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The chemical composition of tick cuticles acts as a barrier to pathogens and may limit infection by entomopathogenic fungi. This study characterized the cuticular neutral lipids (NL) and hydrocarbons (HCs) of four ixodid ticks that are widely distributed in Brazil. HC extracts were analyzed by gas chromatography-mass spectrometry and used to challenge Beauveria bassiana IP361 and Metarhizium robertsii IP146; the effect of cuticular extracts in fungal growth were evaluated by disk diffusion and conidial viability assays. In addition, conidial germination on the tick cuticle was evaluated by scanning electron microscopy, and NL from ticks treated with fungi were assessed by thin layer chromatography. Six HCs were exclusively identified in Amblyomma sculptum . Additionally, cuticle extracts from Dermacentor nitens and A. sculptum inhibited the growth of M. robertsii IP146 and reduced conidial germination of B. bassiana IP361 to 70% and 49%, respectively; the same extracts also produced cytotoxic effects, with conidial death above 30% and 60%. Electron micrographs showed a delayed germination of conidia incubated for 48 h or 72 h on D. nitens and A. sculptum . The lipid profile of A. sculptum treated with fungi was not significantly altered; triacylglycerol was not detected in the cuticle extracts of any other tick species. Finally, A. sculptum and D. nitens cuticles have lipid components that may limit the development of M. robertsii.

Due to the fundamental role that sand flies (Diptera: Psychodidae) have in Leishmania spp. (Kinetoplastida: Trypanosomatidae) transmission, the objective of this study was to evaluate the ecology and spatial distribution of species circulating in the municipality of Seropédica, Rio de Janeiro, Brazil. A total of 17 371 specimens were collected between August 2016 and July 2017, and five species were identified: Nyssomyia intermedia (Lutz & Neiva, 1912), Migonemyia migonei (França, 1920), Nyssomyia whitmani (Antunes & Coutinho, 1939), Pintomyia pessoai (Coutinho & Barretto, 1940), and Pintomyia fischeri (Pinto, 1926). In relation to the number of sand flies collected, we observed a significant difference between rural and periurban areas and between the neighbourhoods. We observed that N. intermedia has a relatively greater adaptation to the peridomiciliary environment. This is the first report of Pi. pessoai and Pi. fischeri (only females) in Seropédica as well as the first record of the frequency of phlebotomine species focusing on some ecological indices and the spatial distribution of sand flies. The results presented here aimed to provide an insight into the spatial population dynamics of phlebotomine species in low endemic areas of American Tegumentary Leishmaniasis (ATL) in Seropédica, highlighting important areas for entomological surveillance., (© 2021 The Royal Entomological Society.)

This cross-sectional study aims to investigate the epidemiology and spatial distribution of hemotropic Mycoplasma spp. and Mycoplasma haemocanis in dogs from Rio de Janeiro, Brazil. Blood samples were collected at random from 437 household dogs. An epidemiological questionnaire was completed concerning the host characteristics as well as the environments in which they lived. A positivity frequency of 17.84% (78/437) was found for Mycoplasma spp. and 2% (9/437) for M. haemocanis in Rio de Janeiro, Brazil, through molecular detection based on the 16S rRNA sequence. According to the present study, dogs that live in households with the presence of rodents (odds ratio [OR] = 9.93; p-value = 0.02; confidence interval [CI]: 1.34-73.66) and wild animals (OR = 1.91; p-value = 0.03; CI: 1.06-3.42) are more likely to be infected with Mycoplasma spp.. Also, dogs with tick infestation (OR = 6.47; p-value = 0.007; CI: 1.63-25.60) have more chances to become infected with M. haemocanis. The spatial analysis disclosed a positive correlation between the Mycoplasma presence and tick infestation (global Moran index = 0.82; pseudo-p-value =0.001). The epidemiological findings support the hypothesis of Rhipicephalus sanguineus s.l. as the vector of M. haemocanis in the studied region and provide insightful information to prevent the Mycoplasma spp. infection in dogs from Rio de Janeiro., (Copyright © 2020. Published by Elsevier B.V.)

