Molecular Events in Response to Triclosan-Induced Oxidative Stress in CRISPR/Cas9-Mediated p53-Targeted Mutants in Daphnia magna.

Triclosan (TCS), a widely used antimicrobial agent, has been implicated in the oxidative stress induction and disruption of cellular processes in aquatic organisms. As TCS is ubiquitous in the aquatic environment, many previous studies have documented the effects of exposure to TCS on aquatic organisms. Nevertheless, most of the research has concentrated on the molecular and physiological responses of TCS, but there are still limited studies on the function of specific genes and the consequences of their absence. In this study, we focused on p53, a gene that is crucial for molecular responses such as autophagy and apoptosis as a result of TCS exposure. In order to ascertain the role and impact of the p53 gene in TCS-induced molecular responses, we examined the molecular responses to TCS-induced oxidative stress in wild-type (WT) and CRISPR/Cas9-mediated p53 mutant (MT) water fleas. The result has been accomplished by examining changes in molecular mechanisms, including in vivo end points, enzyme activities, adenosine triphosphate release rate, and apoptosis, to determine the role and impact of the p53 gene on TCS-induced molecular responses. The results indicated that the sensitivity of MT water fleas to TCS was greater than that of WT water fleas; however, the difference in sensitivity was significant at short exposures within 48 h and decreased toward 48 h. Accordingly, when we confirmed the oxidative stress after 24 h of exposure, the oxidative stress to TCS exposure was stronger in the MT group, with an imbalance of redox. To identify the mechanisms of tolerance to TCS in WT and MT Daphnia magna, we checked mitochondrial and ER-stress-related biomarkers and found an increase in apoptosis and greater sensitivity to TCS exposure in the MT group than in the WT. Our results suggest that the absence of p53 caused alterations in molecular processes in response to TCS exposure, resulting in increased sensitivity to TCS, and that p53 plays a critical role in response to TCS exposure.

Assessing perceptions of flea and tick infestation risk in Southeast Queensland, Australia.

Social cognitive models are useful for improving our understanding of the relationship between people's health beliefs and disease risks and have been widely employed in human medicine. In veterinary science, there is little research about owners' perceptions of pet health and parasitic infections/infestations, despite the risk of transmission of zoonotic pathogens by ectoparasites. This study investigated the effects of demographic factors on pet owner's perception of ectoparasite infestation risk and behaviours related to animal health in southeast Queensland, Australia employing the Health Belief Model (HBM). A total of 241 dog owners were surveyed in 2018. The electronic survey was generated through SurveyMonkey and distributed utilising UQ SVS and Royal Society for the Prevention of Cruelty to Animals (RSPCA) email networks, informative posters and brochures placed in selected veterinary clinics, and in-person visits to dog parks. The relationship between dog owners' perception of flea and tick infestation risk was assessed using an adaptation of the Health Belief Model, social cognitive framework for health protection. Most survey respondents were between 26 and 45 years of age (40%), identified themselves as females (83%) and had a bachelor's degree (43%). Our results indicate that female owners had greater level of health concern related to ectoparasites compared to males. Similarly, owners aged 18 to 25, with a grade 10 to 12 level of attained education and living predominately in a rural setting showed greater level of health concern related to ectoparasite infestations. The results also indicated that cat owners had on average, higher levels of health concern than dog owners. Our study indicates that the perceptions of pet owners towards perception of flea and tick infestation risk varies demographically, and owner education is an important predictor of prevention behaviours towards tick and flea infestation of pets.

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.

Are Rattus rattus fleas invasive? Evaluation of flea communities in invasive and native rodents in Chile.

