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The control of the sheep blowfly relies on the use of insecticides. There have been several reports of in vitro and in vivo resistance to the most widely-used flystrike control chemical, dicyclanil. A recent report also described in vitro resistance to imidacloprid in a strain collected from a single property over three consecutive seasons that also showed resistance to dicyclanil. The present study aimed to use in vitro assays to examine five field-collected blowfly strains to determine if this co-occurrence of resistance to dicyclanil and imidacloprid was present more widely in field strains and to also measure resistance patterns to the other currently-used flystrike control chemicals. Each of the strains showed significant levels of resistance to both dicyclanil and imidacloprid: resistance factors at the IC50 of 9.1-23.8 for dicyclanil, and 8.7-14.1 for imidacloprid. Resistance factors at the IC95 ranged from 16.5 to 53.7, and 14.6-24.3 for dicyclanil and imidacloprid, respectively. Resistance factors were up to 8.5 for cyromazine at the IC95. Resistance to dicyclanil and imidacloprid was suppressed by co-treatment with the cytochrome P450 inhibitor, aminobenzotriazole, implicating this enzyme system in the observed resistances. We discuss the implications of the co-occurrence of resistance to dicyclanil and imidacloprid on insecticide rotation strategies for blowfly control. We also discuss the roles of insecticide resistance, environmental factors (e.g. rainfall), operational factors (e.g. insecticide application technique) and other animal health issues (e.g. scouring / diarrhoea) that together will impact on the likelihood of flystrike occurring at an earlier time point than expected after insecticide application.

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BACKGROUND: The sheep blowfly, Lucila cuprina, is a myiasis-causing parasite responsible for significant production losses and welfare issues for the Australian sheep industry. Control relies largely on the use of insecticides. The pyrimidine compound, dicyclanil, is the predominant control chemical, although other insecticides also are used, including imidacloprid, ivermectin, cyromazine and spinosad. We investigated in vitro resistance patterns and mechanisms in field-collected blowfly strains. RESULTS: The Walgett 2019 strain showed significant levels of resistance to both dicyclanil and imidacloprid, with resistance factors at the IC50 of 26- and 17-fold, respectively, in in vitro bioassays. Co-treatment with the cytochrome P450 inhibitor, aminobenzotriazole, resulted in significant levels of synergism for dicyclanil and imidacloprid (synergism ratios of 7.2- and 6.1-fold, respectively), implicating cytochrome P450 in resistance to both insecticides. Cyp12d1 transcription levels were increased up to 40-fold throughout the larval life stages in the resistant strain compared to a reference susceptible strain, whereas transcription levels of some other cyp genes (6g1, 4d1, 28d1) did not differ between the strains. Similar resistance levels also were observed in flies collected from the same property in two subsequent years. CONCLUSION: This study indicates that in vitro resistance to both dicyclanil and imidacloprid in this field-collected blowfly strain is likely mediated by cytochrome P450, with Cyp12d1 implicated as the enzyme responsible; however, it remains possible that another P450 also may be involved. A common resistance mechanism for the two drugs has important implications for drug rotation strategies designed to prolong the useful life of flystrike control chemicals. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Late in 2017, field samples of the Australian sheep blowfly, Lucilia cuprina, were submitted by sheep producers from three states of Australia (South Australia, Victoria and New South Wales). Some were collected by submitters concerned about shortened periods of flystrike protection from dicyclanil based products. Neonate larval offspring from the NSW field samples survived and successfully completed their life cycles following exposure to dicyclanil and cyromazine at susceptible discriminating concentrations in vitro. The in vivo study reported here used dicyclanil resistant neonate larvae to assess the flystrike protection provided by a cyromazine jetting fluid and a number of dicyclanil based spray-on products, when applied to sheep six weeks after shearing. The two dicyclanil resistant blowfly strains used in this study showed in vitro resistance ratios, at the LC50, of approximately 13- and 25-fold relative to a dicyclanil and cyromazine susceptible strain. Compared to the levels of resistance that L. cuprina has developed to other insecticides these are relatively low, however, three dicyclanil based spray-on products (active ingredient 12.5 g/L, 50 g/L and 65 g/L) had protection periods reduced by 73%, 78% and 69% respectively when compared to the maximum protection periods claimed by the manufacturer. A 50% and a 33% reduction in protection period was also observed to a cyromazine and an ivermectin based jetting fluid respectively. In contrast, protection periods were attained or exceeded regardless of the treatment used against field derived dicyclanil susceptible neonate larvae. For the first time we confirm that dicyclanil resistance enables the completion of the L. cuprina life cycle following flystrike initiation on dicyclanil or cyromazine treated sheep when insecticide levels are considered high and protective. This study also provides in vivo information on the effect of dicyclanil resistance on the protection provided by a product with an active ingredient belonging to an unrelated insecticide group. Dicyclanil resistance is of major concern to the Australian sheep industry.