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals' leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick's immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female's body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gené's organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes' disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Despite the importance of fat body in metabolism of arthropods, studies in ticks are scarce. This study evaluated the lipid composition and activation of extracellular signal-regulated protein kinase (ERK) and AMP-activated protein kinase (AMPK) enzymes in Rhipicephalus microplus fat body after infection with different isolates of the fungus Metarhizium anisopliae sensu lato (Metschnikoff, 1879) Sorokin, 1883. The isolates CG 32, GC 112, GC 148, GC 347, and GC 629 were inoculated as viable or non-viable conidia in the ticks. The engorged females were dissected, and their fat bodies were collected 24 and 48 h after infection. The lipid composition was assessed by thin layer chromatography, and enzyme activation was detected by Western blotting with antibodies against p-AMPK and p-ERK. The study showed increased levels of triacylglycerol 24 and 48 h and fatty acid after 48 h after inoculation with different isolates of viable fungi in the tick's hemocoel. Detection of the active form of ERK was demonstrated only after inoculation with non-viable conidia of all isolates tested. The active form of AMPK, only isolate CG 112 was able to activate with viable or non-viable conidia, whereas isolates CG 32 and CG 629 were able to activate with non-viable conidia. This study provides the first report about changes in important metabolic pathways in ticks infected with entomopathogenic fungi and suggests that the lipid content is modulated by non-usual pathways. However, further studies may be necessary for a better elucidation of this interaction.

Metarril SP Organic is a product based on the fungus Metarhizium anisopliae, which was developed for controlling agricultural pests. The present study evaluated the effect of Metarril SP Organic plus 10% mineral oil, for controlling Rhipicephalus microplus under field conditions. Three groups were formed: Control group, which received no treatment; Oil control group, which was bathed only with water, Tween 80 and mineral oil; and Metarril group, bathed in the oil-based formulation Metarril SP Organic. Two treatments per group were performed and to verify the effect of the treatments, all R. microplus ticks between 4.5 and 8.0mm in length on the left side of the cattle were counted on days +7, +14 and +21 after each treatment, and a sample of engorged females was collected for evaluation of biological parameters. The Metarril SP Organic oil formulation showed efficacy ranging from 8.53 to 90.53%. The average efficacy of the oil-based formulation of Metarril SP Organic was 75.09 and 46.59% compared with the groups Control and Oil control, respectively. There were no significant changes in biological parameters of engorged R. microplus females collected from animals. Although there was no significant difference in the amount of ticks between the Oil control and Metarril groups, it is believed that the association of mineral oil with Metarril SP Organic product is effective in R. microplus tick control in field. Thus, this association has potential to be used in strategic control programs of cattle tick., (Copyright © 2016. Published by Elsevier B.V.)

Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Pr1 is a subtilisin-like protease produced by Metarhizium spp. entomopathogenic fungi, and it is recognized as heavily involved in the initial steps of the fungal invasion of arthropod-host cuticles. In the current study, correlation was sought between mortality of tick larvae and conidial Pr1 levels of one Metarhizium anisopliae senso latu (s.l.) isolate (CG 148). Conidia with different levels of pr1 gene expression and enzymatic activity were obtained by producing them on either artificial medium (to yield low Pr1 activity) or on Rhipicephalus microplus cadavers (to yield high Pr1 activity). Conidial proteolytic activity was assessed using N-suc-ala-ala-pro-phe-ρNA as the chromogenic substrate, and pr1 expression was profiled by qPCR using three genes (gpd, try, and tef) as reference genes. Pr1 enzymatic (proteolytic) activity on conidia obtained from tick cadavers was 36 U mg(-1) in comparison to 4 U mg(-1) on conidia from PDA medium. Also, pr1 gene expression level was ten times higher in conidia from tick cadavers compared to PDA medium. Bioassays of M. anisopliae s.l. CG 148 spores with elevated Pr1 proteolytic activity and gene expression levels did not demonstrate increased virulence (= significant change percent mortality of tick larvae). The minimal levels of Pr1 on conidia produced on artificial medium was adequate to afford high levels of virulence, and the elevated amounts of the enzyme on tick-cadaver-produced conidia did not induce elevated larval mortality. As long as some Pr1 activity was present, fungal virulence of isolate CG 148 against tick larvae was not elevated by increased levels of conidial Pr1.