High Predatory Capacity of a Novel Arthrobotrys Oligospora Variety on the Ovine Gastrointestinal Nematode Haemonchus Contortus (Rhabditomorpha: Trichostrongylidae)
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pathogens Article High Predatory Capacity of a Novel Arthrobotrys oligospora Variety on the Ovine Gastrointestinal Nematode Haemonchus contortus (Rhabditomorpha: Trichostrongylidae) Fabián Arroyo-Balán 1,2,* , Fidel Landeros-Jaime 3, Roberto González-Garduño 4, Cristiana Cazapal-Monteiro 5 , Maria Sol Arias-Vázquez 5 , Gabriela Aguilar-Tipacamú 6, Edgardo Ulises Esquivel-Naranjo 3,* and Juan Mosqueda 1,6,* 1 Immunology and Vaccines Laboratory, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro 76140, Mexico 2 CONACYT-Unidad Regional Universitario de Zonas Áridas, Universidad Autónoma Chapingo, Bermejillo 35230, Mexico 3 Laboratorio de Microbiología Molecular, Unidad de Microbiología Básica y Aplicada, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro 76140, Mexico; [email protected] 4 Centro Regional Universitario Sursureste, Universidad Autónoma Chapingo, Teapa 86800, Mexico; [email protected] 5 COPAR (Control of Parasites), Animal Pathology Department, Veterinary Faculty, Santiago de Compostela University, Campus Universitario, 27002 Lugo, Spain; [email protected] (C.C.-M.); [email protected] (M.S.A.-V.) 6 C.A. Salud Animal y Microbiologia Ambiental, Facultad de Ciencias Naturales, Universidad Autónoma de Citation: Arroyo-Balán, F.; Querétaro, Av. de las Ciencias s/n Col Juriquilla, Querétaro 76230, Mexico; [email protected] Landeros-Jaime, F.; González-Garduño, R.; * Correspondence: [email protected] (F.A.-B.); [email protected] (E.U.E.-N.); Cazapal-Monteiro, C.; Arias-Vázquez, [email protected] (J.M.) M.S.; Aguilar-Tipacamú, G.; Esquivel-Naranjo, E.U.; Mosqueda, J. Abstract: With the worldwide development of anthelmintic resistance, new alternative approaches High Predatory Capacity of a Novel for controlling gastrointestinal nematodes in sheep are urgently required. In this work, we identified Arthrobotrys oligospora Variety on the and characterized native nematode-trapping fungi. We collected seven isolates of fungi with the Ovine Gastrointestinal Nematode capacity to form adhesive, three-dimensional networks as the main mechanism to capture, kill, and Haemonchus contortus (Rhabditomorpha: Trichostrongylidae). Pathogens 2021, 10, consume nematodes. The nematode-trapping fungi were classified into two groups; the first group 815. https://doi.org/10.3390/ includes the R2-13 strain, showing faster growth, abundant aerial hyphae, scarce conidia production, pathogens10070815 bigger conidia, and it formed a clade with Arthrobotrys oligospora sensu stricto. The second comprises the A6, A12, A13, R2-1, R2-6, and R2-14 strains, showing a growth adhering to the culture medium, Academic Editor: Abdul Jabbar forming little aerial hyphae, smaller conidia, and these formed a sister clade to A. oligospora. Except for the R2-6 strain, conidia production was induced by light. In all the strains, the predatory capacity Received: 7 May 2021 against the sheep gastrointestinal nematode Haemonchus contortus was greater than 58% compared Accepted: 22 June 2021 with the control group. The A6 and A13 strains were the most active against the infective H. contortus Published: 29 June 2021 third instar (L3) larvae, with an average capture capacity of 91%. Altogether, our results support evidence for a novel A. oligospora variety with high nematode-trapping activity and promissory in Publisher’s Note: MDPI stays neutral helminthic control. with regard to jurisdictional claims in published maps and institutional affil- Keywords: parasitosis; antihelmintic; biocontrol; nematofagous fungi; Arthrobotrys oligospora; light iations. response Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article The parasitic control programs based solely on the administration of pharmacological distributed under the terms and treatments for nematodes affecting the ovine industry in the tropics can be considered a conditions of the Creative Commons temporary solution because they do not act on the free stages of the soil life cycle of the Attribution (CC BY) license (https:// nematodes. Animals can reinfect themselves upon consuming forage contaminated with creativecommons.org/licenses/by/ those stages, and the serious downsides to the use of agrochemical products are their high 4.0/). costs, the presence of resistance [1], and their possible harmful effect on the environment. Pathogens 2021, 10, 815. https://doi.org/10.3390/pathogens10070815 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 815 2 of 14 To solve these problems, a drastic reduction in pharmacological treatments in the coming decades is needed. Therefore, new alternatives or supplementary methods are