The clock gene Cryptochrome 1 is involved in the photoresponse of embryonic hatching behavior in Bombyx mori.

The hatching of insect eggs is a classic circadian behavior rhythm controlled by the biological clock. Its function is considered to impose a daily rhythm on the embryo, allowing it to hatch within a permissible time window. However, the molecular pathways through which the clock affects embryonic hatching behavior remain unclear. Here, we utilized a clock gene Cryptochrome1 (Cry1) knockout mutant to dissect the pathways by which the circadian clock affects embryonic hatching rhythm in the silkworm. In the Cry1 mutant, the embryo hatching rhythm was disrupted. Under the constant light or constant dark incubation conditions, mutant embryos lost their hatching rhythm, while wild-type embryos hatch exhibiting free-running rhythm. In the light-dark cycle (LD), the hatching rhythm of CRY1-deficient silkworms could not be entrained by the LD photoperiod during the incubation period. The messenger RNA levels and enzymatic activities of Cht and Hel in the mutant embryos were significantly reduced at circadian time 24 (CT24). Transcriptome analysis revealed significant differences in gene expression at CT24 between the Cry1 knockout mutant and the wild-type, with 2616 differentially expressed genes identified. The enriched Gene Ontology pathway includes enzyme activity, energy availability, and protein translation. Short neuropeptide F signaling was reduced in the CT24 embryonic brain of the mutant, the expression of the neuropeptide PTTH was also reduced and the rhythm was lost, which further affects ecdysteroid signaling. Our results suggested that the silkworm circadian clock affects neuropeptide-hormone signaling as well as physiological functions related to hatching, which may regulate the hatching rhythm.

Chemical Composition, Anthelmintic Activity, and Mechanism of Action of Lippia dominguensis Mold. Essential Oil on Haemonchus contortus.

Gastrointestinal nematode parasitism is a major burden to small ruminant production globally, compounded by increasing anthelmintic resistance. Previous studies have identified essential oils (EOs) from the Lippia genus with antiprotozoal and anthelmintic effects. Lippia dominguensis Moldenke (Ld), an endemic specie from the Dominican Republic, has similar popular uses, however, is chemically and pharmacologically yet uncharacterized. Here, we investigated the in vitro anthelmintic activity of LdEO and its ultrastructural effects on eggs and adult nematodes of Haemonchus contortus multidrug-resistant isolated. The GC/MS analysis showed linalool (33.85 %), 1,8-cineole (30.88 %), and δ-terpineol (10.61 %) as the main EO constituents. The LdEO showed an IC50 =0.523 mg/mL in the egg hatch test, and the motility in the adult worm motility test was 95.8 % at 1 mg/mL. The confocal scanning laser microscopy of eggs indicated permeabilization or disruption of egg cell membranes as the possible mechanism of action of LdEO. The scanning electron microscopy of adult worms showed wrinkling, undulations, and cuticular disruptions. The LdEO displayed significant in vitro anthelmintic activity on eggs and adult worms of H. contortus. Additionally, the LdEO showed low oral toxicity in mice at 2,000 mg/kg. Thus, additional in vivo studies are justified to determine its anthelmintic efficacy in small ruminants.

In vitro ovicidal activity of Brongniartia montalvoana against small ruminant gastrointestinal nematodes.

