蚤蝇的昼夜活动节律及睡眠行为研究

蚤蝇是重要的法医昆虫,同时是实验室中遗传、发育和生物测定等研究的重要对象。然而,蚤蝇的昼夜活动节律和睡眠行为及其在脑部的神经网络目前还不清晰。本文通过捕获本地蚤蝇并对其进行分子鉴定,研究了蚤蝇的昼夜活动节律和睡眠行为,同时表征了蚤蝇脑部核心钟神经元和多巴胺神经元。结果表明:蚤蝇在12h光照∶12h黑暗(12L∶12D)条件下不存在对开灯前或关灯前的活动预期,其双峰活动模式是对开关灯的光反应行为。在全黑暗(DD)条件下蚤蝇内源活动周期接近24h。黑腹果蝇神经肽PDF抗体免疫显示蚤蝇脑部核心钟神经元4~5个,不像黑腹果蝇一样存在明显的神经轴突。在睡眠行为上,蚤蝇雄虫和雌虫在整体活动强度、睡眠节律模式、总睡眠上均没有明显差异。相反,雄虫总睡眠次数和晚上睡眠次数低于雌虫,而总睡眠持续时间、晚上睡眠持续时间、总入睡时间和晚上入睡时间高于雌虫。此外,影响睡眠的重要多巴胺神经元在蚤蝇脑部的分布与黑腹果蝇类似。     

Pharmacological characterization of a tyramine receptor from the southern cattle tick,Rhipicephalus (Boophilus) microplus

The southern cattle tick (Rhipicephalus (Boophilus) microplus) is a hematophagous external parasite that vectors the causative agents of bovine babesiosis or cattle tick fever, Babesia bovis and B. bigemina, and anaplasmosis, Anaplasma marginale. The southern cattle tick is a threat to the livestock industry in many locations throughout the world. Control methods include the use of chemical acaricides including amitraz, a formamidine insecticide, which is proposed to activate octopamine receptors. Previous studies have identified a putative octopamine receptor from the southern cattle tick in Australia and the Americas. Furthermore, this putative octopamine receptor could play a role in acaricide resistance to amitraz. Recently, sequence data indicated that this putative octopamine receptor is probably a type-1 tyramine receptor (TAR1). In this study, the putative TAR1 was heterologously expressed in Chinese hamster ovary (CHO-K1) cells, and the expressed receptor resulted in a 39-fold higher potency for tyramine compared to octopamine. Furthermore, the expressed receptor was strongly antagonized by yohimbine and cyproheptadine, and mildly antagonized by mianserin and phentolamine. Tolazoline and naphazoline had agonistic or modulatory activity against the expressed receptor, as did the amitraz metabolite, BTS-27271; however, this was only observed in the presence of tyramine. The southern cattle tick's tyramine receptor may serve as a target for the development of anti-parasitic compounds, in addition to being a likely target of formamidine insecticides.     

Staphylococcus saprophyticus is a pathogen of the cattle tick Rhipicephalus (Boophilus) microplus

During experimental Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infestation on cattle, approximately 5% of engorged female ticks showed symptoms of bacterial infection. The affected ticks were unable to oviposit, secreted a distinctive yellow exudate through the genital orifice and eventually died. Microscopic analysis of tick exudate showed abundant clusters of Gram-positive cocci bacteria that were isolated and cultured on bacteriological medium. Biochemical phenotyping and 16S rRNA ribotyping analysis on cultured bacteria identified it as Staphylococcus saprophyticus. This species was also isolated from healthy tick larvae, indicating that S. saprophyticus is commonly found in ticks during different developmental stages. However, conspicuous symptoms are only found on fully engorged females. Cultured S. saprophyticus induced identical pathological symptoms when the bacteria were experimentally inoculated into healthy ticks, demonstrating it to be the causative agent of the R. microplus infectious lethal disease described in this work.     

Population dynamics of the cattle tick Rhipicephalus (Boophilus) microplus in a subtropical subhumid region of Argentina for use in the design of control strategies

The population dynamics of Rhipicephalus microplus (Ixodida: Ixodidae) in northwest Argentina was analysed to support the design of strategic methods for its control. Both parasitic and non‐parasitic phases were studied. The seasonal activity of R. microplus in its parasitic phase was characterized by three peaks in abundance: the first in mid–late spring; the second in summer, and the third in autumn. The non‐parasitic phase of R. microplus was characterized by a long total non‐parasitic period observed after exposures of females from mid‐summer to early autumn, a short total non‐parasitic period observed after exposures of females from late winter to late spring, a short period of larval longevity in early and mid‐summer, and no hatch of the eggs produced by females exposed in mid‐ and late autumn and winter. Treatments of cattle administered during the period from late winter to late spring will act on small cohorts of R. microplus, preventing the emergence of larger generations in summer and autumn. A 17‐week spelling period starting in late spring and early summer will be necessary to achieve optimal control of R. microplus free‐living larvae. If spelling begins in mid‐ or late summer or in autumn, the required period will be 26–27 weeks.     

