Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development.

The aim of this study was to investigate whether the combination of thymol with eugenol has a synergistic effect on the immature life stages of Rhipicephalus sanguineus sensu lato (s.l.), to evaluate the cost-benefit ratio of using these compounds in combination, and to develop a formulation combining thymol with eugenol with activity on immature stages of R. sanguineus s.l. To evaluate synergism, thymol and eugenol, combined (ratio 1:1) or not, were tested at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL on unfed larvae and nymphs using a larval packet test, and 0.625, 1.25, 2.5, 5.0 and 10 mg/mL on engorged larvae and nymphs using an immersion test. A cost estimate was calculated to produce 1 L of a solution containing a concentration of thymol and eugenol, combined or not, that could cause a tick mortality rate greater than 95 %. Finally, a formulation was developed, consisting of a micellar dispersion containing polymers (MDP), with thymol + eugenol (1:1), at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL, and the activity was evaluated on unfed and engorged larvae and nymphs. For unfed larvae and nymphs, concentrations of 2.5 and 5.0 mg/mL and 2.5, 5.0 and 10.0 mg/mL, respectively, presented synergistic effects. In tests with engorged larvae and nymphs, respective concentrations of 0.625, 1.25 and 2.5 mg/mL and 2.5 and 5.0 mg/mL had synergistic effects. The estimated costs for producing a solution of 1 L with efficacy greater than 95 % was $5.97 using only thymol (15 mg/mL), $ 5.93 using only eugenol (15 mg/mL), and $ 3.97 using thymol + eugenol (1:1 - 5,0 mg/mL). In tests with MDP, the combination of thymol + eugenol resulted in a mortality rate higher than 95 % at concentration of 10 mg/mL for unfed and engorged larvae and nymphs. Thus, the combination of thymol + eugenol, depending on the concentration, has synergistic effects and this combination lowers the cost for the active ingredients thymol and eugenol. The combination of thymol + eugenol in MDP had acaricidal activity against immature life stages of R. sanguineus s.l.

Comparative analysis of spermatids of Rhipicephalus sanguineus sensu lato (Ixodidae) and Ornithodoros rostratus ticks (Argasidae): morphophysiology aimed at systematics.

The phylogenetic relationships among tick species (Acari: Ixodida) have been revisited by several researchers over the last decades. Two subfamilies, Rhipicephalinae (Ixodidae) and Ornithodorinae (Argasidae), deserve special attention. The male reproductive system morphology, as well as the ultrastructure of the germ cells, may provide important information for phylogeny and systematics of metazoan groups, with spermatozoa exhibiting characters that can be used for this purpose. With that information in mind, this study aimed at evaluating, through a comparative analysis, the morphology of the male reproductive systems and germ cells of ticks species Rhipicephalus sanguineus and Ornithodoros rostratus. In order to do that, histology and scanning electron microscopy techniques were used. The results have shown that despite the similarities in the general morphology of the male reproductive system among studied Ixodida so far, there are morphological differences among the species studied herein, mainly the U-shaped testis (ancestral character) in O. rostratus and the pair testes (derived character) in R. sanguineus, and the general morphology of germ cells (spermatids V). Besides that, the morphological changes observed during the spermiogenesis appear to be different between the species studied here, probably characterizing the two families considered. The data generated in this study showed the importance of comparative internal morphology studies, mainly in regard to spermatology, despite the morphological data obtained herein not being enough to product a cladogram (sperm cladistics), it was already possible to observe clear differences among families Argasidae and Ixodidae in regard to the organization of their male reproductive systems and concerning the external morphology of spermatids. Data yet to be obtained through transmission electron microscopy techniques will allow the application of spermiocladistics and spermiotaxonomy as tools for tick systematics.

Entomopathogenic nematodes in insect cadaver formulations for the control of Rhipicephalus microplus (Acari: Ixodidae).

