Lab-on-a-chip and SDS-PAGE analysis of hemolymph protein profile from Rhipicephalus microplus (Acari: Ixodidae) infected with entomopathogenic nematode and fungus.

In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC.

The influence of conidial Pr1 protease on pathogenicity potential of Metarhizium anisopliae senso latu to ticks.

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.

Physiological changes in Rhipicephalus microplus (Acari: Ixodidae) experimentally infected with entomopathogenic fungi.

Carbohydrate metabolism plays an important role in the physiology and maintenance of energy stores within living organisms. However, when organisms are exposed to adverse physiological conditions, such as during pathogenic infection, these organisms begin to use alternative substrates (proteins and lipids) for energy production. This paper studied the carbohydrate metabolism of Rhipicephalus microplus after infection with Beauveria bassiana and Metarhizium anisopliae. The parameters evaluated were glucose concentration, enzymatic activities of lactate dehydrogenase (LDH), alanine aminostransferase (ALT) and aspartate aminostransferase (AST), amounts of uric acid and urea in the hemolymph, and amount of glycogen in the fat body. The results showed changes in nitrogenous products, including an increase in the amount of urea detected 48 h after infection with both fungi. The enzymatic activities of LDH, ALT, and AST were increased after infection. The amount of glucose was increased 24 h after infection with B. bassiana and was reduced 48 h after infection with both fungi. The amount of glycogen in the fat body was reduced at different times of infection with both fungi. These results demonstrate, for the first time, the changes in carbohydrate metabolism of R. microplus after infection with M. anisopliae and B. bassiana and contribute to a better understanding of this host-parasite relationship. Together with knowledge of diseases that affect these ticks and their susceptibility to entomopathogens, an understanding of tick physiology will be necessary for the effective implementation of current biological control methods and will assist in the discovery of new methods to control this ectoparasite.

Neutral lipid composition changes in the fat bodies of engorged females Rhipicephalus microplus ticks in response to fungal infections.

The tick's fat body plays an essential role in energy storage and utilization. This study aimed to analyze the fat body neutral lipid composition in Rhipicephalus microplus engorged females. In the first study (physiological profile of untreated ticks), the lipid analysis took place over the course of 4 days; the engorged females were incubated at optimal conditions and their fat bodies were dissected daily. Fat body lipid analysis after fungal infection with Metarhizium anisopliae sensu lato (s.l.) or Beauveria bassiana s.l. was performed with four groups: one without any treatment, one that was inoculated with a solution of 0.1 % Tween 80 in water, and two groups that were inoculated with M. anisopliae or B. bassiana conidial suspensions. The fat bodies were dissected 24 and 48 h after infection. Lipid analysis was conducted by thin-layer chromatography on a silica plate. The results of the physiological profile showed that the amounts of triacylglycerol (TAG) and free cholesterol (CHO) decreased with time, whereas cholesterol ester (CHOE) increased on the second and fourth days. Following M. anisopliae or B. bassiana infection, there was an increase in the amount of CHO after 24 h, whereas the other lipid classes were not altered. M. anisopliae caused an increase in CHOE and TAG and a reduction in CHO at 48 h after infection; however, B. bassiana infection did not cause significant alterations in the concentrations of these lipids. M. anisopliae and B. bassiana infection changed the fat body metabolism of engorged female R. microplus ticks. This study provides the first report of changes in the neutral lipid composition of the R. microplus fat body.

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples.

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

In vitro efficacy of two commercial products of Metarhizium anisopliae s.l. for controlling the cattle tick Rhipicephalus microplus.

