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.     

Parasitism of Heterotylenchus autumnalis Nickle (Nematoda: Sphaerulariidae) to the Face Fly,Musca autumnalis De Geer (Diptera: Muscidae)

New information is reported on the parasitism of Heterotylenchus autumnalis upon its principal known host, Musca autumnalis. Black to brown spots are produced on the cuticle of all infected host larvae where the nematode penetrated. The principal damage to the host is castration of the female. In laboratory tests nematode larvae were not infective and did not leave the hosts before the female fly was 1 1 days old. Nematode larvae removed from infected male flies infected other hosts, but it is believed that in nature these larvae are unable to leave the host.     

Infectivity of two members of theEntomophthora muscae complex [Zygomycetes: Entomophthorales] forMusca domestica [Dipt.: Muscidae]

Dose-mortality studies were conducted with 2 members of theEntomophthora muscae (Cohn) Fresenius complex from southern California (CA) and Denmark (DA) infecting house flies,Musca domestica L., from southern California. Primary conidia of the DA form were significantly more infective (LC₅₀=34 conidia/mm²) than primary conidia of the CA form (LC₅₀=67 conidia/mm²) for 2–7 days old (‘young’) flies. Infectivity (single experimental trial) for 10–14 day old (‘old’) flies of primary conidia of the CA form (LC₅₀=34 conidia/mm²) was similar to that of the DA form for young flies. Secondary conidia of the CA form were markedly more infective (LC₅₀=0.36 conidia/mm²) than primary conidia of either form. Dose had no significant effect on incubation period for primary conidia of either form, but increased doses resulted in significantly shorter incubation periods for flies exposed to secondary conidia of the CA form.      

Accessibility of blood affects the attractiveness of cattle to horn flies

The burden of infestation of the horn fly, Haematobia irritans (Linnaeus) (Diptera: Muscidae), differs among bovines within the same herd. We hypothesized that these differences might be related to the epidermal thickness of the cattle and the blood intake capacity of the fly. Results showed that dark animals carried more flies and had a thinner epidermis than light‐coloured animals, which was consistent with the greater haemoglobin content found in flies caught on darker cattle. Similarly, epidermal thickness increased with body weight, whereas haemoglobin content decreased. Overall, we suggest that accessibility of blood is a factor that partially explains cattle attractiveness to flies.     

The control of a fly nuisance problem in a woodland area adjacent to Rutland Water

The proposed development of a caravan park in Burley Wood was postponed in 1979 due to the presence of Hydrotaea irritans (Fall.). This species causes intense irritation to man by feeding upon skin secretions and is also of veterinary importance. The fly population was monitored for 10 weeks using a number of trapping methods. The efficiency of one of these, the malaise trap, was found to be greatly increased by the controlled release of carbon dioxide. Used together with habitat modification and locally applied insecticides and repellants this is considered potentially valuable in ameliorating the problem. It is likely that the development of Rutland Water will increase the local range of the fly.     

The use of visual information by house flies,Musca domestica (Diptera: Muscidae), foraging in resource patches

Summary House flies, Musca domestica, respond to visual contrasts on the substrate if a resource is associated with the contrasting patterns. Visible resource patch boundaries serve as a signal to flies that they are about to leave a rewarding patch. Searching flies respond to such visual information by walking along the resource patch boundary and turning back into the patch at its edge. This edge detection and response serve as a mechanism for flies with visual cues to stay in a rewarding patch and locate more resources within it. The intensity of their response correlates with the quality of the resource. In the absence of visual cues, patch shape affects foraging success; flies find more resources in circular than in linear resource distributions. The effects of visual cues, however, render patch shape unimportant. Various substrate contrasts are effective as resource information for flies: dark (e.g., green) figures on bright (e.g., white) backgrounds or bright figures on dark backgrounds. Responses to substrate contrasts measured in this study indicate that, over the short term, house flies can learn a visual cue associated with a food source.     

