In vitro efficacy of extracts of Melia azedarach against the tick Boophilus microplus

The efficacy of ripe fruit extracts of Melia azedarach L. (Rutales: Meliaceae) was evaluated against the tick, Boophilus microplus (Canestrini) (Acari: Ixodidae). Ripe fruits of M. azedarach dried and powdered were extracted by Soxhlet apparatus successively using hexane, CHCl3 and 96% aqueous ethanol. Larvae and engorged females were immersed in decreasing concentrations from 0.25% to 0.015% of each extract. The mortality of larvae was evaluated 24, 72 and 168 h after treatment. The effectiveness of treatment against engorged females was assessed by measuring egg production. All tested extracts caused mortality of B. microplus larvae, with higher mortality rates observed in CHCl3 (100%) and hexanic extract (98%) than in ethanolic extract (50%) 168 h after treatment. The mortality was dependent on concentration and on time after treatment. Similarly hexanic and CHCl3 extracts showed higher effectiveness (varying from 14% to 100%) against B. microplus engorged females than ethanolic extract (varying from 0% to 46%). Melia azedarach extracts did not kill the adult females, but inhibited partially or totally egg production and embryogenesis. These results show that the less polar the extract of M. azedarach ripe fruits the more its effectiveness against larvae and engorged females of B. microplus. This plant may therefore be useful in the control of resistant B. microplus populations.     

胡椒、芒果和黄皮的化感作用潜力

以胡椒、芒果和黄皮作为供体,玉米、黄豆、南瓜、花生、萝卜、稗草、马唐、柱花草为受体,通过种子萌发法和根生长法测定胡椒、芒果和黄皮的化感作用潜力.结果表明,玉米、黄豆、南瓜、稗草和马唐的萌发和根生长都受到3种供体水浸提液的影响,3种供体水浸提液在高浓度下抑制而在低浓度下则促进受体的萌发和根生长;胡椒和芒果根系周围的土壤对玉米萌发和根生长有促进作用而对花生的萌发和根生长则有抑制作用.3种供体的水浸提液通过乙酸乙酯、正丁醇萃取分离成不同极性的三相后分别对玉米、萝卜和柱花草进行处理.结果表明,胡椒和芒果浸提液的水相与正丁醇相对受体的抑制作用明显强于乙酸乙酯相,表明胡椒和芒果的化感物质具有较强的极性.     

Nonvolatile chemical cues affect host-plant ranking by gravid Polygonia c-album females

In a multiple-choice test, the preference of egg-laying Polygonia c-album (comma butterfly) females was studied for oviposition on plants bearing surrogate leaves treated with crude methanol extracts obtained from leaves of seven host-plant species: Humulus lupulus, Urtica dioica, Ulmus glabra, Salix caprea, Ribes nigrum, Corylus avellana, and Betula pubescens. The ranking order of surrogate leaves treated with host-plant extracts corresponded well to that reported on natural foliage, except R. nigrum. Thus, host-plant choice in P. c-album seems to be highly dependent on chemical cues. Moreover, after two subsequent fractionations using reversed-phase chromatography the nonvolatile chemical cues residing in the most polar water-soluble fractions evidently provided sufficient information for egg-laying females to discriminate and rank between the samples of more and less preferred plants, since the ranking in these assays was similar to that for natural foliage or whole methanol extracts, while the physical traits of the surrogate leaves remained uniform.     

Genetic similarity and variability between natural populations and laboratory colonies of North American Boophilus (Acari: Ixodidae)

Four natural populations and 4 laboratory colonies of Boophilus microplus (Canestrini) and a laboratory colony of Boophilus annulatus (Say) were subjected to electrophoretic analysis to determine levels of genetic similarity and genetic variability. Populations of B. microplus exhibited high genetic similarity (I = 0.984 +/- 0.012) indicating that all populations share a relatively undifferentiated gene pool. Host vagility is proposed as an important mechanism promoting gene flow in ectoparasites. Levels of genetic variability for B. microplus (h = 0.092 +/- 0.008; P = 0.330 +/- 0.050) were within levels reported for other arthropods. The average genetic identity of 0.716 +/- 0.013 between B. microplus and B. annulatus is of a level characteristic of sibling species. Diagnostic isozymes between laboratory colonies will facilitate identification of these morphologically similar species.     