Efficacy of winter–spring strategic control against Rhipicephalus (Boophilus) microplus infestations on cattle in an area with ecological conditions highly favourable for the tick in northeast Argentina

This work was performed to test the efficacy of winter–spring control strategies against Rhipicephalus (Boophilus) microplus (Ixodida: Ixodidae) infestations on cattle in the area ecologically most favourable for the development of this tick in Argentina. Two control schemes using three and four annual applications of acaricides, respectively, were evaluated. Animals in Group 1 were treated with ivermectin 3.15% on day 0, fluazuron on day 34, and fipronil on day 85. Animals in Group 2 were treated with ivermectin 3.15% on day 0, fluazuron on day 34, flumethrin on day 85, and fipronil on day 114. Animals in Group 3 represented the control group. Both treatment schemes provided appropriate levels of efficacy against R. microplus and also prevented the occurrence of the major peak in the frequency of this tick in autumn. The two treatment schemes were similar in terms of efficacy and thus the addition of a fourth treatment does not seem to confer any further advantage. The results of this work indicate that these strategic control methods provide appropriate levels of control against R. microplus.     

1,8-cineole and castor oil in sodium lauryl ether sulphate disrupt reproduction and ovarian tissue of Rhipicephalus (Boophilus) microplus

Essential and fixed oils have been researched as alternatives to chemical acaricides. The activity of volatile compounds from essential oils (1,8-cineole, citral and eugenol) at 1.0% (w/v) and fixed oil (castor oil) at 0.3% (w/v) dissolved in 2.0% (v/v) dimethyl sulfoxide (DMSO) + 0.2% (w/v) Tween 80^® was assessed against Rhipicephalus microplus using immersion tests. 1,8-cineole (29.0%) and castor oil (30.2%) had the highest reproductive inhibition rate. A second experiment was performed to verify the effect of the 1,8-cineole (10.0% w/v) and, or castor oil (0.3% w/v) on tick reproduction using different solubilizing agents. The highest reproductive inhibition was observed for the combination of 1,8-cineole/castor oil (94.1%) and 1,8-cineole in 2.0% (w/v) sodium lauryl ether sulphate (SLES) (92.8%). A third experiment showed morphological changes in R. microplus oocytes at different stages of development, as well as in pedicel cells. The most intense effects were observed when ticks were immersed in the formulation containing 1,8-cineole (10.0% w/v) and castor oil (0.3% w/v) dissolved in 2% (w/v) SLES. These findings highlight the potential of this formulation as an alternative for managing cattle ticks as their cytotoxic effects can reduce R. microplus reproductive success.     

Role of Rusa deer Cervus timorensis russa in the cycle of the cattle tick Boophilus microplus in New Caledonia

Two field experiments were conducted to evaluate the efficiency of Rusa deer in the development of the cattle tick Boophilus microplus in comparison with that of steers in the same pastures and under the same conditions of infestation. No difference was noted between a mixed steer/deer herd and a pure steer or pure deer herd in the infestation pattern of each host, suggesting that attachment to the alternative host is mechanical and not affected by the simultaneous presence of the primary host on the pasture. Deer are capable of producing engorged viable females, with weight and reproductive performances similar to or even better than females fed on steers. For moderate levels (1 million larvae per hectare) and high levels (32 million larvae per hectare) of pasture infestation, tick burdens on steers were not very different (e.g. average 1911 and 2681ticks per m² skin, respectively, on day 24). This may be because of saturation of steer skin sites at the moderate larval dose. Deer harboured 2.7–33 times fewer ticks than steers and produce no engorged females at the moderate larval level and 32 times fewer engorged females than steers at the high larval level. Infestation of deer was dosedependent with averages of 12 and 399ticks per m² skin on day 25 at the moderate and high larval levels, respectively. At a high infestation level of the environment, Rusa deer may contribute, but to a limited extent, to infestation of pastures and, consequently, of cattle. However, their role in sustaining a viable tick population requires further investigation.     

