Engorged Females Research Articles

Phlebotomine Sand Flies in Southern Thailand: Entomological Survey, Identification of Blood Meals and Molecular Detection of Trypanosoma spp.

Simple SummaryPhlebotomine sand flies (Diptera: Psychodidae) are hematophagous insects, and many species serve as vectors of various human and animal pathogens, including Leishmania and Trypanosoma protozoa. In Thailand, the first case of autochthonous leishmaniasis was reported 62 years ago. At present, the number of human cases is increasing in different regions of the country, but most cases are reported from southern Thailand. Therefore, we studied the potential transmission of Leishmania and Trypanosoma by sand flies in three provinces of southern Thailand, and analyzed blood sources of engorged sand fly females. We detected Trypanosoma sp. DNA in Sergentomyia barraudi, S. indica, S. khawi and Idiophlebotomus asperulus but no Leishmania spp. DNA. Moreover, bloodmeal analysis revealed that Trypanopsoma-positive females of S. barraudi and Sergentomyia sp. fed on dogs and humans, respectively. The results of this study contribute to the knowledge of leishmaniasis and trypanosomiasis presence and sand fly feeding behavior in southern Thailand.An entomological survey at rural and cavernicolous localities in four provinces in southern Thailand provided 155 blood-fed females of sand flies (Diptera: Psychodidae) that were identified based on morphological characters as Idiophlebotomus asperulus (n = 19), Phlebotomus stantoni (n = 4), P. argentipes (n = 3), Sergentomyia anodontis (n = 20), S. barraudi (n = 9), S. hamidi (n = 23), S. hodgsoni (n = 4), S. hodgsoni hodgsoni (n = 32), S. indica (n = 5), S. iyengari (n = 2), S. khawi (n = 17), S. silvatica (n = 11) and Sergentomyia sp. (n = 6). The dominant species in this study was S. hodgsoni hodgsoni, which was collected specifically in a Buddha cave. Screening for DNA of parasitic protozoans revealed eight specimens (5.16%) of four species (S. barraudi, S. indica, S. khawi and Id. asperulus) positive for Trypanosoma sp., while no Leishmania spp. DNA was detected. Blood meals of engorged females were identified by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay on a fragment of cytochrome b (cyt b) gene with a success rate 36%, humans, dogs, and rats being determined as sources of blood. Bloodmeal analysis of two Trypanopsoma-positive females (S. barraudi and Sergentomyia sp.) identified blood from dogs and humans, respectively. Our findings indicate that S. barraudi, S. indica, S. khawi and Id. asperulus may be incriminated in circulation of detected Trypanosoma spp.

Effect of the formulation with fipronil and fluazuron on the reproductive biology and ovaries histopathology of Rhipicephalus microplus engorged females.

This study aimed to evaluate the effects of a commercial formulation containing fipronil and fluazuron on the reproductive biology and the morphology of ovaries from Rhipicephalus microplus engorged females. To carry out the study, three calves were artificially infested every 3days with approximately 5000 larvae. On day 0, the animals were treated with a commercial formulation containing fipronil (1.25mg/kg) + fluazuron (2.5mg/kg). Before the application of the acaricide, engorged females of R. microplus were collected to constitute the control group (10 for biology analyses and 20 for histology analyses). After applying the commercial formulation, naturally detached engorged females were recovered on days + 5, + 10, and + 20 (10 engorged females/day) to evaluate their reproductive biology, and on days + 4, + 12, and + 20 (20 engorged females/day) for histological evaluation of the ovaries. Females from the treated groups produced smaller amounts of eggs, exhibiting lower viability when compared to eggs from the control group (p < 0.05). The ovaries of females from all treated groups (+ 4, + 12, and + 20) showed morphological changes, including: cytoplasmic disorganization, cytoplasmic degradation, irregular shape of the oocyte and germinal vesicle, reduction and vacuolization of yolk granules and oocyte disruption. Oocytes were observed in smaller numbers in all stages of development (I, II, III, IV, and V) and greater numbers of indeterminate oocytes were verified in the ovaries of the treated groups when compared to the control group. Therefore, results showed that the commercial formulation containing fipronil and fluazuron affected the reproductive biology, caused morphological changes in the ovaries, and reduced the number of oocytes in R. microplus engorged females.