Mites of the familyMacrochelidae as predators of two species of dung-breeding pest flies

Nine species of macrochelid mites [Macrocheles “glaber” (Müller),M. aestivus Halliday,M. mammifer Berlese,M. peniculatus Berlese,M. robustulus (Berlese),M. merdarius (Berlese),M. peregrinus Krantz,M. eurygaster Krantz,Glyptholaspis confusa (Foà) were tested in the laboratory as predators of 2 species of dung-breeding pest flies (Musca vetustissima Walker,Haematobia irritans exigua de Meijere). The mites varied in their ability to attack the 2 fly species at both egg and larval stages. larger mites were generally more effective predators than the smaller species. The level of predation imposed byM. “glaber” onMusca vetustissima also varied with the developmental stage of both predator and prey.      

Estimation of the effects of buffalo fly (Haematobia irritans exigua) on the milk production of dairy cattle based on a meta-analysis of literature data

Published reports on the effect of buffalo fly Haematobia irritans exigua De Meijere (Diptera: Muscidae) and the closely related horn fly (H. irritans) were examined and analysed using non-linear weighted regression techniques in an attempt to establish the relationship between daily production loss (D), average number of parasites (n) and the average damage per parasite per day (d), and to provide estimates of expected losses in milk yield (MYD) and live-weight gain (LWG) in dairy cattle. A Mitscherlich three-parameter model was used to explain the relationship between the total loss of production attributable to buffalo flies and the average number of flies associated with cattle. This model was significant (P<0.01), with R2 = 20.2% and predicted a threshold number of flies (n = 30) below which no adverse effects would be noted. At a moderate level of infestation (n = 200) dMYD was 2.6 ml/fly/day and dLWG was 0.14 g/fly/day, resulting in estimated daily losses in milk yield (D(MYD)) and live-weight gain (D(LWG)) of 520 ml and 28 g, respectively.     

The role of volatile semiochemicals in mediating host location and selection by nuisance and disease-transmitting cattle flies

The role of volatile semiochemicals in mediating the location and selection within herds of Holstein-Friesian heifers by nuisance and disease-transmitting cattle flies was investigated using coupled gas chromatography-electrophysiology (GC-EAG), coupled gas chromatography-mass spectrometry (GC-MS), electrophysiology (EAG), laboratory behaviour and field studies. Using volatile extracts collected by air entrainment from heifers in the Netherlands, a number of active peaks were located by coupled GC-EAG for Musca autumnalis (de Geer) (Diptera: Muscidae) and Haematobia irritans (L.) (Diptera: Muscidae). Volatile samples were also collected from two heifers in Denmark shown in previous counting experiments to differ significantly in their fly loads. Coupled GC-EAG using Ha. irritans antennae revealed differences in the EAG response to the samples, with additional EAG activity in the sample collected from the heifer with the lower fly load. To identify more EAG active compounds, volatiles were also collected from 48-h-old urine by air entrainment. In total, 23 compounds were located and identified by coupled GC-EAG and GC-MS. Further electrophysiological testing of these compounds with five fly species [M. autumnalis, Ha. irritans, Hydrotaea irritans (L.) (Diptera: Muscidae), Stomoxys calcitrans (L.) (Diptera: Musicidae) and Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae)] showed that only some of the compounds were physiologically active across the range of flies tested. These included 1-octen-3-ol, 6-methyl-5-hepten-2-one, (Z)-3-hexen-1-ol, naphthalene, and all EAG active compounds identified from urine. Compounds showing significant EAG activity were tested for behavioural activity using a wind-tunnel designed for measuring upwind flight behaviour. At certain concentrations, 1-octen-3-ol, 6-methyl-5-hepten-2-one and 3-octanol increased upwind flight, whereas naphthalene, propyl butanoate and linalool reduced upwind flight. In field studies using small herds of heifers ranked according to their fly load, individual slow-release formulations of 1-octen-3-ol and 6-methyl-5-hepten-2-one, when applied to low and high fly loading heifers, reduced fly loads on these individuals. This study provides evidence for the hypothesis that the natural differential attractiveness within herds of Holstein-Freisian heifers, i.e. a single host species, for cattle flies is partly due to differences in volatile semiochemicals emitted from the host. It is suggested that this phenomenon applies to other vertebrate host species and their associated insect pests.     