Relationship between glutathione S-transferase, catalase, oxygen consumption, lipid peroxidation and oxidative stress in eggs and larvae of Boophilus microplus (Acarina: Ixodidae)

Glutathione S-transferases (GSTs) are enzymes that act in excretion of physiologic and xenobiotic substances, protecting cells against chemical toxicity and stress. In this work, we characterized the enzymatic activity of GST in eggs and larvae of cattle tick Boophilus microplus, on different days after oviposition and eclosion. The results showed that the GST activity varied depending on the time elapsed after oviposition and eclosion. Molecules involved in mechanism of protection from oxidative stress are correlated with the increase in GST activity. The oxygen consumption kinetics showed a positive correlation with the increase in GST activity during embryogenesis. A high content of thiobarbituric acid reactive substances were observed in egg and larva extracts, indicating that ticks face high oxidative stress during embryogenesis and aging. In eggs and larvae, GST activity can be correlated to kinetic parameters of oxidative stress such as catalase and glutathione. In addition, GST activity showed strong positive correlation with lipid peroxidation, an indication that it plays a role in oxidant defences in eggs.     

Uniformity of protective antigens among isolates of the cattle tick, Boophilus microplus

Abstract. Gut membrane antigens were extracted from ten isolates of the cattle tick Boophilus microplus; the antigen extracts were probed with bovine antisera and three murine monoclonal antibodies (mAbs) in Western blots and dot-ELISA. The antisera had been obtained from cattle which were vaccinated with larval and gut extracts of B.microplus , and which were subsequently protected (84% and 94% respectively) against challenge with B.microplus. One of the mAbs (QU13) has been demonstrated to precipitate protective antigens from the midgut of B.microplus. Gut antigens from all ten isolates displayed similar reactivity profiles against bovine antisera and also against mAbs in Western blots. The end-point titres of antigens in dot-ELISA showed four-fold variation between isolates against bovine antisera, and also against mAb QUI 3. Larval membrane antigen extracted from N-strain B.microplus reacted with QU13 in dot-ELISA, indicating that protective antigens are common to both larval and adult stages of B.microplus. It was concluded that protective antigens recognized by QUI3 and antigens recognized by sera from protected cattle were conserved between the ten isolates examined, and between life-cycle stages.     

Plant extracts as an alternative to control Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae)

We evaluated the effects of crude extracts from the plantain Plantago lanceolata and the bitter gourd Momordica charantia on the oviposition preference and development of the coffee leaf miner Leucoptera coffeella Guérin-Mèneville & Perrottet under laboratory and/or greenhouse conditions. The ovicidal effects of these extracts were also studied in a greenhouse. Plantago lanceolata and M. charantia extracts also underwent fractionation directed by oviposition tests with the coffee leaf miner. The extracts of both plants reduced L. coffeella oviposition and egg hatching, apparently as a result of action of plant metabolites on the embryo. Adults originating from eggs treated with the extracts exhibited similar survival rates, but a higher female/male ratio. Fecundity was reduced for females obtained from eggs treated with the M. charantia extract. Partial chemical analysis indicated that both extracts produced polar fractions that reduced the oviposition of L. coffeella on coffee leaves under laboratory conditions. The extracts of P. lanceolata and M. charantia have potential for use in the development of new products to control the coffee leaf miner.     

Megaselia scalaris reared on Rhipicephalus (Boophilus) microplus laboratory cultures

Different laboratory cultures of the acarine tick Rhipicephalus (Boophilus) microplus (Canestrini, 1888) (Ixodida: Ixodidae) were infested by small Megaselia scalaris (Loew, 1866) (Diptera: Phoridae) flies. Larvae of this species exhibited opportunistic parasitism predominantly on engorged female ticks, causing severe damage to their cuticle through which the flies were able to reach R. microplus internal organs, on which they fed until developing into pupae in the tick's remains. The flies were kept by continuous propagation on fresh ticks over six generations during which the same parasitoid behaviour was observed. Here we report on an ixodid tick laboratory culture used for rearing M. scalaris.     

Displacement of Boophilus decoloratus by Boophilus microplus in the Soutpansberg region, Limpopo Province, South Africa

Boophilus ticks (n = 25,987) were collected from cattle at 30 communal dip tanks and five commercial farms in the Soutpansberg region, Limpopo Province, South Africa, between May 1999 and December 2001. Only 6.6% were Boophilus decoloratus, while 93.4% were Boophilus microplus, a tick that had not been reported from this area previously. B. microplus was the most common Boophilus tick on communal dip tank cattle, while B. decoloratus was more commonly found on commercial farms. Where the two species occurred together, B. microplus tended to displace B. decoloratus. The displacement was almost complete at the communal dip tanks, while on the commercial farms the population change was still in progress at the end of the survey. The present study demonstrated that a postulated reproductive interference was insufficient in preventing B. microplus from spreading when the climatic conditions were favourable to this species, as the displacement in most of the areas appeared to be rapid and complete.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

