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Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthel

The Australian ruminant livestock industries are faced with the need to control parasitic infectious diseases that can seriously impact the health of animals. However, increasing levels of resistance to insecticides, anthelmintics and acaricides are substantially reducing the ability to control some of these parasites. Here we review the current situation with regard to chemical resistances in parasites across the various sectors of the Australian ruminant livestock industries and assess the level of threat that these resistances pose to the sustainability of these sectors in the short to long terms. We also look at the extent to which testing for resistance occurs across the various industry sectors, and hence how well‐informed these sectors are of the extent of chemical resistance. We examine on‐farm management practices, breeding of parasite‐resistant animals, and non‐chemical therapeutics that may act as short to long term means to reduce the current reliance on chemicals for parasite control. Finally, we look at the balance between the prevalence and magnitude of current resistances and the availability and adoption rates of management, breeding and therapeutic alternatives in order to assess the parasite control outlook for the various industry sectors. [ABSTRACT FROM AUTHOR]

Flystrike remains a serious financial and animal welfare issue for the sheep industry in Australia despite many years of research into control methods. The present paper provides an extensive review of past research on flystrike, and highlights areas that hold promise for providing long‐term control options. We describe areas where the application of modern scientific advances may provide increased impetus to some novel, as well as some previously explored, control methods. We provide recommendations for research activities: insecticide resistance management, novel delivery methods for therapeutics, improved breeding indices for flystrike‐related traits, mechanism of nematode‐induced scouring in mature animals. We also identify areas where advances can be made in flystrike control through the greater adoption of well‐recognised existing management approaches: optimal insecticide‐use patterns, increased use of flystrike‐related Australian Sheep Breeding Values, and management practices to prevent scouring in young sheep. We indicate that breeding efforts should be primarily focussed on the adoption and improvement of currently available breeding tools and towards the future integration of genomic selection methods. We describe factors that will impact on the ongoing availability of insecticides for flystrike control and on the feasibility of vaccination. We also describe areas where the blowfly genome may be useful in providing impetus to some flystrike control strategies, such as area‐wide approaches that seek to directly suppress or eradicate sheep blowfly populations. However, we also highlight the fact that commercial and feasibility considerations will act to temper the potential for the genome to act as the basis for providing some control options. [ABSTRACT FROM AUTHOR]

Background There have been several international reports of macrocyclic lactone (ML)-resistant Parascaris equorum over the past decade, but the resistance status of Australian P. equorum populations is largely unknown. A case of apparent reduced efficacy of ivermectin against P. equorum in Australia was investigated. Methods and Results A faecal egg count reduction test carried out on a group of weanling foals in south-east Queensland showed the efficacy of ivermectin to be 65%. Conclusion The case highlights the need to review current worm control strategies, especially for young horses. [ABSTRACT FROM AUTHOR]