Several plants of the Fabaceae family have been assessed regarding their high nutritional value and anthelmintic properties. The ovicidal effect of the hydroalcoholic extract (Bm-HAE) and subfractions from the aerial parts of Brongniartia montalvoana (Fabaceae) against a mixed strain of gastrointestinal nematodes (GIN) (Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp.) resistant to albendazole sulfoxide, ivermectin and levamisole was evaluated by the egg hatch test (EHT). The Bm-HAE was subjected to liquid-liquid chemical separation with ethyl acetate giving two fractions, an aqueous (Bm-Aq) and an organic (Bm-EtOAct). The purification of the bioactive fraction (Bm-EtOAct) through chromatographic separation resulted in four bioactive subfractions (BmR6, BmR7, BmR8 and BmR10). The treatments were designed as follows: Bm-HAE at 800, 1,500, 3,000 and 6,000 µg/mL, and Bm-Aq, Bm-EtOAct and subfractions (BmR6, BmR7, BmR8 and BmR10) at 100, 200, 400 and 800 µg/mL. Two properly negative controls (distilled water and 2% methanol) and thiabendazole (100 µg/mL) as a positive control were used for each bioassay. The chemical identification of the extract, fractions and subfractions was performed through chromatographic processes like open column chromatography, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC-PDA). Additionally, the GIN eggs exposed to the bioactive compounds were observed through confocal laser scanning microscopy (CLSM). The Bm-HAE showed 99.5% egg hatching inhibition (EHI) at 6,000 µg/mL with a lethal concentration (LC50) of 1110 µg/mL. The Bm-EtOAc fraction displayed 99.1% EHI at 800 µg/mL with LC50 = 180 µg/mL. The ovicidal activity of the four subfractions was similar at 800 µg/mL: BmR6 (92% EHI); BmR7 (100% EHI); BmR8 (97.8%); and BmR10 (99.1%). The HPLC-PDA analysis of the bioactive subfractions allowed identification of p-coumaric acid, ferulic acid and coumarin derivatives as major compounds. The CLSM analysis allowed observation of morphological alterations in unhatched larvae caused by bioactive compounds present in the Bm-EtOAc and BmR10. In addition, the flavonoids eriodyctiol, luteolin and cynaroside were described for the first time for B. montalvoana.

Acaricidal potential of active components derived from Alpinia galanga rhizome oils and their derivatives against Haemaphysalis longicornis (Acari: Ixodidae).

Acaricidal activities and acetylcholinesterase (AChE) inhibitory activities were evaluated of active constituents of the essential oil extracted from Alpinia galanga rhizomes cultivated from India and their derivatives against Haemaphysalis longicornis nymphs. In addition, the effect was investigated of active components of A. galanga oil on egg laying of adult females of H. longicornis and egg hatchability. Of the volatile components identified in A. galanga oil, ethyl cinnamate, ethyl methoxycinnamate, and methyl cinnamate at 0.32 mg/cm2 resulted in 100% mortality, respectively, indicating that the acaricidal activity of the A. galanga oil against H. longicornis nymphs could be attributed to these compounds. To evaluate the structure-activity relationship between cinnamate derivatives and their acaricidal activities, allyl cinnamate, benzyl cinnamate, isopropyl cinnamate, isobutyl cinnamate, and isoamyl cinnamate were selected. Among cinnamate derivatives tested, allyl cinnamate exhibited the most potent toxicity (LC50 = 0.055 mg/cm2) against H. longicornis nymphs. The allyl cinnamate was also tested for AChE activity in vivo in H. longicornis nymphs and was found to affect the AChE activity. Allyl cinnamate at 10-50 mg/mL inhibited egg laying of adult females of H. longicornis by 10-43%. Egg hatching was suppressed completely by treatment with allyl cinnamate at 50 mg/mL, whereas allyl cinnamate was minimally toxic against non-target earthworms, Eisenia fetida. These results suggest that allyl cinnamate can be used as an active ingredient for the development of eco-friendly tick acaricides against H. longicornis, a vector for Sever fever with thrombocytopenia syndrome (SFTS) virus.

Ruta Chalepensis Full Extract and Organic Phases Exhibit Nematocidal Activity Against Haemonchus Contortus Eggs and Infective Larvae (L3).

