Ana Yuridia Ocampo-Gutiérrez

Nematode-trapping fungi have emerged as biological alternatives to address the increasing problem of antiparasitic drug resistance. These fungi develop three-dimensional adhesive networks that capture, kill, and digest nematodes. In addition, they employ other mechanisms to degrade their prey, including proteolytic enzymes. Arthrobotrys oligospora is recognized as one of the most effective biological control agents against both free-living and gastrointestinal parasitic nematodes. However, little is currently known about the proteases associated with its mycelium. The objective of this work was the identification and purification of the 78.9 kDa from A. oligospora. In this study, we provide the first evidence that A. oligospora mycelium exhibits two distinct proteolytic activities, corresponding to 78.9 and 250 kDa, revealed by zymography analysis. These proteases displayed activity over different pH ranges, suggesting distinct biochemical properties. The 78.9 kDa protease was subsequently purified by ion-exchange chromatography and biochemically characterized. Inhibition assays demonstrated that it belongs to the Zn-metalloproteases, a finding subsequently confirmed by mass spectrometry. We also performed a 3D modeling prediction and identified its potential zinc-binding sites. Finally, molecular docking analyses demonstrate that this enzyme exhibits a high binding affinity for chitin, suggesting chitinase activity; thus, it could be a key component in the degradation of nematode structural components during fungal predation.

Haemonchus contortus (Hc) is a parasitic nematode affecting sheep. Arthrobotrys is a group of nematophagous fungi (NF) that capture nematodes with specialized traps. This study evaluated the in vitro predatory ability of Arthrobotrys thaumasia (At) and A. musiformis (Am), as well as the nematocidal activity of their liquid culture filtrates against Hc. NF isolates were obtained by sprinkling soil onto agar plates containing nematodes. Isolates were identified morphologically and molecularly. The predatory activity (PA) was tested on agar plates inoculated with nematodes and fungi; plates inoculated only with nematodes served as controls. After 10 days at 18 °C and 28 °C, larvae were counted. For nematocidal activity (NA) of LCFs, fungi were grown in Czapek–Dox (CzDB) and potato dextrose broths. Two concentrations (100 mg/mL and 130 mg/mL) were tested. Nematodes and LCFs were incubated for 48 or 72 h. PA was analyzed using unpaired Student’s t-tests, and LCFs were evaluated by ANOVA with Tukey’s post hoc test. Taxonomic procedures confirmed the isolates as Am and At. For PA, effectiveness was 74.64% for Am and 83.53% for At. The highest NA of LCF of Am in CzDB was 51% at 100 mg/mL and 76.7% at 130 mg/mL. That of the LCF of At was 22% at the highest concentration. Both isolates show strong potential as biocontrol agents against haemonchosis.

This study assessed the predatory activity (PA) of Dactylellina candida ( Dc ) and Arthrobotrys musiformis ( Am ) against Haemonchus contortus infective larvae ( Hc IL). It also examined the effect of crude extracts from Hc IL, Panagrellus redivivus ( Pr ), and Turbatryx aceti ( Ta ) on liquid cultures of these fungi as potential enhancers of their nematocidal activity against Hc IL. The fungi were cultured in sweet potato dextrose broth (SPDB), and the corresponding nematode crude extract (NCE) was added to flasks containing the fungus. Control groups included fungi without extracts and media alone. Liquid culture filtrates were assessed at three concentrations (100, 50, and 25 mg/mL) against Hc IL during 72 hours at 18–25°C. The highest mortality (86.61%) was observed with Dc stimulated by Hc IL crude extract ( Hc CE) at 100 mg/mL (p < 0.05). Arthrobotrys musiformis showed 47.49% mortality when stimulated with Hc CE. Both fungi increased their nematocidal activity when exposed to Hc CE, demonstrating their potential nematocidal activity against H. contortus.