This study evaluated the efficacy of four entomopathogenic nematode (EPN) strains in insect cadaver formulations against Rhipicephalus microplus and compared the efficacy of the most virulent EPNs applied in cadavers of Galleria mellonella and Tenebrio molitor. In the first experiment, infected G. mellonela larvae were used as the source of EPNs. Engorged females of R. microplus were placed in pots filled with soil and different numbers of G. mellonella larvae infected with one of four species of nematodes. All treatments with EPNs of the genus Heterorhabditis caused significant reduction (p<0.05) in the egg mass weight and hatching percentage of larvae. The EPNs of the genus Steinernema, except for the group exposed to Steinernema carpocapsae ALL, whose source nematodes included six larvae of G. mellonella, caused a significant reduction (p<0.05) in the egg mass weight produced per female. Steinernema feltiae SN applied with two, four, and six cadavers and S. carpocapsae ALL with two cadavers caused a reduction in hatching percentage of larvae of R. microplus (p<0.05). The percentage of control was above 95% in all groups treated with Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 and in the treatment with four larvae infected with S. feltiae SN. The second experiment followed the same methodology, using G. mellonella and T. molitor larvae infected by the two most virulent EPNs. H. bacteriophora HP88 and H. indica LPP1 in different formulations caused reduction in the egg mass weight and hatching percentage of larvae. The percentage of control were 82.4 and 84.9% for H. bacteriophora HP88 and H. indica LPP1, respectively, formulated in T. molitor, and reaching 99.9% in groups formulated with G. mellonella. The EPNs tested in insect cadaver formulation showed pathogenicity to engorged females of R. microplus and EPNs of the genus Heterorhabditis formulated in G. mellonella larvae were more effective.

Compatibilidade de Heterorhabditis amazonensis (Rhabditida: Heterorhabditidae) isolado RSC-5 com diferentes carrapaticidas utilizados no controle de Rhipicephalus microplus (Acari: Ixodidae) / Compatibility of Heterorhabditis amazonensis (Rhabditida: Heterorhabditidae) strain RSC-5 with different acaricides used for the control of Rhipicephalus microplus (Acari: Ixodidae)

O estudo teve como objetivo avaliar a viabilidade de Heterorhabditis amazonensis isolado RSC-5 após exposição a diferentes carrapaticidas utilizados no controle de Rhipicephalus microplus. Foram constituídos seis tratamentos, cada um composto por um produto, sendo cada grupo com 75.000 nematoides (NEPs) em suspensão de 20 mL de solução de diferentes carrapaticidas, em concentração comercial. O controle foi formado por 75.000 NEPs e 20 mL de água destilada, e todos os grupos foram mantidos em câmara climatizada a 25°C. A avaliação do percentual de sobrevivência e infectividade em lagartas Galleria mellonella foi realizada 24 e 72 horas após o início do experimento. A mortalidade de lagartas no teste de infectividade foi analisada após 72 e 120 horas. Com 24 horas de exposição, o percentual de sobrevivência de H. amazonensis RSC-5 não foi significativamente reduzido (p > 0,05) somente na exposição ao princípio ativo deltametrina. O mesmo foi observado no período de 72 horas em relação à associação clorpirifós + cipermetrina + butóxido de piperonila + citronelal. Não foi constatada sobrevivência de nenhum juvenil infectivo nos grupos expostos à associação clorfenvinfós + diclorvós. A exposição ao clorfenvinfós resultou em percentual de sobrevivência inferior a 50% após 72 horas. O potencial de infectar lagartas de G. mellonella foi reduzido apenas no grupo tratado com o princípio ativo clorfenvinfós. Dessa forma, é possível concluir que o princípio ativo clorfenvinfós e a associação clorfenvinfós + diclorvós não foram compatíveis com H. Amazonenses RSC-5, causando redução no percentual de sobrevivência e infectividade dos juvenis desse nematoide. Os outros produtos foram compatíveis, não causando redução na infectividade do isolado testado.(AU)

