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.     

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.      

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.     

Isolation and characterization of a trypsin-like enzyme from the buffalo fly, Haematobia irritans exigua

Abstract. The incorporation of soybean trypsin inhibitor (SBTI) into the diet of the buffalo fly, Haematobia irritans exigua (De Meijere), results in increased mortality and reduced fecundity. A trypsin-like enzyme which binds to SBTI was isolated by affinity chromatography on a Sepharose-SBTI column followed by ion-exchange chromatography. The enzyme was inhibited by benzamidine, phenylmethylsulfonyl fluoride, ovomucoid, leupeptin and a-2 macroglobulin. The enzyme was not inhibited by EDTA or p-chloromecuribenzoic acid and had a broad pH optimum of pH 7–9. Vaccination of sheep produced antibodies specific for the trypsin-like enzyme which inhibited enzyme activity in vitro but did not affect the survival of flies maintained in in vitro culture.     

Acetylcholinesterase from the horn fly (Diptera: Muscidae) II: Biochemical and molecular properties

Purified acetylcholinesterase (AChE) of the horn fly was characterized to elucidate the enzymological, inhibitory, and molecular properties of the enzyme. Maximum activity of the AChE against the substrate acetylthiocholine (ATCh) occurred when reactions were conducted at 37°C and pH 7.5. Km and V_max values were (9.2 ± 0.35) × 10^?6 M and 239.8 ± 10.8 units/mg, respectively, for ATCh and (1.5 ± 0.07) × 10^?5 M and 138.5 ± 5.5 units/mg, respectively, for butyrylthiocholine (BTCh). The activity of AChE decreased when concentrations of ATCh or BTCh were higher than 1 mM. Studies of the interaction of AChE with different inhibitors revealed pl₅₀ values of 8.88 for eserine, 6.90 for BW284C51, and 4.97 for ethopropazine. Bimolecular reaction constants (k_is) for the organophosphorus (OP) anticholinesterases were (2.74 ± 0.14) × 10⁶ M^?1 min^?1 for coroxon, (7.20 ± 0.28) × 10⁵ M^?1 min^?1 for paraoxon, and (2.33 ± 0.12) × 10⁵ M^?1 min^?1 for stirofos. Two major forms of native AChE molecules were found on non-denaturing polyacrylamide gel electrophoresis (PAGE) with Triton X-100, corresponding to bands AChE-2 and AChE-4 found on PAGE without Triton X-100. AChE-2 had an estimated molecular weight of 603,000 and was amphiphilic. AChE-4 had a molecular weight of 147,000 and was hydrophilic. Results of PAGE analyses indicated that the purified enzyme had two bands, one of about 123 kDa and the other greater than 320 kDa, prior to disulfide reduction and only one band at about 54 kDa after reduction on SDS-PAGE. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Dynamics of cypermethrin resistance in the field in the horn fly,Haematobia irritans

The toxicity of cypermethrin to the horn fly Haematobia irritans (L.) (Diptera: Muscidae) was determined for samples collected from untreated herds at a farm in central Argentina from October 1997 to May 2001. Field tests of the efficacy of cypermethrin against horn flies were first carried out at this farm in 1993, when the fly was shown to be susceptible to pyrethroids. Subsequently the horn fly populations on this farm were shown to have become resistant and, since 1997, the use of cypermethrin has been restricted to experimental purposes. In this study, fly samples collected in 1999, 2000 and 2001 were subjected to a polymerase chain reaction (PCR) to detect the presence of a specific nucleotide substitution in the sodium channel gene sequence, which has been associated with target site insensitivity to pyrethroids. This analysis showed that the level of cypermethrin resistance had diminished between 1997 and 2001. However, this was not sufficient to restore the efficacy of this pyrethroid to the level found prior to the onset of resistance. Heterozygous and homozygous resistant flies were detected in all samples of flies subjected to PCR diagnosis of alleles conferring target site resistance.     

Molecular detection of zoonotic bartonellae (B. henselae,B. elizabethae and B. rochalimae) in fleas collected from dogs in Israel

Fleas represent an acknowledged burden on dogs worldwide. The characterization of flea species infesting kennel dogs from two localities in Israel (Rehovot and Jerusalem) and their molecular screening for Bartonella species (Rhizobiales: Bartonellaceae) was investigated. A total of 355 fleas were collected from 107 dogs. The fleas were morphologically classified and molecularly screened targeting the Bartonella 16S–23S internal transcribed spacer (ITS). Of the 107 dogs examined, 80 (74.8%) were infested with Ctenocephalides canis (Siphonaptera: Pulicidae), 68 (63.6%) with Ctenocephalides felis, 15 (14.0%) with Pulex irritans (Siphonaptera: Pulicidae) and one (0.9%) with Xenopsylla cheopis (Siphonaptera: Pulicidae). Fleas were grouped into 166 pools (one to nine fleas per pool) according to species and host. Thirteen of the 166 flea pools (7.8%) were found to be positive for Bartonella DNA. Detected ITS sequences were 99–100% similar to those of four Bartonella species: Bartonella henselae (six pools); Bartonella elizabethae (five pools); Bartonella rochalimae (one pool), and Bartonella bovis (one pool). The present study indicates the occurrence of a variety of flea species in dogs in Israel; these flea species are, in turn, carriers of several zoonotic Bartonella species. Physicians, veterinarians and public health workers should be aware of the presence of these pathogens in dog fleas in Israel and preventive measures should be implemented.     

Resistance of the alga Spermatozopsis acidophila Kalina (Chlorophyta,Volvocales) to heavy metals

Abstract

The toxic effects of 14 chemical elements (prevalently heavy metals) on the alga Spermatozopsis acidophila Kalina (Volvocales, Polyblepharidaceae) have been investigated.

The results obtained show a high resistance to heavy metals, particularly to Cu, in comparison with data found in literature on other algae.

This phenomenon might be interpreted as a consequence of evolutionary adaptation to highly acid environments.