Association of ß-globin polymorphisms and tolerance to haemonchosis in ewes and lambs of different sheep breeds.

Gastrointestinal nematodes (GIN), especially Haemonchus contortus, represent a significant challenge for sheep production. Given the global concern about GIN anthelmintic resistance, alternative control methods able to reduce the dependence on these drugs are highly advisable. Since previous studies have shown that sheep carrying the Hb-A allele of ß-globin are more resistant to H. contortus, this study aimed to investigate the relationship between the different haplotypes (Hb-AA, Hb-AB and Hb-BB) and phenotypes in Santa Inês (SI), Texel (TX) and White Dorper (DO) breeds infected with H. contortus. Blood samples were collected from 180 ewes and 123 lambs of the three breeds for DNA extraction followed by qPCR using a hydrolysis probe to identify the ß-globin haplotypes. Phenotypic data, including fecal egg count (FEC), packed cell volume (PCV), FAMACHA score and body condition score for ewes and lambs, as well as weight gain for lambs, were collected. The genotypic frequencies of ß-globin for ewes and lambs were, respectively: 21.7% and 21.4% Hb-AA, 50% and 50% Hb-AB and 28.3% and 28.6% Hb-BB in SI; 0% and 0% Hb-AA, 18.6% and 9.4% Hb-AB and 81.4% and 90.6% Hb-BB in TX; and 0% and 0% Hb-AA, 13.1% and 0% Hb-AB and 86.9% and 100% Hb-BB in DO. In ewes, mean PCV differed (p<0.05) between the three haplotypes, with higher PCV in Hb-AA animals, followed by Hb-AB and Hb-BB. When considering each breed separately, SI Hb-AA ewes presented higher PCV (p<0.05), highlighting that even in a breed already considered resistant, animals with Hb-AA haplotype showed superior performance. Lambs with the Hb-AA haplotype exhibited a higher (p<0.05) mean PCV compared to those with Hb-AB and Hb-BB. The same pattern was found in SI when analyzing each breed separately. No significant association was found between ß-globin haplotypes and FEC, FAMACHA score, body condition score, or weight gain. Nevertheless, given that anemia is the major clinical sign of haemonchosis, our findings on PCV reinforce that sheep carrying the Hb-A allele of ß-globin are more tolerant to haemonchosis. This study may support the development of a valuable tool, targeting genetic selection for GIN control, reducing the dependence on anthelmintics and boosting sheep production worldwide.

From the laboratory to the field: efficacy of entomopathogenic nematodes to control the cattle tick.

BACKGROUND: The control of ticks is challenged by the resistance of tick populations to chemical acaricides. In this study, we evaluated, under laboratory conditions, the efficacy of Heterorhabditis bacteriophora against Rhipicephalus (Boophilus) microplus engorged females with varying body weights (150, 200, 250, 300 or 350 mg per female) or from eight different geographical populations. We also determined the efficacy of H. bacteriophora for tick control under field conditions. RESULTS: R. microplus engorged females with varying body weights exposed to 150 juveniles of H. bacteriophora resulted in a high control efficacy (97.5% to 98.4%). Tests with females from different geographical populations comprised eight tick strains treated with H. bacteriophora and their respective control groups. The biological parameters of females exposed to nematode treatments did not differ significantly and resulted in 89% to 99% of control efficacy. Trials conducted under field conditions were performed in field plots with Megathyrsus maximus grass. Treatment groups received eight cadavers of Tenebrio molitor fully colonized with H. bacteriophora at 1 week prior to the release of female ticks, whereas control groups were untreated. On the first day of the experiment, six engorged females were distributed in each plot. On day 42 and day 63, the apical portion of the grasses with R. microplus larvae were collected and quantified. The population of R. microplus larvae was reduced up to 73.1% in plots treated with H. bacteriophora at day 63 after treatment. CONCLUSION: R. microplus engorged females with varying body weights or from different geographical populations were highly susceptible to H. bacteriophora. The field test demonstrated the efficacy of H. bacteriophora in reducing R. microplus larvae in infested pastures. © 2022 Society of Chemical Industry.

Rhamnolipids and essential oils in the control of mosquito-borne tropical diseases.

