Influence of egg density on larval development and adult body size of cat fleas (Ctenocephalides felis).

Fleas (Siphonaptera) are holometabolous insects with larval and adult stages that exhibit vastly different ecologies from each other. Adult fleas are parasitic and feed exclusively on the blood of a vertebrate host, whereas flea larvae do not live on hosts and consume dried faecal blood from adult fleas. Because flea larvae rely on adult flea faeces for food, excrement and eggs must fall in the same location; thus, larval density is likely high in these restricted habitats. However, the influence of larval density on the subsequent adult stage has not been examined. In the present study, we utilized egg density to investigate density-dependent effects on larval development and adult body size in the cat flea (Ctenocephalides felis Bouché) (Siphonaptera: Pulicidae). Specifically, eggs were collected to create three different larval densities (n = 50, 100 and 150 per 56.7 cm2), and hatched larvae from all groups were fed an excess amount of adult faecal pellets. Larval development was measured by recording the proportion of eggs that developed to the pupal stage and the proportion of eggs that reached adulthood (eclosion). The body size of eclosed adults was quantified for both sexes using head length and length of the total body. We found that the number of eggs had no effect on the proportion of larvae that pupated or the proportion of larvae that eclosed; however, higher egg densities resulted in larger body sizes for both sexes. Overall, these data yield significant insight into how the ecology of larval fleas impacts the biology of the resultant adults.

Immune defense mechanisms against a systemic bacterial infection in the cat flea (Ctenocephalides felis).

A significant amount of work has been devoted towards understanding the cellular and humoral immune responses in arthropod vectors. Although fleas (Siphonaptera) are vectors of numerous bacterial pathogens, few studies have examined how these insects defend themselves from infection. In this study, we investigated the immune defense mechanisms in the hemocoel of cat fleas (Ctenocephalides felis), currently the most important flea pest of humans and many domestic animals. Using model species of bacteria (Micrococcus luteus, Serratia marcescens, and Escherichia coli), we delivered a systemic infection and measured the following: antimicrobial activity of hemolymph, levels of free radicals resulting from the induction of oxidase-based pathways, number of circulating hemocytes, phagocytosis activity of circulating hemocytes, and in vivo bacteria killing efficiency when phagocytosis activity is limited. Our results show that the antimicrobial activity of flea hemolymph increases in response to certain species of bacteria; yet, a systemic infection with the same bacterial species did not influence levels of hydrogen peroxide (H2O2), a reactive intermediate of oxygen, at the same time. Additionally, the number of circulating hemocytes increases in response to E. coli infection, and these cells display strong phagocytic activity against this bacterium. Moreover, limiting phagocytosis by injecting polystyrene beads subsequently increases flea susceptibility to E. coli infection when compared to injury controls; however, impairing the cellular immune response itself did not increase flea susceptibility to infection when compared to untreated fleas. Overall, this work yields significant insight into how fleas interact with bacterial pathogens in their hemocoel, and suggests that cellular and humoral immune responses cooperate to combat systemic bacterial infections.

Detection of Bartonella spp. and Rickettsia spp. in cat fleas (Ctenocephalides felis) collected from free-roaming domestic cats in southeastern Georgia, USA.

The cat flea (Ctenocephalides felis) is a competent vector of numerous bacterial pathogens in the genera Bartonella and Rickettsia. In the United States, free-roaming domestic cats (Felis catus) increase the risk of exposure to C. felis for humans and their companion animals. In collaboration with a trap-neuter-return program, we collected fleas from 283 feral/stray cats in southeastern Georgia between May and July of 2020. A total of 3,643 flea specimens were collected, and C. felis was the only flea species recovered from all cats sampled. The mean number of fleas per cat host was highest in the month of June when compared to May and July, and higher in juvenile cats (< 1 year) than the adults (≥ 1 year). Real-time PCR assays were used to test a subset of the collected fleas (n = 468) for the presence of Bartonella spp. and Rickettsia spp. DNA. Among those flea pools tested, 35.2% were positive for genus-specific citrate synthase gene of Bartonella, 16.5% were positive for the genus-specific 17-kDa protein antigen gene of Rickettsia, and none were positive for the species-specific outer membrane protein B gene of Rickettsia typhi. The identification of potential flea-borne pathogens was more frequent from the subset of C. felis collected in May, and female cats had more Bartonella-positive fleas and less Rickettsia-positive fleas than male cats. Overall, the present study provides valuable insights into the frequency of C. felis from outdoor community cats in southeastern Georgia, and highlights the possible risk for human exposure to potential flea-borne pathogens.

