Ultrastructural and Cytotoxic Effects of Metarhizium robertsii Infection on Rhipicephalus microplus Hemocytes

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 108 conidia mL−1). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control

Cuticular Lipids as a First Barrier Defending Ixodid Ticks against Fungal Infection

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

Alterations in the oxidative metabolism of Rhipicephalus (Boophilus) microplus ticks in response to exposure to the insect growth regulator fluazuron

Abstract Aiming to characterize the potential off-target effects of fluazuron on ticks, biochemical analyses were conducted to evaluate changes in the carbohydrate metabolism of Rhipicephalus (Boophilus) microplus ticks after exposure to fluazuron. Hemolymph and fat body were collected from female ticks before and after (4, 8 and 15 days) exposure to fluazuron. Spectrophotometric analyses were done to quantify glucose concentration and lactate dehydrogenase (LDH) activity in the hemolymph and the concentration of glycogen in the tick’s fat body. High Performance Liquid Chromatography (HPLC) was employed to determine the concentration of carboxylic acids in the hemolymph and to evaluate changes in intermediary metabolic processes requiring oxygen consumption. Increases in the levels of LDH activity and lactic acid concentration indicated that fluazuron enhanced fermentative metabolism in ticks. Exposure to fluazuron was also found to increase glucose concentrations in the hemolymph over time, although no significant differences were noted daily. In addition to expanding the body of knowledge about the mode of action of fluazuron, investigations into these mechanisms may also be useful in discovering new and as yet unexplored secondary effects

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity

Abstract Background Mosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated. Methods Larvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR. Results Propagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF. Conclusion By 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae. Graphical Abstrac

Asssociação de Metarhizium anisopliae sensu lato e cipermetrina para controle de Rhipicephalus microplus

Rhipicephalus microplus é responsável por grandes perdas econômicas na pecuária brasileira. Com o intuito de controlar esse artrópode, o uso indiscriminado de produtos químicos é amplamente empregado, acarretando em prejuízos ao meio ambiente, aos animais e humanos, além de contribuir para o aparecimento de cepas de carrapatos resistentes. Assim, o emprego de produtos químicos associados a entomopatógenos surge como uma alternativa para aumentar a eficácia do controle de carrapato e reduzir os danos causados pelo uso inapropriado de produtos químicos. Desta forma, o objetivo do presente estudo foi de avaliar a eficiência da associação de Metarhizium anisopliae e cipermetrina sobre estágios imaturos de R. microplus. O tratamento dos grupos foi realizado através da imersão de ovos e larvas em 1mL de suspensão aquosa do fungo, solução de cipermetrina, suspenção fúngica associada à cipermetrina e um grupo controle. Os parâmetros biológicos avaliados foram o percentual de eclosão e o percentual de mortalidade das larvas. Observou se que o fungo quando utilizado sozinho em menor concentração (106 conídios mL-1) ou a cipermetrina também em menor concentração (25 ppm) não foram capazes de alterar os parâmetros biológicos quando comparados ao grupo controle. No entanto, a associação do fungo a 106 conídios mL-1 com cipermetrina em menor concentração, foi capaz de reduzir o percentual de eclosão pela metade e causar o dobro de mortalidade quando comparado ao uso do fungo e cipermetrina isoladamente. Desta forma, conclui se que a associação de M. anisopliae com cipermetrina é eficaz no controle de estágios imaturos de R. microplus

Oil-based formulations containing arthropodpathogenic fungi to control Rhipicephalus microplus

