Formamidine structure and detachment of the cattle tick Boophilus microplus

Evaluation of the effectiveness of topically applied chlordimeform, 14 other formamidines, and 5 sulfur-containing related nonformamidine compounds in causing female Boophilus microplus ticks to detach from mice enabled activity to be related to structure. Five compounds were inactive and 15, including 2 sulfur-containing nonformamidines and 1 sulfur-containing formamidine were active at doses ranging from 0.0005 to 2.0 μg/tick. The most active compounds were the N-monomethyl formamidines, BTS-27271 [N′-(2,4-dimethylphenyl)-N-methylformamidine], and C-8520 (demethylchlordimeform). Among the analogs of chlordimeform tested, those with alkyl substitutions at the amino nitrogen decreased in effectiveness in the order, monomethyl (demethylchlordimeform), monoethyl, mono-n-butyl, mono-i-butyl, mono-i-propyl, dimethyl (chlordimeform), and di-n-propyl. Inactive compounds resulted from the replacement of the chlorine at ring position 4 of the aryl moiety of chlordimeform by bromine or hydrogen or by the conversion of the NCH amidino moiety to the NHCSN < thiourea moiety.Detachment due to chlordimeform was antagonized by piperonyl butoxide but that due to its N-monomethyl analog, a known metabolite of chlordimeform in ticks, was synergized by the same compound. These effects on the detaching response parallel those reported elsewhere concerning synergism and antagonism of toxic responses of B. microplus to formamidines.BTS-27271, which was the most effective formamidine in causing detachment after topical application to ticks was moderately effective when injected into mice but its potency relative to chlordimeform was considerably reduced; when sprayed onto cattle BTS-27271 was somewhat more effective in depressing percentage survival of ticks of all stages than chlordimeform.     

Choline acetyltransferase in organophosphorus-resistant and -susceptible strains of the cattle tick, Boophilus microplus

Choline acetyltransferase was demonstrated in homogenate extracts of larvae and adult brains of the cattle tick. The enzyme activity was approximately 15 and 100 μmol of acetylcholine synthesized/g of tissue/hr for larvae and adult brains, respectively. Comparisons of five strains showed that, despite organophosphate selection for acetylcholinesterases with wide differences in activity and inhibition kinetics, the choline acetyltransferase activity was statistically uniform between strains. it is concluded that the two enzymic components of the cholinergic system are controlled independently.     

Induction of hyperactivity in larvae of the cattle tick Boophilus microplus by formamidines and related compounds

The effect of chlordimeform and 18 related compounds on the aggregation behavior of the negatively geotactic larvae of the cattle tick, Boophilus microplus was investigated. Aggregations were treated and hyperactivity in the forms of immediate dispersal, delayed dispersal, and prolonged leg waving evaluated. A marked structure-activity relationship with delayed dispersal resulted; most active were the N-monomethyl formamidines, N′-(2,4-dimethylphenyl)-N-methylformamidine and demethylchlordimeform. Other compounds, including chlordimeform, with structures compatible with metabolism to N-monomethyl formamidines were also active. Delayed dispersal caused by those possessing the N,N-dimethyl moiety was antagonized after inhibition of oxidative N-demethylation to N-monomethyl analogs by piperonyl butoxide. Since the N-monomethyl moiety had already been reported as important in the killing action of the formamidines in cattle tick larvae, the possibility of a relationship between delayed dispersal and acaricidal effectiveness was examined and percentage mortality at 24 hr found to correlate positively with the rate of onset of dispersal.Delayed dispersal was not characteristic of the responses to other acaricides such as lindane, allethrin, carbaryl, and coumaphos. In addition, the monoamine oxidase inhibitors, tranylcypromine, pargyline, and nialamide, did not induce delayed dispersal and showed no lethal effects.     