The ovicidal and larvicidal effect of a full aqueous extract (FE) and two phases: an aqueous (Aq-Ph) and an ethyl acetate (EtOAc-Ph) from Ruta chalepensis (Rc) stems and leaves against Haemonchus contortus (Hc) were assessed. The egg hatching inhibition (EHI) assay and larval mortality (LM) test were performed by triplicate in 96-well micro-titration plates (n=4 wells). The FE against Hc eggs and larvae was assessed at 1.25, 2.5, 5, 10 and 20 mg/mL; and 30, 60, 90, 120,150 and 200 mg/ mL, respectively. The ovicidal effect of Aq-F and EtOAc-F was assessed at 1.25, 2.5, 5, 10 and 20 mg/mL. Plates were incubated at 28 °C for 48 (ovicidal assay) and 72 h (larvicidal assay). The EHI results were considered based on the mean number of eggs hatching failure after 48 h exposure. The LM was recorded after 72 h exposure to the Rc phases and expressed as mortality percentage. The Rc FE caused 96 and 100% EHI at 10 and 20 mg/mL, respectively; meanwhile, 74% LM was recorded at 200 mg/mL (p<0.05). The Aq-Ph showed 78.5% EHI at 2.5 mg/mL. Likewise, the EtOAc-Ph caused 100% EHI in almost all concentrations. Eighteen compounds including alkaloids, cumarins, triterpens, flavonoids, tannins, saponins and sterols were identified by GC-MS analysis. The results indicate that the aqueous extract from Rc possesses bioactive compounds with in vitro nematocidal activity against Hc; mainly in the EtOAc-Ph. Further studies should be performed to elucidate those compounds searching for alternative methods of control of the sheep haemonchosis.

Toxicities of the copper and zinc oxide nanoparticles on Marshallagia marshalli (Nematoda: Trichostrongylidae): evidence on oxidative/nitrosative stress biomarkers, DNA damage and egg hatchability.

This study investigated the in vitro anthelmintic activity of copper oxide (CuO) and zinc oxide (ZnO) nanoparticles (NPs) against Marshallagia marshalli. The in vitro study was based on an egg hatch assay, adult and larvae motility inhibition assays, DNA damage, intensity protein profile along with several oxidative/nitrosative stress biomarkers including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), protein carbonylation (PCO), malondialdehyde (MDA), total antioxidant status (TAS) and nitric oxide (NO) content. Different concentrations of CuO-NPs and ZnO-NPs (1, 4, 8, 12 and 16 ppm) were used to assess anthelmintic effects on three stages of M. marshalli life cycle - that is, eggs, larvae and adult parasites for 24 h. The results indicated that CuO-NPs and ZnO-NPs played a significant role as anthelminthics, and the effect was dependent on time and concentration. The concentrations of 12 and 16 ppm of CuO-NPs and 16 ppm of ZnO-NPs resulted in the induction of oxidative/nitrosative stress (decreased SOD, GSH-Px and CAT, and increased MDA, PCO and NO), increased DNA damage, inhibition of adult and larval motility, egg hatch and low intensity of protein bands following sodium dodecyl sulphate-polyacrylamide gel electrophoresis, compared to control. It was concluded that CuO-NPs and ZnO-NPs could be utilized as novel and potential agents for the control and treatment of M. marshalli infection, and they have the pharmacological potential to be studied in vivo for further utilization in treating parasitic infections.

Endophytic Beauveria bassiana increases galling of 'Rutgers' tomato roots with Meloidogyne incognita.

Beauveria bassiana is endophytic in many plant species and has been shown to protect host plants against insect pests and plant pathogens. However, less is known about its activity against plant-parasitic nematodes. In vitro and plant assays were conducted to determine the effect of B. bassiana 11-98 (Bb) on Meloidogyne incognita (root-knot nematode; RKN). Beauveria bassiana was confirmed as an endophyte in 'Rutgers' tomato and colonization patterns of Bb in 'Rutgers' (highly susceptible to RKN) were compared with those in 'Mountain Spring' (less susceptible to RKN). In greenhouse tests with 'Rutgers' at 30 and 60 days after treatment (DAT) with RKN and Bb, there were few differences in plant growth variables among treatments in repeated trials. However, RKN root galling and egg count/root system were enhanced in plants treated with Bb at 60 DAT. In an in vitro assay with egg masses from greenhouse tests, the percentages of hatched eggs, and mobile and immobile nematodes did not differ significantly for RKN and RKN+Bb treatments. The presence of viable Bb from roots was confirmed by collecting egg suspensions from root galls and plating them on selective medium. Colonies of Bb were verified on agar medium, but no parasitism of RKN eggs was observed. Research is needed to investigate factors responsible for increased galling by RKN in the presence of endophytic Bb in 'Rutgers' tomato.

