https://orcid.org/0000-0002-9262-3111
Figures
Abstract
Nematophagous fungi offer a sustainable alternative for controlling nematode infections in small ruminants. The aims were to isolate and characterize both morphological and molecular nematophagous fungi from soil, to assess their predatory activity and the nematocidal activity of their liquid culture filtrates (LCF) against Haemonchus contortus infective larvae (L3), and to identify the protease activity of the LCF and mycoconstituents. The isolated and characterized Arthrobotrys oligospora was identified using both morphological and molecular techniques, with a similarity of 98%. Additionally, the isolated strain showed 89% phylogenetic similarity in the phylogenetic tree concerning the Arthrobotrys order. The A. oligospora isolate exhibited 72.06% predatory activity, and the liquid filtrate demonstrated 96.10% nematocidal activity at 100 mg/mL after 48 hours post-exposure against H. contortus infective larvae. Regarding enzyme activity, A. oligospora showed metalloprotease and cysteine-protease activities, and the zymogram revealed that these activities were higher under acidic conditions (pH 5).
Citation: de la Crúz-Crúz HA, Higuera-Piedrahita RI, Alcalá-Canto Y, Zamilpa A, Ocampo-Gutiérrez AY, Arango-de la Pava LD, et al. (2026) Predatory activity and nematocidal compounds released into liquid culture filtrates as attack strategies of a Mexican strain of Arthrobotrys oligospora against Haemonchus contortus infective larvae. PLoS One 21(3): e0341469. https://doi.org/10.1371/journal.pone.0341469
Editor: Wesley Lyeverton Correia Ribeiro, Federal University of Ceara: Universidade Federal do Ceara, BRAZIL
Received: September 23, 2025; Accepted: January 7, 2026; Published: March 12, 2026
Copyright: © 2026 Alejandro de la Crúz-Crúz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The manuscript contains all raw data required to replicate the results of our study.
Funding: This study was supported by the Secretariat of Science, Humanities, Technology, and Innovation (SECIHTI) for the scholarship awarded to Professor Héctor Alejandro de la Cruz (registration number 713914). This research was also supported by the SECIHTI scholarship (Frontier Sciences Project-2023, scholarship number CF-2023-I-2309) awarded to Dr. Pedro Mendoza de Gives. This work also received funding from the Program to Support Research and Technological Innovation Projects from General Directorate of Academic Personnel Affairs of the National Autonomous University of Mexico (UNAM DGAPA-PAPIIT) (grant no. 200324) “Molecular docking study of two lignans, 3-dimethoxy-isoguayacin and norisoguayacin, obtained from Artemisia cina against COX-2,” received by Dr. Rosa Isabel Higuera Piedrahita. This study was also supported by the Research Chair Program of the Faculty of Higher Studies Cuautitlán (Cátedra FESC) (grant no. CI2428) awarded to Dr. Rosa Isabel Higuera Piedrahita.
Competing interests: The authors have not competing interests.
Introduction
Gastrointestinal parasitic nematodes (GIN) are among the most significant concerns in the livestock industry worldwide. This has led to a considerable deterioration not only in animal health but also to an enormous economic impact on the livestock industry worldwide [1–3]. In addition, the typical strategy used for ages against these parasitic diseases involves the frequent and continuous administration of chemically synthesized anthelmintic drugs (CSAD), which help mitigate animal damage and indirectly enhance their productivity. However, several disadvantages make this system an overlay. In this regard, the frequent and continuous use of CSAD in animals triggers the development of mutations in one or more genes of the parasites, enabling them to overcome the lethal effect of CSAD and become resistant to these drugs [4–6]. Additionally, the use of such CSADs can lead to residues of these chemicals remaining in meat, milk, and by-products intended for human consumption, posing a potential contaminant risk and a threat to public health [7]. Likewise, CSAD is eliminated by the treated animals through urine and feces, which ultimately reach the soil, where it can affect non-target beneficial microorganisms that comprise the soil microbiome [8] and even impact aquifers, resulting in devastating environmental consequences for water fauna [7]. To summarize, the use and misuse of CSAD can threaten soil microbiota and affect human health. The growing problem of anthelmintic resistance in the parasites, together with other disadvantages, has provoked a bad reputation for the use of these drugs as a unique method of control. This problem requires exploring other alternative methods different from the use of CSAD, such as management systems, i.e., grazing rotation [8,9], grazing alternation of species [10], use of plant/plant metabolites with anthelmintic activity [11–13], vaccines [14,15], biological control using natural nematode enemies such as the nematophagous fungi [16,17]. Moreover, products are derived from the secondary metabolism of nematophagous fungi [18]. Nematode trapping fungi (NTF) are microorganisms found in soil that are considered the primary natural enemies of nematodes in various environments [19]. NTF possesses the capability to be a saprophytic microorganism. However, in the proximity of nematodes, they transform their mycelia into trapping devices designed to capture nematodes, as well as specialized ones that feed on their tissues [16,18,20]. The genus Arthrobotrys is one of the most widely studied NTF that has displayed several biological capabilities, including as a bio-regulator of nematode populations [19], cellulose-degrading enzyme activity [21], endophyte activity [20], apoptotic activity enhancer of cancer cells [22], producer of nematocidal secondary metabolites [23], among other essential vital activities. This study was designed to isolate and characterize both morphological and molecular nematophagous fungi from soil and to assess their predatory activity and the nematocidal activity of their liquid culture filtrates (LCF) against Haemonchus contortus infective larvae (L3). Additionally, we investigated the protease activity of LCF.
