In Vivo Screening for a Promising Antiparasitic Agent Against Neobenedenia melleni in Epinephelus fuscoguttatus♀ × E. lanceolatus♂ and Identification of Its Potential Target

Computer-aided drug screening of anti-Neobenedenia melleni drugs based on annexin B1 in farmed pearl grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus ♂)

Monogenean capsalids of the genus Neobenedenia are widespread parasites of wild and farmed marine fish and represent a major threat to grouper mariculture in China, whose production reached approximately 294,000 tonnes in 2025. The development of effective drugs to control and prevent these infections is therefore urgently needed. Annexins, which have been identified in Neobenedenia and other parasites, differ markedly from their host counterparts, making them potentially attractive targets for antiparasitic therapeutics. Here, we performed a computer-aided drug discovery screen, employing Neobenedenia melleni annexin B1 as the molecular target against a library of 1,456,161 small molecules. The three-dimensional structure of annexin B1 was first predicted using AlphaFold 3, SWISS-MODEL, and I-TASSER, and the most accurate model (AlphaFold) was selected for structure-based virtual screening. In vivo validation of eleven compounds identified abamectin (Aba) as the most effective anti-Neobenedenia agent, achieving complete parasite elimination at 0.16 mg/L. Owing to its low toxicity to the host grouper (24-h LC50 = 0.254 mg/L), abamectin was selected for further investigation. Abamectin exhibited potent anthelmintic activity against N. melleni, with a 24-h bath exposure yielding an EC50 of 0.033 mg/L and complete parasite elimination at 0.16 mg/L, corresponding to a therapeutic index of approximately 7.7. To elucidate the antiparasitic mechanism, we performed long-timescale (1000 ns) molecular dynamics simulations of the annexin B1-abamectin complex, enabling atomic-level analysis of the essential protein motions involved in their interaction. The interaction profile between annexin B1 and abamectin was dominated by hydrophobic contacts and water bridges, involving residues TYR-210, GLU-214, GLU-244, and SER-247, which path a way for further drug optimization.

Integrated Phytochemical, Computational and In Vitro Evaluation of the Oocysticidal Activity of Pleurotus ostreatus Extract Against Cryptosporidium parvum.

Toxicity and Morphological Alterations Caused By Azadirachtin in the Stingless Bee Nannotrigona testaceicornis Lepeletier, 1836 (Insecta: Hymenoptera: Apidae: Meliponini

Nannotrigona testaceicornis are essential pollinators for biodiversity maintenance and agricultural production. However, during foraging in agroecosystems, they may be exposed to residues of bioinsecticides used for pest management. Thus, this study evaluated the toxicity of the commercial product AzaMax® (azadirachtin) to the stingless bee N. testaceicornis. Adult worker bees were exposed to this bioinsecticide by contact and ingestion at concentrations recommended for strawberry crops, and mortality was assessed after 24, 48, and 72 h. Morphological alterations of the midgut and changes in chromatin structure in brain cells were evaluated in the surviving stingless bees. Exposure resulted in low mortality, precluding the estimation of the LC₅₀, but revealed significant sublethal effects, such as disorganization of the midgut epithelium, loss of digestive cells, reduction of regenerative cells, and disappearance of the peritrophic membrane. An increase in chromatin condensation was detected at different exposure times and concentrations, suggesting disruption of gene regulatory processes. These findings indicate that although azadirachtin does not induce immediate lethality in N. testaceicornis, it may compromise digestive physiology and genomic integrity, potentially affecting individual performance and long-term colony maintenance. The results highlight the importance of considering sublethal parameters in bioinsecticide risk assessments, as they may reveal hidden and ecologically relevant impacts that are not detected by mortality alone. By incorporating both lethal and sublethal endpoints, this research contributes to the development of safer agricultural practices and supports the conservation of stingless bees, which are key pollinators in tropical ecosystems.

Ocimum basilicum essential oil controls Haemaphysalis longicornis via multi-modal toxicity, enzyme inhibition, and synergy with hexythiazox

Haemaphysalis longicornis is an important ectoparasite infesting livestock, particularly cattle and sheep, causing significant economic losses and serving as a vector for multiple zoonotic pathogens. The widespread development of acaricide resistance necessitates the exploration of alternative control strategies with novel mechanisms of action. The essential oil (EO) of Ocimum basilicum L. demonstrates potential as a multi-target botanical acaricide against the hard tick Haemaphysalis longicornis. GC-MS analysis identified eugenol (47.75%) as the major constituent. Bioassays revealed significant, dose-dependent contact toxicity against larvae (LC₅₀ = 18.47 mg/mL), nymphs (LC₅₀ = 34.93 mg/mL), and adults (LC₅₀ = 27.75 mg/mL). Purified eugenol exhibited 1.3–1.9-fold greater potency than the whole EO. The EO also showed strong fumigant activity and significantly suppressed fecundity by inhibiting oviposition and reducing egg hatchability. Mechanistic studies indicated that the acaricidal effect may be associated with the inhibition of key metabolic and neurological enzymes, including acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione S-transferase (GST). Furthermore, a 2:8 (hexythiazox: oil) formulation exhibited significant synergism against nymphs (Co-toxicity coefficient = 128.35), enabling a reduction in synthetic acaricide dosage. This work elucidates a multi-modal action mechanism of O. basilicum EO against H. longicornis through contact toxicity, fumigation, reproductive suppression, and enzyme inhibition. The synergistic interaction with hexythiazox provides a sustainable, plant-based strategy for controlling tick infestations in livestock, showing potential for addressing the critical challenge of acaricide resistance in animal production systems.

