Isolation and characterization of polymorphic microsatellite loci for the horn fly, Haematobia irritans (L.) (Diptera: Muscidae)

The horn fly, Haematobia irritans (L.) (Diptera: Muscidae), is a cosmopolitan livestock pest that has caused a great negative impact on the animal production sector throughout the world. Here, we describe 10 polymorphic microsatellite loci isolated from H. irritans. The number of alleles found ranged from two to eight per locus and the expected heterozygosity from 0.1421 to 0.7702. These loci are potentially useful for the fine-scale genetic characterization of horn fly populations and provide fundamental information for pest management and planning of control programs.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Susceptibility to diazinon in populations of the horn fly,Haematobia irritans (Diptera: Muscidae), in Central Brazil

From October 2000 to April 2001, insecticide bioassays were conducted in 18 ranches from 10 counties in the states of Mato Grosso and Mato Grosso do Sul, in Central Brazil. Horn flies from wild populations were exposed to diazinon-impregnated filter papers immediately after collection on cattle, and mortality was recorded after 2 h. A high susceptibility to diazinon was observed in all tested populations. The LC50s ranged from 0.15 to 0.64 micro g/cm2, and resistance ratios were always lower than one (ranging 0.1-0.6). Pyrethroid products, most applied by backpack sprayers, have been used since the horn fly entered the region, about 10 years ago. The high susceptibility observed to diazinon indicates that this insecticide (as probably other organophosphate insecticides) represents an useful tool for horn fly control and resistance management, particularly in pyrethroid-resistant populations.     

Ultraviolet irradiation of cattle in relation to diapause in the horn fly,Haematobia Irritans (L.) (Diptera: Muscidae)

It has been demonstrated that the percentage of diapause in the pupae of the horn fly, HAEMATOBIA IRRITANS,an external blood-sucking parasite, is reduced beyond the effect of photoperiod alone by exposure of host cattle to ultraviolet.

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Zusammenfassung Es wurde gezeigt, dass der Prozentsatz der in Diapause gehenden Puppen der Hornfliege, HAEMATOBIA IRRITANS,eines blutsaugenden Ektoparasiten des Rindes, über den Einfluss der Photoperiode hinaus durch UV-Exponierung des Wirt-Rindes vermindert ist.

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Resume On démontre ici que le taux de larves de la mouche des cornes,HAEMATOBIA IRRITANS, entrant en nymphose est diminué par une exposition de l'hÔte aux rayons ultra-violets et cela au-delà de l'influence exercée par la photo-période.La mouche des cornes est un parasite extérieur des bovins parasite, qui leur suce le sang.

     

Identification and characterization of a cDNA encoding the acetylcholinesterase of Haematobia irritans (L.) (Diptera: Muscidae).

A 2217-nucleotide cDNA presumptively encoding acetylcholinesterase (AChE) of the horn fly, Haematobia irritans (L.) was sequenced. The open reading frame (ORF) encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 95% identity and 98% similarity to the AChE of Musca domestica (L.). Structural features characteristic of the M. domestica and Drosophila melanogaster AChEs are conserved in the H. irritans AChE. The M. domestica and D. melanogaster AChEs are target sites for organophosphate inhibition as previously shown (Walsh et al. 2001. Biochem. J. 359: 175-181, Kozaki et al. 2002. Appl. Entomol. Zool. 37: 213-218), suggesting that this H. irritans AChE2 may be the target site for organophosphate.     

Dynamics of Haematobia irritans irritans (Diptera: Muscidae) infestation on Nelore cattle in the Pantanal, Brazil

From June 1993 to May 1995, horn fly counts were conducted twice a month on untreated Nelore cattle raised extensively in the Pantanal. Horn fly population showed a bimodal fluctuation and peaks were observed every year after the beginning (November/December) and at the end (May/June) of the rainy season, which coincided with mid-late spring and mid-late fall, respectively. Horn flies were present on cattle throughout the year in at least 64% of the animals. Mean horn fly numbers on animals did not exceed 85 flies/cow during peaks and were under 35 flies/cow in most of the remaining periods. The highest infestations (population peaks) were short and dropped suddenly within two weeks. Less than 15% of the animals in both herds could be considered as "fly-susceptible" - showing consistently higher infestations, or "fly-resistant" - showing consistently lower infestations.     