Control of the buffalo fly Haematobia irritans exigua in India, using the pyrethroids deltamethrin and fenvalerate

The effectiveness of two pyrethroids, fenvalerate and deltamethrin, against the fly Haematobia irritans exigua de Meijere (Diptera: Muscidae) on buffalo was considered in a field trial. Fenvalerate provided 100% control for 1, 2 and 4 weeks at concentrations of 0.03, 0.04 and 0.05%, respectively. Concentrations of 0.01 and 0.02% were less effective. One hundred percent control of this fly was obtained with deltamethrin for 2, 3 and 6 weeks at concentrations of 0.003, 0.004 and 0.005%, respectively. Deltamethrin concentrations of 0.001% and 0.002% achieved fly control for only 1-2 weeks, respectively.     

Molecular characterization,relatedness of <Emphasis Type="Italic">Haematobia irritans</Emphasis> (horn fly) populations,by RAPD-PCR

Haematobia irritans is a hematophagous parasite of cattle that causes significant economic losses in many parts of the world, including Brazil. In the present work, one American, four Brazilian populations of this species were studied by Random Amplified Polymorpht DNA (RAPD) to assess basically genetic variability within and between populations. Ten different decamer random primers were employed in the genomic DNA amplification, yielding 117 fragments in the five H. irritans populations. In Drosophila prosaltans, used as an outgroup, 81 fragments were produced. Forty-three of these fragments were shared by both species. Among the H. irritans samples, that from Rio Branco (Acre State, Brazil) produced the smallest numbers of fragments, polymorphic bands. This high genetic homogenity may be ascribed to its geographic origin (in the Northwest of Brazil), which causes high isolation, low gene flow, unlike the other Brazilian populations, from the South Central region, in which cattle trade is very intensive. Marker fragments (exclusive bands) detected in every sample enabled the population origin to be characterized, but they are also potentially useful for further approaches such as the putative origin of Brazilian populations from North America. Similarity indices [Nei & Li, 1979, Proc. Natl. Acad. Sci. USA 76: 5269–5273], phylogenetic trees, rooted by using the outgroup, produced by the Phylogenetic Analysis using Parsimony (PAUP 4.0-Swofford, 2001) program showed the closest relationships between flies from São José do Rio Preto, Turiúba (both from São Paulo State, Brazil) while flies from the geographically distant Rio Branco showed the greatest differentiation relative to the others.     

Studies on the possible role of cattle nuisance flies, especially Hydrotaea irritans, in the transmission of summer mastitis in Denmark

The summer mastitis pathogens Actinomyces pyogenes, Peptococcus indolicus, Bacteroides melaninogenicus ss. levii, Fusobacterium necrophorum and Streptococcus dysgalactiae were isolated from the polyphagous symbovine dipterans Hydrotaea irritans (Fallén) and Morellia sp. caught around dairy heifers on pasture, but not from the haematophagous species Haematobia irritans (L.), Haematobosca stimulans (Meigen), Culicoides sp. and Simulium sp. Secretions from clinical cases of summer mastitis proved to be sources of summer mastitis bacteria for more than 3 weeks despite antibiotic treatment and teat amputation. Taking into account the seasonal activity pattern of Hydrotaea irritans and its topographical distribution on grazing cattle, it appears evident that this fly may play a central role in the establishment and maintenance of the bacterial contamination with summer mastitis pathogens on the teats of healthy cattle. In the present study the survival of A.pyogenes and P.indolicus for 7 days in experimentally infected Hydrotaea irritans, as demonstrated by the recovery of these microorganisms from agar plates exposed to live infected flies, is described. However, experimental transmission of summer mastitis from sick to healthy heifers by Hydrotaea irritans proved unsuccessful.     

