Tutuila were conducted in June 2012, and was followed by molecular screening of samples. One species of flea, Ctenocephalides felis, and one tick species, Rhipicephalus sanguineus, were collected and tested for Bartonella and Rickettsia species via PCR. Bartonella clarridgeiae and an unnamed spotted fever group Rickettsia were detected in the fleas; the Rickettsia species was previously reported from Ctenocephalides spp. from Egypt, Thailand, USA, and the Republic of the Marshall Islands. None of the ticks were positive for Bartonella or Rickettsia species. This is the first report of flea-borne Rickettsia and B. clarridgeiae, considered an emerging human pathogen in New Zealand, from American Samoa. Ectoparasite-borne infections are easily misdiagnosed or ignored as their symptoms are often vague and similar to other illnesses (e.g., measles, dengue). Our results indicate a potential threat to human and animal health as infected fleas were collected from household pets.     

Detection of Rickettsia felis,Rickettsia typhi,Bartonella Species and Yersinia pestis in Fleas (Siphonaptera) from Africa

Little is known about the presence/absence and prevalence of Rickettsia spp, Bartonella spp. and Yersinia pestis in domestic and urban flea populations in tropical and subtropical African countries.

Methodology/Principal findings

Fleas collected in Benin, the United Republic of Tanzania and the Democratic Republic of the Congo were investigated for the presence and identity of Rickettsia spp., Bartonella spp. and Yersinia pestis using two qPCR systems or qPCR and standard PCR. In Xenopsylla cheopis fleas collected from Cotonou (Benin), Rickettsia typhi was detected in 1% (2/199), and an uncultured Bartonella sp. was detected in 34.7% (69/199). In the Lushoto district (United Republic of Tanzania), R. typhi DNA was detected in 10% (2/20) of Xenopsylla brasiliensis, and Rickettsia felis was detected in 65% (13/20) of Ctenocephalides felis strongylus, 71.4% (5/7) of Ctenocephalides canis and 25% (5/20) of Ctenophthalmus calceatus calceatus. In the Democratic Republic of the Congo, R. felis was detected in 56.5% (13/23) of Ct. f. felis from Kinshasa, in 26.3% (10/38) of Ct. f. felis and 9% (1/11) of Leptopsylla aethiopica aethiopica from Ituri district and in 19.2% (5/26) of Ct. f. strongylus and 4.7% (1/21) of Echidnophaga gallinacea. Bartonella sp. was also detected in 36.3% (4/11) of L. a. aethiopica. Finally, in Ituri, Y. pestis DNA was detected in 3.8% (1/26) of Ct. f. strongylus and 10% (3/30) of Pulex irritans from the villages of Wanyale and Zaa.

Conclusion

Most flea-borne infections are neglected diseases which should be monitored systematically in domestic rural and urban human populations to assess their epidemiological and clinical relevance. Finally, the presence of Y. pestis DNA in fleas captured in households was unexpected and raises a series of questions regarding the role of free fleas in the transmission of plague in rural Africa, especially in remote areas where the flea density in houses is high.     

Bartonella species in fleas from Palestinian territories: Prevalence and genetic diversity

Bartonellosis is an infectious bacterial disease. The prevalence and genetic characteristics of Bartonella spp. in fleas of wild and domestic animals from Palestinian territories are described. Flea samples (n=289) were collected from 121 cats, 135 dogs, 26 hyraxes and seven rats from northern (n=165), central (n=113), and southern Palestinian territories (n=11). The prevalent flea species were: Ctenocephalides felis (n=119/289; 41.2%), Ctenocephalides canis (n=159/289; 55%), and Xenopsylla sp. (n=7/289; 2.4%). Targeting the Intergenic Transcribed Spacer (ITS) locus, DNA of Bartonella was detected in 22% (64/289) of all fleas. Fifty percent of the C. felis and 57% of the Xenopsylla sp. contained Bartonella DNA. DNA sequencing showed the presence of Bartonella clarridgeiae (50%), Bartonella henselae (27%), and Bartonella koehlerae (3%) in C. felis. Xenopsylla sp. collected from Rattus rattus rats were infected with Bartonella tribocorum, Bartonella elizabethae, and Bartonella rochalimae. Phylogenetic sequence analysis using the 16S ribosomal RNA gene obtained four genetic clusters, B. henselae and B. koehlerae as subcluster 1, B. clarridgeiae as cluster 2, while the rat Bartonella species (B. tribocorum and B. elizabethae) were an outgroup cluster. These findings showed the important role of cat and rat fleas as vectors of zoonotic Bartonella species in Palestinian territories. It is hoped that this publication will raise awareness among physicians, veterinarians, and other health workers of the high prevalence of Bartonella spp. in fleas in Palestinian territories and the potential risk of these pathogens to humans and animals in this region.     