ABSTRACT. Angelo I.C., Diniz-Neto H.C., Santos S.V., Perinotto W.M.S., Quinelato S., Santos H.A., Gôlo P.S., Camargo M.G., Marciano A.F. & Bittencourt V.R.E.P. [Oil-based formulations containing arthropodpathogenic fungi to control Rhipicephalus microplus.] Formulações oleosas contendo fungos artropodopatogênicos para o controle de Rhipicephalus microplus. Revista Brasileira de Medicina Veterinária, 37(Supl.1):18-24, 2015. Departamento de Epidemiologia e Saúde Pública, Universidade Federal Rural do Estado do Rio de Janeiro, BR 465, Km 7, Seropédica, RJ 23897-970, Brasil. E-mail: [email protected] Rhipicephalus microplus ticks are responsible for economical losses estimated in U$ 3.24 billions per year in Brazil. Arthropodpathogenic fungi are an important alternative to the chemical control of this arthropod; however to be efficient in the field, formulations must be developed to protect conidia against environmental stresses. The present study compared the efficacy of mineral or vegetal oil-based formulations containing Metarhizium anisopliae s.l. or Beauveria bassiana s.l. as well as their association to control R. microplus engorged females. M. anisopliae and B. bassiana isolates were cultivated in Potato Dextrose Agar medium and maintained under optimal conditions. Single fungus oil-based formulations were prepared with the following proportions: 84% aqueous fungal suspension; 15% sterile oil; 1% Tween 80. Associated fungal formulations were prepared as follows: 42% aqueous M. anisopliae suspension; 42% aqueous B. bassiana suspension; 15% sterile oil and 1% Tween. Engorged females were immersed in 1 mL oil formulations. The following biological parameters were evaluated: initial weigh of engorged female; weight of egg mass; egg production index; nutritional index and percentage of tick control. There was no difference in the percentage of tick control when M. anisopliae s.l. and/or B. bassiana s.l. were tested in mineral oil. Although, when vegetable oil-based formulations were tested, only M. anisopliae s.l. formulations (both single and associated) had satisfactory control percentage (41.97% and 40. 13%) while single fungus B. bassiana s.l. formulation yield a very low percentage of tick control (10.88%). Accordingly, the use of different oils (mineral or vegetable oils) may affect in different ways the arthropodpathogenic fungi efficacy to control R. microplus. Therefore, different oils should be tested in formulations to control ticks, as well as their interactions with different fungal isolates what will contribute to the development of a promising alternative to control this important parasite

Association between Metarhizium anisopliae sensu lato and cypermethrin to control Rhipicephalus microplus

ABSTRACT. de Paulo J.F., Ferreira J.R.T., Marciano A.F., de Freitas, M.C. Coutinho-Rodrigues C.J.B., Camargo M.G., Angelo I.daC. Bittencourt V.R.E.P., Gôlo P.S. [Association between Metarhizium anisopliae sensu lato and cypermethrin to control Rhipicephalus microplus.] Associação de Metarhizium anisopliae sensu lato e cipermetrina para o controle de Rhipicephalus microplus. Revista Brasileira de Medicina Veterinária, 38(supl. 3):66-74, 2016. Departamento de Parasitologia Animal, Anexo 1, Instituto de Veterinária, Universidade Federal Rural Rio de Janeiro, BR 465 Km 47, Seropédica, RJ 23890-000, Brasil. E-mail: [email protected] Rhipicephalus microplus is responsible for large economic losses in Brazilian livestock. In order to control this arthropod, the indiscriminate use of chemical products may be widely used, leading to damages to the environment, animals and humans, besides contributing to the appearance of resistant tick populations. The use of chemicals associated with biological entomopathogens drives an alternative to increase the effectiveness of tick control and reduce de damage caused by the inappropriate use of chemicals. Accordingly, the aim of the present study was to evaluate the efficiency of Metarhizium anisopliae and cypermethrin association on immature stages of R. microplus ticks. The groups were exposed by immersing the egg mass and larvae in 1 ml of fungal aqueous suspension, cypermethrin solution, cypermethrin associated to the fungal suspension and Tween 80 0.1% (v/v) (control). The biological parameters evaluated were: larval hatching percentage and larval mortality percentage. It was observed that both the fungus in a low concentration (106 conidia mL-1) or cypermethrin in a low concentration (25 ppm) were not able to change the biological parameters in comparison to the control group. Despite this, when associated the fungus at 106 conidia mL-1 with cypermethrin in a low concentration, together they were able to reduce larval hatching percentage and increase mortality in comparison to the single use of fungus or cypermethrin. Accordingly, it is concluded that the association of M. anisopliae with cypermethrin is a promising alternative to control of immature stages of R. microplus