Nematode-trapping fungi are saprophytic organisms that can transform their mycelium into a parasitic lifestyle, forming traps to capture and feed on nematodes. Although this transition is triggered by environmental conditions, the genetic regulation of this metabolic shift remains unclear. This study assessed the effects of nutritional stress on mycochemical synthesis, trap morphogenesis, and Aomae1 gene expression in Arthrobotrys oligospora and Arthrobotrys musiformis. Fungal biomass was subjected to the following three-stage successive culture model: (i) nutrient-rich (Czapek–Dox broth), (ii) nutritional stress (water), and (iii) media enriched with live prey (Haemonchus contortus Hc-L₃). Samples were taken for molecular analysis, and liquid culture filtrates (LCFs) were recovered for chromatographic identification of mycochemical groups. To assess trap formation (traps/cm²), mycelia from each culture model was transferred to water agar plates and defied with Hc-L₃. Results showed a significant bioenergetic trade-off. Both starvation and larval presence induced a downregulation of mycochemical synthesis, which resulted in the total loss of nematocidal activity in LCfs, while triggering a morphogenetic response. Arthrobotrys musiformis showed the most aggressive phenotype with 3.8-fold increase in trap formation and a massive 429.05-fold overexpression of Aomae1 under predatory challenge. While A. oligospora showed a similar but less pronounced trend (2.4-fold increase in trap formation and 44.48-fold Aomae1 overexpression), our findings suggest that Aomae1 expression plays a critical role in the metabolic switch that regulates and redirects energy resources, prioritizing mechanical trapping mechanisms over secondary metabolism during nutrient scarcity. These findings highlight Aomae1 as a possible key activator for virulence, which offers strategic targets for the optimization of biocontrol agents against gastrointestinal nematodes in livestock.

This study aimed to explore and identify soil-dwelling nematophagous fungi (NF) from the “El Texcal” Ecological Reserve in Morelos, Mexico, and evaluate their potential as biological control agents against Haemonchus contortus infective larvae (HcL3), a major parasitic threat in livestock systems. The fungi were isolated from soil using the sprinkling of soil on water agar plates. The identification of NF was achieved using morphological identification keys, which was corroborated by molecular procedures using the PCR technique in the ITS4 and ITS5 regions. The nematocidal effects occasioned by these NF were examined through their predatory activity (PA) against HcL3 on water agar plates, and additionally, the larval mortality attributed to their liquid filtrates (LFs) was assessed at three different concentrations (25, 50, and 100 mg/mL) on 96-well microtiter plates. Two NF were identified and classified as two species of Dactylellina genus, namely D. haptospora (Dh) and D. phymatopaga (Dp). The PA exhibited by these NF were 94.79% for Dh and 68.88% for Dp; while their LFs showed 27.83% mortality for Dh and 32.86% for Dp at the highest concentration assessed. While the PA was notably high, the moderate larvicidal effect of the LF suggests that their efficacy may primarily rely on direct physical interaction rather than metabolite-mediated toxicity. The high PA demonstrated by these two isolates of NF indicates that they could be effective candidates for biological control agents against HcL3.

This study evaluated the effectiveness of Duddingtonia flagrans chlamydospores and an Artemisia cina hexane extract in reducing Haemonchus contortus fecal egg counts and larvae in periparturient ewes. This study involved five groups of four ewes: a control group, an ivermectin group, an A. cina oral extract group, a D. flagrans group, and a combined treatment group. Treatments began two weeks before delivery, with ivermectin administered 15 days before delivery. Fecal samples were collected every fifteen days to estimate parasite egg counts per gram of feces (EPG) and assess larvae reductions. The results showed very low EPG values for ivermectin and D. flagrans treatments (175 and 150, respectively). The control and combined treatment groups had EPG values rise to 3000 and 4100 by day 15. The EPG values for the A. cina group reached 850 and 533 in later samplings. Throughout the study, the D. flagrans and A. cina groups maintained low EPG values, with the highest recorded values being 50 and 0, respectively. All treatments significantly reduced the larvae in the fecal cultures: D. flagrans (97.4% reduction), ivermectin (91.4%), Artemisia cina (89.9%), and the combined treatment (84.3%).

