Antidiabetic action of the Chinese formula Shouhuitongbian and the underlying mechanism associated with alteration of gut microbiota.

BACKGROUND: The prevalence and incidence of type 2 diabetes mellitus (T2DM) have dramatically increased. The intestinal flora and its derived metabolites are demonstrated to play vital roles in the etiology and onset of T2DM. Shouhuitongbian (SHTB) is a traditional Chinese formula to treat constipation. SHTB is composed of seven herbs and components of Colla corii asini (CCA) that are obtained from the hide of Equus asinus L.. Some of herbs in SHTB such as Aloe vera (L.) Burm.f., Cassia obtusifolia L., fruits of Lycium barbarum L., and Citrus aurantium L. have shown to improve insulin resistance (IR) and T2DM in early reports. We hypothesized that SHTB composed of these herbs has antidiabetic effects. The antidiabetic efficacy and mechanism of action of SHTB have not been previously reported. HYPOTHESIS/PURPOSE: To demonstrate the antidiabetic effect and elucidate the underlying mechanisms of SHTB from the perspective of gut microbiota. STUDY DESIGN: The main compounds were identified and quantified by high-performance liquid chromatography (HPLC)-mass spectrometry analysis. High fat diet (HFD)-fed mice and db/db mice were used to assess the antidiabetic effects and the mechanism of SHTB. The underlying mechanisms were evaluated by enzyme-linked immunosorbent assay (ELISA), western blot analysis, quantitative real time polymerase chain reaction (qPCR) analysis, 16S rRNA high-throughput sequencing, and targeted metabolome analysis. METHODS: HFD-fed mice and db/db mice were orally treated with the standard positive drug metformin (100 mg/kg/d) and with SHTB (200 and 100 mg/kg/d), which was chemically characterized according to the European Medicine Agency (EMA) guidelines. The beneficial effects of SHTB were studied by homeostasis model assessment of insulin resistance (HOMA-IR) index, oral glucose tolerance test (OGTT), insulin tolerance test (ITT), total cholesterol (T-CHO), triglyceride (TG), and inflammation. Subsequently, 16S rDNA-based high-throughput pyrosequencing and GC-MS-based targeted metabolomics profiling were performed to analyze the gut microbiota composition and metabolites profile in the gut, respectively. Moreover, the mammalian target of rapamycin complex 1 (mTORC1) / insulin receptor substrate 1 (IRS-1) / phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) pathway was evaluated via qPCR and western blot. RESULTS: Chemically characterized SHTB, in which six markers were quantified, effectively alleviated glucose intolerance and IR, ameliorated lipid metabolism dysfunction, and reduced inflammation. In addition, 16S rDNA sequencing found that SHTB reshaped the composition of intestinal flora, as indicated by the enrichment of Akkermansia and Parabacteroides in both HFD-fed and db/db mice. Moreover, SHTB enhanced the intestinal production of short-chain fatty acids (SCFAs) and branched short-chain fatty acids (BSCFAs), and reduced the levels of the fecal and circulating branched-chain amino acids (BCAAs). The IRS-1/PI3K/AKT signaling pathway was upregulated after treatment with SHTB. CONCLUSION: Orally administration of SHTB effectively improved IR and reduced hyperglycemia in mice. Treatment with SHTB regulated the gut BCAAs-mTORC1/IRS-1/PI3K/AKT axis by enhancing the BCAAs catabolism in the gut, which attenuated the deleterious effect of BCAAs on the IRS-1 signaling pathway.

Discovery of antifungal secondary metabolites from an intestinal fungus Fusarium sp.

Intestinal fungi, which are important parts of the gut microbiota, have the ability to produce specialized metabolites that significantly contribute to maintaining the balance of the gut microbiota and promoting the health of the host organism. In the present study, two new glycosides, including fusintespyrone A (1) and cerevisterolside A (4), as well as ten known compounds were isolated from the intestinal fungus Fusarium sp. LE06. The structures of the new compounds were elucidated by a combination of spectroscopic methods, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), along with chemical reactions and calculations of NMR and ECD spectra. Compounds 1-3 showed significant growth inhibition against Aspergillus fumigatus, Fusarium oxysporum, and Verticillium dahliae with MIC values in the range of 1.56-6.25 µg ml-1.