Reactive oxygen species-mediated immunity against bacterial infection in the gut of cat fleas (Ctenocephalides felis).

Fleas (Order Siphonaptera) transmit numerous bacterial pathogens that cause severe human diseases (e.g., cat scratch disease, flea-borne spotted fever, murine typhus, plague). Because initial entry of these infectious agents occurs while blood feeding, the immune response in the flea gut is considered to be the first line of defense against invading microbes. However, relatively few studies have identified the flea immune molecules that effectively resist or limit infection in the gut. In other hematophagous insects, an immediate immune response to imbibed pathogens is the generation of reactive oxygen species (ROS). In this study, we utilized cat fleas (Ctenocephalides felis) to investigate whether oral infection with a well-known insect bacterial pathogen (Serratia marcescens) induces ROS synthesis in the flea gut, and whether production of ROS provides a defense mechanism against microbial colonization. Specifically, we treated fleas with an antioxidant to limit the number of free radicals in the digestive tract prior to infection, and then measured the following: S. marcescens infection loads, hydrogen peroxide (ROS) levels, and mRNA abundance of ROS signaling pathway genes. Overall, our data shows that ROS levels increase in response to infection in the flea gut, and that this increase helps to strengthen the flea immune response through the microbicidal activity of ROS.

Fleas (Siphonaptera) Parasitizing Peridomestic and Indigenous Mammals in Panamá and Screening of Selected Fleas for Vector-Borne Bacterial Pathogens.

In total, 341 fleas belonging to 16 species were collected from 78 host mammals belonging to 10 species in Panamá from 2010 to 2016. The cat flea, Ctenocephalides felis (Bouché) predominated on domestic dogs and was also recorded from domestic cats, the raccoon, Procyon lotor (Linnaeus) and the common opossum, Didelphis marsupialis Linnaeus. The largest number of flea species (7) was recorded from D. marsupialis and the most common flea on that host was the ctenophthalmid, Adoratopsylla intermedia copha Jordan. One Oriental rat flea, Xenopsylla cheopis (Rothschild), was collected from D. marsupialis. Native rodents were parasitized by indigenous ceratophyllid, rhopalopsyllid, and stephanocircid fleas. The Mexican deermouse, Peromyscus mexicanus (Saussure), was parasitized by six species of ceratophyllids belonging to the mostly Central American genera, Baculomeris, Jellisonia, Kohlsia and Plusaetis. The long-tailed singing mouse, Scotinomys xerampelinus (Bangs), was parasitized by Plocopsylla scotinomi Tipton and Méndez, the only species of stephanocircid flea known from Central America. Twenty-six pools of extracted flea DNA representing 5 flea species (C. felis, Pulex echidnophagoides (Wagner), Pulex simulans Baker, A. intermedia copha, and P. scotinomi) and 79 individual fleas were all real-time polymerase chain reaction negative for Rickettsia felis, Rickettsia typhi, and Bartonella henselae.

Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection.

Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes-such as cecropin A, Lysozyme C1, Stat-A, and Tep1-is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation-possibly via transstadial immune priming-but that it confers both costs and benefits to the emerged adults.

Immunity of fleas (Order Siphonaptera).

The immune response of arthropod vectors plays a key role in the spread and transmission of vector-borne diseases. Although fleas transmit several human pathogens (e.g., Bartonella henselae, Rickettsia felis, R. typhi, and Yersinia pestis), few studies have examined how these vectors respond to infection. In hematophagous arthropods, imbibed pathogens must survive the hostile environment of blood meal digestion, which includes proteolytic digestive enzymes, protease inhibitors and expression of genes associated with protection of epithelial linings. Additionally, insect epithelial cells exhibit local immune defense against ingested pathogens by producing antimicrobial peptides and reactive oxygen species. This review details these and other aspects of insect immunity as it relates to fleas, with an emphasis on the gut immune response to two blood-borne pathogens, R. typhi and Y. pestis.