Biological Parameters of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Fed on Rabbits,Sheep, and Cattle

In order to determine the effect of various hosts on feeding performance of Rhipicephalus (Boophilus) microplus, we used 3 mammalian species as hosts, cattle (Qinchuan), sheep (T an), and rabbits (Japanese white rabbit) for infest-ing ticks. Five hundreds of R. microplus larvae were exposed to each animal (3 animals/host species). Tick recoveries were 11.0%, 0.47%, and 5.5% from cattle, sheep, and rabbits, respectively. The averages of tick feeding periods were not significantly different on cattle, sheep, and rabbits, 28.8, 25.3, and 26.7 days, respectively. The average weights of individual engorged female from cattle, sheep, and rabbits were 312.5, 219.1, and 130.2 mg, respectively and those of egg mass weights each to 85.0, 96.6, and 17.8 mg. The highest egg hatching rate was in the ticks from cattle (96.0%), fol-lowed by those from rabbits (83.0%) and sheep (19.2%). These data suggest that rabbits could be as an alternative host to cultivate R. microplus for evaluating vaccines and chemical and biological medicines against the tick in the laboratory, although the biological parameters of ticks were less than those from cattle.     

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W., Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525–532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, V.I., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66, 331–341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high β sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae.     

A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity

The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, cysteine endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.     

Changing patterns of vitellin-related peptides during development of the cattle tick Boophilus microplus

The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.     

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fluazuron in Argentina

The aim of this work is to report the presence of resistance to fluazuron in a population of Rhipicephalus microplus in Argentina. The evidence was obtained from field and in vitro trials. In the field trial, cattle infested with ticks was treated with two commercial formulations of fluazuron. The in vitro trial (adult immersion test, AIT) was performed by using technical grade fluazuron. In the field trial, there were no significant differences between the treated and control groups between days 2 and 34 post-treatment. The only exceptions (treated group I in day 14 post-treatment, treated group II in days 23 and 29 post-treatment) had a significantly lower tick load than the untreated group, but the efficacy was not higher than 70%. Viable engorged females were collected on both groups of treated bovines in all counts, and the production of viable larvae was not precluded with the application of the two commercial formulations of fluazuron evaluated in this study. The results obtained with the in vitro assay (AIT) also indicate that the R. microplus population tested in this work has a higher level of resistance to fluazuron than another susceptible field strain. The integrated analysis of the field and in vitro trials clearly reveals the emergence of resistance to fluazuron in a R. microplus population from Argentina. This diagnosis of resistance does not imply that the fluazuron has lost its functionality at a regional scale, but it highlights the need to establish control strategies that minimize the use of this drug in order to preserve its functionality as an acaricide.

     

Development of enzyme linked immunosorbent assays to measure Bm86 antigen of Boophilus microplus (cattle tick) and to detect anti-Bm86 antibodies in serum samples

The immunization of cattle with the Boophilus microplus Bm86 antigen has been successful for the control of cattle tick infestations. To monitor the Bm86 production process and to measure the anti-Bm86 antibody titers in vaccinated cattle, mAb-based ELISA were developed and validated. The development of both immunological methods is essential to obtain a product with high quality and immunogenic properties and to monitor the immunological protection induced in vaccinated cattle against B. microplus.     

A cysteine endopeptidase from tick (Rhipicephalus (Boophilus) microplus) larvae with vitellin digestion activity

The hard tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite. R. microplus free-living stage comprises egg development, hatching, and subsequent larval development until encountering a host. In order to complete the embryological development, this tick relies on yolk reserve substances, mainly vitellin (Vt), which is still present in the larval stage. The present study demonstrates presence and digestion of Vt in unfed R. microplus larvae. An increasing proteolytic activity is observed in larval development, as well as a decrease in total protein and in Vt content. Partial purification and characterization of a R. microplus larval cysteine endopeptidase (RmLCE) with Vt-degrading activity is also described. RmLCE has optimal activity at 37 °C at pH 5.0, being unstable at pH ≥ 7.5. This enzyme is active upon fluorogenic peptide substrates and is able to degrade Vt, its putative natural substrate. These results indicate that RmLCE has a role in supporting the nutritional needs of unfed R. microplus larva through Vt proteolysis, allowing survival until the first blood meal.     

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 ??mol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.     