A comparative study of the reactivity of acetylcholnesterases of the cattle tick Boophilus microplus and cattle erythrocytes with organophosphorus and carbamate inhibitors

The kinetic constants, K_a (affinity) k₊₂ (acylation), k_a (second order, acylation), and k₊₃ (deacylation) were determined for the reaction of a number of organophosphorus (OP) and carbamate inhibitors with acetylcholinesterase (AChE) from inhibitor-sensitive Yeerongpilly (Y) OP susceptible ticks, relatively insensitiveBiarra (B) and Ridgelands (R) OP-resistant ticks and bovineerythrocytes (Bov). B, R, and Bov AChE exhibited lower affinity for inhibitors than Y AChE. K₊₂ (carbamylation and phosphorylation) decreased in the order Bov ? Y ? B ? R. For inhibitors with markedly different leaving groups there was no clear-cut linear relationship between rates of alkaline hydrolysis and enzymatic hydrolysis (acylation). Decarbamylation and dephosphorylation were rate limiting. The ratios of Y to B to R AChE for V_max and catalytic center activity (substrate hydrolysis) and k₊₃ (carbamate hydrolysis) were very similar. This was a requirement if a lowered deacylating ability was the cause of the reduced catalytic hydrolysis of substrate observed for B and R AChE. Changes to the catalytic sites of B and R AChE are discussed. A strong parallel of these results is drawn with those obtained for spider mite AChE.     

A simple binding mechanism accounts for the temperature-dependent toxicity of cis-permethrin to larvae of the cattle tick, Boophilus microplus

The action of cis-permethrin, which evinced a strong negative correlation of toxicity with temperature in larvae of the pyrethroid-susceptible Yeerongpilly and pyrethroid-resistant DDT-R and Malchi strains of the cattle tick, was accounted for by a simple binding mechanism. The mechanism assumed that a single pyrethroid molecule binds to a single site and inactivates it, and that the equilibrium constant which describes the ensuing equilibrium between concentrations of free pyrethroid, unoccupied site, and inactivated site then varies with temperature according to the van't Hoff equation. From van't Hoff plots of the data the enthalpies of binding of cis-permethrin to sites in larvae of each of the above strains were calculated to be ?87, ?92, and ?102 kJ mol^?1, respectively. It was concluded from these results that the kdr-type insensitivity (≈6×, in terms of resistance) observed in DDT-R and Malchi ticks could not be explained by altered binding sites at the site of action. It was suggested that the insensitivity might be explained by altered access to the site of action.     

Carboxylesterases from Boophilus microplus hydrolyze trans-permethrin

An esterase which hydrolyzes the ester bond of trans-permethrin has been partially purified from homogenates of larvae of the cattle tick. The final (gel filtration) step showed two distinct peaks of p-nitrophenylbutyrate-hydrolyzing activity. The trans-permethrin-hydrolyzing activity of the lower-molecular-weight enzyme cochromatographed with the p-nitrophenylbutyrate-hydrolyzing activity of that enzyme. Little trans-permethrin hydrolysis was observed in the high-molecular-weight peak. The yield of the low-molecular-weight enzyme increased on extraction of the homogenates with Triton X-100. Inhibition studies using the low-molecular-weight enzyme and trans-permethrin as substrate indicated that hydrolysis was due largely to a carboxylesterase (EC 3.1.1.1).     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Substrate-specificity and toxicological significance of pyrethroid-hydrolyzing esterases of mouse liver microsomes