Laboratory studies of host finding,acceptance and suitability of the dung-breeding fly,Haematobia thirouxi potans [Dipt.: Muscidae], byAleochara sp. [Col.: Staphylinidae]

Aleochara sp. from southern Africa is a potential candidate for introduction to Australia for control of the Australian buffalo fly,Haematobia irritans exigua De Meijere. Aspects of its host searching and acceptance behaviour were studied in South Africa usingHaematobia thirouxi potans (Bezzi), to assess, in part, its potential value as a biological control agent and to provide a basis for the development of a mass rearing technique. First instars ofAleochara sp. were found to parasitize most effectively those hosts that had buried themselves in the substrate, probably by orienting to a chemical cue and using the tunnels made by the post-feeding larvae. Lower rates of parasitization occurred in pupae placed on the surface of the substrate or buried by the experimenters. They would accept, parasitize and develop on all ages ofH. thirouxi potans pupae, but showed reduced survival in the very oldest ones. Although there was no difference in relative acceptability among hosts of different ages, a higher proportion of younger hosts were parasitized when these had buried themselves, probably due to a tendency of the parasitoids to follow fresher tunnels into the substrate. Survival was high (>80%) at relative humidities from 10–91% but was less than 20% at an R.H. of 100%. In rearing trials, the most economical ratio of parasitoid eggs: hosts was 1∶1.5, with 70% of the parasitoids developing successfully to adults.      

Partial strategic tick control within a herd of European breed cattle in the state of Rio Grande do Sul,southern Brazil

A trial is described, in the state of Rio Grande do Sul, southern Brazil, as one of a series suggested to investigate the effects of strategic but selective acaricide treatments of cattle within herds against Boophilus microplus. They are aimed at considering the repercussions of farmer attempts at immediate reductions in acaricide costs and the potential for creation of ‘refugia’ of untreated ticks. Half (Group 1) of a small experimental herd of European breed heifers were treated strategically against ticks, three times during the late spring–early summer and twice during autumn (southern hemisphere), with an injectable avermectin endectocide, designed to act directly against the first and third generations of parasitic B. microplus per ‘cattle tick year’ at this site, respectively. The consequent levels of infestations on all of the member cattle in their common pasture were monitored. Group 1 showed low to zero tick counts during the 28-day treatment interval periods and up to ca. 14 days after the last of such a series. Treated cattle, however, became re-infested outside of these periods and to levels that would be considered as unacceptable by farmers in the state. The untreated cattle (Group 2) showed infestations at generally higher levels, than their contemporaries, within and outside of the treatment periods. There were thus ample sources of larvae in the pasture, derived principally from falling, untreated engorged female ticks, re-infesting both the treated and untreated cattle. Advantages of maintaining chemically untreated cattle ticks within a herd, compared to their disadvantages as contaminants to classical strategic control procedures, merit re-evaluation, especially in relation to the recent, world-wide resurgence of acaricide resistance in B. microplus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transmission of MdSGHV among adult house flies, Musca domestica (Diptera: Muscidae), occurs via oral secretions and excreta

The MdSGHV is a double-stranded DNA virus that replicates in the salivary glands of infected adult house flies. Transmission of this non-occluded, enveloped virus is believed to be mediated orally via deposition and consumption of oral secretions composed of salivary gland secretions and crop contents. In this study, transmission electron micrographs of crops from infected flies showed numerous enveloped virions in the crop lumen adjacent to the cuticular intima, as well as on the hemocoel side in close vicinity to muscle cells. Oral treatments of newly emerged flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44% infection. Virus released via oral secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66% infection. Using quantitative real-time PCR, MdSGHV DNA was quantified in oral secretions and excreta obtained from viremic flies. Between 2 and 4 days post-infection (dpi), viral copy numbers in oral secretions increased exponentially and from 5 to 21 dpi each infected fly released an average 10⁶ MdSGHV copies per feeding event. Excreta samples collected overnight from individual infected flies at 5 dpi contained an average 6.5 × 10⁵ viral copies. Low but detectable infection rates were produced when newly emerged flies were challenged with excreta samples. In summary, evaluation of the quantity and infectivity of MdSGHV released by individual infected house flies clearly showed that deposition of oral secretions and excreta onto a shared food substrate is the main route of natural MdSGHV transmission among adult house flies.     