The diseases transmitted by mosquito vectors are a great public health issue. Thus, effective vector control becomes the main strategy to reduce their prevalence. However, insecticide resistance has become a huge concern for the mitigation of mosquitoes; here, we propose the use of rhamnolipids in emulsion with clove oil against Aedes aegypti and Culex quinquefasciatus. The toxicity of rhamnolipids and clove oil to two species of mosquitoes transmitting tropical diseases was investigated. After 24 h, the LC50 was 140 mg/L when rhamnolipids were used and 154 mg/L when clove oil was used against Aedes aegypti larvae. In the case of Culex quinquefasciatus, the LC50 was 130 mg/L for rhamnolipids and 19 mg/L for clove oil. When the concentrations of the upper limits of one of the solutions (rhamnolipid or clove oil) were mixed, 100% mortality was obtained after 24 h. The bioassay of insecticidal action for solutions of rhamnolipids and clove oil in the lower limit, upper limit, and lethal concentration 50 to determine the effect on 50% of the population (KD50) achieved low results from KD50 to the upper limit compared to the other concentrations for both Aedes aegypti and Culex quinquefasciatus. The rhamnolipids and clove oil at the upper limit concentration had the greatest repellent activity against the two mosquito species. Bioassays using different concentrations of rhamnolipids revealed variations in the morphology of the intestinal epithelium (800 mg/L). A concentration of 900 mg/L led to the most severe morphological changes in the organization of the epithelium and the cells lining the intestines of these larvae. When larvae were exposed to a concentration of 1000 mg/L, the marginalization of chromatin in the nucleus of epithelial cells was very severe, indicating the onset of cell death.Key points• The toxicity of rhamnolipids and clove oil has a larvicidal, insecticidal, and repellent effect.• The combination of concentrations of these compounds enhances their action.• Different concentrations of rhamnolipids led to severe morphological changes in the organization of the epithelium and the cells and the intestines of larvae.

Thymol: Effects on reproductive biology and Gene's organ morphology in Rhipicephalus sanguineus sensu lato engorged females (Acari: Ixodidae).

Thymol is a monoterpene with proven acaricidal activity on different tick species and life stages. The objective of this work was to evaluate the effect of thymol on the reproductive biology of engorged females of Rhipicephalus sanguineus sensu lato and the morphological changes caused in the cells of the tubular and accessory glands of Gené's organ. The females were exposed to thymol by immersion. Seven groups were established: group I (distilled water), group II (30% ethanol), group III (thymol 1.25 mg/mL), group IV (thymol 2.5 mg/mL), group V (thymol 5.0 mg/mL), group VI (thymol 10.0 mg/mL), and group VII (thymol 20.0 mg/mL), with 20 replicates for each treatment. The ticks were kept in a BOD incubator at 27 ±â€¯1 °C and RH > 80 ±â€¯10%. Ten females from each group were evaluated daily until death, and the remaining ten were kept in the incubator under the same conditions for five days and then dissected for the removal of Gené's organ to note possible damage to cell morphology by scanning electron microscopy (SEM) and hematoxylin-eosin (HE) techniques. Thymol demonstrated 63% and 98% efficacy in groups VI (10.0 mg/mL) and VII (20.0 mg/mL), respectively. In these groups, the cells of the tubular and accessory glands of Gené's organ showed signs of damage: irregular eosin staining, rupture and deformation of the cellular limit, presence of fragmented nuclei, changes in cytoplasmic homogeneity and areas with deformation (folds) in the basal region.

Thymol action on cells and tissues of the synganglia and salivary glands of Rhipicephalus sanguineus sensu lato females (Acari: Ixodidae).

Thymol is a monoterpene present in plants of the families Lamiaceae, Verbenaceae and Apiaceae. Despite its proven acaricidal activity, little is known about the mechanism of action of thymol in ticks. Thus, the aim of this study was to perform a morpho-histochemical analysis of the synganglion and salivary glands of partially engorged females of the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.), exposed to thymol at different concentrations. Five groups were established: Control Group I (distilled water), Control Group II (ethanol 30%), Group III (thymol 1.25 mg/mL), Group IV (thymol 2.5 mg/mL) and Group V (thymol 5.0 mg/mL). The females were exposed to the treatments by the immersion method and subsequently kept in a climatic chamber (27 ± 1 °C and relative humidity 80 ± 10%) for five days. After this period, the synganglion and salivary glands were removed, and the hematoxylin/eosin morphological technique was applied. The von Kossa staining method with counterstaining neutral red was performed on the salivary glands. The results showed that females exposed to thymol had damaged synganglia, with pyknotic nuclei and vacuoles in the cortex and subperineurial regions, as well as rupture of the neural lamellae. The salivary glands showed type I acini with a dilated lumen. Cells with extremely vacuolated cytoplasm and fragmented nuclei were observed in type II and III acini. Type II acini of the females exposed to thymol revealed different calcium staining when compared to the Control Groups I and II. We therefore conclude that the salivary glands and synganglion are subject to changes in morphology and calcium levels when exposed to thymol at concentrations of 1.25, 2.5 and 5.0 mg/mL, demonstrating that this monoterpene has acaricidal potential on partially engorged females of R. sanguineus (s.l.).