Control of the sheep blowfly relies largely on the use of insecticides applied prophylactically in advance of expected fly activity. However, the blowfly has shown an ability to develop resistance to some of these insecticides. Recent reports of the co-occurrence of resistance to both dicyclanil and imidacloprid in in vitro bioassays with field-collected fly strains has raised the possibility that the two resistances may represent cross-resistance linked by a common mechanism. We investigated this by imposing insecticide selection pressure on larvae of two insecticide-resistant field strains over a number of generations using either dicyclanil or imidacloprid and then measuring changes in sensitivity to both the selecting chemical and the alternate chemical. Larvae selected over six generations with each chemical showed significant increases in resistance to the selecting chemical: resistance ratios at the IC 50 5.5 - 8.1-fold higher for dicyclanil, and 3.1 - 3.8-fold for imidacloprid. The larvae also showed significant increases in levels of resistance towards the alternate chemical: resistance ratios 2.6 - 3.1-fold higher towards dicyclanil following selection with imidacloprid, and 2.2 - 3.2-fold higher towards imidacloprid following selection with dicyclanil. The increases in resistance to both chemicals after exposure to either suggests a common mechanism of resistance, at least in our laboratory-selected populations. Assays with the cytochrome P450 inhibitor aminobenzotriazole showed that this synergist was able to remove the increased resistance to both compounds in strains selected with either compound, suggesting that cytochrome P450 is responsible for the resistance observed to both chemicals. Our results confirm that cross-resistance exists between dicyclanil and imidacloprid in the sheep blowfly, and hence the two compounds should be considered as related entities in insecticide rotation strategies for flystrike control., Competing Interests: Declaration of Competing Interest One author (NR) is a paid employee of Elanco Australasia Pty Limited, who provided funding for this study. All other authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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 IC 50 of 9.1-23.8 for dicyclanil, and 8.7-14.1 for imidacloprid. Resistance factors at the IC 95 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 IC 95 . 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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Andrew Kotze reports financial support was provided by Elanco Australasia. Andrew Kotze reports financial support was provided by Commonwealth Scientific and Industrial Research Organisation. Nicholas Rolls and Sarah George are paid employees of Elanco Australasia. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Hookworms are the most common intestinal nematode parasites of dogs in Australia. The control of these parasites relies mostly on regular deworming with anthelmintics, with pyrantel-based dewormers being a relatively low cost and readily-available option for dog owners. Pyrantel resistance in canine hookworms in Australia was first reported in 2007, however pyrantel-based dewormers are still used against hookworm infection in dogs across Australia. The present study was conducted to evaluate the efficacy of pyrantel against hookworms infecting dogs housed in a shelter facility in Southeast Queensland which receives rescued or surrendered animals from greyhound rescue centres and dog shelters across this region. A total of 10 dogs were examined using the faecal egg count reduction test (FECRT). There was no reduction in FEC in any of the dogs following pyrantel treatment, with drug efficacies ranging from -0.9% to -283.3%. Given that these dogs originated from various sites across Southeast Queensland, the present study suggests that pyrantel resistance is widespread in this region, and hence this anthelmintic may not be a useful option for treatment of hookworm infections in dogs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Limacodidae is a family of lepidopteran insects comprising >1500 species. More than half of these species produce pain-inducing defensive venoms in the larval stage, but little is known about their venom toxins. Recently, we characterised proteinaceous toxins from the Australian limacodid caterpillar Doratifera vulnerans, but it is unknown if the venom of this species is typical of other Limacodidae. Here, we use single animal transcriptomics and venom proteomics to investigate the venom of an iconic limacodid, the North American saddleback caterpillar Acharia stimulea. We identified 65 venom polypeptides, grouped into 31 different families. Neurohormones, knottins, and homologues of the immune signaller Diedel make up the majority of A.stimulea venom, indicating strong similarities to D. vulnerans venom, despite the large geographic separation of these caterpillars. One notable difference is the presence of RF-amide peptide toxins in A. stimulea venom. Synthetic versions of one of these RF-amide toxins potently activated the human neuropeptide FF1 receptor, displayed insecticidal activity when injected into Drosophila melanogaster, and moderately inhibited larval development of the parasitic nematode Haemonchus contortus. This study provides insights into the evolution and activity of venom toxins in Limacodidae, and provides a platform for future structure-function characterisation of A.stimulea peptide toxins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Control of flystrike on sheep relies on the use of insecticides. The present study used in vitro assays to examine the potential for increasing the efficacy of synthetic pyrethroids against sheep blowfly larvae using the synergist piperonyl butoxide (PBO). We examined the potency of alpha-cypermethrin (ACP) / PBO combinations against a reference insecticide-susceptible strain (LS) and a field-derived strain showing resistance to dicyclanil and imidacloprid. Co-treatment of the insecticide-susceptible strain with ACP/PBO resulted in increasing levels of synergism as the PBO concentration was increased, with synergism ratios (SRs) of up to 114-fold. Treatment with PBO/ACP combinations at ratios of 20:1 and 5:1 resulted in significant levels of synergism: SRs of 13.5- and 7.6-fold, respectively. However, the levels of synergism were significantly less for the insecticide-resistant strain: SRs of 4.6- and 2.6-fold for the 20:1 and 5:1 ratios, respectively. The resistant strain showed no resistance to ACP when administered alone, however, was 2-fold less sensitive than the LS strain to the toxic effects of PBO alone. This insensitivity to PBO was removed by co-treatment with the P450 inhibitor aminobenzotriazole, suggesting an increased level of P450-mediated metabolism of the PBO in this strain compared to the LS strain, and hence providing a likely explanation for the reduced synergistic efficacy of PBO on ACP toxicity in the resistant strain. While PBO was able to synergise ACP with both of the blowfly strains examined here, the reduced synergistic efficacy observed with the field-derived insecticide-resistant strain lessens the potential usefulness of such a combination for blowfly control in the field., Competing Interests: Declaration of Competing Interest N.R. is a paid employee of Elanco Australasia Pty Ltd, who provided funding for this study. A.K. has no conflict of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 IC 50 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., (© 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Parasitic worms are serious pests of humans, livestock, and crops worldwide. Multiple management strategies are employed in order to reduce their impact, and some of these may affect their genome and population allelic frequency distribution. The evolution of chemical resistance, ecological changes, and pest dispersal has allowed an increasing number of pests to become difficult to control with current management methods. Their lifestyle limits the use of ecological and individual-based management of populations. There is a need to develop rapid, affordable, and simple diagnostics to assess the efficacy of management strategies and delay the evolution of resistance to these strategies. This study presents a multilocus, equal-representation, whole-genome pooled single nucleotide polymorphisms (SNPs) selection approach as a monitoring tool for the ovine nematode parasite Haemonchus contortus. The SNP selection method used two reference genomes of different quality, then validated these SNPs against a high-quality recent genome assembly. From over 11 million high-quality SNPs identified, 334 SNPs were selected, of which 262 were species-specific, yielded similar allele frequencies when assessed as multiple individuals or as pools of individuals, and suitable to distinguish mixed nematode isolate pools from single isolate pools. As a proof-of-concept, 21 Australian H. contortus populations with various phenotypes and genotypes were screened. This analysis confirmed the overall low level of genetic differentiation between populations collected from the field, but clearly identifying highly inbred populations, and populations showing genetic signatures associated with chemical resistance. The analysis showed that 66% of the SNPs were necessary for stability in assessing population genetic patterns, and SNP pairs did not show linkage according to allelic frequencies across the 21 populations. This method demonstrates that ongoing monitoring of parasite allelic frequencies and genetic changes can be achieved as a management assessment tool to identify drug-treatment failure, population incursions, and inbreeding signatures due to selection. The SNP selection method could also be applied to other parasite species., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