Materials and methods
Location
This study was conducted at the Laboratory of Helminthology, CENID-SAI, INIFAP, in Jiutepec, Morelos, Mexico, and at Laboratory 3, Unit of Multidisciplinary Research, FES-Cuautitlán, UNAM, in Cuautitlán Izcalli, Mexico.
Sampling site
Soil sampling was conducted at the Sayil archaeological zone, located in the Santa Rosa Municipality of the Puuc Region, Yucatán, Mexico. Fifty grams of soil, 10 cm deep and 30 cm from the trunk of an Enterolobium cyclocarpum (Jacq.) leguminous tree. Griseb, also known as the “Pich tree” or “Guanacaste” (Fig 1), was collected. To carry out the sampling, a special permit was processed before the National Service of Agrifood ®Health, Safety and Quality (SENASICA) with an approval date of May 2023.
Biological material
Fungal isolation.
The process of fungal isolation was conducted with utmost precision and care. Five milligrams of soil were delicately sprinkled on the surface of water agar plates (3 plates), and a 5 ml aqueous suspension containing 600 specimens of the free-living nematode Panagrellus redivivus was added to each plate to enhance the growth of nematophagous fungi [24]. After a week, the surface of the plates was meticulously examined under a stereomicroscope, searching for the formation of trapping devices and trapped nematodes, as well as taxonomic structures typical of nematophagous fungi. Fungi were then transferred to sterile water agar plates, and pure cultures were obtained by transferring to new sterile water agar plates [25].
Morphological taxonomic identification.
The morphological identification of fungi was performed by observing structures of taxonomic importance under a light microscope (Leica® ZEISS DM6, Wetzlar, Hesse, Germany) at magnifications of 25x, 40x, 75x, and 100x. The length and width of twenty-five conidia and conidiophores were randomly measured, and a range of these measurements was recorded. The presence of branched or unbranched conidiophores, the number of conidial septa, the kind of trapping devices, and the presence or absence of chlamydospores were also considered. Our findings were compared with those reported in specialized taxonomic keys to establish the genus and species of our isolate, finally [26,27].
Molecular identification.
The strain was produced in Czapek-Dox Agar plates for 14 days at room temperature (18–25°C). After this time, abundant cottony mycelium grew on the surface of the plates. Mycelia were collected from culture plate surfaces using a sterile needle and divided into 1.5 ml Eppendorf tubes. DNA extraction was achieved by grinding the mycelia using lysis beads and spinning down at 4000 rpm for 5 minutes. The Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) was used. The DNA genomic quantification was conducted using an IMPLEN spectrophotometer (NanoPhotometer® NP80). DNA amplification was achieved. By PCR using the ITS4 (50-GGAAGTAAAAGTCGTAACAAGG-30) and ITS5 (50- TCCTCCGCTTATTGA-TATGC-30) primers, with a C1000 Touch® Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR conditions were established as follows: initial denaturation at 94 °C for 3 minutes; 35 cycles of denaturation at 94 °C for 1 minute, annealing at 42 °C for 90 seconds, and extension at 72 °C for 90 seconds, followed by a final extension stage at 72 °C for 5 minutes. The size of the amplicons was confirmed using a 1.5% agarose gel.