Tick infestation level interferes with spray formulation (organophosphate + pyrethroid) efficacy against Rhipicephalus microplus

Due to the scarcity of information regarding the control of Rhipicephalus microplus on bovines with different tick burdens, tick counts and the efficacy of a commercial spray formulation (aspersion bath with organophosphate + pyrethroid) were evaluated for animals with a low and high tick burdens for 35 days in a stall test. Thirty-two crossbreed Bos taurus indicus x Bos t. taurus experimentally infested with R. microplus larvae were divided into four groups: T01 high infestation control, T02 high infestation treated, T03 low infestation control, and T04 low infestation treated. The bovines were kept individually in pens and R. microplus females were collected daily. All data were statistically analyzed with a significance level of 5% (P ≤ 0.05). For linear regressions, variables with P≤0.05 and the highest coefficients of determination (R2 ≥ 0.70) were considered the best descriptors. The reliability level was 95%. Tick counts differed (P<0.0001) between T01 and T02 (high infestation control vs. treated), except at day 2 (P >0.05) post-treatment, and efficacy ranged from 85.2 to 50.6%. The number of collected engorged females was lower (P<0.0001) for T04 than for T03 (low infestation treated vs. control) animals from day 3 to 29, and efficacy ranged from 95.2 to 69.8%. In addition, tick burden and efficacy were negatively correlated for both the group of animals with a high tick burden (r = -0.5256; p = 0.0012; R² = 0.2762) and the group of animals with a low tick burden (r = -0.9817; p < 0.0001; R² = 0.9638). In conclusion, a high tick burden on bovines decreases the efficacy of the tested spray acaricide.

Acaricidal Efficacy of Plants from Ecuador, Ambrosia peruviana (Asteraceae) and Lepechinia mutica (Lamiaceae) against Larvae and Engorged Adult Females of the Common Cattle Tick, Rhipicephalus microplus.

Control measures against common cattle tick Rhipicephalus microplus are of the upmost importance because of considerable, deleterious impact on a farm’s economy. Due to resistance phenomena to synthetic acaricides being a constraint in affected farms, the search for plant derivatives as acaricides has increased dramatically in recent years. In this work, essential oils obtained from two Ecuadorian plants, Ambrosia peruviana and Lepechinia mutica (EOAp, EOLm), traditionally used as insecticides in indigenous communities, were studied on larvae and engorged females at the parasitic stages of R. microplus. Larvae and females were treated with five (0.0625, 0.125, 0.25, 0.50 and 1%) and six concentrations (0.125, 0.25, 0.50, 1, 2 and 4%), respectively, of each EOsAp/Lm. A 98–99% larval mortality was achieved with 0.5% of both EOsAp/Lm. EOAp inhibited oviposition and egg hatching up to 82% and 80%, respectively, and had an overall efficacy of 93.12%. Efficacy of EOLm was 72.84%, due to the low influence of EOLm on reproductive parameters. By steam distillation and GC-MS analysis, γ-Curcumene was identified as the main constituent (52.02%) in the EOAp and Shyobunol (10.80%) in EOLm. The results suggest that major components of both essential oils should be further studied as promissory acaricides against R. microplus.

Characterisation of Salivary Gland Proteins and P-18 Gene of Camel Ticks from Bikaner

The main objective of this study was to investigate the various salivary gland proteins and gene analysis of camel ticks Hyalomma dromedarii found on one-humped camels (Camelus dromedarius) in Bikaner city, Rajasthan, India. For this purpose, Salivary glands were collected from 5 adult ticks (engorged females). Various protein analysis from salivary gland using SDS-PAGE for determination of protein bands and their molecular weights for each fraction was done. The molecular weight of resolved major bands ranged from 14.4 to 96.0 KDa. Out of them large majority of protein molecular weight was around 18 KDa. This low molecular weight band of around 18 KDa was detected in the all 5 fractions of salivary gland of Hy. dromedarii ticks. The protein gene P–18 of Hy. dromedarii of present study had 90.4% sequence identity with that of Hy. asiaticum from China.