An esterase or esterases in acetone powder preparations of mouse liver microsomes hydrolyze the cyclopropanecarboxylate ester linkage of pyrethroid insecticide chemicals derived from primary alcohols. The rate of cleavage of (+)-trans-chrysanthemates with various alcohol moieties decreases in the following order: 5-propargyl-2-furylmethyl; 5-benzyl-3-furylmethyl (bioresmethrin); 3-phenoxybenzyl; tetrahydrophthalimidomethyl esters. The hydrolysis rate of benzylfurylmethyl esters with various acid moieties decreases in the order: (+)- or (?)-trans-chrysanthemate; (+)-trans-ethanochrysanthemate; tetramethylcyclopropanecarboxylate; (+)- or (?)-cis-chrysanthemate or (+)-cis-ethanochrysanthemate. The trans-isomers of chrysanthemates and ethanochrysanthemates are hydrolyzed from 2.6- to more than 50-fold more rapidly than the corresponding cis-isomers. This enzyme system does not hydrolyze secondary alcohol esters, i.e., allethronyl (+)-trans- and (+)-cis-chrysanthemates.On intraperitoneal administration to mice, the (+)-trans-chrysanthemate and -ethanochrysanthemate of benzylfurylmethanol are of very low toxicity relative to the corresponding (+)-cis-isomers and the tetramethylcyclopropanecarboxylate. S,S,S-tributyl phosphorotrithioate (DEF) pretreatment increases the toxicity of these five compounds by 2.6- to more than 188-fold, with the exception of bioresmethrin whose toxicity is not altered. When the toxicity is increased, it is probably the result of esterase inhibition since DEF strongly inhibits the esterase activity of fresh liver microsomes while the mixed-function oxidase system remains active. The oxidase system metabolizes the chrysanthemates more rapidly than the ethanochrysanthemates of benzylfuryl-methanol. Depending upon the pyrethroid involved, the esterase or the mixed-function oxidase system, or both may be responsible for limiting the toxicity of these pyrethroids to mice.     

Comparative inhibition of the juvenile hormone esterases from Trichoplusia ni,Tenebrio molitor, and Musca domestica

Twenty-seven compounds were screened as potential inhibitors of juvenile hormone esterases. Of these compounds O-ethyl-S-phenyl phosphoramidothiolate provided the best inhibition for the cabbage looper, Trichoplusia ni (Hubner), and the yellow mealworm, Tenebrio molitor L., while the juvenile hormone esterases of the house fly, Musca domestica L., were best inhibited by a juvenoid carbamate (1-(m-phenoxy-N-ethyl carbamate)-3,7-dimethyl-7-methoxy-2E-octene). The inhibition patterns of T. ni and T. molitor are similar, while those of M. domestica are relatively different. Further studies on the juvenile hormone and α-napthyl acetate esterases of T. ni showed that they could be differentially inhibited. Diisopropyl phosphorofluoridate and an alkyl trifluoromethyl ketone selectively inhibit the hydrolysis of α-naphthyl acetate and juvenile hormone, respectively, while O-ethyl-S-phenyl phosporamidothiolate inhibits both enzymes. The juvenile hormone esterases of T. ni also appear to be unique enzymes that are selective for juvenile-hormone-like molecules. The in vivo inhibition of T. ni juvenile hormone esterases by O-ethyl-S-phenyl phosphoramidothiolate slows the in vivo hydrolysis of juvenile hormone and results in delayed pupation and malformed larvae that resemble larval-pupal intermediates. Thus, the esterases involved in juvenile hormone metabolism appear to be important in juvenile hormone regulation.     

Inhibition of permethrin-hydrolyzing esterases from Wiseana cervinata larvae

Inhibition of permethrin-hydrolyzing enzymes from larvae of the porina moth Wiseana cervinata has been examined in vivo and in vitro. Significant inhibition was shown by carbaryl and pirimiphos-methyl. 1-Dodecylimidazole substantially inhibited permethrin hydrolysis only in liver insects. The triphenylmethane dye tetrabromophenolphthalein was a moderate inhibitor only in vitro. TMDM (bis(N-dimethyl-4-aminophenyl)methane) had little effect on hydrolysis. These observations extend the range of species and substrates for which the triphenylmethane dyes and 1-dodecylimidazole are useful inhibitors of insecticide metabolism.     