Transstadial transmission of larval hemocoelic infection negatively affects development and adult female longevity in the mosquito Anopheles gambiae.

During all life stages, mosquitoes are exposed to pathogens, and employ an immune system to resist or limit infection. Although much attention has been paid to how adult mosquitoes fight infection, little is known about how an infection during the larval stage affects the biology of the resultant adult. In this study, we investigated whether a bacterial infection in the hemocoel of the African malaria mosquito, Anopheles gambiae, is transstadially transmitted from larvae to adults (both females and males), and whether immune stimulation in the hemocoel as a larva alters development or biological traits of the adult. Specifically, larvae were injected in the hemocoel with either fluorescent microspheres or Escherichia coli, and the following traits were examined: transstadial transmission, larval development to adulthood, adult survival, and adult body size. Our results show that transstadial transmission of hemocoel contents occurs from larvae to pupae and from pupae to adults, but that bacterial prevalence and intensity varies with age. Injury, immune stimulation or infection decreases the proportion of larvae that undergo pupation and eclosion, infection decreases the longevity of adult females, and treatment has complex effects on the body size of the resultant adults. The present study adds larval hemocoelic infection to the known non-genetic factors that reduce overall fitness by negatively affecting development and adult biological traits that influence mosquito vector competence.

Effect of Rickettsia felis Strain Variation on Infection, Transmission, and Fitness in the Cat Flea (Siphonaptera: Pulicidae).

Rickettsia felis is a human pathogen transmitted by the cat flea, Ctenocephalides felis (Bouché) (str. LSU), as well as an obligate symbiont of the parthenogenic booklouse Liposcelis bostrychophila (Badonnel) (str. LSU-Lb). The influence of genetic variability in these two strains of R. felis on host specialization and fitness and possible resulting differences on infection and transmission kinetics in C. felis is unknown. Utilizing an artificial host system, cat fleas were exposed to a R. felis str. LSU-Lb-infected bloodmeal and monitored for infection at 7-d intervals for 28 d. Quantitative real-time PCR was used to determine rickettsial load and infection density in newly exposed cat fleas, and transmission frequency between cat fleas. The effect of persistent R. felis infection on cat flea F1 progeny was also assessed. At 7 d postexposure 76.7% of the cat fleas successfully acquired R. felis str. LSU-Lb. In R. felis str. LSU-Lb-exposed cat fleas, the mean infection load (6.15 × 106), infection density (0.76), and infection prevalence (91/114) were significantly greater than R. felis str. LSU infection load (3.09 × 106), infection density (0.68), and infection prevalence (76/113). A persistent R. felis str. LSU-Lb infection was detected for 28 d in adult cat fleas but neither female:male ratio distortion nor vertical transmission was observed in F1 progeny. While infection kinetics differed, with higher intensity associated with R. felis str. LSU-Lb, no distinct phenotype was observed in the F1 progeny.

Rickettsia felis, an Emerging Flea-Borne Rickettsiosis.

Rickettsia felis is an emerging insect-borne rickettsial pathogen and the causative agent of flea-borne spotted fever. First described as a human pathogen from the USA in 1991, R. felis is now identified throughout the world and considered a common cause of fever in Africa. The cosmopolitan distribution of this pathogen is credited to the equally widespread occurrence of cat fleas (Ctenocephalides felis), the primary vector and reservoir of R. felis. Although R. felis is a relatively new member of the pathogenic Rickettsia, limited knowledge of basic R. felis biology continues to hinder research progression of this unique bacterium. This is a comprehensive review examining what is known and unknown relative to R. felis transmission biology, epidemiology of the disease, and genetics, with an insight into areas of needed investigation.

Transmission mechanisms of an emerging insect-borne rickettsial pathogen.