Detection and identification of Bartonella sp. in fleas from carnivorous mammals in Andalusia,Spain

A total of 559 fleas representing four species (Pulex irritans, Ctenocephalides felis, Ctenocephalides canis and Spilopsyllus cuniculi) collected on carnivores (five Iberian lynx Lynx pardinus, six European wildcat Felis silvestris, 10 common genet Genetta genetta, three Eurasian badger Meles meles, 22 red fox Vulpes vulpes, 87 dogs and 23 cats) in Andalusia, southern Spain, were distributed in 156 pools of monospecific flea from each carnivore, and tested for Bartonella infection in an assay based on polymerase chain reaction (PCR) amplification of the 16 S–23 S rRNA intergenic spacer region. Twenty‐one samples (13.5%) were positive and the sequence data showed the presence of four different Bartonella species. Bartonella henselae was detected in nine pools of Ctenocephalides felis from cats and dogs and in three pools of Ctenocephalides canis from cats; Bartonella clarridgeiae in Ctenocephalides felis from a cat, and Bartonella alsatica in Spilopsyllus cuniculi from a wildcat. DNA of Bartonella sp., closely related to Bartonella rochalimae, was found in seven pools of Pulex irritans from foxes. This is the first detection of B. alsatica and Bartonella sp. in the Iberian Peninsula. All of these Bartonella species have been implicated as agents of human diseases. The present survey confirms that carnivores are major reservoirs for Bartonella spp.     

Novel evidence suggests that a ‘Rickettsia felis‐like’ organism is an endosymbiont of the desert flea,Xenopsylla ramesis

Fleas are acknowledged vectors and reservoirs of various bacteria that present a wide range of pathogenicity. In this study, fleas collected from wild rodents from the Negev desert in southern Israel were tested for RickettsiaDNA by targeting the 16S rRNA (rrs) gene. Thirty‐eight Xenopsylla ramesis, 91 Synosternus cleopatrae and 15 Leptopsylla flea pools (a total of 568 fleas) were screened. RickettsiaDNA was detected in 100% of the X. ramesis and in one S. cleopatrae flea pools. None of L. algira flea pools was found positive. All positive flea pools were further characterized by sequencing of five additional genetic loci (gltA, ompB, ompA, htrA and fusA). The molecular identification of the positive samples showed all sequences to be closely related to the ‘Rickettsia felis‐like’ organisms (99–100% similarities in the six loci). To further investigate the association between ‘R. felis‐like’ and X. ramesis fleas, ten additional single X. ramesis adult fleas collected from the wild and five laboratory‐maintained X. ramesis imago, five larva pools (2–18 larvae per pool) and two egg pools (18 eggs per pool) were tested for the presence of ‘R. felis‐like’ DNA. All samples were found positive by a specific ompAPCR assay, confirming the close association of this Rickettsia species with X. ramesis in all its life stages. These results suggest a symbiotic association between ‘Rickettsia felis‐like’ and X. ramesis fleas.     

Prevalence and infection load dynamics of Rickettsia felis in actively feeding cat fleas

Background

Rickettsia felis is a flea-associated rickettsial pathogen recurrently identified in both colonized and wild-caught cat fleas, Ctenocephalides felis. We hypothesized that within colonized fleas, the intimate relationship between R. felis and C. felis allows for the coordination of rickettsial replication and metabolically active periods during flea bloodmeal acquisition and oogenesis.

Methodology/Principal Findings

A quantitative real-time PCR assay was developed to quantify R. felis in actively feeding R. felis-infected fleas. In three separate trials, fleas were allowed to feed on cats, and a mean of 3.9×10⁶ R. felis 17-kDa gene copies was detected for each flea. A distinct R. felis infection pattern was not observed in fleas during nine consecutive days of bloodfeeding. However, an inverse correlation between the prevalence of R. felis-infection, which ranged from 96% in Trial 1 to 35% in Trial 3, and the R. felis-infection load in individual fleas was identified. Expression of R. felis-infection load as a ratio of R. felis/C. felis genes confirmed that fleas in Trial 3 had significantly greater rickettsial loads than those in Trial 1.

Conclusion/Significance

Examining rickettsial infection dynamics in the flea vector will further elucidate the intimate relationship between R. felis and C. felis, and facilitate a more accurate understanding of the ecology and epidemiology of R. felis transmission in nature.     

Bartonellosis,an increasingly recognized zoonosis

Cat scratch disease is the most common zoonotic infection caused by Bartonella bacteria. Among the many mammals infected with Bartonella spp., cats represent a large reservoir for human infection, as they are the main reservoir for Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. Bartonella spp. are vector‐borne bacteria, and transmission of B. henselae by cat fleas occurs mainly through infected flea faeces, although new potential vectors (ticks and biting flies) have been identified. Dogs are also infected with various Bartonella species and share with humans many of the clinical signs induced by these infections. Although the role of dogs as source of human infection is not yet clearly established, they represent epidemiological sentinels for human exposure. Present knowledge on the aetiology, clinical features and epidemiological characteristics of bartonellosis is presented.     