The aim of this study was to assess the viability of Heterorhabditis amazonensis strain RSC-5 after exposure to different acaricides used for Rhipicephalus microplus control. Six treatment groups were formed, one for each product. Each group was composed of 75,000 nematodes in a 20 mL solution of different acaricides, at commercial concentration. The control group was formed by the same number of nematodes in 20 mL of distilled water. All the groups were kept in a climate-controlled chamber at 25°C. The percentage of survival and infectivity in Galleria mellonella caterpillars were determined 24 and 72 hours after the beginning of the experiment. The mortality of the caterpillars in the infectivity test was assessed 72 and 120 hours. After 24 hours of exposure, only the active ingredient deltamethrin did not significantly reduce the survival percentage of H. amazonensis RSC-5 (p > 0.05). The same was observed after 72 hours of exposure to the combination of chlorpyriphos + cypermethrin + piperonyl butoxide + citronellal. There was no survival of infective juveniles in the groups exposed to the combination of chlorphenvinphos + dichlorvos. The exposure to chlorphenvinphos for 72 hours resulted in 50% of mortality. The potential to infect G. mellonella caterpillars was only reduced in the group treated with the active ingredient chlorphenvinphos. Chlorphenvinphos and the combination of chlorphenvinphos + dichlorvos were not compatible with H. amazonensis RSC-5, causing a reduction in the survival and infectivity of juveniles of this nematode, while the other products were compatible, causing no reduction in the infectivity of this isolate.(AU)

Acaricidal activity of essential oil from Lippia sidoides on unengorged larvae and nymphs of Rhipicephalus sanguineus (Acari: Ixodidae) and Amblyomma cajennense (Acari: Ixodidae).

The aims of this work were to identify the compounds and to investigate the acaricidal activity of the essential oil of Lippia sidoides for unengorged larvae and nymphs of Rhipicephalus sanguineus and Amblyomma cajennense. The oil was analyzed by gas chromatography and gas chromatography/mass spectrometry. In total, 22 compounds comprising 98.5% of the total peak area were identified. The major constituent of the essential oil was thymol (69.9%). The acaricidal activity against larvae and nymphs was assessed using a modified larval packet test. In all experiments, oils were tested at concentrations of 2.35, 4.70, 9.40 14.10 and 18.80 mg/mL. The mortalities of larvae and nymphs of R. sanguineus were 20.6, 47.8, 73.6, 99.5 and 99.0% and 12.0, 50.0, 76.3, 96.0 and 96.1%, respectively. For larvae and nymphs of A. cajennense the rates of mortality were 41.9, 63.3, 77.8, 82.5 and 100.0% and 0.0, 32.8, 64.8, 71.1 and 94.0%, respectively. The LC 90 values of the L. sidoides oil were 11.56 and 12.97 mg/mL for larvae and nymphs of R. sanguineus and 15.70 and 18.52 mg/mL for larvae and nymphs of A. cajennense, respectively. The essential oil from L. sidoides has acaricidal activity on unengorged larvae and nymphs of R. sanguineus and A. cajennense.

Chemical composition and acaricidal activity of essential oil from Lippia sidoides on larvae of Dermacentor nitens (Acari: Ixodidae) and larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae).

The aim of this work was to identify the compounds and to investigate the acaricidal activity of the essential oil from the leaves of Lippia sidoides on Rhipicephalus microplus and Dermacentor nitens. The oil was obtained by hydrodistillation and analyzed by gas chromatography (GC/FID) and gas chromatography/mass spectrometry. In total, 15 compounds comprising 99.97 % of the total peak area were identified. The main constituent of the essential oil was thymol (67.60 %). The acaricidal activity was assessed by the modified larval packet test, with oil concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 µl/ml, and by the female immersion test with concentrations of 10.0, 20.0, 40.0, 60.0, and 80.0 µl/ml. The mortality of the R. microplus and D. nitens larvae was greater than 95 % starting at concentrations of 10.0 and 20.0 µl/ml, respectively. In the test with the engorged females, the L. sidoides essential oil starting at a concentration of 40.0 µl/ml caused a significant reduction (p < 0.05) in the values of the egg mass weight and egg production index. The viability of the eggs was affected in all the treated groups, with significantly lower hatching rates (p < 0.05) in relation to the control group. The control percentages at concentrations of 10.0, 20.0, and 30.0 µl/ml were 54, 57, and 72 %, and reached 100 % at the highest two concentrations (60.0 and 80.0 µl/ml). Therefore, it can be concluded that the essential oil from the leaves of L. sidoides has acaricidal activity on R. microplus and D. nitens.