Cypermethrin inhibits egg development in the ixodid tick, Amblyomma hebraeum

The pyrethroid insecticide, cypermethrin (CyM), stimulates vitellogenesis in Ornithodoros moubata (an argasid tick) by stimulating the release of the normal vitellogenesis-inducing factor (a neuropeptide) and subsequent release of the vitellogenic hormone [19]. Here we examine the effects of CyM on egg development in the ixodid tick, Amblyomma hebraeum. Ovary weight, oocyte size, and vitellin content of the ovary were measured after CyM treatment; in partially fed ticks, none of these parameters were affected significantly. However, CyM treatment caused an inhibition of ovary development, as well as reduction of both hemolymph 20-hydroxyecdysone (20E; the vitellogenic hormone in this species) and vitellogenin (Vg)-concentrations in engorged ticks. In addition, the degree of salivary gland degeneration (which is triggered by 20E) was slightly reduced in CyM-treated engorged ticks. These results indicate that CyM acts differently in Amblyomma compared to Ornithodoros. Instead of stimulating vitellogenesis, CyM inhibits egg development perhaps in part as a result of inhibiting release of 20E.     

A standardised in vivo and in vitro test method for evaluating tick repellents

The threat of transmission of Lyme borelliosis and tick-borne encephalitis by ixodid ticks has resulted in an increasing number of tick repellents coming onto the market. To allow proper evaluation of the efficacy of different types of compounds and their formulations, there is a need for standardised methods for testing ticks repellents. Ticks show a marked negative geotactic response following contact with a potential host, i.e., they climb up in order to locate attachment and feeding sites, whereas exposing ticks to repellents induces positive geotaxis, i.e., ticks walk downwards or drop off the treated host or substrate. We describe here complementary tests that employ these geotactic responses to evaluate repellents: one in vitro on a warm glass plate and the other on the lower human leg (shin). The compounds tested were DEET, EBAAP, icaridin, capric acid, lauric acid, geraniol, citriodiol, citronella essential oil and lavender essential oil, all non-proprietary ingredients of widely distributed tick repellent formulations.     

SPRI-231菌株活性组分分离纯化及抑菌作用简报

从广西南宁地区采集的土壤里筛选出一株能够产生抗菌活性物质的链霉菌株(streptomyces), 菌号为SPRI-231。通过溶剂浸提、大孔树脂吸附、柱层析、薄层层析、高效液相色谱等方法分离得到两个具有抑菌活性的化合物,分别命名为SPRI-231(Ⅰ)和SPRI-231(Ⅱ),经过UV、IR、ESI-MS、ESI-HRMS、元素分析等鉴定方法,判定其分子式分别为C51H91N3O21和C52H93N3O21。活体和离体生物活性测定发现其具有较好的抗菌效果,500 mg/L SPRI-231(Ⅱ)精提物对黄瓜灰霉病菌和黄瓜炭疽病菌的抑制率分别为83.02%和90.40%。     

Effects of the avermectin, MK-243, on ovary development and salivary gland degeneration in the ixodid tick, Amblyomma hebraeum

Injection of the avermectin analogue, MK-243, into engorged female Amblyomma hebraeum Koch resulted in reduced ovary weight, oocyte length, and ovary vitellin content. There was no significant reduction in hemolymph vitellogenin concentration in MK-243 treated ticks. Although MK-243 was previously shown to markedly reduce hemolymph 20E-concentration, injection of 20E, the vitellogenic hormone in this tick, did not reverse the effects of MK-243 on ovary development. These data suggest that MK-243 may exert its inhibition of egg development more at the level of vitellogenin uptake than vitellogenin synthesis. MK-243 also reversed salivary gland degeneration slightly, probably via its inhibitory effect on 20E-synthesis.     