Metarhizium anisopliae: influence of pH on enzyme activity and control of Rhipicephalus microplus ticks

ABSTRACT. Marciano A.F., Coutinho-Rodrigues C.J.B., Perinotto W.M.S., Camargo M.G., Gôlo P.S., Sá F.A., Quinelato S., Freitas M.C., Angelo I.C., Nogueira M.R.S. & Bittencourt V.R.E.P. [Metarhizium anisopliae: influence of pH on enzyme activity and control of Rhipicephalus microplus ticks.] Metarhizium anisopliae: influência do pH na atividade enzimática e no controle de Rhipicephalus microplus. Revista Brasileira de Medicina Veterinária, 37(Supl.1):85-90, 2015. Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 47, Seropédica, RJ 23897-970, Brasil. E-mail: [email protected] Rhipicephalus microplus ticks are one of the major agents causing substantial losses to livestock worldwide. In the search for alternative control strategies, both in vitro and in vivo use of the arthropodpathogenic fungus Metarhizium anisopliae has shown promising results against this ectoparasite. During host colonization, protease production by M. anisopliae is considered one important virulence factor once it is directly related to the active penetration process carried by the fungus on the full host cuticle. Nevertheless, limitations as environmental pH may modulate the proteases production and/or activity, as well as, the fungal virulence. The current study aimed evaluate the virulence and total protease activity of M. anisopliae CG 148 sensu lato (s.l.). Fungal aqueous suspensions or 5% mineral oil formulations were used in different pH ranges (5, 7, or 9). Suspensions and formulations were prepared using a pH meter and adjusted to 108 spores mL-1. In the bioassay, four groups were formed for each pH range: the aqueous fungal suspension, the oil-based fungal formulation and their respective controls (aqueous and oil-based), totaling 12 groups. Engorged females were immersed for 3 minutes and maintained under optimal conditions for evaluation of biological parameters. Total protease activity of the artificial medium (after filtration of mycelia) was assessed by azocasein hydrolysis at 72 hours of incubation in minimal medium supplemented with 1% R. microplus cuticle. Oil-based formulations did not yield significant alterations in the fungal virulence or enzyme activity. Nevertheless, the alkaline pH at aqueous suspensions reduced total protease activity and negatively influenced fungal virulence, reducing 1.5 times tick control percent. Based on these results, it is suggested that excessive alkalinity in strictly aqueous media may potentially interfere in the in vitro arthropodpathogenic fungal virulence

EFICIÊNCIA in vitro DE UMA FORMULAÇÃO OLEOSA DE Metarhizium anisopliae SENSU LATO NO CONTROLE DE Rhipicephalus microplus