The effects of two different products - Metarril® SP Organic (dry conidia) and Metarril® SC Organic (emulsifiable concentrated conidia in vegetable oil) - on eggs, larvae and Rhipicephalus microplus engorged females were here explored. Three concentrations (108, 107, and 106 conidia mL-1) for both products were prepared in water + 0.1% Tween® 80 (v/v); afterward, bioassays were carried out for all R. microplus stages by immersion in suspensions (Metarril® SP) or formulations (Metarril® SC). Metarril® SP suspensions showed low efficacy and did not affect biological parameters of treated engorged females; for eggs and larvae, only slight decreases in hatchability and larvae population were observed. Despite a delay in germination, Metarril® SC presented better results; for females, reductions in Egg Mass Weight (EMW) and Egg Production Index (EPI) were reported. On eggs, 108 conidia mL-1 increased Incubation Period (IP), shortened Hatching Period (HP) and decreased hatchability by up to 61%; for larvae, 107 and 108 conidia mL-1 reached 99.6 and 100% larval mortality respectively, 10 days after fungal exposure. Thus, further studies involving the use of oil-based formulations for ticks such as Metarril® SC need to be performed, especially to control the most susceptible stages (eggs and larvae).

The ecology of horse cyathostomin infective larvae (Nematoda-Cyathostominae) in tropical southeast Brazil.

Experimental studies about the recovery, survival and migration to pasture of cyathostomin infective larvae (L(3)) from fresh feces depositions were conducted from February 2005 to March 2007 in a tropical region of southeast Brazil. Grass and feces were collected weekly at 8 a.m., 1 and 5 p.m. and processed by the Baermann technique. Multivariate analysis (principal components method) showed the influence of time and environmental variables on the number of infective larvae recovered from the feces and pasture. In the rainy period (October-March), more infective larvae were recovered on the feces and grass apex. In contrast, in the dry period (April-September), the recovery was higher only on the grass base, as well as the L(3) survival on feces and grass. More larvae were recovered at 8 a.m., except from the grass apex, where the highest recovery was at 1 p.m. Few studies investigating the seasonal transmission of equine cyathostomin have been conducted in South American tropical climates. These results demonstrate that in tropical conditions L(3) are available on feces and pasture throughout the year. Knowledge of climatic influences on the development and survival of L(3) is crucial to designing integrated parasite control programs that provide effective protection while slowing the development of anthelmintic resistance.

Commercial formulation of Metarhizium anisopliae for the control of Rhipicephalus microplus in a pen study.

The present study evaluated, for the first time, the effect of the commercial formulation Metarril(®) SP Organic of Metarhizium anisopliae plus 10% mineral oil to control Rhipicephalus microplus in a pen study. Three groups were formed with six animals each: the first group was exposed to Metarril(®) plus 10% mineral oil and 1% Tween 80; the second group was exposed to sterile distilled water, mineral oil and Tween 80 (oil control group); and the third group received no treatment (control group). The fungal formulation contained 1 × 10(8)conidiaml(-1). Each animal was sprayed with 3L of formulation. Fallen ticks were counted daily and a sample of 20 engorged females per day was incubated for assessment of biological parameters. Throughout the study period, Metarril(®) oil-based formulation showed an efficacy ranging from 19.20% to 67.39% in comparison with the control group; and from 8.18% to 61.38% in comparison with the oil control group. The average efficacy of Metarril(®) oil-based formulation was 47.74% and 40.89% in comparison with control and oil control groups, respectively. Changes in the biological parameters of engorged R. microplus females were observed in the first three days after treatment, with a significant reduction in hatching percentage and egg production index. We concluded that Metarril(®) SP Organic plus 10% mineral oil was efficient against R. microplus in pen studies. However, further in vivo studies are required to increase the efficacy and to establish a protocol for the use of this product in the field against the cattle tick.

Virulence potential of Metarhizium anisopliae s.l. isolates on Rhipicephalus (Boophilus) microplus larvae.

The use of entomopathogenic fungi to control arthropods has been reported worldwide for decades. The aim of the present study was to evaluate the virulence of 30 Metarhizium anisopliae s.l. Brazilian isolates from different geographical regions, hosts or substrates on the larvae of Rhipicephalus (Boophilus) microplus ticks under in vitro conditions to the selection of virulent isolates in order to be further used in biological control programs. The current study confirmed the lethal action of M. anisopliae s.l. isolates on R. (B.) microplus larvae with different mortality levels, usually directly proportional to the conidia concentration. No relationship was found between the origin of the isolate and its virulence potential or between the virulence potential and conidia production. Three isolates (CG 37, CG 384 and IBCB 481) caused a high percentage of larval mortality, reaching LC(50) at 10(6) conidia ml(-1), thus requiring a lower conidia concentration to cause an approximately 100% larval mortality. The results of this study suggest that these three isolates are the most promising for use in programs aimed at microbial control in the field.