Acaricidal activity of essential oils from Curcuma zedoaria and Alpinia zerumbet rhizomes against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

ABSTRACT This study aimed to determine the chemical composition and evaluate the acaricidal activity of essential oils (EOs) from Curcuma zedoaria and Alpinia zerumbet rhizomes against Rhipicephalus microplus in vitro. EOs were obtained by hydrodistillation and chemical characterization was performed by gas chromatography coupled with mass spectrometry (GC-MS). The acaricidal activity of each EO was firstly evaluated on larvae by larval packet test at concentrations of 2.5, 5.0, 10.0, and 15.0 mg/mL. Through the result of the treatment on larvae, where only the EO from C. zedoaria caused mortality, it was realized the adult immersion tests at concentrations 10.0, 20.0, 40.0, 60.0, and 80.0 mg/mL for R. microplus engorged females. The GC-MS analysis of EOs showed 1,8-cineole (43.49%), curzerenone (13.40%) and camphor (12.29%) as major components in C. zedoaria, and 4-terpineol (22.27%), 1,8-cineole (16.51%), terpinene (10.75%), p-cymene (9.24%) and sabinene (9.18%) in A. zerumbet. Regarding the acaricidal activity, both in R. microplus larvae and engorged females, only the EO from C. zedoaria was effective, causing 100% larval mortality at a concentration of 15 mg/mL and 98.6% efficacy on engorged females at a concentration of 80 mg/mL. Thus, our results provide support for further investigation of C. zedoaria EO compounds as novel natural products to control R. microplus.

Efficiency of tick biotherapic on the control of infestation by Rhipicephalus (Boophilus) microplus in Dutch dairy cows.

Background: cattle tick Rhipicephalus (Boophilus) microplus poses serious problems for farmers in Brazil, especially because the parasite easily develops resistance to pesticide agents. For this reason, together with other factors including environmental, human and animal contamination and costs, alternative approaches have been sought for. Aims: this study sough to evaluate the efficiency of a tick biotherapic on tick-infested cows. Methods: 34 dairy Dutch cows were divided in 2 groups: one group received 100g/day of mineral salt supplement impregnated with tick biotherapic 12cH for 6 months, and then in alternate days with tick biotherapic 30cH to complete 28 months of treatment; the other group (control) received only the mineral salt supplement. After 28 months of treatment, engorged Rhipicephalus (boophilus) microplus females were collected in both groups, counted and weighed; in vitro tests were carried out to assess mass of ticks; egg mass; egg-hatching rate; and reproductive efficiency. Results: There was significant difference between both groups for all parameters evaluated; tick-mass (p = 0.0008); egg mass (p=0.0044); egg-hatching rate (p= 0.0017); and reproductive efficiency (p = 0.0044). Conclusion: treatment with tick biotherapic significantly decreased the mass of engorged females, deposition and hatching rate of eggs, resulting consequently in the decrease of the reproductive efficiency of ticks.

Cloning, expression, and function of ferritins in the tick Haemaphysalis flava

The full-length cDNA of two ferritins of Haemaphysalis flava were cloned after which recombinant Hf-FER1 and Hf-FER2 were expressed and their function was analyzed. In addition, RNA interference (RNAi) based on the injection of Hf-fer1 or Hf-fer2 dsRNA into fully engorged female ticks was performed. The cDNA encoding Hf-FER1 is 834 bp in length. It contains an iron-responsive element in the 5ʹ untranslated region and encodes 174 amino acid residues. The full-length cDNA of Hf-FER2 contains 696 bp and encodes 199 amino acids, including a putative signal peptide sequence. Hf-FER1 and Hf-FER2 both have the ferroxidase iron center and the ferrihydrite nucleation center. The evolutionary relationship of Hf-FER1 and Hf-FER2 was established, and the predicted quaternary structures were assembled as typical spherical shells composed of 24 subunits which was demonstrated by nature PAGE. Real-time PCR showed that Hf-fer1 and Hf-fer2 were expressed in all developmental stages, with the highest expression in fully engorged females. The expression of Hf-fer1 and Hf-fer2 were relatively high in unfed larvae. Hf-fer1 was expressed in all tissues and was especially abundant in the salivary glands of fully engorged females. In contrast, the highest levels of Hf-fer2 were found in the midgut of fully engorged females, and no expression was found in the salivary glands of this life stage. Both recombinant Hf-FER1 and Hf-FER2 had iron-binding capabilities. Silencing of both Hf-fer1 and Hf-fer2 affected fecundity. Compared to the control, the percentage of ticks that laid eggs in the Hf-fer1 and Hf-fer2 RNAi groups was 73.3% and 66.7%, respectively. The silenced ticks that laid eggs had lower egg weight to body weight ratios, and the eggs had abnormal morphologies. The hatchability of eggs with normal morphology in the Hf-fer1 and Hf-fer2 silenced groups was 47.8% and 22.8%, respectively, which was significantly different from the control group (P < 0.005). These findings indicate that Hf-FER1 and Hf-FER2 play important roles in the iron storage of H. flava.