Co-invasion, characterized by the simultaneous introduction of hosts and parasites with the latter establishing themselves in native hosts, is a phenomenon of ecological concern. Rattus rattus, a notorious invasive species, has driven the extinction and displacement of numerous avian and mammalian species and serves as a key vector for diseases affecting both humans and wildlife. Among the parasites hosted by R. rattus are fleas, which exhibit obligate parasitic behaviour, a generalist nature and high prevalence, increasing the likelihood of flea invasion. Simultaneously, invasive species can serve as hosts for native parasites, leading to potential amplification or dilution of parasite populations in the environment. In Chile, R. rattus has been present since the 17th century because of the arrival of the Spanish colonizers through the ports and has spread throughout urban, rural and wild Chilean territories. This study aims to evaluate whether co-invasion of native fleas of invasive rats occurs on native rodents in Chile and to determine whether black rats have acquired flea native to Chile during their invasion. For this, we captured 1132 rodents from 26 localities (20° S-53° S). Rattus rattus was found coexisting with 11 native rodent species and two species of introduced rodents. Among the native rodents, Abrothrix olivacea and Oligoryzomys longicaudatus exhibited more extensive sympatry with R. rattus. We identified 14 flea species associated with R. rattus, of which only three were native to rats: Xenopsylla cheopis, Leptopsylla segnis and Nosopsyllus fasciatus. These three species presented a higher parasite load in black rats compared to native fleas. Leptopsylla segnis and N. fasciatus were also found associated with native rodent species that cohabit with R. rattus. The remaining species associated with R. rattus were fleas of native rodents, although they were less abundant compared to those associated with native rodents, except for Neotyphloceras pardinasi and Sphinctopsylla ares. Although there has been evidence of flea transmission from rats to native species, the prevalence and abundance were relatively low. Therefore, it cannot be definitively concluded that these fleas have established themselves in native rodent populations, and hence, they cannot be classified as invasive fleas. This study underscores R. rattus' adaptability to diverse environmental and geographical conditions in Chile, including its capacity to acquire fleas from native rodents. This aspect has critical implications for public health, potentially facilitating the spread of pathogens across various habitats where these rats are found.

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.

Impacts of climate change on the potential distribution of Pulex simulans and Polygenis gwyni.

Pulex simulans and Polygenis gwyni are vectors of many flea-borne diseases. They were widely recorded in the United States and Mexico between 1970 and 2000. Maximum entropy models were used to explore the habitats of both fleas under different climate scenarios to provide the scientific basis for the surveillance and control of flea-borne diseases. We screened climate variables by principal component analysis and Pearson's correlation test and evaluated model performance by ROC curve. ArcMap was used to visualize expressions. Under current climatic conditions, the medium and highly suitable areas for P. simulans are estimated to be 9.16 × 106 km2 and 4.97 × 106 km2, respectively. These regions are predominantly located in South America, along the Mediterranean coast of Europe, the southern part of the African continent, the Middle East, North China, and Australia. For P. gwyni, the medium and highly suitable areas under current climatic conditions are approximately 4.01 × 106 and 2.04 × 106 km2, respectively, with the primary distribution in North China extending to the Himalayas, near the Equator in Africa, and in a few areas of Europe. Under future climate scenarios, in the SSP3-7.0 scenario for the years 2081-2100, the area of high suitability for P. simulans is projected to reach its maximum. Similarly, in the SSP2-4.5 scenario for 2061-2080, the area of high suitability for P. gwyni is expected to reach its maximum. Under global climate change, there is a large range in the potential distribution for both fleas, with an overall upward trend in the area of habitat under future climate scenarios. Governments should develop scientific prevention and control measures to prevent the invasive alien species flea.

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.

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.

Structure of compound and component communities of fleas parasitic on small mammals in six different regions as revealed by environmental-based co-occurrence geometry analyses.

We inferred the patterns of co-occurrence of flea species in compound (across all host species) and component (across conspecific hosts) communities from six regions of the world (Mongolia, Northwest Argentina, Argentinian Patagonia, West Siberia, Slovakia, and South Africa) using the novel eigenvector ellipsoid method. This method allows us to infer structural community patterns by comparing species' environmental requirements with the pattern of their co-occurrences. We asked whether: (a) communities are characterized by species segregation, nestedness, or modularity; (b) patterns detected by the novel method conform to the patterns identified by traditional methods that search for non-randomness in community structure; and (c) the pattern of flea species co-occurrences in component communities is associated with host species traits. The results of the application of the eigenvector ellipsoid method suggested that the co-occurrence of flea species was random in all compound communities except in South Africa, where this community demonstrated a tendency to be nested. Flea species co-occurrences were random in many component communities. Species segregation was detected in the flea community of one host, whereas the flea communities of 14 hosts from different regions appeared to be nested. No indication of a modular structure in any community was found. The nestedness of flea component communities was mainly characteristic of hosts with a low relative brain mass. We concluded that the application of this novel method that combines data on species distribution and their environmental requirements allows better identification of the community structural patterns and produces more reliable results as compared with traditional methods.

Functional similarity affects similarity in partner composition in flea-mammal networks.