BACKGROUND: Vector-borne pathogens must overcome arthropod infection and escape barriers (e.g. midgut and salivary glands) during the extrinsic incubation period (EIP) before subsequent transmission to another host. This particular timespan is undetermined for the etiological agent of flea-borne spotted fever (Rickettsia felis). Artificial acquisition of R. felis by blood-feeding cat fleas revealed dissemination to the salivary glands after seven days; however, this length of time is inconsistent with co-feeding studies that produced infectious cat fleas within 24 h of infection. In the current study, we demonstrated that an alternative mechanism is responsible for the early-phase transmission that typifies flea-borne R. felis spread. METHODS: Co-feeding transmission bioassays were constructed to assess temporal dynamics of R. felis amongst cat fleas, including exposure time to produce infectious fleas and association time to transmit infection to naïve fleas. Additional experiments examined the proportion of R. felis-exposed cat fleas with contaminated mouthparts, as well as the likelihood for cat fleas to release R. felis from their mouthparts following exposure to an infectious bloodmeal. The potential for mechanical transmission of R. felis by co-feeding cat fleas was further examined using fluorescent latex beads, as opposed to a live pathogen, which would not require a biological mechanism to achieve transmission. RESULTS: Analyses revealed that R. felis-infected cat fleas were infectious to naïve fleas less than 24 h after exposure to the pathogen, but showed no rickettsial dissemination to the salivary glands during this early-phase transmission. Additionally, the current study revealed that R. felis-infected cat fleas must co-feed with naïve fleas for more than 12 h in order for early-phase transmission to occur. Further evidence supported that contaminated flea mouthparts may be the source of the bacteria transmitted early, and demonstrated that R. felis is released from the mouthparts during brief probing events. Moreover, the use of fluorescent latex beads supports the notion that early-phase transmission of R. felis is a mechanical mechanism. CONCLUSIONS: Determination of the transmission mechanisms utilized by R. felis is essential to fully understand the vulnerability of susceptible vertebrate hosts, including humans, to this pathogen.

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.

Oil sands naphthenic acids: a review of properties, measurement, and treatment.

The Alberta oil sands contain one of the world's largest reserves of oil - over 169 billion barrels of bitumen are economically recoverable with current extraction technologies. Surface mining and subsequent hot water extraction of bitumen from the ore generates about nine cubic meters of raw tailings per cubic meter of oil. Oil sands facilities are required to operate under a policy of zero water discharge, resulting in ponds containing more than one billion cubic meters of tailings, a mixture of sand, fines and process-affected water. Process-affected water contains numerous organic compounds, including naphthenic acids (NAs), which have been identified as the primary source of acute toxicity of process-affected water. Developments in analytical techniques, aerobic biodegradability, and treatment via chemical oxidation (ozone) of NAs are reviewed. The field continues to be challenged by the lack of a cost-effective, accurate analytical technique for NAs or an understanding of all the organic constituents in process-affected water that may be contributing to observed toxicity and thus requiring treatment.

Indigenous microbes survive in situ ozonation improving biodegradation of dissolved organic matter in aged oil sands process-affected waters.

The oil sands industry faces significant challenges in developing effective remediation technologies for process-affected water stored in tailings ponds. Naphthenic acids, a complex mixture of cycloaliphatic carboxylic acids, have been of particular concern because they concentrate in tailings ponds and are a component of the acutely toxic fraction of process water. Ozone treatment has been demonstrated as an effective means of rapidly degrading naphthenic acids, reducing process water toxicity, and increasing its biodegradability following seeding with the endogenous process water bacteria. This study is the first to examine subsequent in situ biodegradation following ozone pretreatment. Two aged oil sands process-affected waters from experimental reclamation tailings ponds were ozonated to reduce the dissolved organic carbon, to which naphthenic acids contributed minimally (<1mgL(-1)). Treatment with an ozone dose of 50mgL(-1) improved the 84d biodegradability of remaining dissolved organic carbon during subsequent aerobic incubation (11-13mgL(-1) removed from aged process-affected waters versus 5mgL(-1) when not pretreated with ozone). The ozone-treated indigenous microbial communities were as capable of degrading organic matter as the same community not exposed to ozone. This supports ozonation coupled with biodegradation as an effective and feasible treatment option.

Turtle ectoparasites from the Pacific coastal region of Colombia / Ectoparasitos asociados a tortugas en la costa pacifica de Colombia

This study provides an update on the ectoparasites (ticks and leeches) associated with Rhinoclemmys annulata and provides new accounts on the ectoparasites associated with R. nasuta, R. melanosterna and Kinosternon leucostomum from the Pacific coast of Colombia. The presence of Amblyomma sabanerae on R. nasuta and R. melanosterna provided two new host records for the tick species. Also, the documentation of A. sabanerae from the Department of Valle del Cauca represents a new department record for the species in Colombia. Placobdella ringueleti was identified from R. nasuta and K. leucostomum, which represents a new host record for the leech species, as well as a significant extension of the known range.