Life cycle of the tick, Haemaphysalis (Kaiseriana) bispinosa (Acari: Ixodidae) under laboratory conditions

ABSTRACT Haemaphysalis (Kaiseriana) bispinosa is one of the widest–spread tick species in Southern Asia vectoring pathogens to wildlife and livestock animals. Understanding biological parameters of this species is of great importance, particularly when setting control plans or for epidemiological studies under laboratory conditions. The aim of this study was to figure out the biological aspects of H. (K.) bispinosa under laboratory conditions. Three continuous generations of this tick have been recorded. The study resulted in overall means of 10.6 ± 2.46, 8.7 ± 2.68, and 14.6 ± 2.39 days for pre-oviposition period, oviposition, and egg incubation, respectively. Larvae needed means of 4.10 ± 1.24 and 7.50 ± 1.57 days for feeding and pre–moulting periods, respectively, whereas nymphs required means of 5.10 ± 1.32 and 10.90 ± 2.39 days for feeding and pre–moulting periods, respectively. Adult females engorged for a mean of 8.0 ± 0.79 days. The entire life cycle averaged of 71.0 ± 12.0 days. Adult females weighted an average of 47.6 ± 14.8 mg, which produced egg masses that averaged 10.9 ± 3.5 mg. The oviposition rate was 557 ± 179.1 eggs/female with a peak oviposition on day 4. Hatchability rate with an average of 94.3 ± 2.19% was recorded. Significant linear correlations between weight of engorged females and weight of egg mass, and with number of eggs laid were recorded (r = 0.90; P > 0.01; r = 0.89; P > 0.01, respectively). The life cycle of this tick species was ambiguous over the past, and finally has been cleared; therefore, it could be used in the future biological, epidemiological, and control investigations concerning this tick species.

Acaricidal Properties of Bio-Oil Derived From Slow Pyrolysis of Crambe abyssinica Fruit Against the Cattle Tick Rhipicephalus microplus (Acari: Ixodidae).

Slow pyrolysis is a process for the thermochemical conversion of biomasses into bio-oils that may contain a rich chemical composition with biotechnological potential. Bio-oil produced from crambe fruits was investigated as to their acaricidal effect. Slow pyrolysis of crambe fruits was performed in a batch reactor at 400°C and chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS). The bio-oil collected was used in bioassays with larvae and engorged females of the cattle tick Rhipicephalus microplus. Biological assays were performed using the larval packet test (LPT) and adult immersion test. The GC-MS of crambe fruit bio-oil revealed mainly hydrocarbons such as alkanes and alkenes, phenols, and aldehydes. The bio-oil in the LPT exhibited an LC90 of 14.4%. In addition, crambe bio-oil caused female mortality of 91.1% at a concentration of 15% and a high egg-laying inhibition. After ovary dissection of treated females, a significant reduction in gonadosomatic index was observed, indicating that bio-oil interfered in tick oogenesis. Considering these results, it may be concluded that slow pyrolysis of crambe fruit affords a sustainable and eco-friendly product for the control of cattle tick R. microplus.

The Absence of Abdominal Pigmentation in Livestock Associated Culicoides following Artificial Blood Feeding and the Epidemiological Implication for Arbovirus Surveillance.