Rhipicephalus microplus é um ectoparasito responsável por grandes perdas econômicas na pecuária bovina. Na tentativa de diminuir o emprego de produtos químicos e os problemas por eles acarretados, a elaboração de formulações de entomopatógenos vem se destacando nas pesquisas de formas alternativas no combate deste carrapato. Assim o objetivo deste trabalho foi avaliar a eficácia de formulações oleosas de um produto comercial de M. anisopliae (Metarril WP Organic®) em diferentes concentrações de óleo mineral, sobre ovos, larvas e fêmeas ingurgitadas do carrapato R. microplus. Para realização deste estudo, foram formados oito grupos: um grupo controle aquoso, tratado apenas com água destilada estéril e Tween 80 a 0,1%; um grupo tratado com a suspensão aquosa de Metarril WP Organic®; três grupos controle contendo 1, 3 ou 5% de óleo mineral, e três grupos tratados com as formulações de Metarril WP Organic® contendo 1, 3 ou 5% de óleo mineral. As formulações possuíam a concentração de 108 conídios/mL. O tratamento consistiu na imersão dos espécimes em um ml da suspensão testada durante três minutos. Os parâmetros biológicos das fêmeas ingurgitadas foram significativamente alterados pelas formulações oleosas de Metarril WP Organic®, sendo observado um percentual de controle de 97,35% no grupo tratado com a formulação fúngica contendo 5% de óleo mineral. No tratamento de ovos, as formulações oleosas de Metarril WP Organic® reduziram em até 55 vezes a eclosão das larvas quando comparadas aos grupos controle. No tratamento de larvas, a mortalidade foi de 100% no quinto dia após o tratamento com as formulações fúngicas contendo 3 e 5% de óleo mineral. Estes resultados sugerem que as formulações de Metarril WP Organic® contendo 1, 3 ou 5% de óleo mineral são eficientes, in vitro, contra os está- gios de desenvolvimento do carrapato R. microplus

Hemocytes quantification in Rhipicephalus microplus engorged females infected by Beauveria bassiana s.l.

ABSTRACT. Freitas M.C., Coutinho-Rodrigues C.J.B., Perinotto W.M.S., Nogueira M.R.S., Chagas T.T., Marciano A.F., Camargo M.G., Quinelato S., Gôlo P.S., Sá F.A. & Bittencourt V.R.E.P.[Hemocytes quantification in Rhipicephalus microplus engorged females infected by Beauveria bassiana s.l.] Quantifica- ção de hemócitos em fêmeas ingurgitadas de Rhipicephalus microplus infectadas por Beauveria bassiana s.l. Revista Brasileira de Medicina Veterinária, 37(Supl.1):63- 70, 2015. Departamento de Parasitologia Animal, Instituto de Veterinária, Anexo 1, Universidade Federal Rural do Rio de Janeiro, Campus Seropédica, BR 465 Km 7, Seropédica, RJ 23897-970, Brasil. E-mail: [email protected] Rhipicephalus microplus tick is an ectoparasite with a great negative impact for veterinary medicine. Under massive infestations cattle may present severe anemia; besides that, ticks can transmit pathogenic agents causing host death. Among the methods used to alternatively control ticks is the use of the arthropodpathogenic fungus Beauveria bassiana s.l. that has been considered promising due to the fungus capacity of penetration of full cuticle in all tick developmental stages, colonizing them and causing tick death. Arthropod immune response is basically composed of humoral and cellular components. Several cell types that actively work against microbial infection are present within the hemolymph. The aim of the present study was to evaluate the amount and identify hemocytes present in the hemolymph of R. microplus engorged females artificially inoculated with B. bassiana senso latu (s.l.) conidia (isolate CG 206). Fungal suspension (1 × 108 conidia/mL) or Tween 0.01% (control group) were inoculated in R. microplus engorged females. Hemolymph collections were made through the female dorsal region (for quantitative analysis) and the distal section of the legs (for qualitative analysis) 24, 48 and 72 hours after fungal inoculation. Hemocytes quantification was performed using a Neubauer chamber. Qualitative analysis was carried out by a smear of hemolymph on slides. Data were submitted to Mann-Whitney test for comparison of averages on a significance level of 5% (p <0.05). Comparison between unexposed females and females exposed to B. bassiana s.l. showed that hemolymph from the fungus treated group presented significant reduced amount of hemocytes 24 hours after fungal inoculation. Total amount of hemocytes 48 hours after fungal inoculation was 49.5 times lower in comparison with the amount observed 24 after fungal exposure. The following hemocytes were identified in tick females’ hemolymph: prohemocytes, granulocytes, plasmatocytes, spherulocytes and possible oenocytoids. Based on these results, it is concluded that inoculation of B. bassiana s.l. isolate CG 206 caused cellular alteration in the hemolymph of R. microplus females showing reduction of total amount of hemocytes 48 hours after fungal inoculation