Horn fly (Diptera: Muscidae) resistance to organophosphate insecticides

Insecticidal ear tags impregnated with organophosphate (OP) insecticides were used each year from 1989 to 1998 at Rosepine, LA. Weekly fly counts were conducted to evaluate control efficacy of the treatments, and bioassays were conducted at least twice per year to measure fly susceptibility to OP and pyrethroid insecticides. Between 1989 and 1992, the efficacy of 20% diazinon-impregnated ear tags was reduced from >20 to just 1 week of control. A high risk of control failure was observed when a resistance frequency of approximately 5% was measured in pre-season bioassays. Resistance to diazinon, fenthion, ethion, pirimiphos-methyl, and tetrachlorvinphos was observed. Esterase activity toward alpha-naphthyl acetate was significantly higher in flies collected at Rosepine in 1997 than in flies from a laboratory colony and from a susceptible field population.     

Acetylcholinesterase mutation in diazinon-resistant Haematobia irritans (L.) (Diptera: Muscidae)

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.     

Development of treated targets for controlling stable flies (Diptera: Muscidae)

Electrocution techniques were used to determine if treated targets similar to those used for tsetse control could be developed for stable fly control. In a series of two experiments, a half blue and half black (UK) 1 m2 target constructed of trigger cotton poplin was determined to be acceptable for development studies. In the first experiment, an average of 350 stable flies per hour (maximum 794 flies in 1 h) was collected using the UK target. A time-delayed circuit trial using untreated UK targets demonstrated that stable flies remained on or around the targets for at least 30 s. Two experiments were conducted with time-delayed circuits and UK targets treated with 0.1% lambda-cyhalothrin. In the first experiment, the number of flies collected using the 30 s on/off treated target treatment was not different from the number of flies collected using the other treatments. In the second experiment, the number of flies collected using the 30 s on/off treated target treatment was not different than the untreated target continuous or 30 s on/off treatments, but was significantly lower than the treated target continuous treatment. The number of flies collected with UK trigger targets was significantly higher than that for alsynite cylinder traps in two experiments. The mean number of flies collected during 22 1h assays using targets was 6.1-fold higher than that for alsynite traps, and the mean number of flies collected during 40 3 h using the targets also was 6.1-fold higher than that for alsynite traps. The results of this study indicate that treated cloth targets may be a viable addition for stable fly control programs.     

Seasonal dynamics of synbovine flies (Diptera: Muscidae) during the grazing period in Jarov

Synbovine flies and the seasonal pattern of their occurrence were studied throughout the grazing period in the cattle pastures of the Strakonice district. Skufin's traps were used for catching the flies. The total number of dipterous insects caught during the series of trappings in two- to three-week intervals was 2096. The family Muscidae (23 species) was represented most amply in the sample catches (Musca autumnalis 65.9%, Hydrotaea Meteorica 20.4%, Hydrotaea irritans 4.2%, Haematobosca stimulans 2.6%). The maximum occurrence of haematophagous species was recorded at the beginning and at the end of the grazing season. The maximum occurrence of secretophagous species was recorded in June.     

Host finding and feeding in Hydrotaea irritans (Diptera, Muscidae): the role of chemical senses

Hydrotaea irritans is commonly considered to be the primary vector for the bacteria which cause summer mastitis in cattle. A behavioural physiology approach was used to investigate potential host odours (kairomones) that may be used by the fly in finding its host and to determine which substrates or sites on the host may be utilised in feeding. Attractant odours include CO2 and butyric, propionic and acetic acids: the latter three are also produced by the bacteria causing summer mastitis. When milk, slaver, nasal secretion, mastitis secretion and blood were offered to flies as feeding substrates only the last three produced significant increases in feeding duration in comparison to controls offered distilled water.     

Distribution, seasonality and relative abundance of Stomoxys calcitrans (stablefly) (Diptera: Muscidae) in New Zealand