Culicoides midges (Diptera: Ceratopogonidae), the vectors of economically important arboviruses such as bluetongue virus and African horse sickness virus, are of global importance. In the absence of transovarial transmission, the parity rate of a Culicoides population provides imperative information regarding the risk of virus dispersal. Abdominal pigmentation, which develops after blood feeding and ovipositioning, is used as an indicator of parity in Culicoides. During oral susceptibility trials over the last three decades, a persistent proportion of blood engorged females did not develop pigment after incubation. The present study, combining a number of feeding trials and different artificial feeding methods, reports on this phenomenon, as observed in various South African and Italian Culicoides species and populations. The absence of pigmentation in artificial blood-fed females was found in at least 23 Culicoides species, including important vectors such as C. imicola, C. bolitinos, C. obsoletus, and C. scoticus. Viruses were repeatedly detected in these unpigmented females after incubation. Blood meal size seems to play a role and this phenomenon could be present in the field and requires consideration, especially regarding the detection of virus in apparent “nulliparous” females and the identification of overwintering mechanisms and seasonally free vector zones.

Monthly dynamics of the cold-adapted one-host biological north form of Hyalomma scupense under the influence of the warm summer subtype of the Mediterranean climate in Turkey.

The one-host biological north form / ecotype of Hyalomma scupense Schulze, 1919 (Acari, Ixodidae) is reported for the first time in Turkey herein. Following the first detection of the tick, a longitudinal field study was carried out to fill gaps in the data concerning its biological features. This study also aimed to determine how the monthly activation dynamics of this relatively cold climate-adapted species is characterized under the influence of the warm summer subtype of the Mediterranean climate. During this study, which was carried out on a monthly basis in 2014, H. scupense was found on cattle from 5 out of 18 villages screened in Thrace (the European part of Turkey). The field study revealed that i) this north ecotype of H. scupense exhibits winter one-host behavior beginning in October (with larval stages) and ending in April (with engorged adults); ii) engorged females detach from the cattle, drop on the floors of barns during night and accumulate on piled bedding contaminated with slurry manure; iii) in the late spring, engorged females lay eggs, and larvae hatch in the same area; and iv) larvae become active in autumn as the weather grows cooler. The results indicated that although one-host H. scupense is known to be distinctly adapted to cold conditions, it can also be effectively established in relatively temperate regions and complete its life cycle with some modifications in the timing of its monthly activation dynamics.

Host preference and Leishmania infantum natural infection of the sand fly Phlebotomus perfiliewi in northern Italy

The host preference of hematophagous insects is important in determining the cycle of pathogens that they potentially transmit; for example, sand flies are competent vectors of Leishmania parasites. In this work, we evaluated the host preference of sand flies collected in the Emilia-Romagna region of Italy in 2018 and 2019 in an area in which Leishmania infantum circulates actively. Out of about 30,000 sampled sand flies, we obtained 252 engorged females, which were processed to identify the sources of blood meals. Sampling data collected confirmed a positive phototropism of Phlebotomus (Ph.) perfiliewi respect to Ph. perniciosus and the enhanced efficiency of light traps in collecting engorged females compared with traps baited with carbon dioxide. We identified blood source in 185 females (183 Ph. perfiliewi, two Ph. pernicious). The most bitten animal was the roe deer (49.5%), followed by humans (29.2%), hare (7.1%) and cow (4.7%). Other animals, including wild boar, horse, donkey, porcupine, chicken and red fox, were less represented (<2%), while the blood of dogs and rodents were not detected. In addition, we singly screened engorged females for Leishmania founding 5 positive specimens, fed on roe deer (4) and man (1), providing evidence of parasite circulation in a sylvatic environment, where presence of dogs was not common. These findings suggest the existence of an uncharacterized Leishmania reservoir in the surveyed area.

Artificial resting sites: An alternative sampling method for adult mosquitoes.