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OBJECTIVE: To determine the prophylactic and therapeutic efficacy of Australian-registered insecticide formulations against Old World screwworm (OWS) myiases for potential use in screwworm containment and eradication programs. METHODS: The longevity of protection provided by six insecticidal formulations (subcutaneous ivermectin, doramectin and abamectin, a topically applied aqueous formulation of spinosad, ivermectin controlled-release capsule and a dicyclanil spray-on formulation) was tested using implants of 1st-instar OWS larvae on Javanese thin-tail sheep. Therapeutic efficacy of four formulations (topical ivermectin, chlorfenvinphos/cypermethrin mixture, aerosol spinosad formulation and a formulation containing propetamphos and eucalyptus oil) was tested against 2- and 4-day-old OWS strikes. RESULTS: Both the ivermectin capsule and dicyclanil spray-on formulation gave 100% protection against screwworm implants for the full 12 weeks of the study. Ivermectin, doramectin and abamectin administered SC all gave 100% protection at 3 days post-treatment, but at 2 weeks the protection had become incomplete. Spinosad dipping did not give complete protection at any time. All four therapeutic treatments gave complete resolution of 2-day-old strikes and topical ivermectin, spinosad and chlorfenvinphos/cypermethrin, but not the propetamphos/eucalyptus oil formulation, gave complete resolution of all 4-day-old strikes. CONCLUSION: Dicyclanil spray-on and ivermectin capsule formulations, both registered for use in sheep, but not for cattle or other livestock species, gave much longer protection against screwworm implants than the currently recommended SC ivermectin. Pre-emptive action to facilitate rapid deployment of these formulations in the event of a screwworm incursion is urgently needed.

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Insect growth regulators (IGRs) such as lufenuron, methoprene, and pyriproxyfen have been important tools in the integrated pest management of cat fleas, Ctenocephalides felis (Bouché), for the past two decades. Other IGRs have been registered for the control of termite, dipteran, and lepidopterous pests including chlorfluazuron, cyromazine, dicyclanil, and precocene I, but have been not tested against C. felis. The intrinsic activity of IGRs was determined by exposing larvae to treated larval rearing media. The LC50s of chlorfluazuron, cyromazine, dicyclanil, lufenuron, and precocene I against cat fleas were 0.19, 2.66, 0.04, 0.20, and 10.97 ppm, respectively. The LC95s of chlorfluazuron, cyromazine, dicyclanil, lufenuron, and precocene I were 0.78, 51.24, 0.30, 0.62, and 175.05, respectively. The regression slopes of chlorfluazuron and lufenuron were 2.65 ± 0.24 and 3.40 ± 0.45 (SEM), respectively, and considerably steeper than the other IGRs tested (1.51 to 1.74). The intrinsic activity of IGRs tested is summarized as dicyclanil > chlorfluazuron = lufenuron > cyromazine > precocene I. The responses of the laboratory UCR strain to these IGRs can serve as a baseline of susceptibility until a more susceptible cat flea strain is found. Chlorfluazuron and dicyclanil look like promising candidates against cat fleas.