AIMS: To determine the current distribution, seasonality and relative abundance of Stomoxys calcitrans in New Zealand in order to provide information that could be used to assess risks of transmission of equine infectious anaemia (EIA). METHODS: Adhesive yellow traps were distributed to schools throughout New Zealand and used to detect the presence of S. calcitrans between November 1999 and April 2000 at sites considered likely to be a focus for S. calcitrans breeding and activity. In addition, researchers undertook monthly trapping at six other sites between August 1999 and June 2000 to measure the duration and seasonal periodicity of S. calcitrans flight activity. Veterinary practices and farmers were also surveyed to provide anecdotal evidence of the presence or absence of S. calcitrans, particularly in areas where no flies were trapped by schools. RESULTS: Stomoxys calcitrans was found to occur in both North and South Islands, but principally in locations where dairy farming occurred. The fly was active during most months of the year, except perhaps during July and August, and was particularly active in warmer North Island districts such as the Waikato. Peak activity was recorded from January to May. The fly was more abundant in Northland, Auckland, Waikato, the Marlborough Sounds and Nelson than in other districts, as determined by the number of occasions flies were caught relative to the number of traps set. There are only a few areas, such as around Taupo, the Otago Lakes, Central Otago and the Mackenzie Basin, where S. calcitrans was not trapped or, on the basis of anecdotal evidence, thought not to occur. CONCLUSIONS: Given that other potential vectors of EIA are absent from New Zealand, Taupo, the Otago Lakes, Central Otago and the Mackenzie Basin districts could be considered areas of low risk for EIA transmission to horses. In addition, teachers and school children were capable, in most instances, of supporting a nationwide survey providing the methods were simple and the aims few.     

Studies on the feeding patterns of the sheep headfly Hydrotaea irritans (Diptera, Muscidae) in Great Britain

Serum-precipitin tests carried out on the stomach contents of laboratory maintained and field-caught Hydrotaea irritans showed that a positive result could be obtained for at least seven to 10 days after feeding. Eye, nasal and salivary secretions, even if undiluted, did not give a precipitin response in these tests. Field results from over 3000 flies thus indicate that 20 to 55% of the active fly population had taken a blood meal within the previous 10 days: 75 to 100% of these had fed on cattle blood and 15% or less on sheep blood. Significantly lower feeding levels were obtained from flies collected within plantations, indicating a protracted resting phase in woodland. From "fly feeding areas" high blood-feeding levels were shown from early July, as ovarian development begins. Early feeding rates were similar in both sexes.     

Populational dynamics of Stomoxys calcitrans (Linneaus) (Diptera: Muscidae) in three biocenosis, Minas Gerais, Brazil

Populational flux of the adult phase of Stomoxys calcitrans was observed in the municipal district of Pedro Leopoldo, Minas Gerais, Brazil. Three biocenoses were selected for the study: stable agrobiocenosis, pastural agrobiocenosis and eubiocenosis. The occurrence and the populational flux of the insects, using the Magoon trap for their capture, were established. For each trap located in different biocenoses, a crossbred calf (Bos taurus × Bos indicus) approximately 6-month-old was used as “live bait,” exposed weekly for 48 h in the traps. Of the three agrobiocenoses studied, the stable agrobiocenosis contributed the greatest number of specimens of. S. calcitrans captured, corresponding to 96.9% of the total flies of this species collected. S. calcitrans shows seasonal behavior for approximately 6 months (spring and summer being the rainiest months of the year). The population peaked during the months of November and December. During the months of July and August, there was no capture of flies.     

Chlorfenapyr ear tags to control Haematobia irritans (L.) (Diptera: Muscidae) on cattle

The efficacy of ear tags containing 30% chlorfenapyr (total tag weight=13g) to control natural Haematobia irritans (L.) infestations was evaluated for Holstein heifers in Rafaela, province of Santa Fe, Argentina. A group of heifers (TG) was treated with two ear tags (one tag per ear). A control group (ACG) was maintained in a paddock adjacent to the TG paddock and, a distant control group (DCG) was maintained 700m apart from the other groups. From day 4 to day 98 after treatment, H. irritans infestations of ACG were significantly higher (P<0.05, test of Kruskal-Wallis) than the corresponding infestation of TG, but significantly lower than infestation in DCG, probably due to the proximity of TG. The chlorfenapyr control period, with an efficacy higher than 90% to reduce horn fly populations, lasted for 9 weeks when TG infestation was compared to fly numbers in ACG, but increased to 12 weeks in comparison to DCG. The results of this study show that ear tags impregnated with chlorfenapyr are a useful alternative to pyrethroids and organo-phosphate compounds for horn fly control.     

Toxicity of cypermethrin and diazinon to Haematobia irritans (Diptera: Muscidae) in its American southern range

Synthetic pyrethroids are considered currently less effective than organophosphate insecticides for controlling Haematobia irritans on cattle in its American southern range. To test the validity of this hypothesis, horn fly populations from 95 cattle herds in central-northern Argentina and southern Brazil were evaluated to determine 50% lethal concentrations (LC(50)) to cypermethrin and diazinon, respectively. Horn flies were exposed to filter papers and glass vials treated with cypermethrin and diazinon, respectively, and LC(50) values were determined using probit analysis. Two and 78 fly populations showed LC(50) values indicating susceptibility and resistance to cypermethrin, respectively, in comparison to historical LC(50) values obtained when the horn fly populations were susceptible to cypermethrin. Five fly populations had an undetermined resistance status to cypermethrin. The diazinon tests showed no obvious indications of H. irritans resistance. Thirty nine percent of farmers still rely on pyrethroids to control horn fly populations on cattle which represents a decreasing proportion from a few years ago when horn fly control was carried out using pyrethroids almost exclusively. Organophosphates could provide control for horn flies resistant to pyrethroids; however, the reliance on chemicals alone to control H. irritans is not considered a proper approach for the future.     