The role of HiTI, a serine protease inhibitor from Haematobia irritans irritans (Diptera: Muscidae) in the control of fly and bacterial proteases

Blood-sucking arthropods are vectors responsible for the transmission of several pathogens and parasites to vertebrate animals. The horn fly Haematobia irritans irritans (Diptera: Muscidae) and the tick Boophilus microplus are important hematophagous ectoparasites that cause losses in cattle production. A serine protease inhibitor from a thorax extract of the fly H. irritans irritans (HiTI) was previously isolated, characterized and cloned. In the present study we described the expression, purification, and characterization of the recombinant HiTI (rHiTI) and its possible role in the control of different endogenous and bacterial proteases. rHiTI was successfully expressed using the pPIC9 expression vector with a yield of 4.2 mg/L of active rHiTI. The recombinant HiTI purified by affinity chromatography on trypsin-Sepharose had a molecular mass of 6.53 kDa as determined by LS-ESI mass spectrometry and inhibition constants (Kis) similar to those of native HiTI for bovine trypsin and human neutrophil elastase of 0.4 and 1.0 nM, respectively. Purified rHiTI also showed inhibitory activity against the trypsin-like enzyme of H. i. irritans using its possible natural substrates, fibrinogen and hemoglobin; and also inhibited the OmpT endoprotease of Escherichia coli using fluorogenic substrates. The present results confirm that HiTI may play a role in the control of fly endogenous proteases but also suggest a role in the inhibition of pathogen proteases.     

Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae)

The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin-Sepharose has a molecular mass of 7029Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI-Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein.     

Azadirachtin as a larvicide against the horn fly, stable fly, and house fly (Diptera: Muscidae)

Effects of azadirachtin, a triterpenoid extracted from neem seed, Azadirachta indica A. Juss., were similar to those of insect growth regulators against the immature stages of the born fly, Haematobia irritans (L.), the stable fly, Stomoxys calcitrans (L.), and the house fly, Musca domestica L. When an ethanolic extract of ground seed was blended into cow manure, LC50 and LC90's for larval horn flies were 0.096 and 0.133 ppm azadirachtin, respectively. An emulsifiable concentrate (EC) had an LC50 for larval horn flies of 0.151 ppm and an LC90 of 0.268 ppm. For larval stable flies, the EC formulation had an LC50 of 7.7 ppm and an LC90 of 18.7 ppm azadirachtin in manure. Against larval house flies, the LC50 and LC90 were 10.5 and 20.2 ppm, respectively. When the EC formulation was administered orally to cattle at a rate of greater than or equal to 0.03 mg azadirachtin per kg of body weight per day or when ground neem seed was given as a daily supplement of greater than or equal to 10 mg seed per kg body weight, horn fly development in the manure was almost completely inhibited. In contrast, ground seed mixed in cattle feed at the rate of 100-400 mg seed per kg of body weight per day caused less than 50% inhibition of stable flies in the manure.     

Long-term study of Haematobia irritans (Diptera: Muscidae) seasonal distribution in central Argentina with focus on winter fly abundance.