This is the first record of the fungus Flavocillium subprimulinum in Mexico. The isolate was taxonomically characterised and cultured in potato dextrose broth (PDB), Czapek–Dox broth (CzDoxB), and sweet potato dextrose broth (SPDB) to obtain its filtrates (FLCF). The nematocidal activity (NA) of three FLCF concentrations was assessed against Haemonchus contortus L3. Protease activity (PA) was assessed with SDS-PAGE, followed by a zymogram. The NA of the FLCF reached 94.43% in PDB and 95.82% in CzDoxB, respectively, at 100 mg/mL. Lower mortality (64%) was found in SPDB at 100 mg/mL. SDS-PAGE showed bands (in PBS) of ~25, ~40, and ~55 kDa. The zymogram showed protein bands (PBs) with PA in the media, including PBs of ~14, ~40, and ~55 kDa. This study establishes the basis for exploring the potential use of this fungus against H. contortus, which is considered the most pathogenic parasite affecting lambs.

Haemonchus contortus (Hc) is a parasite affecting small ruminants worldwide. Arthrobotrys musiformis (Am) is a nematode-trapping fungi that captures, destroys and feeds on nematodes. This study assessed the predatory activity (PA) and nematocidal activity (NA) of liquid culture filtrates (LCF) of Am against Hc infective larvae (L3), and additionally, the mycochemical profile (MP) was performed. Fungal identification was achieved by traditional and molecular procedures. The PA of Am against HcL3 was performed in water agar plates. Means of non-predated larvae were recorded and compared with a control group without fungi. LCF/HcL3 interaction was performed using micro-tittering plates. Two media, Czapek–Dox broth (CDB) and sweet potato dextrose broth (SPDB) and three concentrations, were assessed. Lectures were performed after 48 h interaction. The means of alive and dead larvae were recorded and compared with proper negative controls. The PA assessment revealed 71.54% larval reduction (p < 0.01). The highest NA of LCF was found in CDB: 93.42, 73.02 and 51.61%, at 100, 50 and 25 mg/mL, respectively (p < 0.05). Alkaloids and saponins were identified in both media; meanwhile, coumarins were only identified in CDB. The NA was only found in CDB, but not in SPDB. Coumarins could be responsible for the NA.

The nematocidal activity of an Oxalis tetraphylla hydroalcoholic extract against the nematode Haemonchus contortus (Hc) was assessed in vitro and the major compounds associated with nematocidal activity were identified. One hydroalcoholic extract was obtained from O. tetraphylla stems and leaves (Ot HE-SLE). The in vitro lethal concentrations (LC50 and LC90) against both eggs and exsheathed Hc infective larvae (L3) were assessed. Ot HE-SLE showed a potent ovicidal activity (LC50 = 0.213 mg/mL; LC90 = 0.71 mg/mL) and larvicidal effect (LC50 = 28.01 mg/mL; LC90 = 69.3 mg/mL). Later on, the extract was bipartitioned to obtain an ethyl acetate phase (EtOAc-Ph) and an aqueous phase (Aq-Ph). Both phases were assessed against Hc eggs at 0.25 and 1.0 mg/mL concentrations. The results with EtOAc-Ph showed 93.6% ovicidal activity, while 96.6% was recorded with Aq-Ph at 48 h post-confrontation (PC). In the case of larvicidal activity, both phases were assessed at 28 mg/mL; Aq-Ph showed >80% larvicidal activity 24 and 72 h PC, while EtOAc-Ph did not show important activity. HPLC analysis showed the presence of coumaric acid and flavonols. Flavonol compounds were the major compounds and were associated with the nematocidal activity. Additionally, the Aq-Ph that showed the highest activity was purified, and the fraction F3 showed the highest nematocidal activity.