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Clonorchis sinensis is a Group-I bio-carcinogen, associated with cholangiocarcinoma (CCA). The hamster is the only experimental model of C. sinensis-mediated CCA, but we oblige another animal model. The present study intended to develop a C. sinensis (Cs) mediated CCA model using C3H/He mice, co-stimulated with N-nitrosodimethyl-amine (NDMA) and dicyclanil (DC). The mice were divided into 8 groups with different combinations of Cs, NDMA, and DC. Six months later the mice were sacrificed and subjected to gross and histopathological examination. The body weights were significantly reduced among the groups treated with 2 or more agents (eg. Cs+NDMA, Cs+DC, NDMA+DC, and Cs+NDMA+DC). In contrast, liver weight percentages to body weight were increased in above groups by 4.1% to 4.7%. A Change of the spleen weight was observed only in Cs+NDMA group. Though C. sinensis infection is evident from hyperplastic changes, only 1 worm was recovered. T wo mice, 1 from Cs and the other from Cs+DC group, showed mass forming lesions; 1 (281.2 mm(3)) from the Cs group was a hepatocellular adenoma and the other (280.6 mm(3)) from the Cs+DC group was a cystic mass (peliosis). Higher prevalence of gray-white nodules was observed in Cs group (42.9%) followed by Cs+NDMA+DC group (21.4%). The mice of the Cs+NDMA+DC group showed hyper-proliferation of the bile duct with fibrotic changes. No characteristic change for CCA was recognized in any of the groups. In conclusion, C3H/He mice produce no CCA but extensive fibrosis when they are challenged by Cs, NDMA, and DC together.

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Clonorchis sinensis is a Group-I bio-carcinogen, associated with cholangiocarcinoma (CCA). The hamster is the only experimental model of C. sinensis-mediated CCA, but we oblige another animal model. The present study intended to develop a C. sinensis (Cs) mediated CCA model using C3H/He mice, co-stimulated with N-nitrosodimethyl-amine (NDMA) and dicyclanil (DC). The mice were divided into 8 groups with different combinations of Cs, NDMA, and DC. Six months later the mice were sacrificed and subjected to gross and histopathological examination. The body weights were significantly reduced among the groups treated with 2 or more agents (eg. Cs+NDMA, Cs+DC, NDMA+DC, and Cs+NDMA+DC). In contrast, liver weight percentages to body weight were increased in above groups by 4.1% to 4.7%. A Change of the spleen weight was observed only in Cs+NDMA group. Though C. sinensis infection is evident from hyperplastic changes, only 1 worm was recovered. T wo mice, 1 from Cs and the other from Cs+DC group, showed mass forming lesions; 1 (281.2 mm3) from the Cs group was a hepatocellular adenoma and the other (280.6 mm3) from the Cs+DC group was a cystic mass (peliosis). Higher prevalence of gray-white nodules was observed in Cs group (42.9%) followed by Cs+NDMA+DC group (21.4%). The mice of the Cs+NDMA+DC group showed hyper-proliferation of the bile duct with fibrotic changes. No characteristic change for CCA was recognized in any of the groups. In conclusion, C3H/He mice produce no CCA but extensive fibrosis when they are challenged by Cs, NDMA, and DC together.

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OBJECTIVE: To determine whether a cyromazine-resistant strain of Lucilia cuprina was able to establish strikes sooner than a susceptible strain on cyromazine- or dicyclanil-treated sheep. METHODS: Groups of 7 sheep were treated with cyromazine or dicyclanil in accordance with label directions. Beginning 5 weeks after treatment, 5 sheep from each group were challenged by implantation of neonate larvae belonging to the cyromazine-resistant strain 'Nimmitabel-selected' and the susceptible blowfly strain 'Field 2011' according to standard larval implant technique. The implant sites were alternated between the shoulder, mid-back and rump within the treatment zone. Similarly, implants of the resistant and susceptible strain larvae were alternated at each challenge such that the strain implanted on the left side of the sheep at one challenge was implanted on the right side at the next. Challenges were conducted at 3-weekly intervals until the susceptible larvae formed strikes on at least 2 of the 5 sheep in a treatment group or until 29 weeks after treatment. RESULTS: Sheep treated with cyromazine or dicyclanil were protected from flystrike by the cyromazine-susceptible strain for periods consistent with, or longer than, the registered product label claims. The cyromazine-resistant strain created strikes several weeks sooner after treatment than did the susceptible strain. Accordingly, the protection periods provided by cyromazine and dicyclanil against the resistant strain were reduced from 14 and 18-24 weeks to <8 weeks and <11 weeks, respectively. CONCLUSION: Resistance, even in the pure-breeding resistant strain, was not so severe as to cause treatment failure with cyromazine or dicyclanil, but was sufficient to reduce the protection period provided. It is recommended that producers adopt management practices that minimise the development of resistance to these and other compounds.