In vitro screening of 51 birdsfoot trefoil (Lotus corniculatus L.; Fabaceae) strains for anti-parasitic effects against Haemonchus contortus.

Secondary plant compounds have shown bioactivity against multi-drug resistant Haemonchus contortus in small ruminants. This study screened 51 strains of birdsfoot trefoil (BFT, Lotus corniculatus) crude aqueous extracts (BFT-AqE) for anti-parasitic activity in vitro against egg hatching, and of those 51 strains, 13 were selected for further testing of motility of first (L1) and third stage (L3) larvae, and exsheathment of L3. Proanthocyanidin content ranged between 1.4 and 63.8 mg PAC g-1 powder across the 51 BFT strains. When tested against egg hatching, 21 of the 51 aqueous extracts had an EC50 of 1-2 mg powder mL-1, 70% of the strains were >90% efficacious at 6 mg powder mL-1 and 11 of the strains were 100% efficacious at 3 mg powder mL-1 BFT-AqE. Across the 13 strains tested against L3, efficacy ranged from 0 to 75% exsheathment inhibition, and 17 to 92% L3 motility inhibition at a concentration of 25 mg powder mL-1 BFT-AqE. There was no correlation between the PAC content of BFT powders and the anti-parasitic activity of aqueous extracts, therefore other secondary compounds may have contributed to the observed anti-parasitic effects. Further testing of BFT using bioactivity-driven fractionation and screening of BFT populations for the identified anti-parasitic compounds is needed.

Effects of different extracts of three Annona species on egg-hatching processes of Haemonchus contortus.

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.

Ovicidal in vitro activity of 2-aryl-3-(2-morpholinoethyl)thiazolidin-4-ones and 2-aryl-3-(3-morpholinopropyl)thiazolidin-4-ones against Fasciola hepatica (Linnaeus, 1758).

Although there is a variety of biological activity reports regarding compounds derived from thiazolidin-4-ones, no data related to ovicidal activity against trematodes, particularly Fasciola hepatica are available. Since there are reports about anthelmintic resistance in F. hepatica, new drugs are required. Thus, this study evaluated ovicidal action in vitro against F. hepatica eggs in two systematic series of thiazolidin-4-ones: 2-aryl-3-(2-morpholinoethyl)thiazolidin-4-ones (1a-h) and 2-aryl-3-(3-morpholinopropyl)thiazolidin-4-ones (2a-h) at different concentrations (20, 2, 0.2, 0.02 and 0.002 µg/ml). The egg hatch assay (EHA) was used to evaluate the ovicidal action property of such compounds. In addition, potential negative effects of the compounds on metabolic activity of bovine kidney (MDBK) cells were evaluated by determining mitochondrial dehydrogenase activity. The eggs used in the EHA were obtained from parasites removed from the liver of cattle, which were discarded by slaugh after sanitary inspection. The results of EHA showed that compounds 2a-h exhibited ovicidal activity, especially compounds 2b which showed 90% ovicidal activity and viability of 93% MDBK cells at the concentration of 2 µg/ml; and 2e with 96-99% ovicidal activity at 0.2 µg/ml, 0.02 µg/ml and 0.002 µg/ml. The results show the potential of compound 2b to continue the studies in the production of new compounds with anthelmintic action.

An in vitro approach to evaluate the nutraceutical value of plant foliage against Haemonchus contortus.