Identification and Characterization of Peptaibols as the Causing Agents of Pseudodiploöspora longispora Infecting the Edible Mushroom Morchella.

Pseudodiploöspora longispora (previously known as Diploöspora longispora) is a pathogenic fungus of Morchella mushrooms. The molecular mechanism underlying the infection of P. longispora in fruiting bodies remains unknown. In this study, three known peptaibols, alamethicin F-50, polysporin B, and septocylindrin B (1-3), and a new analogue, longisporin A (4), were detected and identified in the culture of P. longispora and the fruiting bodies of M. sextelata infected by P. longispora. The primary amino sequence of longisporin A is defined as Ac-Aib1-Pro2-Aib3-Ala4-Aib5-Aib6-Gln7-Aib8-Val9-Aib10-Glu11-Leu12-Aib13-Pro14-Val15-Aib16-Aib17-Gln18-Gln19-Phaol20. The peptaibols 1-4 greatly suppressed the mycelial growth of M. sextelata. In addition, treatment with alamethicin F-50 produced damage on the cell wall and membrane of M. sextelata. Compounds 1-4 also exhibited inhibitory activities against human pathogens including Aspergillus fumigatus, Candida albicans, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, and plant pathogen Verticillium dahlia. Herein, peptaibols are confirmed as virulence factors involved in the invasion of P. longispora on Morchella, providing insights into the interaction between pathogenic P. longispora and mushrooms.

Simplicillium sinense sp. nov., a novel potential pathogen of tinea faciei.

Simplicillium species are widely distributed with a broad spectrum of hosts and substrates. Generally, these species are entomopathogenic or mycoparasitic. Notably, some isolates of Simplicillium lanosoniveum and Simplicillium obclavatum were obtained from human tissues. In this study, two fungi were isolated from the annular itchy patch of infected skin of a 46-year-old man with diabetes mellitus. Based on a combination of morphological characteristics and phylogenetic analysis, a novel species, Simplicillium sinense, was introduced herein. It morphologically differs from the remaining Simplicillium in the size of phialides and conidia. Additionally, it grows slowly on YPD at 37°C. Antimicrobial susceptibility testing presented that this fungus is resistant to most azole antifungals. Therefore, the diagnosis of tinea faciei was made, and after 2 weeks of being treated with oral terbinafine (250 mg, once a day) and topical terbinafine cream for 1 month, the rash was mainly resolved and no recurrence happened after 6 months of follow-up. Herein, Simplicillium sinense was introduced as a new fungal taxon. Meanwhile, a case of superficial infection caused by S. sinense was reported. So far, it is the third Simplicillium species obtained from human tissue. Meanwhile, terbinafine is recommended as the first-line antifungal treatment against Simplicillium infection.

Immunogenic molecules associated with gut bacterial cell walls: chemical structures, immune-modulating functions, and mechanisms.

Interactions between gut microbiome and host immune system are fundamental to maintaining the intestinal mucosal barrier and homeostasis. At the host-gut microbiome interface, cell wall-derived molecules from gut commensal bacteria have been reported to play a pivotal role in training and remodeling host immune responses. In this article, we review gut bacterial cell wall-derived molecules with characterized chemical structures, including peptidoglycan and lipid-related molecules that impact host health and disease processes via regulating innate and adaptive immunity. Also, we aim to discuss the structures, immune responses, and underlying mechanisms of these immunogenic molecules. Based on current advances, we propose cell wall-derived components as important sources of medicinal agents for the treatment of infection and immune diseases.

Proposal of a new family Pseudodiploösporeaceae fam. nov. (Hypocreales) based on phylogeny of Diploöspora longispora and Paecilomyces penicillatus.