Variation in population density of horn flies (Haematobia irritans irritans) (L.) (Diptera: Muscidae) in Nellore cattle (Bos indicus)

The population density of horn flies was evaluated in the year 1998 in the municipality of Ara?atuba, S?o Paulo Brazil, in relation to temperature and rainfall conditions. Two lots of 30 Nellore steers (Bos indicus) were used which had no insecticidal treatment and were naturally infested with horn flies. The infestations were assessed by two counting methods, i.e., the traditional estimate method and the filming method. The highest fly frequencies were recorded in spring, summer, autumn and the lowest frequencies were recorded in winter. The increase in fly number was positively correlated (P<0.05) with rainfall.     

Some effects of temperature on the adults, eggs and pupae of Stomoxys calcitrans Linnaeus (Diptera: Muscidae)

Adults could only live and reproduce to their full capacity at temperatures between 20 degrees C and 30 degrees C. At 15 degrees C the females laid no eggs, the adult life span was relatively short and the reproductive capacity of females kept at 35 degrees C was low. The thermal histories of the flies had no apparent effect on their later reactions to temperature in any of the parameters tested. The viability rates of S. calcitrans eggs exposed to temperatures between 10 degrees C and 40 degrees C exceeded 84%, but 45 degrees C was lethal. The optimum temperatures for incubation of the eggs was 30 degrees C. Pupae of S. calcitrans seemed to tolerate temperatures between 20 degrees C and 30 degrees C, but their mortalities increased markedly outside this temperature range. Tests showed that pupal mortalities increased linearly with increasing periods of exposure to a temperature of 15 degrees C.     

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae)

The present study assessed the pathogenic effect of isolates E9, IBCB425 and IBCB159 of the Metarhizium anisopliae fungus, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) against eggs and larvae of the horn fly Haematobia irritans. Eggs were inoculated with suspensions containing 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ of the fungal isolates and observed after 48 h to determine viability. In the larvae study, eggs were allowed to hatch into fresh bovine feces that had been treated with 10⁸, 10⁷ or 10⁶ conidia mg feces⁻¹. In both studies, 5 days after initial procedures, all formed pupae were transferred to an incubator at 27 ± 0.5 °C until the emergence of the adult flies was complete. The M. anisopliae isolates did not cause the death of H. irritans eggs, but they did promote the death of larvae that hatched from treated eggs, and therefore increased the total mortality. Isolate E9 promoted 100% mortality of treated larvae at a concentration of 10⁸ conidia ml⁻¹. For the B. bassiana isolates, no activity was observed against insect eggs or larvae. Both I. fumosorosea isolates promoted significant mortality (p < 0.05) of eggs at every concentration of conidia. Isolate CG195 of I. farinosa increased the mortality of larvae and pupae that hatched from treated eggs and promoted significant total mortality (p < 0.05) of the insect at every concentration of conidia.     

Validation of a subjective counting method for a horn flies (Haematobia irritans irritans) (Diptera: Muscidae) population in a cattle herd

A trained observer direct count method to measure horn fly population was evaluated to determine the reliability (inter-observer agreement) and its validity when compared with a "gold standard" method (video film). All the counts were performed with the animals restrained in a chute in a single herd. A direct count of horn flies by each of two observers on opposite sides of the animal was made. In addition a videotape recording of the counting surface on each animal was made. Horn flies were counted on 80 cows in the morning twice monthly from 22 October 1999 to 24 March 2000. The correlation between observer counts was high as was the correlation between observer counts and counts made from a videotape recording. Direct counts by trained observers were highly reliable and had good validity. However, videotape recording can be useful when the horn fly population is high, because it is faster in the field than the direct count method. The results of this study suggest that the direct count method can be used with confidence under field conditions.     