Nutraceutical plants provide nutrients for the animal as well as secondary compounds that can affect the biology and survival of gastrointestinal nematodes (GIN). Current screening of nutraceutical plants is based on in vitro evidence of anthelmintic (AH) activity against different life stages of GIN, but nutritional information is omitted or scarce. This study proposes an integral in vitro screening protocol to identify the nutraceutical value of the foliage from plant species consumed by small ruminants, using Haemonchus contortus as a biological model. The leaves from Acacia collinsii, A. pennatula, Bunchosia swartziana, Gymnopodium floribundum, Havardia albicans, Leucaena leucocephala, Lysiloma latisiliquum, Mimosa bahamensis, Piscidia piscipula, and Senegalia gaumeri were evaluated for their chemical composition and in vitro digestibility. Acetone:water extracts (70:30) from leaves of each plant were evaluated using the egg hatch assay and larval exsheathment inhibition assay. Respective effective concentrations 50% (EC50) were determined for each assay. The ten plant species showed good nutritional value for ruminants, including crude protein (> 10%), metabolizable energy (> 2.9 MJ/kg DM), and varied CT content (from 1.0 to 37.6%). The best AH activity against H. contortus eggs (EC50 = 401.8 µg/mL) and L3 (EC50 = 83.1 µg/mL) was observed for S. gaumeri extract. Although all the plant species showed in vitro nutraceutical potential, the leaves of S. gaumeri had the best values. The proposed in vitro protocol showed to be useful for the integral assessment of the nutraceutical potential of different plant species as it included the nutritional value and the AH activity against eggs and L3 in the selected plant species.

Is there a negative association between the content of condensed tannins, total phenols, and total tannins of tropical plant extracts and in vitro anthelmintic activity against Haemonchus contortus eggs?

In vitro studies using plant extracts suggest a relationship between their polyphenol contents and their anthelmintic (AH) activity against Haemonchus contortus. High polyphenol content appears to increase the efficacy of plant extracts against H. contortus as assessed by the larval exsheathment inhibition assay (LEIA) while appearing to reduce the AH efficacy measured using the egg hatch assay (EHA). In addition, some plants lack AH activity. Therefore, the present study investigated the relationship between the contents of condensed tannins (CT), total phenols (TP), and total tannins (TT) in methanol:water extracts (70:30) obtained from ten tropical plant species consumed by small ruminants as well as their AH activity against H. contortus evaluated by LEIA and EHA. Extracts of Acacia collinsii, Lysiloma latisiliquum, Havardia albicans, Senegalia gaumeri, Mimosa bahamensis, Piscidia piscipula, Acacia pennatula, Gymnopodium floribundum, Leucaena leucocephala, and Bunchosia swartziana were examined. Positive correlations were found between the effective concentration 50% (EC50) (EHA) of extracts and their CT (r = 0.6809, P < 0.05, n = 10) and TP (r = 0.9152, P < 0.05, n = 10) content, suggesting that their concentration negatively affected AH activity against eggs. Based on the LEIA, there was no significant association between the EC50 and the CT, TP, or TT of all extracts evaluated. Thus, if sheep and goats consume a complex feed mixture with high amounts of CT, TP, and TT, it might be difficult to observe an AH effect against H. contortus egg hatching. However, the AH effect upon L3 establishment might be feasible.

Laboratory evaluation on a potential birth control diet for fruit fly sterile insect technique (SIT).

Sterile insect technique (SIT) is one of the most effective fruit fly control technologies. Irradiation has been used to sterilize male fruit flies before release to the field to compete with the wild males for females. Imagine an environmental and cost effective method using a rearing diet that can make insects sterile indefinitely, by feeding for 7days before release. This could replace costly irradiation process. A potential birth control diet was evaluated on fertility, mating, survival, and protein analysis for fruit fly species in Hawaii. Insects were continuously fed an agar diet with lufenuron (LFN) for 7d after emergence and then switched to a control diet to simulate the actual field condition. The influence on egg hatch was dose dependent. With dose of 2-4mg/g in the diet, egg hatch from LFN-fed was almost 100% suppressed for 24 experimental days if adults of Ceratitia capitate (Widemann), Bactrocera dorsalis (Hendel), and B. latifrons (Hendel) continued to feed on LFN diet. B. cucurbitae (Coquillett) was not affected by LFN. However, egg hatch from LFN fed B. latifrons and B. dorsalis were suppressed for at least 2weeks after switching to the control diet at 7d. Egg hatch did not recover >4% up to 24d. Proteome analysis revealed that ABD-4 protein was under expressed by 70-83% on LFN fed females and males of B. latifrons and B. dorsalis while Pbprp2 protein was significantly over expressed by 6-12 fold on LFN fed males only. These two proteins were not expressed in C. capitata and B. cucurbitae. Therefore, this report focused more on B. latifrons and B. dorsalis. This finding suggested a great potential for one alternative to sterilize fruit flies for SIT without irradiation.