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OBJECTIVE: To demonstrate the protection of Merino sheep from flystrike by Lucilia cuprina with cyromazine or dicyclanil in an implant study and in the field. METHODS: In the implant study, sheep were treated with cyromazine or dicyclanil and implanted with 1st-stage larvae from a newly isolated field strain of L. cuprina (CYR-LS) or a reference strain (DZR50), then assessed over 3 days and compared with the implants on untreated control sheep. In the field study, weaner lambs were treated with cyromazine or dicyclanil and monitored weekly for flystrike over 18 weeks of grazing on the same farm from which the L. cuprina were isolated. RESULTS: Implant study: cyromazine (6%) provided effective protection against CYR-LS and DZR50 L. cuprina for a minimum of 13 and 10 weeks, respectively. Dicyclanil (5%) provided at least 18 weeks' protection against both strains. Field study: only 1 of 386 lambs in the cyromazine-treated group was struck in the first 14 weeks of the trial. No strikes occurred in the 198 sheep treated with dicyclanil (5%). Rainfall, temperature and flytrap data indicated consistent fly pressure during the study. CONCLUSIONS: Based on the results of these studies, there was no evidence of reduced susceptibility to cyromazine or dicyclanil and the periods of protection of sheep against L. cuprina were unaffected and consistent with the registered label claims.

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OBJECTIVE: Compare breech-strike on Merino ewes and hoggets that were mulesed, had breech and tail clips applied ('clipped') or left unmulesed and treated strategically with long-acting insecticide. DESIGN: A cohort study on two farms in southern Victoria. METHODS: Three treatment groups were established at lamb marking in 2008 and 2009 on each farm. The unmulesed group was treated with a long-acting insecticide in early spring. The prevalence of breech-strike and key risk factors, including presence of dag, urine stain and breech wrinkle, were compared between groups. RESULTS: Breech-strike was detected from October to December on 1.9% and 7.2% of mulesed, and on 14.8% and 12.5% of clipped ewes, respectively, on each farm. Thus, clipped ewes had a relative risk of breech-strike 7.8- and 1.7-fold that of mulesed ewes. Unmulesed ewes treated with insecticide had similar or less breech-strike compared with mulesed ewes (3.4% and 1.4%), but significantly more dag, stain and breech wrinkle. From January, breech-strike on unprotected unmulesed ewes was 8.5% and 2.8%, compared with 3.5% and 0% on mulesed ewes. CONCLUSION: Early-season treatment of unmulesed sheep prevented most breech-strikes during spring and early summer on both hoggets and breeding ewes, confirming it as a short- to medium-term option for the control of breech-strike. Some benefits were associated with the use of clips but, to control breech-strike, clipped sheep should be treated the same as unmulesed sheep. Genetic selection to reduce the prevalence of dag will be required for future strategies to control breech-strike on Merino sheep in south-eastern Australia.

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A method for the determination of cyromazine and dicyclanil residues on greasy wool was developed with HPLC and confirmed with HPLC-MS/MS.The cyromazine and dicyclanil residues on greasy wool were extracted with 1% trichloroacetic acid solution with ultrasonic, and cleaned up by MCX SPE column.The HPLC separation was performed on a Hypersil NH_2 using water-acetonitrile (V/V) as the mobile phase with gradient elution and detected at the wavelength of 214 nm.The corroboration method of HPLC-MS/MS was used with electro-spray ionization of positive ion mode.The [ M + H ]~+ and characteristic ions of dicyclanil were m/z 191.0, 150.0 and 163.0, and cyromazine were m/z 167.0, 85.0 and 125.0.The linear ranges of cyromazine and dicyclanil were 0.05-5.0 mg/L.There were good linear relationships between the peak area and concentration in the linear range.The correlation coefficient was 0.9999.The detection limit of cyroma zine was 0.02 mg/kg, and dicyclanil was 0.01 mg/kg.The average recoveries of cyromazine and the dicycla nil were 95.0%-99.9% and 83.6%-92.2%, respectively.