Treated Filter Paper Bioassay for Detection of Insecticide Resistance in Haematobia irritans exigua de Meijere (Diptera: Muscidae)

This paper reports on the standardisation of an assay, whereby buffalo flies Haematobia irritans exigua de Meijere from a laboratory population which had not been exposed to insecticides for 8 years were confined in Petri dishes containing filter paper treated with known concentrations of insecticide. Mortality was assessed after 2 and 4 h and mean LC₅₀ and LC₉₉ values for each of 11 insecticides were calculated. the bioassay technique had acceptable repeatability and was simple to perform. Field populations of northern Queensland buffalo fly were assayed using the technique and were found to have high levels of pyrethroid resistance and little or no organophosphate resistance.     

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.     

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.     

Pathogenicity of three formulations of entomopathogenic fungi for control of adult Haematobia irritans (Diptera: Muscidae)

Powder formulations of three species of entomopathogenic fungi were evaluated for their pathogenic effect upon adult horn flies, Hematobia irritans (L.) (Diptera: Muscidae). Flies were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. (strain GHA), Metarhizium anisopliae (Metschnikoff) Sorokin (strain ESCI), and Paecilomyces fimosoroseus (Wize) Brown & Smith (strain ARSEF 3581) in the laboratory. At 4 d postexposure, flies treated with B. bassiana had an average of 98.4% mortality versus 43.5% from treatment with M. anisopliae and 13.0% from treatment with P. fiimosoroseus. At 7 d postexposure, flies treated with B. bassiana had an average of 100.0% mortality compared with 73.0% from treatment with M. anisopliae and 33.3% from treatment with P.fumosoroseus. Mean lethal time (LT50) was 2.70, 4.98, and 7.97 d for B. bassiana, M. anisopliae, and P. fiumosoroseus, respectively. Entomopathogenic fungi such as B. bassiana and M. anisopliae may have the potential for controlling populations of horn flies. These studies indicate that B. bassiana and M. anisopliae were not only pathogenic to adult horn flies, but they caused mortality in a short time.     

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.     

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 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.     

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.     

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.     

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.     

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.     

Chemical composition and fumigant toxicity of the essential oils from 16 species of Eucalyptus against Haematobia irritans (Diptera: Muscidae) adults

Oils extracted from various species of Eucalyptus (Eucalyptus badjensis Beuzev & Welch, Eucalyptus badjensis x Eucalyptus nitens, Eucalyptus benthamii variety dorrigoensis Maiden & Cambage, Eucalyptus botryoides Smith, Eucalyptus dalrympleana Maiden, Eucalyptus fastigata Deane & Maiden, Eucalyptus nobilis L.A.S. Johnson & K. D. Hill, Eucalyptus polybractea R. Baker, Eucalyptus radiata ssp. radiata Sieber ex Spreng, Eucalyptus resinifera Smith, Eucalyptus robertsonii Blakely, Eucalyptus rubida Deane & Maiden, Eucalyptus smithii R. Baker, Eucalyptus elata Dehnh, Eucalyptus fraxinoides Deane & Maiden, E. obliqua L'Hér) were obtained by hydrodistillation. The chemical composition of essential oils was determined by gas chromatography coupled to mass spectrometry. Essential oils were mainly composed of 1,8-cineole, alpha-pinene, alpha-terpineol, 4-terpineol, and p-cymene. Vapors from these essential oils and their major components were found to be toxic to Haematobia irritans (L.) (Diptera: Muscidae) adults. An aliquot of each oil was placed in a cylindrical test chamber, and the number of knocked down flies was recorded as a function of time. Knockdown time 50% was then calculated. Results showed that essential oil of E. polybractea had the highest knockdown activity of 3.44 min. A correlation was observed between the content of 1,8-cineole in the Eucalyptus essential oils and the corresponding toxic effect.     

Evaluation of phloxine B as a photoinsecticide on immature stages of the horn fly, Haematobia irritans (L.) (Diptera: Muscidae)

The use of photoactive substances for controlling adult or immature stages of insect pests is an attractive alternative to chemical insecticides. Phloxine B is an environmentally friendly xanthene derivative that is safe for mammals but toxic for dipterans. In this study we tested the effect of phloxine B as a phototoxic larvicide against immature stages of the blood-sucking horn fly, Haematobia irritans (L.). The mortality rate of phloxine B was very low in the dark during the larval stage (100 h) unless a 0.5-mM dye concentration was used. However, a high mortality rate was attained when larvae III were transferred to containers exposed to 5000 lux during the last 2 h before pupariation. This was concentration-dependent up to 0.1-mM phloxine B. After a 2-h larval exposure to light the phloxine B 50% lethal concentration was 0.043 mM. These results indicate that H . irritans larvae are very sensitive to this dye, which in turn seems a promising component for larvicide formulations to control horn flies.     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.