An assessment of benzimidazole resistance against caprine nematodes in Central India.

Current status of resistance to benzimidazole (BZ) group of anthelmintic drugs against caprine nematodes in Central India at Amanala goat farm, Jabalpur, Madhya Pradesh (M. P.), was systematically investigated using faecal egg count reduction (FECR) test and egg hatch test (EHT). Besides, allele-specific PCR (AS-PCR) was deployed to ascertain the susceptible genotype (alleles) especially of the Haemonchus contortus. Randomly selected 30 goats, irrespective of age and sex, were divided into three groups of 10 each, to serve as treated and untreated controls. It was ensured that the animals were not administered with an anthelmintic drug for the past 3 months prior to undertaking the study, and faecal egg counts were estimated. FECR test evidenced fenbendazole resistance by partial elimination (24.90%) copro-egg counts in the treated group of animals vis-à-vis controls with a lower confidence interval of -26%. Further, EHT revealed ED-50 value of 0.335 µg of thiabendazole/ml, confirming benzimidazole resistance in the animals of that farm. AS-PCR showed that 62% of H. contortus larvae were homozygous resistant (rr), 24% heterozygous (rS) and 14% homozygous susceptible (SS). The genotypic frequencies of three genotypes (rr, rS and SS) were significantly (P < 0.01) different. The prevalence of benzimidazole resistance allele (r) was also significantly (P < 0.01) higher (74%) as compared to susceptible allele (S) (26%). The resistance to benzimidazole has been discussed while emphasizing improved managemental practices designed to reduce exposure of the goat population to parasites, minimize frequency of anthelmintic use at optimum dose and rotational use of different chemical groups of medicines with different mode of action, so as to overcome and combat the upcoming problem in the field.

Control of Nematodirus spp. infection by sheep flock owners in Northern Ireland.

BACKGROUND: To address a lack of information on the control of ovine helminth parasites in Northern Ireland (NI), a number of research projects have been undertaken, dealing with gastrointestinal nematodes, tapeworms and liver fluke. This investigation concerns Nematodirus and concentrates on three aspects of disease: farm management strategies for its control, derived from the results of a Questionnaire; the efficacy of treatment used by farmers, as determined by a coprological survey; and the hatching requirements of Nematodirus eggs, that is, whether prolonged chilling is a pre-requisite for hatching. RESULTS: A Questionnaire was sent to 252 sheep farmers in NI in March 2012 (covering the years 2009-2012) and replies were received from 228 farmers. Under-dosing, inaccurate calibration of equipment and inappropriate product choice were poor practices identified. Following this survey, the efficacy of treatment of Nematodirus spp. in sheep flocks was evaluated in April and May 2012. Sampling kits were sent to 51 flock owners, all of whom returned pre- and post-anthelmintic dosing faecal samples to the laboratory for analysis. At the time of treatment, 41 flocks were positive for Nematodirus (as diagnosed by the presence of eggs). Reduced benzimidazole efficacy was detected in 35.7% of flocks tested (n = 28). Although only involving a small number of flocks, reduced efficacy of levamisole treatment was detected in 50%, of avermectins in 33% and of moxidectin in 75% of flocks tested (n = 2, 6 and 4, respectively). In the egg hatch experiment, carried out under "chilled" and "non-chilled" conditions, 43% of the eggs in the "non-chilled" group were able to hatch, compared to 100% in the "chilled" group. CONCLUSIONS: The identification of inefficient control strategies argues for continued education of stockholders, in order to improve their management programmes. This is particularly important where the practices might impact on the development of anthelmintic resistance, which has been shown to exist on NI farms. The appropriate choice of anthelmintic is a vital part of this plan. The ability of eggs to hatch under non-chilled conditions demonstrates a flexibility in hatching behaviour. This may represent an adaptation to climate change and account for the recent emergence of a second, autumnal peak of infection.