The QIAquick gel extraction kit (QIAGEN) was used, and the procedure was performed with purified products, following the manufacturer’s instructions. Genomic DNA was sequenced at the Institute of Biotechnology of the National Autonomous University of Mexico (IBT-UNAM) using an Applied Biosystems sequencer. The sequences were aligned using the NCBI BLAST program (www.ncbi.nlm.nih.gov/blast/) accessed on April 30, 2024 (Basic Local Alignment Search Tool).
Nematodes
Panagrellus redivivus.
A strain of the free-living nematode P. redivivus was commercially obtained as a commercial food for fish from Heber Martinez Pateiro, Mexico. This strain was reproduced by culturing sterile wet oat flakes in plastic bowls covered with gauze, incubating at room temperature (26–28°C) for seven days. After this period, nematodes were recovered from bowls using the Baermann funnel technique for 24 h [26,28]. Recovered nematodes were sieved through a 100 µm mesh and resuspended in sterile distilled water.
Haemonchus contortus infective larvae (L3).
A four-month-old male lamb, artificially infected with 5,000 L3 H. contortus infective larvae (FESC strain) previously dewormed, was used as a donor animal for nematode eggs. After 18 days of the pre-patent period, feces were directly collected from the rectum of this animal to prepare fecal cultures in plastic bowls by the modified Corticelli-Lai technique [29]. The H. contortus strain (FESC strain) It has been isolated and maintained at the Faculty of Higher Studies Cuautitlán, and has also been characterized with heterozygous genes for benzimidazole resistance [30].
The sheep donor for H. contortus strain was maintained under controlled conditions following the Norma Oficial Mexicana (Official Mexican Standard) with official rule number NOM-052-ZOO-1995 (http://www.senasica.gob.mx). Additionally, the experimental protocol was approved by the Internal Committee for Care and Use of Experimental Animals (CICUAE-FESC) at Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, under protocol number C24_25 (S1 Fig).
Predatory activity assessment.
The nematode/fungus confrontation was performed in plastic petri dishes containing water agar. Ten plates (60 *15 mm) were inoculated with the mycelia of the fungal isolate and incubated for seven days at room temperature, 25–28°C. The other ten plates containing only the medium (without fungus) were used as controls. One milliliter of an aqueous suspension containing 600 H. contortus infective larvae was deposited on the surface of each plate of both groups. The whole plates were incubated for ten days at the temperature mentioned above. After incubation, the agar from each plate was removed and placed on a Baermann funnel system to recover non-trapped larvae from the treated group and whole larvae from the control group plates. The larvae quantification was achieved using the same procedure previously described. Results were based on the means of recovered and standard deviation larvae from both experimental groups. The reduction percentage of recovered larvae attributed to the predatory effect of the fungus was estimated using the ABBOTT formula [31]:
Where:
PA% =Predatory activity (percentage).
RLc = Mean of recovered larvae in plates with no fungi.
RLt = Mean of recovered larvae in plates with fungi.
Statistical analysis
The ANOVA test following by Tukey test, which analyzed data, proved that the means of recovered larvae in the two experimental groups were the dependent variables. The SAS statistical package, SAS 9.0, was used.
Liquid filtrate obtaining.
The fungal isolate grew on water agar plates for seven days at room temperature (25–28°C). A small cylinder plug (1 x 1 cm) was taken from the fungal cultures and deposited in 500 mL flasks containing 150 mL of Czapek-Dox broth®. Flasks were incubated under static conditions at the same temperature described above for 21 days. After this period, the liquid from the cultures was filtered using three different filtration systems: a coffee filter, Whatman paper No. 4, and finally, a 0.45 μm and 0.22 μm filter system (KIMBLE® ULTRA-WARE®, Mainz, Germany). Liquid culture filtrates were frozen using a blast freezer (BIOBASE®, Shangdong, China). After the freezer material was lyophilized using a conventional lyophilizer (LABCONCO®, Kansas, USA). Lyophilized LCFs were kept at room temperature (25–28°C) until use.
Assessment of the lethal activity of liquid culture filtrates against Haemonchus contortus larvae.