This review article provides an historical perspective on some of the major research advances of relevance to ruminant livestock gastrointestinal nematode control over the last 50 years. Over this period, gastrointestinal nematode control has been dominated by the use of broad-spectrum anthelmintic drugs. Whilst this has provided unprecedented levels of successful control for many years, this approach has been gradually breaking down for more than two decades and is increasingly unsustainable which is due, at least in part, to the emergence of anthelmintic drug resistance and a number of other factors discussed in this article. We first cover the remarkable success story of the discovery and development of broad-spectrum anthelmintic drugs, the changing face of anthelmintic drug discovery research and the emergence of anthelmintic resistance. This is followed by a review of some of the major advances in the increasingly important area of non-pharmaceutical gastrointestinal nematode control including immunology and vaccine development, epidemiological modelling and some of the alternative control strategies such as breeding for host resistance, refugia-based methods and biological control. The last 50 years have witnessed remarkable innovation and success in research aiming to improve ruminant livestock gastrointestinal nematode control, particularly given the relatively small size of the research community and limited funding. In spite of this, the growing global demand for livestock products, together with the need to maximise production efficiencies, reduce environmental impacts and safeguard animal welfare - as well as specific challenges such as anthelmintic drug resistance and climate change- mean that gastrointestinal nematode researchers will need to be as innovative in the next 50 years as in the last., (Copyright © 2021 Australian Society for Parasitology. All rights reserved.)

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M-PONTX-Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC 50 values of 2.8-5.6 μM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides., Competing Interests: The authors declare no competing interest.

Anthelmintic resistance is a significant threat to livestock production systems worldwide and is emerging as an important issue in companion animal parasite management. It is also an emerging concern for the control of human soil-transmitted helminths and filaria. An important aspect of managing anthelmintic resistance is the ability to utilise diagnostic tests to detect its emergence at an early stage. In host-parasite systems where resistance is already widespread, diagnostics have a potentially important role in determining those drugs that remain the most effective. The development of molecular diagnostics for anthelmintic resistance is one focus of the Consortium for Anthelmintic Resistance and Susceptibility (CARS) group. The present paper reflects discussions of this issue that occurred at the most recent meeting of the group in Wisconsin, USA, in July 2019. We compare molecular resistance diagnostics with in vivo and in vitro phenotypic methods, and highlight the advantages and disadvantages of each. We assess whether our knowledge on the identity of molecular markers for resistance towards the different drug classes is sufficient to provide some expectation that molecular tests for field use may be available in the short-to-medium term. We describe some practical aspects of such tests and how our current capabilities compare to the requirements of an 'ideal' test. Finally, we describe examples of drug class/parasite species interactions that provide the best opportunity for commercial use of molecular tests in the near future. We argue that while such prototype tests may not satisfy the requirements of an 'ideal' test, their potential to provide significant advances over currently-used phenotypic methods warrants their development as field diagnostics., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Existing diagnostic methods for the parasitic gastrointestinal nematode, Haemonchus contortus, are time consuming and require specialised expertise, limiting their utility in the field. A practical, on-farm diagnostic tool could facilitate timely treatment decisions, thereby preventing losses in production and flock welfare. We previously demonstrated the ability of visible-near-infrared (Vis-NIR) spectroscopy to detect and quantify blood in sheep faeces with high accuracy. Here we report our investigation of whether variation in sheep type and environment affect the prediction accuracy of Vis-NIR spectroscopy in quantifying blood in faeces., Methods: Visible-NIR spectra were obtained from worm-free sheep faeces collected from different environments and sheep types in South Australia (SA) and New South Wales, Australia and spiked with various sheep blood concentrations. Spectra were analysed using principal component analysis (PCA), and calibration models were built around the haemoglobin (Hb) wavelength region (387-609 nm) using partial least squares regression. Models were used to predict Hb concentrations in spiked faeces from SA and naturally infected sheep faeces from Queensland (QLD). Samples from QLD were quantified using Hemastix® test strip and FAMACHA© diagnostic test scores., Results: Principal component analysis showed that location, class of sheep and pooled versus individual samples were factors affecting the Hb predictions. The models successfully differentiated 'healthy' SA samples from those requiring anthelmintic treatment with moderate to good prediction accuracy (sensitivity 57-94%, specificity 44-79%). The models were not predictive for blood in the naturally infected QLD samples, which may be due in part to variability of faecal background and blood chemistry between samples, or the difference in validation methods used for blood quantification. PCA of the QLD samples, however, identified a difference between samples containing high and low quantities of blood., Conclusion: This study demonstrates the potential of Vis-NIR spectroscopy for estimating blood concentration in faeces from various types of sheep and environmental backgrounds. However, the calibration models developed here did not capture sufficient environmental variation to accurately predict Hb in faeces collected from environments different to those used in the calibration model. Consequently, it will be necessary to establish models that incorporate samples that are more representative of areas where H. contortus is endemic.