Effect of oil-based formulations of acaripathogenic fungi to control Rhipicephalus microplus ticks under laboratory conditions.

The formulations of acaripathogenic fungi to control ticks have been widely studied. The present study evaluated the efficacy of oil-based formulations of Metarhizium anisopliae sensu lato (s.l.), isolate Ma 959, and Beauveria bassiana, isolate Bb 986, on different Rhipicephalus microplus stages, comparing the efficacy between aqueous suspensions and 10, 15 and 20% mineral oil formulations. Twelve groups were formed: one aqueous control group; three mineral oil control groups, at 10, 15 or 20%; two aqueous fungal suspensions of M. anisopliae s.l. or B. bassiana; and three formulations of M. anisopliae (s.l.) or B. bassiana containing 10, 15, and 20% mineral oil. To prepare aqueous suspensions and oily formulations, fungal isolates were cultivated on rice grains in polypropylene bags. The conidial suspensions and formulations had a concentration of 10(8)conidia/mL. Bioassays were repeated twice. After treatment, the following biological parameters of engorged females were evaluated: hatching percentage, egg production index, nutritional index, and percentage of tick control. The following parameters were evaluated in the bioassays with eggs: period of incubation, period of hatch, and hatching percentage. Mortality was evaluated in bioassays with larvae. M. anisopliae s.l. and B. bassiana oil-based formulations were more effective than aqueous suspensions against R. microplus eggs, larvae and engorged females, however, there was no significant difference between the three oil concentrations used. M. anisopliae s.l. and B. bassiana formulated in mineral oil reached 93.69% and 21.67% efficacy, respectively, while M. anisopliae s.l. and B. bassiana aqueous suspensions attained 18.70% and 1.72% efficacy, respectively. M. anisopliae s.l. oil-based formulations caused significant effects in all biological parameters of engorged females while B. bassiana oil-based formulations modified significantly the nutritional index only. Eggs treated with M. anisopliae s.l. and B. bassiana oil-based formulations showed hatching rates that decreased 102.5 and 3.65 times, respectively. In the bioassay with larvae, M. anisopliae s.l. oil-based formulations caused nearly 100% mortality five days after treatment, while larva treated with B. bassiana oil-based formulations reached 100% mortality at day 20 after treatment. Larva from oil-based control groups showed mortality at day 15 after treatment, which indicated a possible toxic effect of the oil for this R. microplus stage. The results showed that the fungal mineral oil formulations tested were more effective than the aqueous suspension. Oil-based formulations at 10, 15 and 20% enhanced the activity of M. anisopliae s.l. Ma 959, and B. bassiana Bb 986, isolates against R. microplus eggs, larvae, and engorged females tick. Mineral oil was effective as an adjuvant in formulations of M. anisopliae s.l., Ma 959, and B. bassiana, Bb 986, for the control of R. microplus under laboratory conditions.

Seasonal dynamics of cyathostomin (Nematoda - Cyathostominae) infective larvae in Brachiaria humidicola grass in tropical southeast Brazil.

The ecology of cyathostomin larvae was evaluated in different seasons, from July 2007 to June 2008, in the municipality of Seropédica, Rio de Janeiro state, southeastern Brazil. Samples of feces and grass were collected every 15 days at 8 AM and 5 PM and the infective larvae were recovered by the Baermann technique. Leaves of the grass Brachiaria humidicola were cut to 20 cm, which is the length containing most of the larvae. The highest number of larvae was recorded at 8 AM the winter (8300 L(3)kg(-1)dm) and spring (5300 L(3)kg(-1)dm). These results demonstrate that climate conditions can affect the recovery of larvae and that rain and temperature contributed to the migration and survival of the larvae, which were available throughout the year in the study area.