During a field survey of cultivated Morchella mushroom diseases, Diploöspora longispora and Paecilomyces penicillatus, causal agents of pileus rot or white mould disease were detected, which resulted in up to 80% of yield losses. Multi-locus phylogenic analysis revealed that the fungi were affiliated in a distinct clade in Hypocreales. We further constructed a phylogenetic tree with broader sampling in Hypocreales and estimated the divergence times. The D. longispora and P. penicillatus clades were estimated to have diverged from Hypocreaceae around 129 MYA and Pseudodiploösporeaceae fam. nov is herein proposed to accommodate species in this clade. Two new genera, i.e. Pseudodiploöspora and Zelopaecilomyceswere, were introduced based on morphological characteristics and phylogenic relationships of Diploöspora longispora and Paecilomyces penicillatus, respectively. Five new combinations - Pseudodiploöspora cubensis, P. longispora, P. fungicola, P. zinniae, and Zelopaecilomyces penicillatus - were proposed.

Genome and transcriptome reveal lithophilic adaptation of Cladophialophora brunneola, a new rock-inhabiting fungus.

Rock-inhabiting fungi (RIF) are slow-growing microorganisms that inhabit rocks and exhibit exceptional stress tolerance owing to their thick melanised cell walls. This study reports the identification of a novel rock-inhabiting fungus, Cladophialophora brunneola sp. nov. which was isolated from a karst landform in Guizhou, China, using a combination of morphological and phylogenetic analyses. The genome of C. brunneola was sequenced and assembled, with a total size of approximately 33.8 Mb, encoding 14,168 proteins and yielding an N50 length of 1.88 Mb. C. brunneola possessed a larger proportion of species-specific genes, and phylogenomic analysis positioned it in an early diverged lineage within Chaetothyriales. In comparison to non-RIF, C. brunneola displayed reduction in carbohydrate-active enzyme families (CAZymes) and secondary metabolite biosynthetic gene clusters (BGCs). Transcriptome analysis conducted under PEG-induced drought stress revealed elevated expression levels of genes associated with melanin synthesis pathways, cell wall biosynthesis, and lipid metabolism. This study contributes to our understanding of the genomic evolution and polyextremotolerance exhibited by rock-inhabiting fungi.

Polysaccharides from Lyophyllum decastes reduce obesity by altering gut microbiota and increasing energy expenditure.

Polysaccharides are known to confer protection against obesity via modulation of gut microbiota. To expand our knowledge of mushroom-derived prebiotics, we investigated the structural characteristics and anti-obesity effects of Lyophyllum decastes polysaccharides. Two heteroglycans were purified and characterized. The isolated polysaccharides effectively reduced obesity and the related disorders in the diet-induced obese (DIO) mice. An altered gut microbiota with enrichments of Bacteroides intestinalis and Lactobacillus johnsonii and an increase of secondary bile acids were detected in the polysaccharide-treated mice. Supplementation of B. intestinalis and L. johnsonii prevented the obesity and hyperlipidemia in DIO mice, demonstrating their causal linkage to the efficacy of polysaccharides. An enhancement of energy expenditure in the brown adipose tissues due to up-regulation of the secondary bile acids-activated TGR5 pathway was deduced to be one of the mechanisms underlying the effect of polysaccharides. These results confirmed Lyophyllum decastes-derived polysaccharides as new prebiotics for preventing and treating obesity.

Discovery of Anti-MRSA Secondary Metabolites from a Marine-Derived Fungus Aspergillus fumigatus.

Methicillin-resistant Staphylococcus aureus (MRSA), a WHO high-priority pathogen that can cause great harm to living beings, is a primary cause of death from antibiotic-resistant infections. In the present study, six new compounds, including fumindoline A-C (1-3), 12ß, 13ß-hydroxy-asperfumigatin (4), 2-epi-tryptoquivaline F (17) and penibenzophenone E (37), and thirty-nine known ones were isolated from the marine-derived fungus Aspergillus fumigatus H22. The structures and the absolute configurations of the new compounds were unambiguously assigned by spectroscopic data, mass spectrometry (MS), electronic circular dichroism (ECD) spectroscopic analyses, quantum NMR and ECD calculations, and chemical derivatizations. Bioactivity screening indicated that nearly half of the compounds exhibit antibacterial activity, especially compounds 8 and 11, and 33-38 showed excellent antimicrobial activities against MRSA, with minimum inhibitory concentration (MIC) values ranging from 1.25 to 2.5 µM. In addition, compound 8 showed moderate inhibitory activity against Mycobacterium bovis (MIC: 25 µM), compound 10 showed moderate inhibitory activity against Candida albicans (MIC: 50 µM), and compound 13 showed strong inhibitory activity against the hatching of a Caenorhabditis elegans egg (IC50: 2.5 µM).