Survey of flea infestation in cats in Spain

Fleas are a common cause of feline skin disorders as well as vectors of zoonotic diseases. This study evaluated the flea species infesting domestic cats in Spain and assessed factors influencing their distribution. Fleas from 217 cats from 57 localities in Spain were identified and associations between abundance, and host‐dependent, host habitat and environmental factors were examined. Variations in infracommunity and component community structure were also explored. Three species were present, of which Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) was the most abundant (98.4%), followed by Ctenocephalides canis (Curtis) (1.1%) and Pulex irritans (L.) (Siphonaptera: Pulicidae) (0.5%). Overall abundance and abundances of both C. felis and C. canis were higher on farms than in apartments, but overall flea abundance and abundances of both C. felis and C. canis were lower in rural than urban environments. Overall abundance and C. felis abundance were lower during the warmest months, and mean annual rainfall was positively correlated with overall, C. felis and C. canis abundances. No relationship between the number of species per cat and any host, habitat or physiographical variable was found. Species richness was not correlated with mean annual temperature or rainfall. Flea abundance was mainly associated with host habitat and environmental factors.     

Cofeeding intra‐ and interspecific transmission of an emerging insect‐borne rickettsial pathogen

Cat fleas (Ctenocephalides felis) are known as the primary vector and reservoir of Rickettsia felis, the causative agent of flea‐borne spotted fever; however, field surveys regularly report molecular detection of this infectious agent from other blood‐feeding arthropods. The presence of R. felis in additional arthropods may be the result of chance consumption of an infectious bloodmeal, but isolation of viable rickettsiae circulating in the blood of suspected vertebrate reservoirs has not been demonstrated. Successful transmission of pathogens between actively blood‐feeding arthropods in the absence of a disseminated vertebrate infection has been verified, referred to as cofeeding transmission. Therefore, the principal route from systemically infected vertebrates to uninfected arthropods may not be applicable to the R. felis transmission cycle. Here, we show both intra‐ and interspecific transmission of R. felis between cofeeding arthropods on a vertebrate host. Analyses revealed that infected cat fleas transmitted R. felis to naïve cat fleas and rat fleas (Xenopsylla cheopis) via fleabite on a nonrickettsemic vertebrate host. Also, cat fleas infected by cofeeding were infectious to newly emerged uninfected cat fleas in an artificial system. Furthermore, we utilized a stochastic model to demonstrate that cofeeding is sufficient to explain the enzootic spread of R. felis amongst populations of the biological vector. Our results implicate cat fleas in the spread of R. felis amongst different vectors, and the demonstration of cofeeding transmission of R. felis through a vertebrate host represents a novel transmission paradigm for insect‐borne Rickettsia and furthers our understanding of this emerging rickettsiosis.     

Survey of Flea Infestation in Dogs in Different Geographical Regions of Iran

Medically important arthropods, including fleas, play an important role in causing clinical disorders and disease in man and domestic animals. This study was conducted to determine the seasonal flea infestations for domestic dogs from different geographic regions of Iran. A total of 407 fleas, belonging to 5 different species, were recovered from 83 domestic dogs from 3 regions. There was a distinctive pattern of species distribution and infestations with the highest infestation rates observed in a temperate climate and higher rainfall. Additionally, fleas were observed over all seasons, except February and March, with the highest infestation rate observed in August (24.7%) and the lowest rate in January (1.7%). They also parasitize dogs with a different spectrum of species. The cat flea, Ctenocephalides felis (67.5%), exhibited the highest prevalence among all flea species found on dogs. Thus, climatic conditions and seasonal patterns impact on flea infestation and must be considered in developing control programs.     

Identification and characterization of two arginine kinases from the parasitic insect Ctenocephalides felis

Arginine kinase (ATP:l-arginine ω-N-phosphotransferase, EC2.7.3.3.; AK) is an enzyme crucial for the energy metabolism of insects and other invertebrates, that has known allergenic potential in humans and that has been proposed as a pesticidal drug target. Here we report the identification, cDNA cloning, genomic gene structure and functional expression of AK genes from Ctenocephalides (C.) felis (cat flea). In contrast to other insect species investigated so far, C. felis possesses two AK genes, cfak1 and cfak2, encoding the functional enzymes CfAK1 and CfAK2 that can be distinguished by their guanidino substrate specificity and the kinetic parameters for their natural substrates. Molecular modelling on CfAK1 and CfAK2 based on the Limulus polyphemus AK X-ray structure (Zhou et al., 1998) and substrate docking studies provide a potential rational for the observed specificities. Evidence is provided that adult fleas express predominantly CfAK1 as an abundant soluble protein, and that in vivo in C. felis, the AK metabolites are present in concentration ranges relevant for this enzyme.