Virulence,proteolytic and lipolytic activities of Brazilian Beauveria bassiana s.l. isolates (Hypocreales: Clavicipitaceae) to Rhipicephalus microplus ticks (Acari: Ixodidae)

Beauveria bassiana s.l. is a cosmopolitan fungus used in the control of different species of arthropods. The current study explored the virulence for ticks, proteolytic and lipolytic activities of 10 Brazilian B. bassiana s.l. isolates. For this purpose, Rhipicephalus microplus biological parameters was evaluated after immersion of the engorged females in fungal suspension (10⁸ conidia mL^?1) and the enzymatic activities were performed posteriorly the fungal growth in minimal medium. After the biological assays, five isolates changed all parameters analysed with highest efficacy of approximately 61% (CG 206) and 66% (CG 481). However, we observed that the most virulent isolates did not show the highest enzymatic activities. Interestingly, CG 500, considered an isolate of intermediate efficacy, demonstrated higher enzymatic activities than the other isolates in four of five analyses (total protease, Pr1, Pr2 and lipase; p?相似文献   

Therapeutic and persistent efficacy of a single injection treatment of ivermectin and moxidectin against Boophilus microplus (Acari: Ixodidae) on infested cattle

The effectiveness of a single treatment with either ivermectin or moxidectin was determined by administering a single subcutaneous injection of each endectocide at 200 g per kg body weight to cattle infested with all parasitic developmental stages (adults, nymphs, and larvae) of Boophilus microplus (Canestrini). The percentage reduction in the number of females that reached repletion following treatment (outright kill) was 94.8 and 91.1% for ivermectin and moxidectin, respectively. In addition, the reproductive capacity of the females that did survive to repletion was reduced by >99%, regardless of the endectocide. Based on these two factors, the therapeutic level of control obtained against ticks on the cattle at the time of treatment was 99.0 and 99.1% for ivermectin and moxidectin, respectively. Engorged females recovered from either group of treated cattle weighed 3-times less than untreated females, and the egg masses produced by treated females weighed 5–8-times less than egg masses produced by untreated females. Partitioning of data into three separate 7-d post-treatment intervals allowed for an estimation of the efficacy of each endectocide against each individual parasitic development stage (adult, nymph, and larva). Results indicated that both endectocides were 99.7% effective against ticks that were in either the adult or nymphal stage at the time of treatment. However, the level of control against ticks in the larval stage of development at treatment was significantly lower at 97.9 and 98.4% for ivermectin and moxidectin, respectively. Analysis of the persistent (residual) activity of the two endectocides indicated that neither material provided total protection against larval re-infestation for even 1-wk following treatment. Against larvae infested 1–4 wk following treatment, the level of control with moxidectin ranged from 92.4% (1 wk) to 19.5% (4 wk). These control levels were higher at each weekly interval than for ivermectin, which ranged from 82.4% (1 wk) to 0.0% (4 wk). The potential for the use of these injectable endectocide formulations in the US Boophilus Eradication Program is discussed.This paper reports the results of research only. Mention of a commercial or proprietary product in this paper does not constitute an endorsement by the U.S. Department of Agriculture. In conducting the research described in this report, the investigators adhered to protocol approved by the USDA-ARS Animal Welfare Committee. The protocol is on file at the USDA-ARS, Knipling-Bushland U.S. Livestock Insects Laboratory, Tick Research Unit, Kerrville, TX. The U.S. Governments right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.This revised version was published online in May 2005 with a corrected cover date.     

Anti-tick monoclonal antibody applied by artificial capillary feeding in Rhipicephalus (Boophilus) microplus females

The tick Rhipicephalus microplus is an ectoparasite harmful to livestock, a vector of disease agents that affects meat and milk production. However, resistance to acaricides reflects the need for alternative tick control methods, among which vaccines have gained increasing relevance. In this scenario, monoclonal antibodies can be used to identify and characterize antigens that can be used as vaccine immunogens. Capillary tube artificial feeding of partially engorged R. microplus females with monoclonal antibodies against proteins from the gut of tick were used to test the effects of immunoglobulins in the physiology of the parasite. The results of artificial feeding showed that female ticks over 25mg and under 60 mg in weight performed better in the artificial feeding process, with a 94-168% weight increase after 24h of feeding. Results showed that artificial feeding of ticks proved to be a viable technique to study the effects of antibodies or drugs in the physiology of the parasite. One monoclonal antibody (BrBm2) induced decreased oviposition. Moreover, the antigen recognized by BrBm2 was identified as a 27-kDa protein and immunolabeled on digestive vesicles membranes of digestive cells of partially and fully engorged females.