The nematode/LCF confrontation was conducted in 96-well plastic microtitering plates. Three different concentrations of LCF were established: 25, 50, and 100 mg/mL. A hundred microliters of each concentration and 100 mL of an aqueous suspension containing 500 H. contortus infective larvae were deposited in the corresponding well (n = 4). Czapek-Dox broth® (fungus-free), 10% ivermectin, and sterile distilled water were used as controls. The plate was incubated at room temperature (25–28°C) for 72 h. After incubation, the number of dead and live larvae in each well of the entire experiment was counted by taking five 40-ml aliquots from each well. The results were considered when calculating the means of dead and live larvae in all the treatments (three concentrations and their controls). The mortality percentages attributed to the effect of LCF were estimated using the ABBOTT formula, as follows:
Where:
LMP = Larval mortality percentage.
MRDL Mean of recovered dead larvae.
MRLL Mean of recovered live larvae.
Statistical design.
A completely random analysis was performed using an ANOVA analysis, with dead and live larvae as the dependent variables. The Tukey complementary method was used. The SAS statistical package, SAS 9.0, was used.
Microscopic analysis.
A set of microphotographs was recorded of both the predatory activity and the effect of LCF on H. contortus larvae to visualize the fungal predatory activity and the significant damage exerted by LCF on the nematodes. Photographic material was captured using a Leica DM6 B compound microscope (Wetzlar®, Germany).
Zymography assays.
Proteolytic activities were determined according to the method described by Ramírez-Rico et al. [32]. The technique utilized 10% polyacrylamide gels copolymerized with 0.2% bovine gelatin (Sigma-Aldrich®). The protein concentration was adjusted to 20 μg of LCFs of Arthrobothrys oligospora for electrophoresis. Electrophoresis was performed at 100 V for 2 hours at 4°C, without treating the samples with β-mercaptoethanol or boiling them. After electrophoresis, the gels were washed twice with a 2.5% (v/v) Triton X-100 solution (Sigma-Aldrich®) for 30 minutes and then incubated overnight at 37°C with different buffer solutions: 100 mM sodium acetate, 2 mM CaCl2 (pH 5.0); 100 mM Tris, 2 mM CaCl2 (pH 7.0); and 100 mM Na2CO3-NaHCO3, 2 mM CaCl2 (pH 9.0) (all from Sigma-Aldrich®). Finally, the gels were stained with 0.5% (w/v) Coomassie Brilliant Blue R-250 (Bio-Rad®, Feldkirchen, Germany). Proteolytic activities were detected as clear bands against a blue background. The gels were washed until the proteolytic bands were visible.
Characterization of proteolytic activity using protease inhibitors.
The LCF of A. oligospora was pre-incubated for 1 hour at 22°C with different inhibitors under constant agitation for the protease inhibition assays, following the technique described by Ramírez-Rico et al. [32]. The inhibitors were used at the following concentrations: 10 mM p-hydroxymercuri benzoate (pHMB) for cysteine proteases; 5 mM phenylmethyl-sulfonyl fluoride (PMSF) for serine proteases; and 5 mM EGTA, 5 mM EDTA, or 10 mM o-phenanthroline for metalloproteases (all obtained from Sigma-Aldrich®). Samples were loaded onto 10% SDS-PAGE gels copolymerized with 0.2% bovine gelatin (Sigma-Aldrich®) and subjected to electrophoresis at 4°C in an ice bath at 100 V for 2 hours. Subsequently, the gels were washed twice with 2.5% (v/v) Triton X-100 solution (Sigma-Aldrich®) for 30 minutes, incubated overnight with 100 mM Tris-2 mM CaCl2 (pH 7.0), and stained as previously described.
Results
Traditional morphological taxonomy
Macroscopically examining the plates containing the fungal isolate growing on water agar showed a whitish, cottony growth, forming concentric mycelial rings (Fig 1A). The microscopic observation of the fungus revealed the presence of erect and straightforward conidiophores with initial apical conidial cluster formation (Fig 1B). In older cultures, several conidial clusters formed along the conidiophores (Fig 1A). Additionally, chlamydospores, three-dimensional adhesive nets, and trapped nematodes were observed (Fig 1D–1F).
Table 1 shows the measurements of taxonomic importance for nematophagous fungi, i.e., the length and width of conidia and the length of conidiophores. After comparing the structures and measurements with those described in specialized taxonomic keys, we concluded that our isolate corresponded to Arthrobotrys oligospora.
Molecular taxonomy
The analysis of fungal DNA sequences, followed by alignment and comparison to other isolates reported in the National Center for Biotechnology Information, determined that our isolation corresponds to A. oligospora, confirming the results obtained from the traditional morphological analysis. Data on query coverage, similarity percentages, and GenBank accession numbers are presented in Table 2.