Mosquito collections are commonly conducted with baited traps predominantly attracting host-seeking females. In contrast, resting sites are generally colonized by a broader range of the mosquito population, including a higher proportion of males and blood-engorged females. This study evaluates the sampling success of different artificial resting sites, attached to a deciduous or coniferous tree at different heights. As standard sampling method, carbon dioxide-baited Biogents Sentinel traps (BG traps) were operated in parallel. BG traps caught a higher number of specimens compared to the resting sites. However, the proportion of blood-engorged females and males was higher in resting sites. More Culiseta spp. specimens were collected in resting sites compared to BG traps, but less Aedes spp. specimens. In general, fewer specimens and species were recorded in small resting sites and at top height level compared to medium or large resting sites at medium or ground level. The proportion of males was highest at the ground, while the proportion of engorged females was highest at medium and top level. Due to the higher proportion of blood-engorged females, artificial resting sites are especially useful for studies of host-feeding patterns or xenosurveillance. Low costs and efforts allow a cost-effective increase of the number of resting sites per sampling site to collect more mosquitoes.

Combination of cypermethrin and thymol for control of Rhipicephalus microplus: Efficacy evaluation and description of an action mechanism

The cattle tick Rhipicephalus microplus is one of the most important ectoparasites in the tropical and subtropical regions of the world. Synthetic pyrethroids are widely used to control this tick, and the selection of resistant populations is a huge problem worldwide. The activity of thymol, a natural monoterpene, free or in combination with other compounds, has been demonstrated against different species of ticks. However, the mode of action is not fully understood. The present study aimed to evaluate the efficacy and the potential mode of action of the combination of cypermethrin and thymol on ticks from two populations with different levels of susceptibility to cypermethrin (low and high susceptibility). The isolated acaricidal activity of cypermethrin and thymol on larvae was carried out in different concentrations. The combination with different concentrations of cypermethrin and fixed concentrations of thymol (1300µg/mL for the low susceptibility population; 690µg/mL for the high susceptibility population) were performed. Adult engorged females were divided into five experimental groups (n=20): 1) Control group untreated; 2) Control group: 2.0% (v/v) DMSO; 3) Thymol group: 1300µg/mL thymol; 4) Cypermethrin group: 3700µg/mL cypermethrin; 5) Association of cypermethrin (3700µg/mL)+thymol (1300µg/mL). A subgroup was used to study the efficacy of the reproductive parameters and another subgroup, with ten adults from each treatment, was used to quantify thymol and cypermethrin by HPLC chromatographic analysis. All compounds tested were effective on larvae from both populations, and the combination with thymol decreased the LC50 of cypermethrin (232.4 to 52.7µg/mL) on the low-susceptibility population. The combination of thymol and cypermethrin was effective in both populations of R. microplus (reproductive performance of engorged females) when compared to the untreated control group, even with higher percent control values (pop. 1: 93.5±5.6% and pop. 2: 92.7±1.1%) than the group treated only with cypermethrin (pop. 1: 87.3±7.3% and pop. 2: 83.5±1.2%). From the HPLC analyzes, a higher concentration of cypermethrin (pop. 1: 30.3±6.9 and pop. 2: 45.4±17.7ng/mg) was detected in the tissues of engorged females treated with the combination compared to analyte concentrations in groups treated with cypermethrin only (pop. 1: 12.4±4.4 pop. 2: 25.5±9.4ng/mg). This was the first study to investigate the acaricidal efficacy of the combination of thymol+cypermethrin on R. microplus and demonstrate that the presence of thymol increases the concentration of cypermethrin in the internal tissues of engorged females through a possible mechanism for increasing the penetration of cypermethrin at the cuticular level.

Microbiome analysis of the midguts of different developmental stages of Argas persicus in China

Argas persicus is an ectoparasite of poultry. The bacterial community structure and the pathogenic bacteria associated with different developmental stages of A. persicus have implications for control. Argas persicus were collected from chickens in the city of Jiuquan in Gansu, China. Bacterial DNA was extracted from the midgut contents of blood engorged larvae, nymphs and adult females. The V3-V4 hypervariable regions of 16S rRNA genes were sequenced using the IonS5™XL platform. Identification of Rickettsia spp. and detection of Coxiella burnetii were performed using PCR on target genes. The bacterial diversity within larvae was the highest and the bacterial diversity within nymphs was greater than that of adults. At different classification levels, seven bacterial phyla were common phyla, 27 genera were common genera, and 18 species were common species in the three samples. At the phylum level, Proteobacteria showed a marked predominance in all samples. Rickettsia, Stenotrophomonas, Spiroplasma, and Coxiella were the dominant bacteria at the genus level. The Rickettsia species in A. persicus was identified as Rickettsia hoogstraalii and the Coxiella species was identified as a Coxiella-like endosymbiont. Additionally, some bacterial species such as Pseudomonas geniculata, Sphingomonas koreensis, and Acinetobacter haemolyticus were reported here for the first time in A. persicus.