The World Health Organization (WHO) has defined moderate-to-heavy intensity (M&HI) infections with soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the two hookworms, Ancylostoma duodenale and Necator americanus) based on specific values of eggs per gram of stool, as measured by the Kato-Katz method. There are a variety of novel microscopy and DNA-based methods but it remains unclear whether applying current WHO thresholds on to these methods allows for a reliable classification of M&HI infections. We evaluated both WHO and method-specific thresholds for classifying the M&HI infections for novel microscopic (FECPAKG2, McMaster and Mini-FLOTAC) and DNA-based (qPCR) diagnostic methods. For this, we determined method-specific thresholds that best classified M&HI infections (defined by Kato-Katz and WHO thresholds; reference method) in two multi-country drug efficacy studies. Subsequently, we verified whether applying these method-specific thresholds improved the agreement in classifying M&HI infections compared to the reference method. When we applied the WHO thresholds, the new microscopic methods mainly misclassified M&HI as low intensity, and to a lesser extent low intensity infection as M&HI. For FECPAKG2, applying the method-specific thresholds significantly improved the agreement for Ascaris (moderate → substantial), Trichuris and hookworms (fair → moderate). For Mini-FLOTAC, a significantly improved agreement was observed for hookworms only (fair → moderate). For the other STHs, the agreement was almost perfect and remained unchanged. For McMaster, the method-specific thresholds revealed a fair to a substantial agreement but did not significantly improve the agreement. For qPCR, the method-specific thresholds based on genome equivalents per ml of DNA moderately agreed with the reference method for hookworm and Trichuris infections. For Ascaris, there was a substantial agreement. We defined method-specific thresholds that improved the classification of M&HI infections. Validation studies are required before they can be recommended for general use in assessing M&HI infections in programmatic settings., Competing Interests: The FECPAKG2 technology was produced and distributed by Techion Group Ltd, of which ET is an employee and GM is managing director. Both hold stocks in Techion Group Ltd. The Mini-FLOTAC device is a commercial product distributed by GC, LR and MPM through the University of Napoli Federico II. However, their affiliations did not play any role in the preparation and submission of this manuscript. All other authors declared that they have no competing interests.

Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and diverse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula , exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus , the fruit fly Drosophila melanogaster , and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure-activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types., Competing Interests: The authors declare no conflict of interest.

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase αE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp αE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid ( Myzus persicae ). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy., Competing Interests: Competing interest statement: G.J.C., C.J.J., and N.L., are inventors on a US patent application (62/443,825) for the described synergists.

Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC 50 4.9 μM and 16.1 μM, respectively, in a drug-susceptible isolate) with no existing cross-resistance in two multidrug resistant isolates and a monepantel-resistant isolate. Combinations of pentamidine with commercial anthelmintics showed additive activity, with no significant synergism detected. Pentamidine and diminazene showed different life-stage patterns of activity; both were active against early stage larvae in development assays, but only diminazene was active against the infective L3 stage in migration assays. This suggests some differences in uptake of the two drugs across the nematode cuticle, or differences in the nature and expression patterns of their molecular targets. As pentamidine and diminazene have been reported to be potent inhibitors of mammalian acid-sensing ion channels (ASIC), we tested the activity of known ASIC inhibitors against H. contortus to probe whether these channels may represent potential anthelmintic targets in nematodes. Remarkably, the spider-venom peptide Hi1a, a potent inhibitor of ASIC1a, inhibited H. contortus larval development with an IC 50 of 22.9 ± 1.9 μM. This study highlights the potential use of diarylamidines as anthelmintics, although their activity needs to be confirmed in vivo. In addition, our demonstration that ASIC inhibitors have anthelmintic activity raises the possibility that this family of ion channels may represent a novel anthelmintic target., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Breeding for resistance to gastrointestinal nematodes (GIN) in sheep relies largely on the use of worm egg counts (WEC) to identify animals that are able to resist infection. As an alternative to such measures of parasite load we aimed to develop a method to identify animals showing resistance to GIN infection based on the impact of the infection on blood parameters. We hypothesized that blood parameters may provide a measure of infection level with a blood-feeding parasite through perturbation of red blood cell parameters due to feeding behaviour of the parasite, and white blood cell parameters through the mounting of an immune response in the host animal. We measured a set of blood parameters in 390 sheep that had been exposed to an artificial regime of repeated challenges with Trichostrongylus colubriformis followed by Haemonchus contortus. A simple analysis revealed strong relationships between single blood parameters and WECs with correlation coefficients -0.54 to -0.60. We then used more complex multi-variate methods based on supervised classifier models (including Bayesian Network) as well as regression models (Lasso and Elastic Net) to study the relationships between WECs and blood parameters, and derived algorithms describing the relationships. The ability of these algorithms to classify sheep GIN resistance status was tested using the WEC and blood parameters collected from a different group of 418 sheep that had acquired natural infections of H. contortus from pasture. We identified the most resistant and most susceptible animals (10% percentiles) of this group based on WECs, and then compared the identities of these animals to the identities of animals that were predicted to be most resistant and most susceptible by our algorithms. The models showed varying abilities to predict susceptible and resistant sheep, with up to 65% of the most susceptible animals and 30% of the most resistant animals identified by the Elastic Net model algorithms. The prediction algorithms derived from female sheep data performed better than those for male sheep in some cases, with the predicted animals accounting for up to 50-60% of the actual resistant and susceptible female animals. Heritability values were calculated for blood parameters and the aggregate trait descriptions defined by the novel prediction algorithms. The aggregate trait descriptions were moderately heritable and may therefore be suitable for use in genetic selection strategies. The present study indicates that multivariate models based on blood parameter data showed some ability to predict the resistance status of sheep to infection with H. contortus., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