Subsequently, the phylogenetic tree (Fig 2) showed that the evolutionary relationships between taxa were determined using the Maximum Likelihood method. This analysis included 43 nucleotide sequences corresponding to the Arthrobotrys, Dactylellina, and Drechslerella genera. All ambiguous positions were removed for each pair of sequences using the pairwise removal option. The final dataset consisted of 522 base pairs. The phylogenetic analyses were conducted in IQTtree® (version 2.3.6). The best substitution model (SYM + I + G4) was calculated using JModelTest (version 2.1.10).
Our strain, Arthrobotrys oligospora (NCBI: isolate PP741577), is highlighted in red. Bootstrap values are shown in bold.
Predatory activity assessment
Table 3 shows the fungal predatory activity. The NF was divided into Group 1 (larvae and fungus) and Group 2 (larvae only). Additionally, the mean number of H. contortus larvae recovered after ten days of fungal predation on the nematode, as well as the percentage reduction in larvae attributed to its nematocidal capacity, is also observed.
Lethal activity of liquid culture filtrates against Haemonchus contortus larvae
Table 4 shows the percentage of H. contortus infective larvae that died after exposure to A. oligospora filtrates at concentrations of 100, 50, and 25 mg/mL. The mortality rate decreased from 90.10% to 36.35%, and the filtrate exhibited a concentration-efficacy relationship after 48 hours of exposure.
Microscopic analysis
Fig 3 shows the damage observed in H. contortus infective larvae exposed to A. oligospora filtrates (100 mg/mL) after 48 hours. Larvae exhibited changes in their internal structures, particularly with the loss of intestinal cell architecture and a remarkable degree of tissue disorganization. Additionally, a notable loss of movement in the larvae was observed, eventually resulting in them remaining motionless. At lower concentrations, no cell damage was observed in the infective larvae.
Arthrobotrys oligospora proteolytic activities and their stability over a wide pH range
To evaluate the proteolytic activity present in the A. oligospora filtrate, we performed zymography assays and evaluated the activity at different pHs. Our results revealed several proteolytic activities (Fig 4). At pH 5, three highly active proteases are mainly observed with molecular weights of 75, 90, and 140 kDa, and a less intense activity of 250 kDa (Fig 4a). At pH 7, a higher number of proteolytic activities were found compared to pH 5, with molecular weights of 37, 45, 55, 75, 90, 100, 140, 155, and 250 kDa (Fig 4b). In the case of pH 9, the same proteases are observed as in pH 7, but the high molecular weight activities between 75 and 140 kDa show greater proteolytic activity (Fig 5c).
Twenty micrograms of proteins derived from the culture filtrates of A. oligospora were loaded onto 10% polyacrylamide gels copolymerized with 0.2% bovine gelatin. The following buffers were evaluated to determine the activation pH: (a) pH 5.0, (b) pH 7.0, and (c) pH 9.0. In the first lane of each pH evaluated, the reference molecular weight marker (Mw) is observed. The arrows indicate the proteolytic activities evaluated at each pH.
We performed an incubation of the filtrates before electrophoresis, using the following inhibitors: EDTA (lane 2), EGTA (lane 3), and phenanthroline (lane 4) for metalloproteases; phenylmethylsulphonyl fluoride (PMSF) (lane 5) for serine proteases, and p-hydroxy-mercury-benzoate (pHMB) (lane 6) for cysteine proteases. The sample without inhibitors was used as an experimental control lane (lane 1).
A. oligospora secretes metallo and cysteine proteases
The present study demonstrates that A. oligospora exhibits significant proteolytic activity, which can be modulated with different pH conditions. To determine what type of proteases A. oligospora secretes, we performed proteolytic inhibition assays with different inhibitors and subsequently performed zymograms.
The results revealed that all compounds tested in this study inhibited low molecular weight proteases (37–55 kDa) (Fig 5, lanes 2–6), compared to the control sample without inhibitors (Fig 5, lane 1). However, when we used EDTA, we observed a complete inhibition of 75 kDa activity and a partial inhibition of high molecular weight proteases (>75 kDa) (Fig 5, lane 2). EGTA partially inhibited proteases from 75 kDa onwards (Fig 5, lane 3). However, the inhibition results with pHMB and mainly with phenanthroline showed a complete inhibition of all proteolytic activities of A. oligospora filtrates (Fig 5, lanes 4 and 6). We did not observe inhibition of high molecular weight proteases when we used PMSF (Fig 5, lane 5, S2 Fig).