Additions and corrections to “The Muscidae of Central Europe” II. Two new species of Spilogona (Diptera, Muscidae)

Two species of Spilogona, S. angustigena sp. n. and S. tatrica sp. n., are described from the Czech Republic and Slovakia, respectively. Both species are characterised by a gynaecomorphic shape of the male head, i.e., by a broad frons and the presence of a pair of orbital setae. Three species with this type of head are already known in the Palaearctic region: S. karelica (Tiensuu, 1935) from Russian Karelia, S. lapponica (Ringdahl, 1932) occurring in Norway and Sweden and redescribed here from material collected recently in Russia and Sweden, and S. spinicosta (Stein, 1907) from Tibet. All five species are compared and a new identification key is given. Apart from the external similarity of the male heads, the species under study are hardly closely related and their dichoptic heads may be explained as a homoplasy among similar ecomorpha.     

Evaluating biological control of fire ants using phorid flies: effects on competitive interactions

The effects of the parasitic phorid fly, Pseudacteon tricuspis Borgmeier, on the competitive interactions between the red imported fire ant, Solenopsis invicta Buren, and a native North American ant, Forelius pruinosus (Roger), were investigated in the laboratory. P. tricuspis is a highly host-specific endoparasitoid of S. invicta workers that is currently being reared and released as a biological control agent of S. invicta in the US. We tested the effect of P. tricuspis on the colony growth rate of S. invicta when S. invicta was forced to compete with F. pruinosus for a protein resource (freeze-killed crickets) in laboratory competition arenas. In addition to colony growth rate, we quantified the effect of the phorid flies on the foraging rate of S. invicta. Though S. invicta significantly reduced its foraging rate in the presence of the phorid flies, we did not detect an effect of the flies on colony growth rate. Possible explanations for these results include behavioral compensation by S. invicta for the presence of the flies. We present these laboratory results in light of a literature search indicating that laboratory tests of biological control agent efficacy are good predictors of field efficacy. We conclude that P. tricuspis alone is unlikely to suppress S. invicta populations in the field by reducing their competitive ability.     

Variation in the load of the horn fly, Haematobia irritans, in cattle herds is determined by the presence or absence of individual heifers

The distribution of horn flies, Haematobia irritans (L.) (Diptera: Muscidae), in herds of Danish Holstein-Friesian cattle was investigated in two studies conducted during two field seasons. In the first study, highly significant differences in fly distribution between the most and the least fly-susceptible heifers were observed. In one herd, the mean difference between the most fly-susceptible and the most fly-resistant heifers was 268 Ha. irritans specimens. The highest ratio between upper and lower mean fly number was 64.1:1, whereas the lowest was 3.1:1. In the second year, it was demonstrated that the heifers kept their rank in fly attraction over time. The trial clearly demonstrated that some heifers were attracting flies, whereas others, even in the same herd, only carried a few. In the second study, heifers were moved in and out of herds in an attempt to manipulate fly loads in the herds. In year 1, one herd (herd A) received four fly-resistant heifers from another herd (herd B), resulting in a drop in the mean number of flies, whereas herd B received four fly-susceptible heifers from herd A, resulting in an elevation of the mean number of flies. In year 2, a similar pattern emerged using herds C and D, and when the cattle were later returned to their original herds, the fly loads returned to their original distribution. The data presented here show unequivocally that, for horn flies, there can be considerable differences in fly loads for individual heifers within the Holstein-Friesian breed. Furthermore, the overall fly load within herds can be manipulated, and can be reversed. Thus, the distribution in the number of flies within a herd appears to depend on the number of fly-resistant or fly-susceptible heifers. The possible role of chemical factors emitted by heifers, i.e. volatile semiochemicals, in determining differences in fly loads is discussed, whereby attractants are emitted by fly-susceptible heifers and enable flies to locate their host, and repellents are emitted by fly-resistant heifers such that the flies are actively repelled from the herd.     

Revision of the key characters for the Thricops nigrifrons species-group (Diptera, Muscidae)

An analysis of key characters for the separation of Thricops nigrifrons and Thricops longipes (Diptera, Muscidae) is given. A revised key for Thricops nigrifrons and related species, including two species recently described from the Caucasus, is proposed.