Toxicological And Physiological Responses Of Amblyomma Americamim To Pesticides And Cloning And Characterization Of A Male-speciifc Antimicrobial Peptide From Haemaphysalis Longicornis

Ticks are obligate ectoparasite living by blood of mammals, birds and reptilia,transmit more pathogenic microorganisms than any other arthropod vectors, and areamong the most important vectors of pathogens. On global basis, economic lostcaused by tick and tick-borne diseases is almost billions of dollars. In the post twodecades, tick-borne diseases had increased incidence of and are becoming one of thefocus of public health problem. Unremitting work is making against ticks. In thecurrent study, toxicological, physiological, neurophysiological and molecularbiological research, using Amblyomma americanum and Haemaphysalis longicornis,was made to （1） test the anti-tick activities of three essential oils,（2） investigatephysiological response of the poisoned ticks and the effects of chemical pesticideson neurophysiology and （3） explore the innate immunity of ticks. The results will besignificant for acaricidal research, further understand the ultimate causes of pesticideintoxication and explore the interaction of tick and pathogens and the adaptationmechanism of ticks. Major results of the current study were as followed:1. Toxicological efficacy of essential oils from Lippia graveolens, Pimentadioica, Nepeta cataria L. was tested against A. americanum nymphs. All the threeessential oils showed lethal toxicity. Especially after24h of treatment, highmortalities were observed. LC50and the relationship between dose and mortality of3and6h post-treatment revealed that toxicity of Lippia graveolens was significantlyhigh than Pimenta dioica and Nepeta cataria L. on same time point, toxicity of thelatter two didn’t differ significant.In order to provide basis for subsequent work, the lethal and sublethal dose oftwo chemical pesticides, permethrin and amitraz, against A. americanum females,were also toxicologically tested. The lethal and sublethal doses were0.010%、0.005%and16μg μl^-1、8μg μl^-1for permethrin and amitraz, respectively.2. Changes in the gas exchange and water balance of lone star tick, A.americanum poisoned by permethrin and amitraz, were examined using flow-through respirometry. All control ticks treated by nothing or acetone exhibiteddistinct discontinuous gas exchange cycle （DGC）. The pattern of permethrin-treatedticks was characterized by impetuous ventilation followed by highly frequent DGC;while the ventilation of amitraz and permethrin+amitraz （mixture）-treated ticksremained apparently chaotic. Standard metabolic rates of untreated andacetone-treated ticks were0.461and0.452μl h^-1, respectively, and didn’t differsignificantly from each other （p>0.05）. Acaricides-treated ticks exhibitedsignificant increasing in SMR (p <0.05;1.054,1.392and1.520μl h^-1for permethrin,amitraz and mixture-treated ticks, respectively). Permethrin also induced obviouswater loss in ticks. A mass of water lost in the first1-2h post-treatment wasobserved and coincidence between water loss and CO2emission was foundespecially during DGC periods. In contrast to permethrin-treated ticks, no matchbetween water loss and gas exchange was found in mixture-treated ticks, but greatwater loss was demonstrated throughout the recordings. No obvious water loss wasobserved in control and amitraz-treated ticks. The results suggested that permethrinupset the water maintenance mechanisms of ticks; amitraz overexcited the spiracularcontrolling mechanisms; and thereby, the mixture of permethrin and amitraz resultedin chaotic ventilation and great water loss.3. The activity of leg motor of A. americanum, before and after treated bypermethrin, amitraz and their mixture, was recorded via electrophysiologicaltechnique. Two kinds of spike were detected and were termed as S1and S2. Normalticks show distinct pattern, S1burst was clear and the two kinds of spike differedobviously. Various signals were detected in permethrin, amitraz and mixture treatedticks. Permethrin-treated ticks exhibited frequent and prolonged burst, the burst ofamitraz-treated ticks became disordered generally after treatment and lost the patternfinally. Mixture-treated ticks showed both response of permethrin and amitraz singletreated ticks and the response of1h post-treatment suggested that the nervous systemwas paralyzed.4. A novel gene was identified from a cDNA library of the male accessoryglands of Haemaphysalis longicornis. The full-length cDNA of the gene was349bp, encoding a79-amino acid defensin-like protein and therefore the protein was namedas HlMS-defensin. Reverse transcriptase-PCR results suggested that this gene wasexpressed exclusively in male ticks. The tissue expression pattern and the mRNAlevels of HlMS-defensin during blood feeding were determined using real-time PCR.HlMS-defensin was expressed predominately in the male accessory gland and wasup-regulated during blood feeding. The antimicrobial activity of a synthetic peptidebased on the predicted mature portion of HlMS-defensin was examined against avariety of Gram-positive and Gram-negative bacteria and fungi. It appears that ticksuse the antimicrobial peptide to protect their reproductive tracts from microbialinfections.