Metarhizium anisopliae lipolytic activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection

Lipases secreted by Metarhizium anisopliae, an important biological control agent, could potentially be involved in the host infection process. Here, we present the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity and prevented the infection of the Rhipicephalus (Boophilus) microplus host. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process. The filamentous fungus Metarhizium anisopliae is one of the most important and studied biological agents for the control of several arthropod pests, including the cattle tick Rhipicephalus (Boophilus) microplus. Lipases secreted by M. anisopliae could potentially be involved in the host infection process. This work presents the activity profile during the host infection process and the effect of lipase activity inhibitor ebelactone B on infection. During the course of tick exposure to spores (6-120 h) lipase activity increased from 0.03 ± 0.00 U to 0.312 ± 0.068 U using rho NP palmitate as substrate. In zymograms, bands of lipase activity were detected in ticks treated with spores without inhibitor. The previous treatment of spores with lipase activity inhibitor, ebelactone B, completely inhibited lipolytic activity, at all times specified, and prevented the infection of the R. microplus host. Spores treated with the inhibitor did not germinate on the tick, although this effect was not observed in the culture medium. The results herein presented prove, for the first time, the importance of lipase activity in M. anisopliae host infection process.     

Influence of larval frass extracts on the oviposition behaviour of Monochamus alternatus (Col., Cerambycidae)

Abstract:  The oviposition behaviour and response of adult females of Monochamus alternatus to short lengths (bolts) of Pinus massoniana trunks treated with hexane extracts of larval frass of M. alternatus was investigated in the laboratory. Females gnawed significantly fewer oviposition scars, and deposited significantly fewer eggs, on extract-treated bolts than on control bolts. These results suggest the presence of a possible oviposition deterrent in larval frass of M. alternatus . Hexane extracts of larval frass were analysed by gas chromatography–mass spectrometry, and α -pinene, β -pinene, 3-carene, limonene, longifolene and butylated hydroxytoluene were identified. Experiments with synthetic mixtures indicated that a mixture of monoterpenes and butylated hydroxytoluene exhibited oviposition-deterring activity.     

Comparison of two extraction procedures for the assessment of sediment genotoxicity: implication of polar organic compounds

Four sediment samples (Va?ne Airport VA, Va?ne Center VC, Va?ne North VN and Reference North RN) were collected in the Berre lagoon (France). Sediments were analyzed for polycyclic aromatic hydrocarbons (PAHs) by use of pressurized fluid extraction with a mixture of hexane/dichloromethane followed by HPLC with fluorescence detection analysis. Organic pollutants were also extracted with two solvents for subsequent evaluation of their genotoxicity: a hexane/dichloromethane mixture intended to select non-polar compounds such as PAHs, and 2-propanol intended to select polar contaminants. Sediment extracts were assessed by the Salmonella/microsome mutagenicity test with Salmonella typhimurium TA98+S9 mix and YG1041±S9 mix. Extracts were also assessed for their DNA-damaging activity and their clastogenic/aneugenic properties by the comet assay and the micronucleus test with Chinese Hamster ovary (CHO) cells. The PAH concentrations were 611ngg(-1)dw, 1341ngg(-1) dw, 613ngg(-1)dw and 482ngg(-1)dw for VA, VC, VN and RN, respectively. Two genotoxic profiles were observed, depending on the extraction procedure. All the non-polar extracts were mutagenic for TA98+S9 mix, and VA, VC, VN sediment samples exerted a significant DNA-damaging and clastogenic activity in the presence of S9 mix. All the polar extracts appeared mutagenic for TA98+S9 mix and YG104±S9 mix, and VA, VC, VN were genotoxic and clastogenic both with and without S9 mix. These results indicate that the genotoxic and mutagenic activities mainly originated from PAHs in the non-polar extracts, while these activities came from other genotoxic contaminants, such as aromatic amines and nitroarenes, in the polar extracts. This study focused on the important role of uncharacterized polar contaminants such as nitro-PAHs or aromatic amines in the global mutagenicity of sediments. The necessity to use appropriate extraction solvents to accurately evaluate the genotoxic hazard of aquatic sediments is also highlighted.     

The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: fivefold tandem repetition of a coding region.