Cyclic Tetrapeptides with Synergistic Antifungal Activity from the Fungus Aspergillus westerdijkiae Using LC-MS/MS-Based Molecular Networking.

Fungal natural products play a prominent role in the development of pharmaceuticalagents. Two new cyclic tetrapeptides (CTPs), westertide A (1) and B (2), with eight known compounds (3-10) were isolated from the fungus Aspergillus westerdijkiae guided by OSMAC (one strain-many compounds) and molecular networking strategies. The structures of new compounds were unambiguously determined by a combination of NMR and mass data analysis, and chemical methods. All of the isolates were evaluated for antimicrobial effects, synergistic antifungal activity, cytotoxic activity, and HDAC inhibitory activity. Compounds 1-2 showed synergistic antifungal activity against Candida albicans SC5314 with the presence of rapamycin and weak HDAC (histone deacetylase) inhibitory activity. These results indicate that molecular networking is an efficient approach for dereplication and identification of new CTPs. CTPs might be a good starting point for the development of synergistic antifungal agents.

Exploring Verrucosidin Derivatives with Glucose-Uptake-Stimulatory Activity from Penicillium cellarum Using MS/MS-Based Molecular Networking.

Under the guidance of LC-MS/MS-based molecular networking, seven new verrucosidin derivatives, penicicellarusins A-G (3-9), were isolated together with three known analogues from the fungus Penicillium cellarum. The structures of the new compounds were determined by a combination of NMR, mass and electronic circular dichroism spectral data analysis. The absolute configuration of penicyrone A (10) was corrected based on X-ray diffraction analyses. Bioactivity screening indicated that compounds 1, 2, and 4 showed much stronger promising hypoglycemic activity than the positive drug (rosiglitazone) in the range of 25-100 µM, which represents a potential new class of hypoglycemic agents. Preliminary structure-activity relationship analysis indicates that the formation of epoxy ring on C6-C7 in the structures is important for the glucose uptake-stimulating activity. The gene cluster for the biosynthesis of 1-12 is identified by sequencing the genome of P. cellarum and similarity analysis with the gene cluster of verrucosidins in P. polonicum.

Four new species of Trichoderma in the Harzianum clade from northern China.

The Harzianum clade of Trichoderma comprises many species, which are associated with a wide variety of substrates. In this study, four new species of Trichoderma, namely T. lentinulae, T. vermifimicola, T. xixiacum, and T. zelobreve, were encountered from a fruiting body and compost of Lentinula, soil, and vermicompost. Their colony and mycelial morphology, including features of asexual states, were described. For each species, their DNA sequences were obtained from three loci, the internal transcribed spacer (ITS) regions of the ribosomal DNA, the gene encoding the second largest nuclear RNA polymerase subunit (RPB2), the translation elongation factor 1-α encoding gene (TEF1-α). The analysis combining sequences of the three gene regions distinguished four new species in the Harzianum clade of Trichoderma. Among them, T. lentinulae and T. xixiacum clustered with T. lixii, from which these new species differ in having shorter phialides and smaller conidia. Additionally, T. lentinulae differs from T. xixiacum in forming phialides with inequilateral to a strongly-curved apex, cultural characteristics, and slow growth on PDA. Trichoderma vermifimicola is closely related to T. simmonsii, but it differs from the latter by producing phialides in verticillate whorls and smaller conidia. Trichoderma zelobreve is the sister species of T. breve but is distinguished from T. breve by producing shorter and narrower phialides, smaller conidia, and by forming concentric zones on agar plates. This study updates our knowledge of species diversity of Trichoderma.

Unveiling the Hidden Diversity of Rock-Inhabiting Fungi: Chaetothyriales from China.