Acaricidal and pathogenic effects of the entomopathogenic fungus Beauveria bassiana on engorged females of the fowl tick, Argas persicus (Argasidae).

This study examined the acaricidal, histopathological and genotoxic effects of the entomopathogenic fungus Beauveria bassiana on engorged females of the fowl tick, Argas persicus. The acaricidal effect of B. bassiana (at 106-1010 conidia/mL) was concentration- and time-dependent. At 106 conidia/mL, the accumulated mortality of treated females ranged from 3.33 ± 3.3 to 13.33 ± 3.3% over 21days. However, the mortality was remarkably increased after the application of 1010conidia/mL, ranging from 6.67 ± 3.3 to 80 ± 5.8%. Females treated with various conidial concentrations exhibited complete inhibition of oviposition. Light and electron microscopic examination of the ovary of engorged female ticks at 3, 6 and 9days after treatment with 107 conidia/mL revealed that the oocytes exhibited drastic changes that affected their growth and development. The overall damage observed in the ovary included loss of grape-like appearance, distortion of oocytes, cytoplasmic vacuolation, degeneration of organelles and myelin figure formation. Previtellogenic oocyte progression was completely inhibited as the vitellogenic phase was not observed. Random amplified polymorphic DNA (RAPD-PCR) and inter simple sequence repeat (ISSR) methods were used to assess the genotoxic effects of B. bassiana at 107 conidia/mL on engorged female A. persicus over 3weeks. The lowest percentage of genomic template stability was recorded in paralyzed ticks after 3weeks. The study demonstrated the efficacy of B. bassiana as a biocontrol agent against A. persicus as it interfered with its reproduction, movement and viability and disrupted its normal tissue and DNA integrity.

Toxicity evaluation and chemical composition of Capsicum frutescens for natural control of Asian blue tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

BackgroundIndiscriminate use of synthetic acaricides in the management of veterinary parasites has led to environmental pollution, acaricidal resistance and their residues in the animal products. These problems are directly demanded an alternative acaricidal source for the ticks control and that should be cost-effective, eco-friendly and target specific. The current study demonstrated the acaricidal effects of Capsicum frutescens (fruits) against the cattle tick Rhipicephalus (Boophilus) microplus. In adult immersion test, the effects of the treatment on engorged females were assessed by measuring egg mass production, estimated reproductive factor, and % inhibition of reproduction.ResultsMethanol extract was observed the most effective against adults with LC50 617.54 ppm and LC90 1040.41 ppm. The other target extracts (petroleum ether and hexane) were less effective to the engorged females of R. microplus. Chemical analysis of the potent extract was elucidated by Gas Chromatography–Mass Spectrometry analysis and Cis-13-octadecenoic acid was observed as main compound (43.54%). The simultaneous evaluation of the qualitative chemical screening of the methanol extract showed the presence of tannins, saponins, carbohydrates, steroids, terpenoids, flavonoids, and alkaloids.ConclusionThe study concludes that the methanol extract of C. frutescens fruits revealed the significant acaricidal properties and may be used as safe alternative for tick management.