The larval development assay has been used for many years to measure the sensitivity of the free-living life stages of trichostrongylid nematodes to anthelmintics. The assay has applications in both drug discovery and the diagnosis of drug resistance. We revisited the usefulness of the larval development assay for diagnosis of resistance to levamisole using field-derived isolates of Haemonchus contortus and Trichostrongylus colubriformis showing varying levels of resistance to this drug in vivo. Each of the resistant isolates showed a plateau in their larval development assay dose-response at the highest drug concentrations tested, representing a highly-resistant fraction, amounting to between 6.9 and 55.1% of the populations. The remaining population fractions for the resistant isolates showed IC 50 values from 1.4- to 17.8-fold higher than their corresponding susceptible isolate of the same species. We used a data set from the DrenchRite ® test user manual to derive equations describing the relationship between the IC 50 values for these low / moderate resistance components of each population and in vivo drug efficacy, and then used these equations to estimate the expected in vivo efficacy of levamisole against this population component of each isolate. A combination of this expected efficacy, with the known zero efficacy of the drug in vivo against the highly-resistant population fractions in each isolate, allowed us to calculate a predicted drug efficacy for the whole population for each isolate. The predicted levamisole efficacies for the three resistant H. contortus isolates were 88.8, 84.1 and 43.7%. These compared favourably with the actual efficacy of the drug against these isolates as determined in faecal egg count reduction tests or total worm count studies: 79, 66.3 and 40.6%, respectively. Similarly, for T. colubriformis, predicted efficacies of 82.0 and 1.8% compared favourably with the actual efficacies of 65-92 % and 0%, respectively. This study illustrates the usefulness of the larval development assay as a diagnostic tool for predicting in vivo efficacy of levamisole against H. contortus and T. colubriformis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