We sequenced across all of the gene boundaries in the mitochondrial genome of the cattle tick, Boophilus microplus, to determine the arrangement of its genes. The mtDNA of B. microplus has a coding region, composed of tRNA(Glu) and 60 bp of the 3' end of ND1, that is repeated five times. Boophilus microplus is the first coelomate animal known to have more than two copies of a coding sequence. The mitochondrial genome of B. microplus has other unusual features, including (1) reduced T arms in tRNAs, (2) an AT bias in codon use, (3) two control regions that have evolved in concert, (4) three gene rearrangements, and (5) a stem-loop between tRNA(Gln) and tRNA(Phe). The short T arms and small control regions (CRs) of B. microplus and other ticks suggest strong selection for small genomes. Imprecise termination of replication beyond its origin, which can account for the evolution of tandem repeats of coding regions in other mitochondrial genomes, cannot explain the evolution of the fivefold repeated sequence in the mitochondrial genome of B. microplus. Instead, slipped-strand mispairing or recombination are the most plausible explanations for the evolution of these tandem repeats.     

The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle

A locust wing bioassay, that allowed an entomopathogenic fungus to be removed from host cuticle before penetration, was used to investigate the role of surface lipids and waxes in pre-penetration growth of the specific locust pathogen Metarhizium anisopliae var. acridum. SEM and atomic force electron microscopy showed the impact of the fungus on the architecture of the cuticle surface. Although the fungus can germinate on authentic alkanes as the sole carbon source, only low levels of germination occurred on crude, non-polar wing cuticle extracts, containing a mixture of long-chain n-alkanes and other waxes (identified in particular by gas chromatography and mass spectroscopy). The fungus removed a large proportion of non-polar and polar components during pre-penetration growth on the wing. Polar crude extracts from Schistocerca gregaria hindwings, which contained fatty acids, fatty acid esters, glucose, amino acids and peptides, were strong promoters of germination, and poor germination was observed on a locust hindwing from which the extract had been taken. Thus simple polar compounds, also present on the surface, may be required to stimulate germination before the fungus can make use of a complex mixture of non-polar lipids.     

Natural oviposition-deterring chemicals in female cotton bollworm, Helicoverpa armigera (Hubner)

Observation of the oviposition behavior of cotton bollworm demonstrated the existence of substances that can act as oviposition deterrents. The scales, abdomens and tarsi of the moths, the most likely carrier, were extracted for the substances. An acetone extract exhibiting an oviposition deterrent effect was analyzed using gas chromatography and combined gas chromatography-mass spectroscopy. Three components, 4-methyl-4-hydroxyl-pentanone-2, hexadecanoic acid and (Z)-9-octadecenoic acid, were identified, being present in the ratio 1:2:2. Experiments with synthetic mixtures indicated that a mixture of equal amounts of the two organic acids exhibited similar repellent and deterrent activity to a synthetic mixture of all three components. It is concluded that the natural oviposition-deterring pheromone is a mixture of hexadecanoic acid and (Z)-9-octadecenoic acid.     

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.     

Isolated thallus-associated compounds from the macroalga Fucus vesiculosus mediate bacterial surface colonization in the field similar to that on the natural alga

This study investigated whether surface-associated compounds isolated from the macroalga Fucus vesiculosus had the potential to mediate microbial and/or macrobial epibiosis similar to that on the natural alga. To selectively yield thallus-associated compounds and avoid contamination by intracellular algal compounds, cell lysis was monitored by surface microscopy of algal cells and chemical profiling of algal surface extracts by coupled gas chromatography mass spectroscopy. The optimized extraction resulted in polar and non-polar algal surface extracts. The non-polar surface extract was immobilized in hydrogel, the polar surface extract was homogeneously perfused through the gel to ensure a temporally constant delivery of polar extract components. During a 7 day field trial, bacterial biofilms were formed on control gels and gels featuring polar and/or non-polar extract components. PERMANOVA revealed that bacterial community profiles on controls and on gels featuring polar or non-polar extract were significantly different from the profile on F. vesiculosus, while the profile on the gels bearing both polar and non-polar extracts was not. Moreover, the polar surface extracts inhibited the settlement of barnacle cyprids. Considering the pronounced effects of bacterial biofilms on invertebrate larval settlement, these results suggest that algal surface chemistry affects macrofouling not only directly but also indirectly, via its control of biofilm formation and composition.     

Ultrastructure of the accessory glands of gene's organ in the cattle tick, Boophilus

The organization and ultrastructure of the accessory glands of the cattle tick, Boophilus microplus, are described. The glands consist of two groups of acinar cells situated on either side of Gene's organ. A single acinus consists of from eight to 12 cells and each cell is connected via an individual duct to pores on the dorsal surface of the mouthparts. The position of these pores is such that the secretion of the accessory glands is incorporated into the egg wax during oviposition. Each gland cell has striking quantities of smooth endoplasmic reticulum and numerous Golgi dictyosomes and appears to produce a secretion that is lipoidal in nature. Each cell secretes into its own individual lumen and is connected to a cuticular pore by a duct cell.