actively sought. In nature, there are numerous antagonists of helminths, such as fungi, bacteria, viruses, protozoa, and mites. Among them, nematophagous fungi likely play a role as natural enemies of parasitic nematodes of plants and animals because they can capture, parasitize, or paralyze some free-living nematodes. In a simplified fashion, nematophagous fungi have been traditionally classified into predators or endoparasites [2]. Nowadays, it seems more appropriate to classify them according to how they feed on nematodes and the mechanisms involved in these processes, thus finding fungi with ovicidal [3,4] or larvicidal properties [5]. Nematophagous fungi comprise over 200 species of taxonomically diverse fungi that can capture, kill, and digest nematodes [6]. Noticeably, most studies of nematophagous fungi have focused on predatory species belonging to the genera Arthrobotrys, Duddingtonia, and Monacrosporium as a biocontrol alternative for the use of anthelmintics in infective larvae of parasites in domestic animals [7]. This is because they possess some natural advantages, including a short life cycle, high reproductive ability, production of resistant spores, and survival in the saprophytic phase in the absence of hosts. Additionally, they are not pathogenic for humans, and they reduce parasitic populations rather than wiping them out, which maintains an important immunological stimulus in the animals [8,9]. Most of the studies on biological control of livestock nematodes are centered on Duddingtonia flagrans because it produces a large number of thick-walled chlamydospores that can be added to livestock feed as additives to control parasites, because when administered to animals they pass through the gastrointestinal tract and, when excreted into the environment, they colonize the fecal matter and capture and destroy a significant number of nematode larvae in situ [8,10]. Furthermore, some studies have documented the ability of Arthrobotrys oligospora to successfully feed on larvae in the third stage (L3) of cyathostomins that infect horses in Brazil [11] and some gastrointestinal nematodes (GIN) in sheep and cattle [12,13]. Arthrobotrys species were first described in Europe in 1850 by George Fresenius. A. oligospora was the first species of fungus known to actively capture nematodes [14] and is thought to be the most common nematode-trapping fungus and the most widespread. As a premise, Arthrobotrys species behave as generalist predators [15], indicating great potential in the biocontrol of parasitic nematodes. This study aimed to isolate, identify, and evaluate in vitro the predatory ability of nematophagous fungus strains native from the Mexican state of Querétaro, and provide a first approach that can be used as biological control agents for infective larvae of ruminant gastrointestinal nematodes. 2. Materials and Methods 2.1. Gathering of Samples for Isolation (Area of Study) The study was carried out from June 2018 through September 2018, in three locations in the Mexican state of Querétaro: one in El Marqués (20◦42037.500 N, 100◦15024.600 W) and two in Jalpan de Serra (21◦11031.700 N, 99◦27023.000 W) and (21◦09159.100 N, 99◦21001.700 W). Samples of feces from sheep, cattle and horses were collected from the soil in depth ranges of 0 to 5 cm and in compost, pasture soil and under trees. In total, 55 samples were collected and stored in polyethylene bags, properly labeled, and transported to the facilities of the Microbiology Laboratory of the School of Natural Sciences of the Autonomous University of Querétaro for the isolation and identification of nematophagous fungi. 2.2. Infective Larvae of Haemochus Contortus For obtaining infective larvae (L3) of H. contortus, we utilized fecal samples from a lamb previously infected under experimental conditions with L3 from H. contortus collected and cultivated for 10 days at room temperature [16,17]. Afterward, the larvae were collected using Baerman’s technique and quantified and stored at 4 ◦C before analysis [18]. Pathogens 2021, 10, 815 3 of 14 2.3. Isolation of Nematophagous Fungi Soil samples were processed with the sprinkled plate method outlined by Barron [2,17,19] for isolating nematophagous fungi. In this technique, for each soil sample, five subsamples of 0.5–1 g were collected and placed into 9 cm Petri dishes containing water-agar (WA). One milliliter of suspension containing 100–300 L3 of H. contortus was added using a micropipette. The plates were incubated at room temperature (23–26 ◦C), and from the fourth day onward, they were analyzed daily with an inverted microscope in search of hyphae, conidia, spores, or parasitized nematodes. Observations were maintained for a further week until 28 days after the incubation; the goal was to increase the likelihood of finding a fungal structure.