Rock-inhabiting fungi (RIF) are nonlichenized fungi that naturally colonize rock surfaces and subsurfaces. The extremely slow growth rate and lack of distinguishing morphological characteristics of RIF resulted in a poor understanding on their biodiversity. In this study, we surveyed RIF colonizing historical stone monuments and natural rock formations from throughout China. Among over 1000 isolates, after preliminary delimitation using the internal transcribed spacer region (ITS) sequences, representative isolates belonging to Trichomeriaceae and Herpotrichiellaceae were selected for a combined analysis of ITS and the nuclear ribosomal large subunit (nucLSU) to determine the generic placements. Eight clades representing seven known genera and one new genus herein named as Anthracina were placed in Trichomeriaceae. While, for Herpotrichiellaceae, two clades corresponded to two genera: Cladophialophora and Exophiala. Fine-scale phylogenetic analyses using combined sequences of the partial actin gene (ACT), ITS, mitochondrial small subunit ribosomal DNA (mtSSU), nucLSU, the largest subunit of RNA polymerase II (RPB1), small subunit of nuclear ribosomal RNA gene (SSU), translation elongation factor (TEF), and ß-tubulin gene (TUB) revealed that these strains represented 11 and 6 new species, respectively, in Trichomeriaceae and Herpotrichiellaceae. The 17 new species were described, illustrated for their morphologies and compared with similar taxa. Our study demonstrated that the diversity of RIF is surprisingly high and still poorly understood. In addition, a rapid strategy for classifying RIF was proposed to determine the generic and familial placements through preliminary ITS and nucLSU analyses, followed by combined analyses of five loci selected from ACT, ITS, mtSSU, nucLSU, RPB1, and/or the second subunit of RNA polymerase II gene (RPB2), SSU, TEF, and TUB regions to classify RIF to the species level.

IMA Genome-F 9: Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata.

Draft genomes of the species Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina and Morchella septimelata are presented. Both mating types (MAT1-1 and MAT1-2) of Cercospora beticola are included. Two strains of Coleophoma cylindrospora that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of Aspergillus mulundensis, Coleophoma cylindrospora and Phialophora cf. hyalina has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi.

A new species of Trichoderma hypoxylon harbours abundant secondary metabolites.

Some species of Trichoderma are fungicolous on fungi and have been extensively studied and commercialized as biocontrol agents. Multigene analyses coupled with morphology, resulted in the discovery of T. hypoxylon sp. nov., which was isolated from surface of the stroma of Hypoxylon anthochroum. The new taxon produces Trichoderma- to Verticillium-like conidiophores and hyaline conidia. Phylogenetic analyses based on combined ITS, TEF1-α and RPB2 sequence data indicated that T. hypoxylon is a well-distinguished species with strong bootstrap support in the polysporum group. Chemical assessment of this species reveals a richness of secondary metabolites with trichothecenes and epipolythiodiketopiperazines as the major compounds. The fungicolous life style of T. hypoxylon and the production of abundant metabolites are indicative of the important ecological roles of this species in nature.

Development of a transformation system for Hirsutella spp. and visualization of the mode of nematode infection by GFP-labeled H. minnesotensis.

Hirsutella rhossiliensis and H. minnesotensis are endoparasitic fungi of the second-stage juvenile (J2) of the soybean cyst nematode (Heterodera glycines) in nature. They also parasitize both H. glycines J2 and Caenorhabditis elegans on agar plates. Agrobacterium tumefaciens-mediated transformation conditions were established for these Hirsutella spp. The resulting transformants were similar to the corresponding wild-type strains. The infection processes of H. glycines J2 and C. elegans second larval stage (L2) by H. minnesotensis expressing ZsGreen were microscopically analyzed. Conidia of H. minnesotensis adhered to passing nematodes within 8 h post-inoculation (hpi), formed an infection peg between 8 and 12 hpi, and penetrated the nematode cuticle between 12 and 24 hpi for C. elegans L2 and between 12 and 32 hpi for H. glycines J2. Hyphal proliferation inside of the nematode coelom was observed at approximately 32 hpi for C. elegans L2 and at approximately 40 hpi for H. glycines J2. The fungus consumed the whole body and grew out to produce conidia at approximately 156 and 204 hpi for C. elegans L2 and H. glycines J2, respectively. The efficient transformation protocol and a better understanding of infection process provide a solid foundation for studying the molecular and cellular mechanisms underlying fungal parasitism of nematodes.