Discussion
Traditional morphological taxonomy
Nematophagous fungi share specific general characteristics across different genera and species of nematode-trapping fungi. These morphological similarities can sometimes complicate traditional taxonomic identification. For instance, several species within the Arthrobotrys genus appear quite similar at first sight. However, differences such as the type of conidiophore (whether single or branched), conidium shape and size, trapping devices, and the presence or absence of chlamydospores enable accurate species differentiation.
Our isolate produces single, unbranched, erect conidiophores, a characteristic observed in various nematode-trapping fungi, including Arthrobotrys oligospora, A. musiformis, A. conoides, A. arthrobotryoides, A. dactyloides, and A. javanica [33]. In addition to the type of conidiophores, these species exhibit differences in other traits that help inform our taxonomic diagnosis.
Our isolate developed three-dimensional adhesive nets as trapping devices, a feature also seen in A. oligospora, A. conoides, A. musiformis, and A. arthrobotryoides. However, A. dactyloides is distinguished by producing constricting rings [34], while A. sinensis employs a combination of trapping devices, including both three-dimensional adhesive nets and constricting rings [35].
While A. oligospora and A. conoides share the characteristics of single and erect conidiophores, the stems of the conidiophores in A. oligospora are larger compared to those in A. conoides. Additionally, A. conoides has smaller conidia, measuring 8.4 x 11.8 μm, while A. oligospora’s conidia are larger, measuring an average of 23 x 12.1 μm [36]. A summary of the main morphological characteristics of five species within the Arthrobotrys genus is provided in Table 5.
Molecular taxonomy
The phylogenetic tree was constructed using 43 sequences of related nematophagous fungi, which were previously reported in the NCBI database. This analysis revealed that our sequence is closely related to A. oligospora_920, a relationship that is strongly supported by a bootstrap value of 100. Interestingly, our isolate also shows a close phylogenetic relationship with A. conoides_670, which is found in the same clade. As we noted in the morphological taxonomic identification, these two species are highly similar and share specific characteristics, such as the development of single, erect conidiophores. Both species also produce three-dimensional adhesive nets as trapping devices. Additionally, another species, A. flagrans (or Duddingtonia flagrans), shares some morphological similarities with these two Arthrobotrys species, including the formation of conidia clusters and the same type of trapping devices [42]. The phylogenetic analysis revealed that this species branches off in a distinct part of the evolutionary tree yet remains closely related to others. Beyond our examination of the molecular data confirmed the findings from our morphological analysis. A recent study focused on investigating the functions of mitophagy, conidiation, stress response, and pathogenicity in A. oligospora. It revealed that two isolates of A. oligospora homologs resulted in a close relationship with orthologs from other nematophagous fungi, including D. flagrans [43]. In another study, focused on determining the phylogenetic relationship of D. flagrans with other fungi, it was revealed that both D. flagrans and A. oligospora are grouped in the same clade, indicating that they share the same common ancestor [44].
Predatory activity assessment
The isolate obtained in the present study exhibited significant predatory activity, with a rate higher than 70% against the infective larvae H. contortus, comparable to the predatory behavior shown by other isolates from other countries (Table 6).
The practical application of nematophagous fungi for controlling parasitic nematodes in ruminants involves the oral administration of spores from selected fungal isolates. After passing through the animals’ digestive tracts, these spores are excreted in the feces. Once in the feces, the spores germinate in situ, initiating a morphogenesis process that transforms their mycelia into trapping devices [44]. The most commonly used genus of nematode-trapping fungus for controlling ruminant parasitic nematodes is Duddingtonia flagrans, which is known for its ability to develop a large number of chlamydospores. These chlamydospores are more resistant than simple spores and can survive after passing through the gastrointestinal tract of animals [42]. Nevertheless, A. oligospora spores have also been reported to survive through the gastrointestinal tract of small ruminants, and after reaching the feces, they germinate and exert their predatory activity [51]. Other species have also demonstrated their ability to survive their passage through the digestive process in ruminants while maintaining their predatory activity (Table 7). The A. oligospora isolate used in this study could be a promising candidate for further assessment as a potential biocontrol agent in future experiments.