Nematicidal activity of marcenmicin D2 from Fusarium sp. against Bursaphelenchus xylophilus.

BACKGROUND Pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is a devastating forest disease worldwide, and there is an urgent need for environmentally friendly biocontrol agents. Cyclic lipopeptides from microbial sources exhibit diverse biological activities, but their nematicidal potential against B. xylophilus remains largely unexplored. RESULTS A cyclic lipopeptide, marcenmicin D2, was isolated from the endophytic fungus Fusarium sp. SSY-3. Marcenmicin D2 exhibited potent nematicidal activity against B. xylophilus in a concentration- and time-dependent manner, with a median lethal concentration (LC50) value of 41.6 mg L-1 at 24 h. At 50 mg L-1, it achieved 100% mortality within 48 h and strongly inhibited egg hatching (>96% at 50 mg L-1). The compound rapidly reduced head swing frequency within 3 h, and induced unique morphological abnormalities including aggregation, irregular body bending, coiling and cuticle shrinkage, which were not observed in abamectin-treated nematodes. In treated nematodes, the biochemical evidence of oxidative perturbation (changes in superoxide dismutase, catalase and malondialdehyde) and the transcriptomic signatures, notably the coordinated downregulation of cuticle-related collagens, peroxisomal/lysosomal pathways, cytochrome P450-dependent xenobiotic metabolism and carbohydrate metabolic genes, are consistent with the multifaceted nature of marcenmicin D2's activity against B. xylophilus. CONCLUSION Marcenmicin D2 is a potent cyclic lipopeptide nematicide from Fusarium sp. that acts through disruption of cuticle integrity, impairment of detoxification systems, interference with cellular degradation pathways, and induction of oxidative stress. Its rapid immobilization of B. xylophilus, mechanism distinct from that of abamectin and strong egg-hatching inhibition make it a promising lead compound for sustainable PWD management. © 2026 Society of Chemical Industry.

Multi-stage insecticide potentials of Myristica fragrans against the invasive Aedes albopictus, and putative binding mechanism of myristicin targeting the odorant-binding proteins (OBPs)

The Asian tiger mosquito, Aedes albopictus, is native to Southeast Asia and an invasive species with rapid geographical expansion. Aedes albopictus has contributed to the spread of numerous infectious diseases affecting both humans and animals. This study evaluated the insecticidal potential of Myristica fragrans essential oil (MFEO) from Maluku Island against multiple life stages of Ae. albopictus. MFEO completely inhibited egg hatching from 0.001% (w/v), with treated eggs remaining unhatched throughout a 25-day observation period; scanning electron microscopy revealed structural damage to the exochorionic layer, indicating that disruption of the egg surface impairs embryonic development. In larvicidal assays, MFEO showed rapid, concentration- and time-dependent toxicity, achieving 100% mortality within 30 min at concentrations of 100 ppm and above, comparable to temephos; at 1–50 ppm, complete lethality was reached within 120 min. Against adults, MFEO produced a 30-min knockdown concentration (KD50) of 1.545% and a 24-h lethal concentration (LC50) of 0.270%. MFEO-treated ovitraps received no eggs, whereas controls attracted 78–90 eggs in 48 h. Gas chromatography-mass spectrometry identified myristicin (35.35%) as the principal constituent, with α-terpineol comprising 10.74% and phenol, 2,4,6-tris(1-phenylethyl)- comprising 11.24%. Molecular docking against 19 annotated odorant-binding proteins (OBPs) of Ae. albopictus showed the strongest binding of myristicin to OBP3 (−6.8 kcal/mol), mediated by van der Waals, hydrophobic and polar hydrogen interactions; molecular dynamics validation (CABS-flex) of the myristicin-OBP3 complex yielded a mean root-mean-square fluctuation of 1.468 Å, i.e. below the 2 Å stability threshold. Further docking against acetylcholinesterase (AChE), GABA receptors and voltage-gated sodium channels (VGSC) supported a neurotoxic component. Together, these results indicate that MFEO disrupts multiple life stages through combined chemosensory interference, eggshell damage and neurotoxic action. The multi-target, multi-stage activity observed in MFEO, together with its behavioral deterrent effects, suggests it warrants further investigation as a botanical candidate for integrated vector management, potentially offering an alternative to some conventional neurotoxic insecticides. Further, functional validation by bio-guided fractionation and target-specific assays is recommended.