Functional signal in an interaction network is a phenomenon in which species resembling each other in their traits interact with similar partners. We tested the functional signal concept in realm-specific and regional flea-host networks from four biogeographic realms and asked whether the species composition of (a) host spectra and (b) flea assemblages is similar between functionally similar flea and host species, respectively. Analogously to testing for phylogenetic signal, we applied Mantel tests to investigate the correlation between flea or host functional distances calculated from functional dendrograms and dissimilarities in sets of interacting partners. In all realm-specific networks, functionally similar fleas tended to exploit similar hosts often belonging to the same genus, whereas functionally similar hosts tended to harbour similar fleas, again often belonging to the same genus. The strength of realm-specific functional signals and the frequency of detecting a significant functional signal in the regional networks differed between realms. The frequency of detecting a significant functional signal in the regional networks correlated positively with the network size for fleas and with the number of hosts in a network for hosts. A functional signal in the regional networks was more frequently found for hosts than for fleas. We discuss the mechanisms behind the functional signal in both fleas and their hosts, relate geographic functional signal patterns to the historic biogeography of fleas and conclude that functional signals in the species composition of host spectra for fleas and of flea assemblages for hosts result from the interplay of evolutionary and ecological processes.

Searching for common patterns in parasite ecology: species and host contributions to beta-diversity in helminths of South African ungulates and fleas of South American rodents.

We searched for common patterns in parasite ecology by investigating species and host contributions to the beta-diversity of infracommunities (=assemblages of parasites harboured by a host individual) in helminths of three species of South African ungulates and fleas of 11 species of South American rodents, assuming that a comparison of patterns in distinctly different parasites and hosts would allow us to judge the generality or, at least, commonness of these patterns. We used data on species' composition and numbers of parasites and asked whether (i) parasite species' attributes (life cycle, transmission mode, and host specificity in helminths; possession of sclerotized combs, microhabitat preference, and host specificity in fleas) or their population structure (mean abundance and/or prevalence) and (ii) host characteristics (sex and age) affect parasite and host species' contributions to parasite beta-diversity (SCBD and HCBD, respectively). We found that parasite species' morphological and ecological attributes were mostly not associated with their SCBD. In contrast, parasite SCBD, in both ungulates and rodents, significantly increased with either parasite mean abundance or prevalence or both. The effect of host characteristics on HCBD was detected in a few hosts only. In general, parasite infracommunities' beta-diversity appeared to be driven by variation in parasite species rather than the uniqueness of the assemblages harboured by individual hosts. We conclude that some ecological patterns (such as the relationships between SCBD and parasite abundance/prevalence) appear to be common and do not differ between different host-parasite associations in different geographic regions, whereas other patterns (the relationships between SCBD and parasite species' attributes) are contingent and depend on parasite and host identities.

A RANGE-WIDE ECTOPARASITE SURVEY FOR ALLEGHENY WOODRATS (NEOTOMA MAGISTER).

Allegheny woodrats (Neotoma magister) are karst-specializing rodents that are rare or in conservation need in many states within their current range. Parasitism and habitat fragmentation have been suggested as primary reasons for declining populations. The presence, prevalence, and impact of ectoparasites, including fleas, ticks, and bots, is not fully understood rangewide. We collected Allegheny woodrat ectoparasites across 8 states in their range, identifying parasites via morphological and genetic means. Across contributions from 8 states, we discovered 2 woodrat-specific fleas parasitizing Allegheny woodrats: Orchopeas pennsylvanicus (all contributing states, n = 228) and Epitedia cavernicola (Indiana only, n = 9). The former was a new state record in New Jersey and Ohio. Woodrat specialists Ixodes woodi were morphologically identified as the dominant tick species (n = 38), and our contributions to genetic databases may ease confusion in future efforts. Three generalist species of ticks representing 8 individuals were identified as Dermacentor variabilis, Amblyomma americanum, and Ixodes scapularis. Only 2 bot fly species were recognized in Allegheny woodrats: 1 squirrel bot (Cuterebra emasculator) and 10 individuals of Cuterebra sp. not genetically conspecific to any known eastern U.S. rodent bot. The host specificity for fleas is not surprising, given that previous small-scale surveys and ticks primarily appear to be a mix of genus-specific (Ixodes woodi) and generalist species. There remains uncertainty with bots via morphological and genetic analyses. Our survey presents a wide-ranging baseline survey for Allegheny woodrats across their range, emphasizing the diversity (or specificity) of parasite groups for this species. An understanding of Allegheny woodrats and the health impact of ectoparasites is imperative because they face myriad challenges rangewide, especially considering the bot-driven demise of 1 woodrat in our study. Ectoparasites can have a marked impact on already-declining woodrat populations across their range and should not be overlooked in future surveys.

Metabolomic analysis to understand the mechanism of Ti3C2Tx (MXene) toxicity in Daphnia magna.