Lethal activity of liquid culture filtrates against Haemonchus contortus larvae
The larval mortality of H. contortus, attributed to the lethal effects of the liquid culture filtrates of A. oligospora, showed significant results. Mortality rates ranged from 36.35% at the lowest concentration (25 mg/mL) to over 90% at the highest concentration (100 mg/mL). A clear dependence on concentration was observed. This finding is noteworthy, as the highest percentage of mortality was observed at the highest concentration of the liquid filtrates. It is essential to consider that such a high larval mortality rate was achieved using a liquid culture filtrate that may contain various compounds, which could mask the effects of the molecules responsible for this mortality. Therefore, we would expect to see greater activity with much lower concentrations of the purified compound. In future studies, we plan to elucidate the compound(s) responsible for the nematocidal activity, which could lead to the discovery of a natural compound with nematocidal properties. Different authors report the use of liquid culture filtrates showed un table 8.
Microscopic analysis
The examination of larvae exposed to liquid culture filtrates of A. oligospora under a microscope revealed significant damage, particularly to the internal structures of the larvae. Notably, the intestinal cell architecture was lost only in the larvae that were exposed to the filtrates, while larvae in the control group that were not exposed showed no such damage. This fact suggests that compounds derived from the fungal secondary metabolism present in the liquid filtrates may be responsible for the degradation of the intestinal cells. Exposed larvae gradually reduced their movements until they finally remained motionless until death. Future studies utilizing confocal microscopy, along with immunolocalization markers and fluorophores, could provide more detailed information about the specific sites where the bioactive compounds affect the internal tissues of the larvae.
The mechanism by which A. oligospora filtrates induces the observed damage to the larval intestinal cells, we propose that this is likely a multi-faceted process involving both enzymatic degradation and the direct toxic activity of metabolites. The proteolytic enzymes, particularly the metallo- and cysteine proteases identified in our zymograms, could initiate the process by degrading the proteinaceous components of the larval cuticle, compromising its structural integrity and creating pathways for internal entry [61,62]. Once this barrier is weakened, low molecular weight nematocidal metabolites, such as the polyketide-terpenoid hybrids known to be produced by A. oligospora [23], could passively diffuse into the pseudocoelom. Their lipophilic nature would allow them to cross cell membranes and act directly on internal organs, including intestinal cells, disrupting their architecture and function. Concurrently, the oral route cannot be discounted; larvae likely ingest the filtrate, introducing proteases and metabolites directly into the digestive tract. Ingested enzymes could damage the intestinal epithelium from the lumen, while absorbed metabolites could exert a systemic toxic effect. Therefore, we suggest a synergistic and sequential model: proteolytic attack facilitates the internalization of toxic compounds, and the combined action of ingested enzymes and absorbed metabolites leads to the severe internal disorganization and eventual death of the larva. Future studies using immunohistochemical localization of these proteases and metabolites within the nematode would be invaluable to confirm this proposed model.
Zymography assays
Arthrobotrys oligospora has been reported to produce extracellular proteases that are involved in its nematicidal effect [61,63,64]. Traps are essential components for nematophagous fungi, and their alteration decreases their predation efficiency. A. oligospora, belonging to this group, has been reported to produce extracellular serine proteases that degrade the nematode cuticle, promoting fungal penetration and colonization [51,65,66].
In this work, A. oligospora secretes a large amount of cysteine and metalloproteases of different molecular weights, with proteolytic activity over a wide pH range. Tunlid and Jansson [64] reported in 1991 that A. oligospora secretes proteases, which are inhibited by serine protease inhibitors, aspartate protease inhibitors, and cysteine protease inhibitors. They also observed a significantly decreased immobilization of nematodes captured by the fungus after incubation with serine protease inhibitors, PMSF, antipain, or chymostatin, or the metalloprotease inhibitor phenanthroline [65]. This finding is consistent with the results obtained, which showed inhibition of proteolytic activity with cysteine and metalloprotease inhibitors.
The study of proteases secreted by A. oligospora has focused on two low-molecular-weight serine proteases. Tunlid et al. [65] purified and characterized an extracellular serine protease of 35 kDa (PII) [61,62]. On the other hand, Junwei et al. [67] purified the serine protease XAoz1 with a molecular weight of 50 kDa. Both proteases participate in the nematicidal effect of nematode-trapping fungi. In this study, we detected low molecular weight proteolytic activities, which coincided with the molecular weights reported in both studies, and also found inhibition with PMSF.