There is an increasing interest in the use of combination anthelmintic products for the control of intestinal nematode parasites of livestock. These products are seen as attractive options for parasite control in the face of increasing levels of resistance to the different anthelmintic drug classes, as well as a means to slow the rate at which resistance develops to the individual components of the combination. With the recent introduction of an anthelmintic combination product containing abamectin and monepantel (at 1:12.5), we were interested in measuring the response of drug-susceptible and drug-resistant isolates of Haemonchus contortus to these two drugs alone and in combination, using larval development assays. The GWBII isolate showed resistance to abamectin (12-fold) alongside susceptibility to monepantel. The resistance ratio was reduced from 12- to 3.2-fold when the two drugs were combined. The MPL-R isolate was resistant to both drugs, with resistance factors of 6-fold towards abamectin, and 10.6- and 1008-fold towards monepantel in two sub-populations present in the isolate. This isolate showed 6.4-fold resistance to the drug combination. Hence, for both GWBII and MPL-R, the level of resistance towards the combination was reduced compared to the resistance towards abamectin or monepantel alone, respectively, but was not abolished. However, for GWBII, this in vitro resistance to the drug combination would be expected to have no impact on the in vivo efficacy of the combination drench product as the isolate is resistant to only the abamectin component of the drench, with monepantel remaining effective. On the other hand, the observed in vitro resistance to the combination shown by the MPL-R isolate is derived from significant levels of resistance towards both components separately, and hence may impact on in vivo efficacy of the combination. Isobologram analysis did not find any evidence for a synergistic interaction between the two drugs in larval development assays. We examined the predicted effects of varying the abamectin:monepantel ratio in drug combinations, assuming that the two drugs acted in an additive fashion. For GWBII, resistance to the drug combination was reduced to almost zero as the abamectin:monepantel ratio increased from 1:12.5 to 1:100, reflecting its resistance to only the abamectin component of the combination. For MPL-R, on the other hand, the resistance increased as the relative proportion of monepantel in the combination was increased, reflecting the extreme level of in vitro resistance shown by this isolate to monepantel., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There is a need to investigate new methods of controlling cyathostomins in horses due to increasing anthelmintic resistance amongst these parasites. In a previous study we identified the Australian plant Alectryon oleifolius as having anthelmintic activity towards cyathostomins. This study aimed to isolate and identify the bioactive compound(s) responsible for all or part of this anthelmintic activity and quantify its activity in vitro. The condensed tannin procyanidin A2 was isolated from the plant through a process of bioassay guided fractionation and identified using 1D and 2D nuclear magnetic resonance spectroscopy and high performance liquid chromatography with mass spectrometry. Procyanidin A2 demonstrated significant anthelmintic activity in larval development assays, completely inhibiting development from egg to third larval stage at concentrations as low as 50μg/mL and having an IC 50 value of 12.6μg/mL. Procyanidin A2 also significantly inhibited larval migration at concentrations of 25μg/mL. This study indicates that procyanidin A2 is the principal anthelmintic compound in extracts from A. oleifolius, and further highlights the potential for the use of this plant as a component of cyathostomin control programs in the future., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The use of macrocyclic lactone drugs for control of equine cyathostomins is threatened by increasing levels of resistance. Detection of changes in drug sensitivity is important for effective and sustainable management of cyathostomins, however, at present such detection relies on the use of the faecal egg count reduction test, which is known to be an insensitive method. The present study therefore aimed to examine the use of a 96-well plate larval migration inhibition test for detection of resistance to macrocyclic lactone drugs in cyathostomins. We optimised conditions for migration of larvae, and examined the effects of larval storage time on drug dose responses. The modified test was able to define the sensitivity of cyathostomin isolates to ivermectin and eprinomectin in terms of dose response curves, and IC 50 and IC 95 values. The IC 95 showed much greater consistency than the IC 50 with larvae that had been stored for different periods prior to the test. Comparisons between two isolates, which had both been defined previously as susceptible using faecal egg count reduction tests, showed more variation at the IC 50 compared to the IC 95 . Limitations of the test included the degree of variation in control-well migration despite optimisation of migration incubation conditions, and the need to incorporate a method to determine the species composition of the larval populations to account for possible species differences in drug sensitivity among cyathostomins. Validation of the technique on reference susceptible and resistant isolates of known species composition is still required., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Resistance to the anthelmintic drug monepantel (Zolvix ® ) has emerged in parasitic worms infecting sheep and goats. The mechanism of resistance in these cases is unknown. The drug targets nicotinic acetylcholine receptors belonging to the nematode-specific DEG-3 subfamily. We examined the receptor gene, Hco-mptl-1, in a highly Zolvix ® -resistant and a -susceptible isolate of the parasitic nematode Haemonchus contortus. cDNA coding for the full length receptor protein (Hco-MPTL-1) was present in all clones prepared from a pool of susceptible larvae (21/21 clones) and approximately 50% of those from the resistant isolate (17/33). On the other hand, the remaining clones from the resistant isolate showed various mutations that resulted in truncated predicted proteins, missing at least one transmembrane domain. The most common mutation (11/33 clones) resulted in the retention of intron 15, a premature stop codon, and a truncated protein. Sequencing of intron 15 genomic DNA showed very few SNPs in susceptible larvae and in 12/18 clones from resistant larvae, alongside the presence of at least 17 SNPs in the remaining resistant clones. The present study shows that the highly resistant isolate has a number of mutations in the drug target gene that would most-likely result in a non-functional receptor, thus rendering the larvae insensitive to the drug. The presence of many wild-type sequences in this highly-resistant population suggests that there was a significant presence of heterozygotes in the survivors of the field drench treatment from which the isolate was derived, and hence that at least some of the mutations may be dominant. Alternatively, their presence may be due to the additional influence of mutations at another locus contributing to the resistance phenotype. The presence of multiple separate mutations in the Hco-mptl-1 gene in this viable field-derived worm isolate may at least partly explain why resistance to Zolvix ® has arisen rapidly in the field., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The macrocyclic lactone (ML) drugs are central to the control of equine strongyles but recent international reports raise concerns about reduced efficacy of these drugs against cyathostomins. The objectives of the present study were firstly, to evaluate the efficacy of ML drugs against cyathostomins on a cross-section of Australian horse farms, and secondly, to determine the egg reappearance period (ERP) following treatment of horses with MLs. A total of 419 horses on 43 properties were treated orally with ivermectin, abamectin or moxidectin, at recommended dose rates and drug efficacy was determined using the faecal egg count reduction test. Efficacy of 100% at 14days post-treatment was reported on all of the 43 farms. ERP following ivermectin treatment was 6weeks on two properties and ERP following moxidectin treatment was 12weeks on a third property. These ERPs are shorter than those reported at the time of commercial release of these drugs which likely reflects changing drug susceptibility of the cyathostomin populations tested. Ongoing surveillance of drug efficacy and ERPs should be part of an integrated management approach to equine worm control that prioritises the preservation of anthelmintic efficacy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Histone deacetylase inhibitors (HDACi) are being investigated for the control of various human parasites. Here we investigate their potential as insecticides for the control of a major ecto-parasite of sheep, the Australian sheep blowfly, Lucilia cuprina. We assessed the ability of HDACi from various chemical classes to inhibit the development of blowfly larvae in vitro, and to inhibit HDAC activity in nuclear protein extracts prepared from blowfly eggs. The HDACi prodrug romidepsin, a cyclic depsipeptide that forms a thiolate, was the most potent inhibitor of larval growth, with equivalent or greater potency than three commercial blowfly insecticides. Other HDACi with potent activity were hydroxamic acids (trichostatin, CUDC-907, AR-42), a thioester (KD5170), a disulphide (Psammaplin A), and a cyclic tetrapeptide bearing a ketone (apicidin). On the other hand, no insecticidal activity was observed for certain other hydroxamic acids, fatty acids, and the sesquiterpene lactone parthenolide. The structural diversity of the 31 hydroxamic acids examined here revealed some structural requirements for insecticidal activity; for example, among compounds with flexible linear zinc-binding extensions, greater potency was observed in the presence of branched capping groups that likely make multiple interactions with the blowfly HDAC enzymes. The insecticidal activity correlated with inhibition of HDAC activity in blowfly nuclear protein extracts, indicating that the toxicity was most likely due to inhibition of HDAC enzymes in the blowfly larvae. The inhibitor potencies against blowfly larvae are different from inhibition of human HDACs, suggesting some selectivity for human over blowfly HDACs, and a potential for developing compounds with the inverse selectivity. In summary, these novel findings support blowfly HDAC enzymes as new targets for blowfly control, and point to development of HDAC inhibitors as a promising new class of insecticides., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: There is some evidence that ATP binding cassette (ABC) transporters play a role in resistance to anthelmintics, particularly against macrocyclic lactones. Some anthelmintics, including ivermectin (IVM), have been shown to induce transcription of multiple ABC transporters in nematodes; however, the effects of monepantel (MPL) on transcription of these transporter genes has not been studied., Methods: Larvae of two MPL-susceptible isolates of Haemonchus contortus were exposed to MPL at two concentrations (2.5 and 250 μg/ml) for periods of 3, 6 and 24 h. Transcription levels of sixteen ABC transporter genes were measured at the end of the incubation periods. The consequences of MPL exposure were examined by measuring rhodamine-123 efflux from the larvae, and their sensitivity to subsequent treatment with IVM or levamisole., Results: Multiple ABC transporter genes showed significantly higher transcription in both worm isolates following exposure to MPL at 250 μg/ml for 3, 6 or 24 h, particularly the P-glycoprotein (P-gp) genes pgp-11, pgp-12 and pgp-14. Of these, only pgp-11 maintained the elevated levels 24 h after the end of the drug exposure period. In contrast, there was only a single instance of low-level upregulation as a result of exposure to MPL at 2.5 μg/ml. Larvae exposed to MPL at 250 μg/ml showed an increased efflux of rhodamine-123 and a proportion of the larval population showed an ability to subsequently tolerate higher concentrations of IVM in migration assays. There was no increased tolerance to IVM following pre-exposure to MPL at 2.5 μg/ml., Conclusions: Exposure of H. contortus larvae to 250 μg/ml MPL results in increased transcription of multiple transporter genes and increased R-123 efflux. The subsequent ability of a proportion of the larvae to tolerate IVM suggests a protective role of ABC transporters across different chemical entities. However, these observations were only made at a concentration of MPL well above that experienced by parasitic life stages in vivo, and hence their significance remains unclear.