Due to their potential release into the environment, the ecotoxicity of Ti3C2Tx (MXene) nanomaterials is a growing concern. Unfortunately, little is known about the toxic effects and mechanisms through which Ti3C2Tx induces toxicity in aquatic organisms. The aim of this study is thus to investigate the toxic effects and mechanisms of Daphnia magna upon exposure to Ti3C2Tx with different sheet sizes (100 nm [Ti3C2Tx-100] and 500 nm [Ti3C2Tx-500]) by employing conventional toxicology and metabolomics analysis. The results showed that exposure to both Ti3C2Tx-100 and Ti3C2Tx-500 at 10 µg/mL resulted in a significant accumulation of Ti3C2Tx in D. magna, but no effects on the mortality or growth of D. magna were observed. However, the metabolomics results revealed that Ti3C2Tx-100 and Ti3C2Tx-500 induced significant changes in up to 265 and 191 differential metabolites in D. magna, respectively, of which 116 metabolites were common for both. Ti3C2Tx-100-induced metabolites were mainly enriched in phospholipid, pyrimidine, tryptophan, and arginine metabolism, whereas Ti3C2Tx-500-induced metabolites were mainly enriched in the glycerol-ester, tryptophan, and glyoxylate metabolism and the pentose phosphate pathway. These results indicated that the toxicity of Ti3C2Tx to D. magna has a size-dependent effect at the metabolic level, and both sheet sizes of Ti3C2Tx can lead to metabolic disturbances in D. magna by interfering with lipid and amino acid metabolism pathways.

Relationships between functional alpha and beta diversities of flea parasites and their small mammalian hosts.

We studied the relationships between functional alpha and beta diversities of fleas and their small mammalian hosts in 4 biogeographic realms (the Afrotropics, the Nearctic, the Neotropics and the Palearctic), considering 3 components of alpha diversity (functional richness, divergence and regularity). We asked whether (a) flea alpha and beta diversities are driven by host alpha and beta diversities; (b) the variation in the off-host environment affects variation in flea alpha and beta diversities; and (c) the pattern of the relationship between flea and host alpha or beta diversities differs between geographic realms. We analysed alpha diversity using modified phylogenetic generalized least squares and beta diversity using modified phylogenetic generalized dissimilarity modelling. In all realms, flea functional richness and regularity increased with an increase in host functional richness and regularity, respectively, whereas flea functional divergence correlated positively with host functional divergence in the Nearctic only. Environmental effects on the components of flea alpha diversity were found only in the Holarctic realms. Host functional beta diversity was invariantly the best predictor of flea functional beta diversity in all realms, whereas the effects of environmental variables on flea functional beta diversity were much weaker and differed between realms. We conclude that flea functional diversity is mostly driven by host functional diversity, whereas the environmental effects on flea functional diversity vary (a) geographically and (b) between components of functional alpha diversity.

Immune Reactions of Vector Insects to Parasites and Pathogens.

This overview initially describes insect immune reactions and then brings together present knowledge of the interactions of vector insects with their invading parasites and pathogens. It is a way of introducing this Special Issue with subsequent papers presenting the latest details of these interactions in each particular group of vectors. Hopefully, this paper will fill a void in the literature since brief descriptions of vector immunity have now been brought together in one publication and could form a starting point for those interested and new to this important area. Descriptions are given on the immune reactions of mosquitoes, blackflies, sandflies, tsetse flies, lice, fleas and triatomine bugs. Cellular and humoral defences are described separately but emphasis is made on the co-operation of these processes in the completed immune response. The paper also emphasises the need for great care in extracting haemocytes for subsequent study as appreciation of their fragile nature is often overlooked with the non-sterile media, smearing techniques and excessive centrifugation sometimes used. The potential vital role of eicosanoids in the instigation of many of the immune reactions described is also discussed. Finally, the priming of the immune system, mainly in mosquitoes, is considered and one possible mechanism is presented.

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.

Fleas of wild carnivores in Romania: diversity, distribution, and host-associations.