Acaricidal activity of the essential oils from Leptospermum scoparium, Origanum vulgare and Litsea cubeba on Rhipicephalus microplus: Influence of the solvents and search for fractions with higher bioactivity

The use of natural products in research on tick control for Rhipicephalus microplus is increasing year by year, with promising results. In this regard, the aim of the present study was to phytochemically characterize the essential oils (EOs) of Leptospermum scoparium, Origanum vulgare and Litsea cubeba, and to evaluate the acaricidal activity of these EOs in solutions prepared using ethanol, dimethylsulfoxide (DMSO) and Tween 80 on larvae and females of R. microplus. In addition, three L. scoparium fractions were also isolated and their acaricidal activity on these larvae and adult females was tested. Gas chromatography and mass spectrometry results showed that cis-calamenene (29.82 %), carvacrol (64.85 %) and geranial (42.44 %) were the majority compounds of L. scoparium, O. vulgare and L. cubeba, respectively. Three fractions were isolated from L. scoparium: A1, rich in sesquiterpene hydrocarbons, and A2 and A3, rich in β-triketones. Bioassays on unfed larvae (immersion test) were performed using all the EOs at concentrations from 2.5 to 10.0 mg/mL; and using the three fractions obtained from L. scoparium EO at concentrations from 0.625 to 10 mg/mL. We observed 100 % mortality of larvae in all treatments with L. scoparium EO at all concentrations (diluted both in DMSO and in ethanol), and in treatments with O. vulgare EO diluted in DMSO. However, L. cubeba EO only gave rise to more than 99 % mortality at a concentration of 10 mg/mL, using the same solvents. For engorged females, the immersion test was performed at concentrations from 2.5 to 10.0 mg/mL. Percentage control greater than 90 % was observed only at the highest concentrations of L. scoparium and O. vulgare EOs diluted in DMSO and ethanol, while L. cubeba EO did not reach 90 % control in any of the treatments. In tests on L. scoparium fractions, larval mortality in the fractions rich in β-triketones (A2 and A3) was above 97 % at a concentration of 2.5 mg/mL, while in the A1 fraction, rich in sesquiterpene hydrocarbons, at the same concentration (2.5 mg/mL), mortality did not reach 22 %. In the adult immersion test, the percentage control was higher than 98 % at the lowest concentration (2.5 mg/mL) of the A1 fraction, while in the treatments with the fractions A2 and A3, the control levels were 16 and 50 %, respectively. Thus, we can conclude that the EOs of L. scoparium, O. vulgare and L. cubeba have acaricidal activity on R. microplus, as also do the fractions derived from L. scoparium EO.

Assessment of lipid profile in fat body and eggs of Rhipicephalus microplus engorged females exposed to (E)-cinnamaldehyde and α-bisabolol, potential acaricide compounds

In the present study, the lipid profile from the fat body and eggs of Rhipicephalus microplus was evaluated after exposure of engorged females to (E)-cinnamaldehyde and α-bisabolol, substances which have acaricide potential according to the literature. Engorged females collected from artificially infested cattle were immersed in a concentration of 10.0 mg/mL of each substance. Dissection of the female fat bodies was performed at different times (72 h and 120 h), for subsequent lipid extraction. In addition, on the fifth day of oviposition, were collected 50.0 ml50.0 mL aliquots of the egg mass of each treatment to perform the same lipid extraction procedure. To assess the lipid profiles, the samples were submitted to the thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GCMS) analysis. Furthermore, an in silico analysis was performed using PASS online® software to predict the possible molecular targets of (E)-cinnamaldehyde and α-bisabolol. As result, the main lipids identified from the fat body were triacylglycerides, fatty acids, and cholesterol, whereas, triacylglycerides (TAG), fatty acids (FA), and cholesterol (CHO) and cholesterol esters (CHOE), were identified in the eggs. The results also showed a significant increase (p < 0.05) of CHO in the fat body in the group exposed to (E)-cinnamaldehyde at 72 h (0.12 μg/fat body) and 120 h (0.46 μg/fat body), in the eggs from females treated with this same substance, there was a significant reduction (p < 0.05) in the amount of CHO (0.21 μg), compared to the water control group (0.45 μg). In the GCMS technique, 5 chemical classes were found, and variations were observed between these substances, mainly hydrocarbons and steroids, in the different groups, and (E)-cinnamaldehyde promoted the greatest changes. From the predictions of the in silico study, 38 and 20 targets were selected, respectively, which are mainly related to alterations in lipid metabolism, immune system and nervous system. This study provides the first report of changes in lipid metabolism of R. microplus exposed to (E)-cinnamaldehyde and α-bisabolol, as well as presenting possible activity on the molecular targets of these substances, expanding knowledge for the potential use of these compounds in the development of botanical acaricides.