Anthelmintic resistance is a major problem in parasitic nematodes of livestock worldwide. One means to counter resistance is to use synergists that specifically inhibit resistance mechanisms in order to restore the toxicity, and hence preserve the usefulness, of currently available anthelmintics. P-glycoproteins (P-gps) eliminate a wide variety of structurally unrelated xenobiotics from cells, and have been implicated in anthelmintic resistance. Crizotinib is a tyrosine kinase inhibitor under development as a cancer therapeutic. The compound also inhibits P-gps, and has been shown to reverse multidrug resistance in cancer cells. We were therefore interested in determining if the compound was able to increase the sensitivity of Haemonchus contortus larvae to ivermectin, as measured by in vitro larval development and migration assays with a drug-resistant and a -susceptible isolate. In migration assays, co-administration of crizotinib increased the toxicity of ivermectin to resistant larvae (up to 5.7-fold decrease in ivermectin IC50), and rendered the resistant larvae equally or more sensitive to ivermectin than the susceptible isolate. On the other hand, co-administration of crizotinib had no effect on ivermectin sensitivity in the susceptible isolate. In development assays, significant increases in the sensitivity of both the resistant (up to 1.9-fold) and susceptible (up to 1.6-fold) larvae to ivermectin were observed, although the magnitude of the observed synergism was less than seen in migration assays, and the resistant larvae retained significant levels of ivermectin resistance. By highlighting the ability of the P-gp inhibitor crizotinib to increase the sensitivity of H. contortus larvae to ivermectin, this study provides further evidence that P-gp inhibitors are potential tools for modulating the efficacy of anthelmintics. In addition, the differences in the outcomes of the two assays, with 'resistance-breaking' effects being much more marked in migration assays, suggest that some life-stage-specific aspects may exist in the interaction of ivermectin with P-gps in the two worm isolates., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Resistance to the amino-acetonitrile derivative monepantel has been reported in several species of gastrointestinal nematodes over recent years. We were interested in the use of in vitro assays with free-living worm life-stages to detect resistance to this drug. We therefore used larval development and larval migration assays to examine dose response relationships for the drug against two susceptible and one resistant isolate of Haemonchus contortus. The resistant isolate was established by laboratory propagation of the survivors of a field treatment with Zolvix(®) that had originally resulted in a drug efficacy of over 99%. Drug efficacy against this field-derived laboratory-propagated resistant isolate in vivo was approximately 15%. The larval development assay proved able to discriminate between the susceptible and resistant isolates, with larvae of the resistant isolate showing an ability to develop at higher drug concentrations than the two susceptible isolates. The resistant isolate showed the presence of two distinct subpopulations, separated by a plateau in the dose-response curve. Sub-population 1 (approximately 40% of the total population) showed a low level of resistance with an IC50 increased approximately 7-fold compared to the baseline susceptible isolate, while sub-population 2 (the remaining 60% of the total population) showed an IC50 increased over 1000-fold compared to the baseline susceptible isolate. This level of resistance is unusually high for any gastrointestinal nematode species in drug dose-response in vitro assays. In contrast, the migration assay could not discriminate between the three isolates, with migration not reduced to zero at any of the drug concentrations tested. This study demonstrates that a larval development assay is able to detect resistance to monepantel in H. contortus, and that resistance can exist in two distinct forms. This suggests that at least two separate monepantel resistance mechanisms are acting within the worm isolate studied here, with one or more mechanisms conferring a much higher level of resistance than the other(s)., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Haemonchus contortus has shown a great ability to develop resistance to anthelmintic drugs. In many instances, resistance has appeared less than 10years after the introduction of a new drug class. Field populations of this species now show resistance to all major anthelmintic drug classes, including benzimidazoles (BZs), imidazothiazoles and macrocyclic lactones. In addition, resistance to the recently introduced amino-acetonitrile derivative class (monepantel) has already been reported. The existence of field populations showing resistance to all three major drug classes, and the early appearance of resistance to monepantel, threatens the sustainability of sheep and goat production systems worldwide. This chapter reviews the history of the development of resistance to the various anthelmintics in H. contortus and examines the mechanisms utilized by this species to resist the effects of these drugs. Some of these mechanisms are well understood, particularly for BZ drugs, while our knowledge and understanding of others are increasing. Finally, we summarize methods available for the diagnosis of resistance. While such diagnosis currently relies largely on the faecal egg count reduction test, which suffers from issues of expense and sensitivity, we describe past and current efforts to utilize cheaper and less laborious phenotypic assays with free-living life stages, and then describe progress on the development of molecular assays to provide sensitive resistance-detection tests., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Australian sheep blowfly, Lucilia cuprina, is an ecto-parasite that causes significant economic losses in the sheep industry. Emerging resistance to insecticides used to protect sheep from this parasite is driving the search for new drugs that act via different mechanisms. Inhibitors of histone deacetylases (HDACs), enzymes essential for regulating eukaryotic gene transcription, are prospective new insecticides based on their capacity to kill human parasites. The blowfly genome was found here to contain five HDAC genes corresponding to human HDACs 1, 3, 4, 6 and 11. The catalytic domains of blowfly HDACs 1 and 3 have high sequence identity with corresponding human and other Dipteran insect HDACs (Musca domestica and Drosophila melanogaster). On the other hand, HDACs 4, 6 and 11 from the blowfly and the other Dipteran species showed up to 53% difference in catalytic domain amino acids from corresponding human sequences, suggesting the possibility of developing HDAC inhibitors specific for insects as desired for a commercial insecticide. Differences in transcription patterns for different blowfly HDACs through the life cycle, and between the sexes of adult flies, suggest different functions in regulating gene transcription within this organism and possibly different vulnerabilities. Data that supports HDACs as possible new insecticide targets is the finding that trichostatin A and suberoylanilide hydroxamic acid retarded growth of early instar blowfly larvae in vitro, and reduced the pupation rate. Trichostatin A was 8-fold less potent than the commercial insecticide cyromazine in inhibiting larval growth. Our results support further development of inhibitors of blowfly HDACs with selectivity over human and other mammalian HDACs as a new class of prospective insecticides for sheep blowfly., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