Impact of direct and indirect application of rising furfural concentrations on viability, infectivity and reproduction of the root-knot nematode, Meloidogyne incognita in Pisum sativum.

The gradual withdraw of several broadly used nematicides from market has enhanced the need to develop sustainable and eco-friendly alternatives with nematicidal properties. Furfural is one of the promising alternatives to fill this need. Baseline information about the impact of furfural on egg hatch, penetration potential and ultrastructure of nematode is lacking. In this study, the reagent-grade (purity ≥ 99.0%) of furfural was applied against Meloidogyne incognita. In vitro tests showed gradual reduction in either the rate of egg hatch or second stage juvenile (J2) viability of M. incognita when immersed in concentrations ranging from 0 to 10.0 µl/ml furfural. The mean EC50 for J2 and egg hatch was 0.37 and 0.27 µl/ml furfural, respectively. Furfural, even at low concentrations, resulted in a considerable suppression in egg hatch. Hatch was <5% after 8 days at 0.63 µl/ml furfural. The same furfural concentrations after 12 h caused 57.25% loss of viability in J2. Moreover, the penetration rate of juveniles to pea roots was suppressed when furfural was even applied at low rates. In pot experiments, furfural was applied as liquid (direct) or vapor (indirect) treatments at rates of 0-1.5 ml/kg soil. Significant reduction in galling, egg production and population density of M. incognita observed when furfural was applied at rates >0.2 ml/kg soil. No adverse effect was detected on plants or free-living nematodes as a result of furfural application. Liquid furfural proved to have superior juvenile-suppressive effect whereas its vapor has such superiority against eggs. Scanning electron microscope (SEM) study showed irregular appearance of the body surface accompanied with some cuticle disfigurement of furfural-treated juveniles. These results indicated that furfural can adversely affect egg hatch, juvenile viability, penetration potential and ultrastructure of M. incognita. Furfural may therefore be of a considerable potential as an appropriate alternative for class I nematicides.

In vivo assessment of closantel ovicidal activity in Fasciola hepatica eggs.

Anthelmintic resistance in livestock parasites is currently a worldwide problem. Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment. Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs. As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable.

Impact of temperature on postdiapause and diapause of the Asian gypsy moth, Lymantria dispar asiatica.

Lymantria dispar asiatica (Vnukovskij) (Lepidoptera: Lymantridae) is one of three gypsy moth subspecies found in East Asia. Understanding the diapause and postdiapause phases of its eggs is important in characterizing its life cycle. The effects of different constant temperatures for different lengths of times on field-collected, postdiapause eggs were tested during the first year. In the second year, the effects of the same treatments on laboratory-raised eggs in diapause were investigated. The effects of temperature on percent egg hatching, time to hatching, and hatching duration were determined. When field-collected eggs were held at 0 and 5°C, they terminated postdiapause within 11 days. The percent hatching tended to decline with an increased duration of exposure at temperatures greater than 5°C. Diapause terminated slowly (> 37 days) and with a high percentage of hatching for postdiapause eggs held at 10°C. There was a positive correlation between temperature and the speed of postdiapause development for field-collected eggs held at constant temperatures between 10 and 25°C. However, the number of days to the first hatch was significantly longer than for eggs treated with lower temperatures before being transferred to 25°C. Freshly oviposited eggs treated at a constant 0 or 5°C for 200 days were unable to develop into pharate larva. However, eggs treated at a constant 20 or 25°C for 200 days developed into pharate larva but did not hatch even after a subsequent chill. This result suggests why L. dispar asiatica is not found in tropical areas and helps us to predict the distribution of the gypsy moth in China.

Triclabendazole resistance in Fasciola hepatica: First report in sheep from the Santa Cruz province, Argentinian Patagonia.