Acaricidal activity of thymol against larvae of Rhipicephalus microplus (Acari: Ixodidae) under semi-natural conditions.

This is the first study to investigate the activity of thymol on Rhipicephalus microplus larvae under semi-natural conditions. For this purpose, tests were conducted in pots with Brachiaria decumbens seedlings containing cattle tick larvae. Thymol, diluted in ethanol 50° GL, was tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/mL, along with the control group treated with the solvent alone. Each treatment was composed of five pots (1 pot = a repetition). The experiment was performed in three steps. On the first day, the larvae were applied at the base of the signalgrass. Twenty-four hours later, approximately 25 mL of the solution was applied with thymol on the top of the vegetation in each pot. The survival of the larvae was measured 24 h after application of the solutions. Each pot was analyzed individually, and the grass fillets contained larvae were cut with scissors, placed in Petri dishes, and taken to the laboratory to count the number of living larvae. At the highest concentrations (10, 15, and 20 mg /mL), the number of live larvae declined by more than 95 % in relation to the control group. The lethal concentration 50 % (LC50) and LC90 values were 3.45 and 9.25 mg/ml, respectively. The application of thymol in semi-natural conditions starting concentration of 10 mg/mL significantly reduced the number of living R. microplus larvae.

Determination of the susceptibility of unengorged larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae) to different methods of dissolving thymol.

The present study evaluated efficacy of thymol against engorged females and unengorged larvae of Rhipicephalus microplus when diluted in 1 % dimethyl sulfoxide (DMSO) (formulation 1) and diluted in hydro-ethanol solutions of 50 % (formulation 2), 40 % (formulation 3), and 30 % (formulation 4). The immersion test was used on the engorged females for all dilution methods, while the larval packet method was applied to the larvae to test formulations 3 and 4 since the other two had already been tested before. Two control groups were established in both experiments; in one, ticks were treated with 1 % DMSO, and in the other, with 50 % ethanol, with ten repetitions for each treatment. The groups were maintained at 27±1 °C and RH>80 ± 10 %. For engorged females, the parameters weight before oviposition, egg mass weight, hatching percentage of larvae, and percentage of control were evaluated, while for larvae, percentage of mortality was evaluated. The egg mass weight of all treatments differed significantly (p<0.05) from the control groups; however, the reduction in egg mass weight was more accentuated (p<0.05) in the treatments with formulations 1, 3, and 4. The values of hatching percentage of the group treated with formulation 2 and control group were statistically similar (p>0.05). The group treated with formulation 2 attained the lowest percentage of control (66 %). In the other treatments, the control percentages were greater than 99 %. In larval test, the mortality was 100 % in all the treatments, and there was no mortality in the control groups.

Association between entomopathogenic nematodes and fungi for control of Rhipicephalus microplus (Acari: Ixodidae).

The aim of the study was to assess the effect of the association of entomopathogenic nematodes and fungi on Rhipicephalus microplus. The nematodes used were Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 and the fungi were Metarhizium anisopliae IBCB 116 and Beauveria bassiana ESALQ 986. In the groups treated with the fungi, the females were immersed for 3 min in a conidial suspension, while in the groups treated with the nematodes, the ticks were exposed to infective juveniles. To evaluate the interaction between entomopathogens, the females were first immersed in a conidial suspension and then exposed to the nematodes. The egg mass weight and hatching percentage values of the groups treated with M. anisopliae IBCB 116 and B. bassiana ESALQ 986 in the two experiments were statistically similar (p > 0.05) to the values of the control group. In the groups treated only with nematodes, there was a significant reduction (p < 0.05) in the egg mass weight, a fact also observed for the hatching percentage of the group treated with H. indica LPP1. In all the groups treated with nematodes in association with fungi, there was a significant reduction (p < 0.05) in the egg mass weight and hatching percentage. The percentage of control of the groups treated with fungi alone varied from 31 to 55%. In the groups treated with nematodes associated or not with fungi, the control percentage was always greater than 90% and reached 100% in the group treated with H. bacteriophora HP88 associated with the fungus M. anisopliae IBCB 116.