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The complete replacement of fishmeal with terrestrial meals did not have a negative impact on the attractiveness, palatability, and apparent digestibility of the formulation. Shrimp were found on average to eat more and have similar appetite revival on the terrestrial meal-based formulation (TM) diet compared to the traditional fishmeal-based formulation (FM) diet. However, methionine (Met) and lysine (Lys) leached out rapidly from the TM diet, and as a result, this initially overfortified diet showed lower levels of those AA in comparison to FM after 60 min immersion. Both dietary Lys and Met were sub-optimal in TM within 120 min of immersion, whereas in comparison, the FM diet supplied consistent levels of EAA for up to 240 min immersion. Nonetheless, shrimp fed fresh TM had significantly higher peak haemolymph concentrations at 30 and 60 min for total AA, Met, and Lys than FM-fed shrimp. The over-supply of CAA far compensated leaching losses, and CAA were well absorbed and used by the shrimp within 120 min, with no obvious signs of asynchronous absorption of CAA to protein-bound AA. However, shrimp fed the TM diet that had leached out for 60 min, had haemolymph concentrations of Met and Lys that were only 41% and 44% of the ones on fresh feed respectively, while there was a negligible effect of leaching on FM. This study provides further insight into the feeding behaviour and bioavailability of dietary amino acids for P. monodon juveniles.

Aquaculture production can be improved by understanding how ingredients can affect feed intake and digestion. This is an area poorly understood for many aquaculture species, including for the shrimp Penaeus monodon. To investigate these processes, we measured feed intake, the rate at which digesta moves through the gastro-intestinal tract, the change of free amino acids (FAA) circulating in the haemolymph following feeding, and how different test ingredients affect these processes in this species. Diets supplemented with microbial Novacq™ (NQ), krill meal (KM) or whole squid meal (SQ) and a control diet were used across two separate experiments. In the first, feed intake was measured in communally housed shrimp for 60, 120 and 360 min to examine the effects of diet on ingestion. Feed intake after 360 min was greater in shrimp offered the SQ diet while no effect was observed at earlier time points. In the second experiment, individually housed shrimp were used to measure feed intake, the rate at which digesta passed through the gut and the change of individual FAA in the haemolymph over a 30 min period. For this experiment, addition of SQ and NQ increased both the rate at which digesta travels through the gut and post-prandial concentration of FAA in haemolymph at 5 min. Krill meal had the highest haemolymph concentration of amino acids at 10 min and all dietary treatments showed a convergence of haemolymph FAA by 30 min. The different ingredients appear to affect the appearance of FAA in haemolymph through different mechanisms. For SQ, the FAA content of the diet was high, being 2.8 times that of the others (27.6 vs. 10.0 g kg−1), and it is hypothesized that the availability of these FAA resulted in the rapid increase in haemolymph observed for this treatment. In contrast, two other ingredients also led to rapid increases in haemolymph FAA. However, for these diets the amount of FAA present was similar to the control indicating that other characteristics of the meals may be causal. Our data indicate that specific ingredients can increase both the rate at which digesta travels through the gut and the rate of absorption of amino acids and that these mechanisms could explain increases in both feed duration and intake.

Although Litopenaeus vannamei is the most produced and researched shrimp species worldwide, there are no published studies evaluating the use of the microbial biomass, Novacq™, in L. vannamei feeds. This study investigated the effects of supplementing Novacq™ at 100 g kg−1 in practical and commercial feeds in postlarvae 12 (PL 12) and juvenile white leg shrimp. Three experiments were carried out. In Experiment 1, PL12 were fed for 21 days with a Control, Novacq™ or Commercial feed. The Novacq feed was equal in PL12 performance to the Commercial diet and superior to the Control (final individual weight = 0.169, 0.167 and 0.087 g; percentage weight gain = 8014, 7956 and 4091% for Novacq™, Commercial and Control feeds, respectively). No statistical differences in survival, coefficient of variation and daily feed offered were observed across dietary treatments. In Experiments 2 and 3 the performance of juvenile shrimp fed practical and Commercial feeds supplemented with Novacq™ for 42 days, respectively. In Experiment 2, Novacq™ feed outperformed the Control feed by improving survival (89 vs. 51%), final individual weight (7.4 vs. 3.5 g), weight gain (7.1 vs. 3.2 g), and percentage weight gain (1997 vs. 898%). In Experiment 3 supplementing Novacq™ into the Commercial feed improved performance (final individual weight = 6.4 vs. 5.5 g, weight gain = 6.0 vs. 5.1 g, and percentage weight gain = 1712 vs 1443%) and reduced the coefficient of variation (19 vs 53%). Shrimp fed feeds containing Novacq™ also displayed numerically reduced daily feed offered and estimated feed conversion ratio values in comparison to the other dietary treatments. Whole-body composition was similar across dietary treatments. In summary, supplementation of Novacq™ in practical and Commercial feeds improved growth and efficiency of PL12 and juvenile shrimp suggesting its validity as a useful feed additive in the nutrition of white leg shrimp.

Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone (Haliotis rubra) processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII)-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 μg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT), activated partial thromboplastin time (aPTT), and thromboelastography (TEG). All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin.

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., Graphical abstract Image 1, Highlights • We measured the insecticidal effects of histone deacetylase inhibitors against the sheep blowfly. • Insecticidal activity correlated with inhibition of HDAC enzyme activity in nuclear extracts. • Romidepsin showed equivalent or greater potency than commercial blowfly insecticides. • Some insights gained into structural requirements for insecticidal HDAC inhibitors. • Potential for HDAC inhibitors as insecticides.

Macrocyclic lactone (ML) drugs inhibit pharyngeal pumping, motility and egg laying in parasitic nematodes. Previous work has indicated that in vitro effects on worm feeding occurred at lower ivermectin concentrations than effects on worm motility, suggesting that the pharynx musculature was a more important target site for the ML drugs than somatic musculature. We have reassessed this issue of relative sensitivity by examining the response of drug-susceptible and -resistant adult Haemonchus contortus worms to abamectin in vitro using both feeding and motility assays. The motility assay involved observation of changes in the form and degree of movement of individual worms in response to the drug. A comparison of the data from the two assays indicated that worm motility was affected at drug concentrations below those required to inhibit feeding. Analysis of the motility data using different levels of sensitivity (varying in the degree to which they accounted for subtle vs. more profound changes in worm motility) provided an explanation as to why earlier reports had observed feeding to be the more sensitive target. Motility IC50 values shifted from being less than feeding IC50s to being greater than the feeding IC50s as the motility assay analysis method became less sensitive. The present study indicates that when sensitive worm motility assessment methods are utilised, worm motility is affected at lower abamectin concentrations than worm feeding, and hence highlights somatic musculature as a more important target site for this ML drug, and most likely for ML drugs in general.

SUMMARYIn this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematodeHaemonchus contortus. We chose as targets five genes that are essential inCaenorhabditis elegans(mitr-1, pat-12, vha-19, glf-1andnoah-1), orthologues of which are present and expressed inH. contortus, plus four genes previously tested by RNAi inH. contortus(ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages ofH. contortuswithEscherichia colithat express dsRNA targetting the test genes; (2) electroporation of dsRNA intoH. contortuseggs or larvae; and (3) soaking adultH. contortusin dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the ‘essential’ genes,Hc-pat-12, Hc-vha-19andHc-glf-1. We recommend theE. colifeeding method for RNAi inH. contortusand provide recommendations for future research directions for RNAi in this species.

While the F200Y SNP in the beta-tubulin gene is most commonly associated with benzimidazole resistance in trichostrongylid nematodes, other SNPs as well as drug efflux pathways have been implicated in the resistance. The relative contributions of all these mechanisms are not understood sufficiently to allow expected drug efficacy to be inferred from molecular data. As a component of developing better means to interpret molecular resistance tests, the present study utilised a drug resistant Haemonchus contortus isolate which possesses two of the principal benzimidazole resistance SNPs (E198A and F200Y) in order to assess the relative degree of resistance conferred by the two SNPs. We exposed larvae to a range of thiabendazole concentrations in in vitro development assays, and collected the surviving L3 larvae at each drug concentration to establish sub-populations showing increasing levels of resistance. We then sequenced the isotype 1 beta-tubulin gene in pooled larval samples, and measured allele frequencies at the two SNP positions. The frequency of the resistance allele at the 198 position increased as the thiabendazole concentration increased, while the frequency of the resistance allele at the 200 position decreased. Genotyping of individual larvae showed that the highest drug concentration was associated with the removal of all genotypes except for homozygous resistance at the 198 position alongside homozygous susceptible at the 200 position. This indicates that, at least for larval life stages, the E198A SNP is able to confer higher levels of resistance to benzimidazole drugs than the F200Y SNP, and that the homozygosity at 198 in the highly resistant individuals is mutually exclusive with heterozygosity or resistant homozygosity at the 200 position. This study illustrates the need to understand the relative contributions of different resistance mechanisms in order to maximise the degree to which molecular tests are able to inform on drug resistance phenotype.

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., Graphical abstract, Highlights • Lucilia cuprina genome contains five histone deacetylase genes. • Significant amino acid differences between insect and human HDACs 4,6 and 11. • Trichostatin highly toxic towards blowfly larvae. • Potential for HDAC inhibitors as insecticides.