BACKGROUND: Fleas are important hematophagous insects, infesting mammals and birds with a worldwide distribution. Fleas of medical importance have been reported from various carnivores worldwide, such as felids, canids, or mustelids. Romania hosts a wide carnivore diversity, but very little is known about flea species that parasitize these animals in Romania. This study aimed to provide a better understanding of the fleas' diversity and their distribution in a relatively large and diverse number of wild carnivore hosts from Romania. METHODS: From 2013 to 2021, 282 carcasses of wild carnivores from different locations in Romania were collected and examined for the presence of ectoparasites. All collected fleas were morphologically identified using specific keys and descriptions. An analysis of the co-occurrence networks was performed. RESULTS: A total of 11 flea species were identified: Pulex irritans (41.09%), Paraceras melis (20.11%), Ctenocephalides felis (7.33%), Ctenocephalides canis (7.83%), Monopsyllus sciurorum (11.11%), Chaetopsylla trichosa (21.96%), Chaetopsylla homoea (5.5%), Chaetopsylla tuberculaticeps (100%), Chaetopsylla rothschildi (13.33%), Chaetopsylla sp. (14.34%), Chaetopsylla globiceps (5.12%), Echidnophaga gallinacea (10%). The statistical analyses showed a significant difference between the infestation of Martes foina with females being more frequently infected than males (66% versus 33%). Paraceras melis infesting Meles meles had a significantly higher prevalence in female badgers than in males (× 2 = 7.7977, P < 0.01) and higher intensities of infestations in males than in females (t = 1.871, P < 0.05). CONCLUSIONS: This is the first large-scale study investigating the distribution and diversity of flea species infesting wild carnivores in Romania. Three flea species were identified for the first time in Romania (E. gallinacea, C. homoea, and C. tuberculaticeps).

Detection of Rickettsia raoultii in Vermipsylla alakurt-Like Fleas of Sheep in Northwestern China.

INTRODUCTION: To date, a total of 2574 validated flea species have been discovered. Vermipsyllidae is a family of fleas that comprises at least eight species. Vermipsylla is a genus of the family Vermipsyllidae within the order Siphonaptera of fleas. Here a novel Vermipsylla species was described, and rickettsial agent was also detected in it. METHODS: A total of 128 fleas were collected directly from 260 pastured sheep in China. Of these, eight representative fleas (four males and four females) were identified by key morphological features. Meanwhile, 120 flea DNAs, including six flea samples for molecular taxonomy, were subjected to Rickettsia spp. DNA detection. The molecular identity of fleas was determined by amplification and sequenmce analysis of four genetic markers (the 28S rDNA genes, the 18S rDNA genes, the mitochondrial cytochrome c oxidase subunit I and subunit II). In addition, five Rickettsia-specific gene fragments were used to identify the species of the rickettsial agents. The amplified products were sequenced and phylogenetically analyzed. RESULTS: The morphological characteristics of the flea species identified in this study were similar to Vermipsylla alakurt, but presented difference in hair number of the metepimeron, the third tergum, the genitals and the tibiae of hind leg. The 18S rDNA, 28S rDNA and COII genetic markers from fleas showed the highest identity to those of V. alakurt, shared 98.45% (954/969), 95.81% (892/931) and 85.86% (571/665) similarities, respectively. However, the COI sequence showed the highest identity to that of Dorcadia ioffi with 88.48% (576/651) similarity. Rickettsia raoutii tested positive in 14.17% (17/120) flea DNA samples. CONCLUSION: Our study reports the detection of R. raoultii in V. alakurt-like fleas infesting sheep in China.

Essential oil pharmaceuticals for killing ectoparasites on dogs.

BACKGROUND: External parasites, particularly ticks and fleas, are among the most common problems affecting dogs. Chemical medicines are commonly used to prevent and eliminate such external parasites, but their improper use can cause adverse reactions, and the toxins they contain may remain in the environment. OBJECTIVES: The objective of this study was to investigate the in vitro efficacy of Zanthoxylum limonella, citronella, clove, peppermint, and ginger essential oils against dog ticks and fleas and to test the sensitivity of dogs' skin to these essential oils. METHODS: The five essential oils were tested for in vitro efficacy against ticks and fleas, and the two most effective essential oils were then tested on the dogs' skin. RESULTS: The results revealed that these five essential oils at 16% concentrations effectively inhibited the spawning of female engorged ticks. In addition, all five essential oils had a strong ability to kill tick larvae at concentrations of 2% upward. Furthermore, 4% concentrations of the five essential oils quickly eliminated fleas, especially clove oil, which killed 100% of fleas within 1 h. A 50%, 90%, and 99% lethal concentration (LC50, LC90, and LC99) for the essential oils on tick larvae in 24 h were found to be low values. LC50, LC90, and LC99 for the essential oils on flea in 1 h was lowest values. Clove oil at 16% concentration was the most satisfactory essential oil for application on dogs' skin, with a low percentage of adverse effects. CONCLUSIONS: This study confirmed the effectiveness of essential oils for practical use as tick and flea repellents and eliminators. Essential-oil-based pharmaceutical can replace chemical pesticides and provide benefits for both consumers and the environment.