Mechanism of action of clenpyrin on the cattle tick Boophilus microplus

Clenpyrin is effective against all stages of development of one-host cattle ticks (Boophilus spp.) including strains resistant to organophosphorus compounds and carbamates. Ticks treated with clenpyrin are quickly immobilised due to a slackening of the muscles. Manometric measurements revealed a marked depression in respiratory gas exchange and a rise in the R Q within 24 h after treatment. Clenpyrin inhibits NADH oxidation and, as a result, all synthesising processes in the tick. The breakdown of proteins taken up with the blood meal is also inhibited. Clenpyrin, however, does not interfere with carbohydrate degradation. These results have been corroborated in detail by determining various substances in treated and untreated ticks: proteins, haematin, guanine, glycogen, glucose, lactate, ATP, lipoids and cuticular wax. Clenpyrin does not inhibit tick proteinase, tick and other acetylcholinesterase, or rat monoamine oxidase; therefore, the mode of action of clenpyrin differs from that of other known tickicides, especially those involving the acetylcholinesterase system.     

Inhibition of oviposition in the cattle tick Boophilus microplus by certain acaricides

Doses of ten acaricides, ranging from 2.5 to 10 μg, induced about 10-60% mortality and inhibited oviposition in engorged Boophilus microplus as well as preventing larval hatching from the eggs. The effects of the acaricides were dose dependent. The efficacy of acaricides in reducing the reproductive potential (inhibition of oviposition × inhibition of hatching/100) was in the following order: dimethoate > dioxathion > naled > diazinon > chlordimeform > carbaryl > trichlorphon > phosphamidon > gamma-HCH > fenitrothion. Most of the acaricides also retarded the oviposition cycle, delaying the peak activity by 4-8 days. The ovarian development in control ticks reached its peak between the 5th and 7th days before decreasing gradually and ceasing completely between the 16-20th days after engorgement. The protein content of the ovaries and the rate of incorporation of [¹⁴C]glycine into the tissues followed a similar pattern. Carbaryl, fenitrothion and naled inhibited these activities. Chlordimeform stimulated [¹⁴C]glycine incorporation and increased the ovarian protein content up to the 13th day before resorption of the oocytes. The eggs from treated ticks were mostly non-viable and contained more protein; those from dioxathion, carbaryl and chlordimeform treatments had a higher dry weight than the control.     

The purification and characterization of esterases from insecticide-resistant and susceptible house flies

Four major esterases in one susceptible (CSMA) and two resistant (Hirokawa, E₁) house fly strains were separated by chromatofocusing. Of the four esterases, those with pI's of 5.1 and 5.3 accounted for 90% of the p-nitrophenyl butyrate hydrolyzing activity in the three house fly strains. They also accounted for 70% (Hirokawa, E₁) and 40% (CSMA) of the paraoxon-hydrolyzing activity as well as 87% (Hirokawa), 39% (E₁) and 66% (CSMA) of the malathion-hydrolyzing activity in microsomes as measured by esterase-antibody interaction. In the Hirokawa strain, the pI 5.1 esterase was the predominant esterase and was more active than that of the the CSMA strain. Different substrate specificities and a different K_m toward acetylthiocholine, as well as different rates of malathion and paraoxon hydrolysis between the Hirokawa and CSMA strains, suggest a qualitative difference in the pI 5.1 esterase. For the pI 5.1 esterase from the E₁ strain, a different substrate specificity, a different K_m for p-nitrophenyl butyrate, a different sensitivity to inhibitors, and a different rate of paraoxon hydrolysis suggest that it is a modified esterase. This esterase is not a phosphorotriester hydrolase, nor does it lack nonspecific esterase activity. It is a modified esterase which has a different substrate specificity when compared to the esterases from the other strains. The molecular weight of the esterases studied was approximately 220,000, with pH optima of about 7.0.The ratio of malathion α-monoacid to β-monoacid formation was about 9.0 for the pI 5.1 and 5.3 esterases and 1.5 for the pI 4.8 and 5.6 esterases. The existence of a higher $\text{α}\text{β}$ ratio for the pI 5.1 and 5.3 esterases and their significant rate of malathion hydrolysis in the Hirokawa strain indicate that an increase in the $\text{α}\text{β}$ ratio in house flies reported was due to the increase in the pI 5.1 esterase in the resistant strain.     

Molecular cloning, expression, and characterization of a phi-type glutathione S-transferase from Oryza sativa

The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its K_m value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.