The seasonal distribution of Haematobia irritans (Linnaeus, 1758) was evaluated at 31 degrees 12' S-61 degrees 29' W, Santa Fe, Argentina from November 1992 to August 2000 by weekly fly counts on 20 Holstein cows not treated against ectoparasites. The analysis was mainly focused on winter fly abundance. Two peaks of abundance were found from spring to autumn. Adults of H. irritans were consistently found on cattle during winter, with increasing numbers from the end of July to late August. The only climatic parameter soundly correlated with the four week period, before each winter fly count, was the mean air temperature with the exception of year 1998. No significant relationship was found between level of cow infestation and relative humidity, saturation deficit and rainfall. Horn fly infestation on cows was lower than 20% from end of June to end of August only in 1995, when mean air temperature was consistently lower than 11 degrees C during the four week period previous to most fly counts. Conversely, the mean temperature was higher than 12 degrees C previous to fly counts in 1998, when most cows remained infested. The results indicate that a proportion (unknown) of immature stages of H. irritans were insensitive to diapause inducing factors and developed through winter.     

Field Survey of Insecticide Resistance in Haematobia irritans exigua de Meijere (Diptera: Muscidae)

A survey of farms in northern New South Wales and southeastern, central, western and northern Queensland was conducted to determine levels of insecticide resistance in populations of buffalo fly Haematobia irritans exigua. A field bioassay using discriminating concentrations of 10 insecticides commonly used for buffalo fly control was used. Resistance to all synthetic pyrethroids tested (cypermethrin, deltamethrin, cyhalothrin, flumethrin and cyfluthrin) was common and widespread in coastal zones, but was lower in inland zones. In contrast, there was no resistance to the organophosphate diazinon and only low levels of resistance to ethion and chlorfenvinfos. Synergism between piperonyl butoxide and cypermethrin was demonstrated.     

Variation in general esterase activity within a population of Haematobia irritans (Diptera: Muscidae).

Control of the horn fly, Hematobia irritans (L.), is generally dependent on chemical insecticides. However, the biology and behavior of the horn fly favors rapid development of insecticide resistance. To prolong the effectiveness of the insecticide option, information is required regarding the mechanisms of insecticide resistance. Metabolic hydrolysis of insecticides by esterases is a detoxification mechanism in many insect species. Measurement of general esterase activity within populations of horn flies may provide a diagnostic tool for resistance management. In this study we evaluated the amount of variation in general esterase activity within female and male horn fly samples from a population that had not been exposed to insecticides for 8 yr. We found considerable variation in general esterase activity within samples of each sex, with females demonstrating the greater variation. The observed variation is thought to be the result of age-structure dynamics within the population. The amount of inherent variation makes it difficult to detect small mean differences between populations, thus limiting the utility of general esterase assays. Thus, effective diagnosis of esterase-mediated resistance mechanisms can only be achieved by the identification of specific detoxification esterases and the design of assays, either biochemical or molecular, for their detection and measurement.     

Dynamics and mechanisms of permethrin resistance in a field population of the horn fly, Haematobia irritans irritans

A study was conducted at the Pressler ranch, near Kerrville, Texas, USA between 2002 and 2006 to determine the dynamics and mechanisms of resistance to permethrin in a field population of the horn fly, Haematobia irritans irritans (L.). Changes of resistance to pyrethroid insecticide associated with use of a pour-on formulation of cyfluthrin in 2002 and use of diazinon ear tags in subsequent years were studied using a filter paper bioassay technique and a polymerase chain reaction assay that detects two sodium channel mutations, kdr and super-kdr resistance alleles. A maximum of 294-fold resistance to permethrin was observed in the summer of 2002. A significant decrease in the resistance level was observed in spring 2003, and resistance continued to decline after animals were treated with diazinon ear tags. In response to pyrethroid treatments, the allelic kdr and super-kdr frequency increased from 56.3% to 93.8% and from 7.5% to 43.8%, respectively in 2002, and decreased significantly in 2003 when the pyrethroid insecticide was no longer used to treat animals. Females were found to have a higher allelic super-kdr frequency than males in 2002, while no difference was detected between males and females in the allelic kdr frequency. There was a significant positive correlation between frequencies of the sodium channel mutations and levels of permethrin resistance, suggesting that the sodium channel mutations, kdr and super-kdr , are the major mechanisms of resistance to pyrethroids in this horn fly population. Results of synergist bioassays also indicated possible contributions of two metabolic detoxification mechanisms, the mixed function oxidases (MFO) and glutathione S-transferases (GST). Compared to a horn fly infestation of an untreated herd, treatments with the pyrethroid pour-on formulation failed to control horn flies at the Pressler ranch in 2002. Sustained control of horn flies was achieved with the use of diazinon ear tags in 2003 and subsequent years.     