Comparison of the reproductive biology between acaricide-resistant and acaricide-susceptible Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The reproductive fitness of Rhipicephalus (Boophilus) microplus (Canestrini) strains resistant to organophosphate (OP), pyrethroid (P), or formamidine (F) acaricides was compared to an acaricide-susceptible (SUS) strain to determine whether the acquisition of resistance affected reproductive fitness in the resistant strains. The SUS strain females had a 3.0 days preoviposition period, a 12.1 days oviposition period, a 22.5 days egg incubation period, a mean of 3670 eggs per female, and a mean percentage egg hatch of 78.1%, which were all remarkably similar to these same parameters reported for this species throughout the world. The reproductive biology of the P-resistant strain (PYR) and the F-resistant strain (FOR) were, for the most part, similar to those of the SUS strain. In the few instances where statistical differences did occur there was little evidence that the variation had any biological basis that could be attributed to a reduction in fitness related to resistance to P or F acaricides. Although the comparison of reproductive parameters of the OP-resistant strain (OPR) and the SUS strain identified statistical differences between the mean egg incubation and oviposition periods, the magnitude of the differences was not sufficient to conclude that the OPR strain was biologically less fit than the SUS strain. However, the OPR strain produced 30% fewer eggs (2562 eggs per female) than the SUS strain (3670 eggs per female) indicating the acquisition of resistance placed the OPR at a selective disadvantage relative to the SUS strain. This coupled with a lower, though non-significant, egg hatch was used to predict there would be a reduction of at least 34.1% in larval numbers available to potentially re-infest subsequent cattle than were available from the SUS strain. These data may aid the development of management strategies that can be used to control OP-resistant ticks.     

Molecular identification of Anaplasma marginale and rickettsial endosymbionts in blood-sucking flies (Diptera: Tabanidae, Muscidae) and hard ticks (Acari: Ixodidae)

In an attempt to identify the main vector and possible transmission routes of Anaplasma spp. in a region of Hungary with high prevalence of ovine and bovine anaplasmosis, DNA was extracted from 316 haematophagous arthropods (individually or in pools), including 4 species of ixodid ticks, 6 species of tabanid flies and hornflies. Midichloria-like organisms were identified with PCR (amplifying a portion of the 16S rRNA gene) and sequencing from Dermacentor marginatus and Ixodes ricinus. Significantly higher 16S positive D. marginatus individuals were collected in March than in April, suggesting earlier questing of ticks that contain rickettsial agents (thus endosymbionts). Midichloria- and Wolbachia-like organisms were also found in randomly caught horse flies (Tabanus bovinus and T. tergestinus) as well as hornflies (Haematobia irritans), respectively, with 97-99% similarity to sequences deposited in the GenBank. Although all ticks were negative in the Anaplasma spp.-specific msp4 PCR, four individuals of T. bovinus collected near to grazing cattle were positive for Anaplasma marginale. The results of the present study provide the first molecular evidence for the potential mechanical vector role of T. bovinus in the transmission of A. marginale, and broaden the range of haematophagous arthropods harbouring Midichloria-like bacteria, for the first time in any Dermacentor or Tabanus species.     

Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties

A 2193-nucleotide cDNA encoding acetylcholinesterase (AChE) of the stable fly, Stomoxys calcitrans (L.) was sequenced and expressed in the baculovirus system. The open reading frame encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 96% and 94% identity to the AChEs of Haematobia irritans (L.) and Musca domestica (L.), respectively. Structural characteristics of M. domestica, H. irritans, and Drosophila melanogaster AChEs were conserved in the S. calcitrans AChE. The recombinant enzyme was inhibited by eserine, coroxon, and paraoxon and exhibited K_m values of 63.9 μM for acetylthiocholine and 96.7 μM for butyrylthiocholine, confirming its biochemical identity as an acetylcholinesterase (EC 3.1.1.7). These data will enable rapid identification and assay for mutations that reduce AChE sensitivity to organophosphate (OP) pesticides, potentially aiding resistance management efforts to prevent fixation of the mutations in pest populations.     

Aspects of the reproductive biology of the southern mullet,Liza richardsoni,from Algoa Bay,South Africa

The reproductive biology of the southern mullet from Algoa Bay was studied between June 1978 and October 1980. Gonadosomatic indices and macroscopic and microscopic examinations of the gonads were used to establish the breeding cycle. The period of greatest sexual development extends from September to March and spawning occurred throughout that period. Spawning probably occurs close in shore. Liza richardsoni exhibited the phenomenon of group synchronism, two distinct oocyte groups being present within a pre-spawning ovary. The proportion of yolkless oocytes exceeded that of vitellogenic oocytes in ripe fish. However, it was not apparent whether all the vitellogenic oocytes would reach maturity. This highlights the need for quantitative histological studies to accompany fecundity estimates. Histological investigations were useful in the clarification of breeding cycle determinations based on gonadosomatic indices and visual macroscopic assessments.