Este estudio proporciona nueva información sobre ectoparásitos (garrapatas y sanguijuelas) encontrados en Rhinoclemmys annulata, y aporta nuevos registros sobre ectoparásitos asociados a R. nasuta, R. melanosterna y Kinosternon leucostomum en la costa pacífica de Colombia. La presencia de Amblyomma sabanerae en R. nasuta y R. melanosterna provee dos nuevos hospederos para esta especie de garrapata. Igualmente el reporte de A. sabanerae amplía el registro de esta especie en Colombia, al departamento del Valle del Cauca. R. nasuta y K. leucostomum, se constituyen en nuevos registros de hospederos para Placobdella ringueleti, e incrementa de manera significativa el rango de distribución geográfico conocido para esta especie de sanguijuela.

Roles of the calcineurin and CaMK signaling pathways in fast-to-slow fiber type transformation of cultured adult mouse skeletal muscle fibers.

Two Ca2+-dependent signaling pathways, mediated by the Ca2+-activated phosphatase calcineurin and by the Ca2+-activated kinase Ca2+/calmodulin-dependent kinase (CaMK), are both believed to function in fast-to-slow skeletal muscle fiber type transformation, but questions about the relative importance of the two pathways still remain. Here, the differential gene expression during fast-to-slow fiber type transformation was studied using cultured adult flexor digitorum brevis (FDB) fibers and a custom minimicroarray system containing 21 fiber type-specific marker genes. After 3 days of culture, unstimulated fibers showed a generally slower gene expression profile; 3 days of electric field stimulation of cultured FDB fibers with a slow fiber-type pattern transformed the fibers to an even slower gene expression profile. Unstimulated FDB fibers overexpressing constitutively active calcineurin featured a slower gene expression profile, except four genes, indicating that transformation occurred, but was incomplete with activation of the calcineurin pathway alone. In both unstimulated FDB fibers and slow-type electrically stimulated FDB fibers, blocking of CaMK pathway with KN93 generated a faster gene expression profile compared with the negative control KN92, indicating that CaMK pathway functions during the transformation induced by both unstimulated culturing and slow fiber-type electrical stimulation. Moreover, neither the calcineurin nor the CaMK pathway alone could maximally activate the transformation, and coordination of the two pathways is required to accomplish a complete fast-to-slow fiber type transformation.

Ca2+ sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers.

Ca(+) sparks are rare in healthy adult mammalian skeletal muscle but may appear when adult fiber integrity is compromised, and occur in embryonic muscle but decline as the animal develops. Here we used cultured adult mouse flexor digitorum brevis muscle fibers to monitor occurrence of Ca(2+) sparks during maintenance of adult fiber morphology and during eventual fiber morphological dedifferentiation after various times in culture. Fibers cultured for up to 3 days retain normal morphology and striated appearance. Ca(2+) sparks were rare in these fibers. At 5-7 days in culture, many of the original muscle fibers exhibit sprouting and loss of striations, as well as the occurrence of spontaneous Ca(2+) sparks. The average rate of occurrence of Ca(2+) sparks is >10-fold higher after 5-7 days in culture than in days 1-3. With the use of fibers cultured for 7 days, application of the Ca(2+) channel blockers Co(2+) or nifedipine almost completely suppressed the occurrence of Ca(2+) sparks, as previously shown in embryonic fibers, suggesting that Ca(2+) sparks may be generated by similar mechanisms in dedifferentiating cultured adult fibers and in embryonic fibers before final differentiation. The sarcomeric disruption observed under transmitted light microscopy in dedifferentiating fibers was accompanied by morphological changes in the transverse (T) tubular system, as observed by fluorescence confocal imaging of both an extracellular marker dye and membrane staining dyes. Changes in T tubule morphology coincided with the appearance of Ca(2+) sparks, suggesting that Ca(2+) sparks may either be a signal for, or the result of, disruption of DHPR-ryanodine receptor 1 coupling.