Managing the horn fly (Diptera: Muscidae) using an electric walk-through fly trap

An electric walk-through fly trap was evaluated for the management of the horn fly, Hematobia irritans (L.), on dairy cattle in North Carolina over 2 yr. The trap relies on black lights and electrocution grids to attract and kill flies that are brushed from the cattle passing through. During the first season, horn fly densities were reduced from >1,400 to <200 flies per animal. Horn fly density averaged 269.2 +/- 25.8 on cattle using the walk-through fly trap twice daily, and 400.2 +/- 43.5 on the control group during the first year. The second year, seasonal mean horn fly density was 177.3 +/- 10.8 on cattle using the walk-through fly trap compared with 321.1 +/- 15.8 on the control group. No insecticides were used to control horn flies during this 2-yr study.     

Effect of horn fly (Haematobia irritans) control on growth and reproduction of beef heifers

The influence of horn fly control with commercially available ear tags was studied on beef replacement heifers (n = 670) for growth and reproductive performance. The study was conducted at five sites in Louisiana over 3 yr. Heifers used were yearling replacement females that were exposed to fertile bulls during a limited spring breeding season that coincided with the horn fly season. In mid to late May of each year, heifers were randomly assigned to one of two horn fly treatments: untreated and treated for horn fly control. The trial continued each year until September or October at the end of fly season. Pregnancy status was determined by rectal palpation. Horn fly populations were controlled on the treated heifers at moderate levels (84%). Total weight gain of treated heifers was 14% greater than for untreated heifers. Horn fly treatment had no effect on pregnancy rate (78% and 75% for untreated and treated heifers, respectively). Treatment differences for weight gain were of greater magnitude for heifers failing to conceive than for heifers that became pregnant. Weight gains of nonpregnant treated heifers were 33% greater than for nonpregnant untreated heifers, whereas weight gains of pregnant treated heifers were 8% greater than for pregnant untreated heifers. In conclusion, horn fly control on yearling beef replacement heifers improved weight gain but had no effect on first exposure reproduction.     

骆驼斯氏副柔线虫病传播媒介西方角蝇和截脉角蝇的幼虫龄期划分

【目的】本研究旨在找出区分西方角蝇Haematobia irritans和截脉角蝇H.titillans幼虫龄期划分标准,为准确鉴定两种角蝇各龄幼虫,研究斯氏副柔线虫在角蝇体内的发育过程,以及制定防控骆驼斯氏副柔线虫病的有效措施等奠定基础。【方法】采用实验室人工孵育两种角蝇幼虫的方法,分别测量不同发育阶段幼虫的虫体长、咽骨体长和咽骨体宽3项指标,利用SPSS Statistics 19.0统计软件对数据进行处理,结合Crosby生长法则和线性回归的方法进行分析,比较两种角蝇幼虫之间差异,以确定两种角蝇幼虫最佳龄期划分标准。【结果】结果表明,两种角蝇的幼虫均分为3龄,咽骨体是两种角蝇幼虫龄期划分的特征性结构,两种角蝇各龄幼虫相同指标的测量值随龄期的增长呈现出相同的增长规律。咽骨体长是划分两种角蝇幼虫龄期的最佳测量指标,咽骨体宽可作为分龄的辅助指标;两种角蝇相邻龄期幼虫的体长变化范围存在相互重叠,不能准确划分角蝇幼虫龄期。【结论】研究表明通过西方角蝇和截脉角蝇幼虫咽骨体的形态特征可简便、快速和准确地鉴定两种角蝇幼虫的龄期。