[Development and migration of cyathostome infective larvae (Nematoda: Cyathostominae) in bermuda grass (Cynodon dactylon) in tropical climate, in Baixada Fluminense, RJ, Brazil]. / Desenvolvimento e migração de larvas infectantes de ciatostomíneos (Nematoda: Cyathostominae) em gramínea coast cross (Cynodon dactylon) em clima tropical, na Baixada Fluminense, RJ, Brasil.

A study following the development and migration of Cyathostominae infective larvae was conducted from July 2003 to November 2004 in tropical climate, Baixada Fluminense, RJ, Brazil. Samples of naturally infected feces were placed on 12 m2 plot each month on a cyathostomin-free "Bermuda grass" pasture (Cynodon dactylon). After Seven days, samples of feces and grass were collected every week at 8 a.m, 1 and 5 p.m., weighed and processed by Baermann technique. Higher survival of L3 was found at dry season, 15 and 12 weeks on feces and sward respectively, at rainy season the survival was smaller. The multivariable analysis of main components was evident the influence of time and environment variables on L3 recovery from feces and grass. Close relationship between time and the number of L3 in feces could be noted, in contrast with L3 in sward. The climatic conditions influenced directly the number of infective larvae. The infective larvae were recovered during three times and the Kruskal-Wallis test did not present significance among them.

Desenvolvimento e migração de larvas infectantes de ciatostomíneos (Nematoda: Cyathostominae) em gramínea coast cross (Cynodon dactylon) em clima tropical, na Baixada Fluminense, RJ, Brasil / Development and migration of cyathostome infective larvae (Nematoda: Cyathostominae) in bermuda grass (Cynodon dactylon) in tropical climate, in Baixada Fluminense, RJ, Brazil

Esse estudo foi realizado no período de julho de 2003 a novembro de 2004, para avaliar o desenvolvimento, a sobrevivência, a migração das larvas infectantes em gramínea "coast cross" (Cynodon dactylon) e o horário de maior disponibilidade, em condições de clima tropical, na Baixada Fluminense, RJ, Brasil. De julho de 2003 a setembro de 2004, massas fecais de equinos naturalmente infectados foram depositadas mensalmente sobre a gramínea. Sete dias após, amostras de fezes e gramínea foram coletadas semanalmente em diferentes horários (8, 13 e 17 horas), pesadas e processadas pela técnica de Baermann. O desenvolvimento, a sobrevivência e a migração das larvas infectantes nas fezes e na gramínea foram observados durante todo o período. A sobrevivência das L3 foi de até 15 semanas nas fezes e 12 semanas na gramínea no período seco e de nove e oito semanas, respectivamente, para o período chuvoso. No período chuvoso, maior número de L3 foi recuperado nas fezes e, no período seco, na gramínea. Condições climáticas influenciaram diretamente o número larvas infectantes. Pela análise multivariada, ficou demonstrado uma forte relação entre o tempo e o número de L3 nas fezes, sendo esta relação menos acentuada para a gramínea. Não se observou diferença significativa entre os horários de coleta.

A study following the development and migration of Cyathostominae infective larvae was conducted from July 2003 to November 2004 in tropical climate, Baixada Fluminense, RJ, Brazil. Samples of naturally infected feces were placed on 12 m² plot each month on a cyathostomin-free "Bermuda grass" pasture (Cynodon dactylon). After Seven days, samples of feces and grass were collected every week at 8 a.m, 1 and 5 p.m., weighed and processed by Baermann technique. Higher survival of L3 was found at dry season, 15 and 12 weeks on feces and sward respectively, at rainy season the survival was smaller. The multivariable analysis of main components was evident the influence of time and environment variables on L3 recovery from feces and grass. Close relationship between time and the number of L3 in feces could be noted, in contrast with L3 in sward. The climatic conditions influenced directly the number of infective larvae. The infective larvae were recovered during three times and the Kruskal-Wallis test did not present significance among them.