Evolution of Chemical Diversity in Echinocandin Lipopeptide Antifungal Metabolites.

The echinocandins are a class of antifungal drugs that includes caspofungin, micafungin, and anidulafungin. Gene clusters encoding most of the structural complexity of the echinocandins provided a framework for hypotheses about the evolutionary history and chemical logic of echinocandin biosynthesis. Gene orthologs among echinocandin-producing fungi were identified. Pathway genes, including the nonribosomal peptide synthetases (NRPSs), were analyzed phylogenetically to address the hypothesis that these pathways represent descent from a common ancestor. The clusters share cooperative gene contents and linkages among the different strains. Individual pathway genes analyzed in the context of similar genes formed unique echinocandin-exclusive phylogenetic lineages. The echinocandin NRPSs, along with the NRPS from the inp gene cluster in Aspergillus nidulans and its orthologs, comprise a novel lineage among fungal NRPSs. NRPS adenylation domains from different species exhibited a one-to-one correspondence between modules and amino acid specificity that is consistent with models of tandem duplication and subfunctionalization. Pathway gene trees and Ascomycota phylogenies are congruent and consistent with the hypothesis that the echinocandin gene clusters have a common origin. The disjunct Eurotiomycete-Leotiomycete distribution appears to be consistent with a scenario of vertical descent accompanied by incomplete lineage sorting and loss of the clusters from most lineages of the Ascomycota. We present evidence for a single evolutionary origin of the echinocandin family of gene clusters and a progression of structural diversification in two fungal classes that diverged approximately 290 to 390 million years ago. Lineage-specific gene cluster evolution driven by selection of new chemotypes contributed to diversification of the molecular functionalities.

Farming of a defensive fungal mutualist by an attelabid weevil.

The mutualism between fungus-growing animals and fungi is a classic example of a complex interspecies association. A handful of insects, notably the well-recognized fungus-farming ants, termites and beetles, have developed advanced agriculture, which includes seeding new gardens with crop propagules, improving growth conditions and protecting and harvesting the fungal crop. More examples, which could be called 'proto-fungiculture', involve fewer adaptations, as exemplified by marine snails that farm intertidal fungi on marsh grass. Recent work has indicated that the solitary leaf-rolling weevil Euops chinensis (family Attelabidae) has a protofarming symbiosis with the mycangial fungus Penicillium herquei (family Trichocomaceae). In this study, we investigated how the weevils create cradles (leaf-rolls) for their offspring and protect the fungal garden. We describe new specialized structures and behaviors that E. chinensis females use for leaf-rolling and fungus inoculation. The fungus P. herquei produces the antibiotic (+)-scleroderolide in laboratory culture and in leaf-rolls, which can serve to inhibit microbial 'weeds' and pests, thus protecting the fungal garden against potential infection. The fungiculture of E. chinensis differs from other advanced insect fungiculture systems because female weevils do not continuously tend the inoculated microbe and do not depend nutritionally on the fungus. The defensive role of the cultivated fungus makes the attelabid weevils exceptional in 'proto-fungiculture' animals.

Parasitism of secondary-stage juvenile of Heterodera glycines and four larva stages of Caenorhabditis elegans by Hirsutella spp.

The fungi Hirsutella rhossiliensis and Hirsutella minnesotensis generally parasitize only plant-parasitic nematodes in nature but parasitize the bacterivorous nematode Caenorhabditis elegans on agar plates. To establish a model system for studying the interaction between fungi and nematodes, we compared the parasitism of the first- to fourth-stage larvae (L1-L4) of C. elegans and second-stage juvenile (J2) of Heterodera glycines by twenty isolates of Hirsutella spp. Although parasitism differed substantially among isolates, both H. minnesotensis and H. rhossiliensis parasitized a higher percentage of H. glycines J2s than of C. elegans larvae. Parasitism of C. elegans L1s was correlated with parasitism of H. glycines J2s. Parasitism of C. elegans by H. rhossiliensis and H. minnesotensis was negatively correlated with larva size and motility, i.e., parasitism was higher for the younger stages. The C. elegans L1 is recommended for studying parasitism of nematodes by H. rhossiliensis and H. minnesotensis.