In the fall of 2022, decreased triclabendazole (TCBZ) efficacy against F. hepatica was suspected in a sheep farm located in the Santa Cruz province, Argentinian Patagonia. Since TCBZ-resistance in F. hepatica has never been reported in this province, this study aimed to confirm potential TCBZ-resistance in F. hepatica and to evaluate the efficacy of closantel (CLO) and nitroxinil (NTX), through faecal egg count reduction test (FECRT), and the efficacy of albendazole (ABZ) through the in vitro egg hatch test (EHT) in sheep. Sixty-eight (68) animals were selected from a herd of eighty (80) female Merino naturally infected with F. hepatica based on eggs per gram of F. hepatica (EPGFh) counts and assigned into four (4) groups (n = 17 per group): Group Control, animals did not receive anthelmintic treatment; Group TCBZ, animals were orally treated with TCBZ (12 mg/kg); Group CLO, animals were orally treated with CLO (10 mg/kg); and Group NTX, animals were subcutaneously treated with NTX (10 mg/kg). The fluke egg output was monitored on days 0 and 21 post-treatment. For the EHT, liver fluke eggs were isolated from faecal samples (approx. 50 g) collected from animals of the control group. TCBZ efficacy against liver fluke was 53.4%, confirming the presence of TCBZ-resistant isolates on the farm. CLO and NTX were highly effective (100%) for the treatment of F. hepatica on this farm. The EHT was carried out in two different laboratories, in which was observed an ABZ efficacy of 95.8 (Bariloche) and 96.5% (Tandil). These results indicate the ABZ susceptibility of this F. hepatica isolate and the inter-laboratory precision of the test.

Assessment of gastrointestinal nematode anthelmintic resistance and acaricidal efficacy of fluazuron-flumethrin on sheep and goat ticks in the North West province of South Africa.

Background and Aim: Anthelmintic resistance (AR) and acaricide resistance (ACR) pose great economic threat to communal livestock raised by rural communities, limiting sustainable production. This study was conducted to assess the occurrence of AR and ACR against nematodes and ticks that infest small ruminants (sheep and goats) from small-scale farming communities in the North West Province of South Africa, as well as document the associated risk factors. Materials and Methods: The study was conducted on small-scale farming locations in two districts of the North West Province, namely, Dr. Ruth Segomotsi Mompati district and Dr. Kenneth Kaunda district, from November 2019 to March 2020. A questionnaire survey based specifically on antiparasitic treatment and related management practices was administered to 86 small-scale farmers. A fecal egg count reduction test (FECRT) was used to determine in vivo AR in small ruminants against benzimidazole (BZD), levamisole, and macrocyclic lactone on nine ruminant farms. Then, deoxyribonucleic acid was extracted from L3 larvae and resistant nematodes were identified using a polymerase chain reaction, targeting the internal transcribed spacer 2 gene. An egg hatch assay (EHA) and a larval mortality assay (LMA) were used to determine in vitro AR against thiabendazole (TBZ and BZD) in the same farms. Acaricide resistance against fluazuron-flumethrin (Drastic Deadline eXtreme) pour-on was assessed using an adult immersion test (AIT) on Rhipicephalus evertsi. Results: Questionnaire results indicated that most farmers (89%) relied solely on anthelmintics. Farmers used visual appraisal to estimate the dosage, which is the primary cause of resistance. The FECRT revealed AR in all the farms. Egg hatch assay results revealed AR development against TBZ in all districts, with >95% of the eggs hatching at variable doses. Larval mortality assay results revealed the development of resistance against BZD, with 50% of L3 larvae surviving at different doses in all farms. Adult immersion test results indicated that fluazuron-flumethrin (>99%) exhibited high acaricidal efficacy against R. evertsi by inhibiting tick oviposition. Conclusion: This investigation found that sheep and goats in the studied areas are developing AR to gastrointestinal parasites. The findings of in vivo tests showed resistance with fecal egg count reduction percentage of <95% or lower confidence limit of <90%. The results of EHA and LMA revealed no evidence of inhibition of egg development and larval mortality, indicating the development of resistance. Acaricide resistance was not detected against fluazuron-flumethrin, which is commonly used in the study areas. Thus, developing management methods for these economically significant livestock nematodes, including teaching small-scale farmers how to properly administer anthelmintics and acaricides to their livestock, is urgently needed.