P-glycoproteins (P-gps) play an important role in the sensitivity of nematodes to anthelmintic drugs. They have been implicated in a number of anthelmintic resistances, particularly for macrocyclic lactone drugs. Hence, inhibition of nematode P-gps has been suggested as a means of reversing some types of anthelmintic resistance. The present study aimed to investigate the ability of the most-recently developed group of P-gp inhibitors (the so-called 'third generation' of inhibitors) including tariquidar, zosuquidar and elacridar, to increase the sensitivity of Haemonchus contortus larvae to various anthelmintics (ivermectin, levamisole and thiabendazole) in vitro. We compared these compounds to some older P-gp inhibitors (e.g. verapamil and valspodar). Larval migration and development assays were used to measure the sensitivity of larvae to anthelmintics alone, or in combination with P-gp inhibitors. Significant increases in sensitivity to ivermectin were observed with zosuquidar and tariquidar in larval migration assays (synergism ratios up to 6-fold). Several of the inhibitors increased the sensitivity of both the drug-resistant and -susceptible isolates (e.g. tariquidar with ivermectin in migration assays, zosuquidar with ivermectin in larval development assays), while others had significant effects on the resistant isolate only (e.g. zosuquidar with ivermectin in migration assays, verapamil with ivermectin in development assays). This suggests that some of the inhibitors interact with P-gps representing intrinsic pathways present across nematode populations with quite different drug sensitivities, while other inhibitors interact with P-gps of significance only to resistant nematodes, and hence most likely representing an acquired resistance mechanism. The study highlights the potential of the third generation of P-gp inhibitors for increasing the sensitivity of nematodes to anthelmintics., (Copyright © 2015 Elsevier B.V. All rights reserved.)

There is some evidence that resistance to levamisole and pyrantel in trichostrongylid nematodes is due to changes in the composition of nicotinic acetylcholine receptors (nAChRs) which represent the drug target site. Altered expression patterns of genes coding for nAChR subunits, as well as the presence of truncated versions of several subunits, have been implicated in observed resistances. The studies have mostly compared target sites in worm isolates of very different genetic background, and hence the ability to associate the molecular changes with drug sensitivity alone have been clouded to some extent. The present study aimed to circumvent this issue by following target site gene expression pattern changes as resistance developed in Haemonchus contortus worms under laboratory selection pressure with levamisole. We applied drug selection pressure to early stage larvae in vitro over nine generations, and monitored changes in larval and adult drug sensitivities and target site gene expression patterns. High level resistance developed in larvae, with resistance factors of 94-fold and 1350-fold at the IC50 and IC95, respectively, in larval development assays after nine generations of selection. There was some cross-resistance to bephenium (70-fold increase in IC95). The expression of all the putative subunit components of levamisole-sensitive nAChRs, as well as a number of ancillary protein genes, particularly Hco-unc-29.1 and -ric-3, were significantly decreased (up to 5.5-fold) in the resistant larvae at generation nine compared to the starting population. However, adult worms did not show any resistance to levamisole, and showed an inverse pattern of gene expression changes, with many target site genes showing increased expression compared to the starting population. A comparison of the larval/adult drug sensitivity data with the known relationships for field-derived isolates indicated that the adults of our selected population should have been highly resistant to the drug if the larval/adult sensitivity relationships were in accordance with previous field isolates. Hence, our selected worms showed a life-stage drug sensitivity pattern quite different to that seen in the field. The present study has highlighted an association between drug target site changes and resistance to levamisole in H. contortus larvae. However, it has also highlighted the artificial nature of the larval selection method with levamisole, as the resistance phenotype and the associated molecular changes were only observed in the drug-pressured life stage. The study therefore reinforces the need for caution in extrapolating larval-based laboratory selection outcomes to field resistances., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lucilia cuprina is a parasitic fly of major economic importance worldwide. Larvae of this fly invade their animal host, feed on tissues and excretions and progressively cause severe skin disease (myiasis). Here we report the sequence and annotation of the 458-megabase draft genome of Lucilia cuprina. Analyses of this genome and the 14,544 predicted protein-encoding genes provide unique insights into the fly's molecular biology, interactions with the host animal and insecticide resistance. These insights have broad implications for designing new methods for the prevention and control of myiasis.

We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)