Chitinases are hydrolytic enzymes involved in the crucial digestion of nematode cell walls, particularly during egg parasitism by nematophagous fungi [68]. It has been reported that A. oligospora secretes chitinases with molecular weights greater than 100 kDa [69]. The serine protease PII can degrade cuticle, where chitin is present [70]. Therefore, the proteases secreted by A. oligospora could also have chitinase activity. Furthermore, the pHMB inhibitor is capable of inhibiting glycosidases to which chitinases belong [70], so the high-molecular-weight proteases in this study may possess this enzymatic activity, which aligns with our proteolytic inhibition results.; however, we did not evaluate the ability of these proteases to degrade chitin. It would be interesting to evaluate in future assays, in addition to their purification and characterization. These proteases could also contribute to the pathogenicity of this fungus, which is involved in the nematicidal effect that traps nematodes, and demonstrate that A. oligospora secretes different types and classes of proteases.
Characterization of proteolytic activity using protease inhibitors
The enzymatic inhibition assay performed reveals the nature of the proteases present, demonstrating the activity of low molecular weight enzymes (37–55 kDa) that were completely inhibited by phenanthroline and pHMB, indicating their dependence on metals and thiol groups, characteristic of metalloproteases and cysteine proteases. Additionally, partial inhibition was observed with EDTA, suggesting the presence of calcium-dependent enzymes. At the same time, PMSF did not affect the high molecular weight proteases, ruling out significant involvement of serine proteases in this range. The results obtained partially contrast with those of Tunlid et al. [61], who identified serine proteases as the main contributors to the nematicidal activity in A. oligospora. This discrepancy could be due to differences in strains or culture conditions, highlighting the enzymatic variability among fungal isolates. Overall, the data emphasize the complexity of A. oligospora’s enzymatic arsenal and its potential for the development of biocontrol agents based on specific enzymes. This finding aligns with previous studies, such as those by Wang et al. [52], which underscores the key role of extracellular proteases in nematode cuticle degradation. The presence of these enzymes in the LCF could explain the high mortality of Haemonchus contortus observed in the study, as they facilitate the penetration and digestion of parasitic tissues by nematophagous fungi
Conclusion
This study comprehensively demonstrates the high biocontrol potential of a Mexican strain of Arthrobotrys oligospora against the parasitic nematode Haemonchus contortus. The fungus employs a dual attack strategy: a direct predatory mechanism with a high trapping efficiency (72.06%), and the release of potent nematocidal compounds into its liquid culture filtrate (LCF), which caused up to 96.10% larval mortality.
The findings significantly advance the field by characterizing the enzymatic arsenal behind this activity, identifying a suite of pH-stable metallo- and cysteine proteases as key virulence factors. Furthermore, the chromatographic profiling suggests that secondary metabolites, including lignans and an ellagic acid derivative, may contribute to the observed lethal effect.
This multi-faceted mode of action—combining physical trapping, enzymatic degradation, and bioactive metabolite production—makes this Mexican A. oligospora isolate a highly promising and sustainable candidate for the development of novel biocontrol agents against livestock nematodes, offering a viable strategy to counteract the growing issue of anthelmintic resistance.
Acknowledgments
This study formed part of the PhD thesis work of MVZ Héctor Alejandro de la Cruz Cruz at the National Autonomous University of Mexico (UNAM), Mexico, under the direction of Pedro Mendoza de Gives and the tutelage of Yazmin Alcalá Canto and Alejandro Zamilpa.
For MSc Elvia Adriana Morales Hipólito and Hugo Cuatecontzi Flores, who supported the UPLC technique at Facultad de Estudios Superiores Cuautitlán-UNAM, México.
Institutional review board statement: The sheep donor for Haemonchus contortus strain was maintained under controlled conditions following the Norma Oficial Mexicana (Official Mexican Standard) with official rule number NOM-052-ZOO-1995 (http://www.senasica.gob.mx, accessed on 7 May 2024) and the Ley Federal de Sanidad Animal (Federal Law for Animal Health) DOF 07-06-2012 were strictly applied (https://www.gob.mx/cms/uploads/attachment/file/118761/LFSA.pdf, accessed on 7 May 2024). Additionally, the experimental protocol was approved by the Internal Committee for Care and Use of Experimental Animals (CICUAE-FESC) at Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, under protocol number C24_25
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