Nematicidal Activity of Cyclotides: Toxicity Against Caenorhabditis elegans.

Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.

Plant-based production of an orally active cyclotide for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead, and an orally active analgesic is demonstrated.

Scalable and Efficient In Planta Biosynthesis of Sunflower Trypsin Inhibitor-1 (SFTI) Peptide Therapeutics.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide which has been effectively employed as a scaffold for engineering a range of peptide therapeutic candidates. Typically, synthesis of SFTI-1-based therapeutics is performed via solid-phase peptide synthesis and native chemical ligation, with significant financial and environmental costs associated. In planta synthesis of SFTI-1 based therapeutics serves as a greener approach for environmentally sustainable production. Here, we detail the methods for the transient expression, production, and purification of SFTI-1-based therapeutic peptides in Nicotiana benthamiana using a scalable and high-throughput approach. We demonstrate that a prerequisite for this is the co-expression of specialized asparaginyl endopeptidases (AEPs) that perform the backbone cyclization of SFTI-1. In our founding study, we were able to achieve in planta yields of a plasmin inhibitor SFTI-1 peptide at yields of ~60 µg/g of dried plant material.

Comparative analysis of cyclotide-producing plant cell suspensions presents opportunities for cyclotide plant molecular farming.

Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem. & Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides.

Production of a structurally validated cyclotide in rice suspension cells is enabled by a supporting biosynthetic enzyme.

MAIN CONCLUSION: We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1b from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC-MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.

Make it or break it: Plant AEPs on stage in biotechnology.

Asparaginyl endopeptidases (AEPs) are cysteine proteases that control a myriad of cellular functions in plants, including maturation of seed storage proteins and programmed cell death. Recently, several noteworthy AEPs have been discovered that primarily function as transpeptidases rather than hydrolases, to instead catalyse the formation of new peptide bonds. These AEPs appear to have evolved for the cyclisation of a large class of plant defence peptides called cyclotides. Here we describe recent insights into the structural differences between AEPs that preference peptide ligation over hydrolysis. This knowledge is instrumental for the deployment of AEP ligases as biotechnological tools for in vitro applications such as protein labelling and or cyclization, and for plant molecular farming applications.

Circular Permutation of the Native Enzyme-Mediated Cyclization Position in Cyclotides.

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis, and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.

A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein.

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1b. These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

Butterfly Pea (Clitoria ternatea), a Cyclotide-Bearing Plant With Applications in Agriculture and Medicine.

The perennial leguminous herb Clitoria ternatea (butterfly pea) has attracted significant interest based on its agricultural and medical applications, which range from use as a fodder and nitrogen fixing crop, to applications in food coloring and cosmetics, traditional medicine and as a source of an eco-friendly insecticide. In this article we provide a broad multidisciplinary review that includes descriptions of the physical appearance, distribution, taxonomy, habitat, growth and propagation, phytochemical composition and applications of this plant. Notable amongst its repertoire of chemical components are anthocyanins which give C. ternatea flowers their characteristic blue color, and cyclotides, ultra-stable macrocyclic peptides that are present in all tissues of this plant. The latter are potent insecticidal molecules and are implicated as the bioactive agents in a plant extract used commercially as an insecticide. We include a description of the genetic origin of these peptides, which interestingly involve the co-option of an ancestral albumin gene to produce the cyclotide precursor protein. The biosynthesis step in which the cyclic peptide backbone is formed involves an asparaginyl endopeptidase, of which in C. ternatea is known as butelase-1. This enzyme is highly efficient in peptide ligation and has been the focus of many recent studies on peptide ligation and cyclization for biotechnological applications. The article concludes with some suggestions for future studies on this plant, including the need to explore possible synergies between the various peptidic and non-peptidic phytochemicals.

Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide.

The backbone cyclic and disulfide bridged sunflower trypsin inhibitor-1 (SFTI-1) peptide is a proven effective scaffold for a range of peptide therapeutics. For production at laboratory scale, solid phase peptide synthesis techniques are widely used, but these synthetic approaches are costly and environmentally taxing at large scale. Here, we developed a plant-based approach for the recombinant production of SFTI-1-based peptide drugs. We show that transient expression in Nicotiana benthamiana allows for rapid peptide production, provided that asparaginyl endopeptidase enzymes with peptide-ligase functionality are co-expressed with the substrate peptide gene. Without co-expression, no target cyclic peptides are detected, reflecting rapid in planta degradation of non-cyclized substrate. We test this recombinant production system by expressing a SFTI-1-based therapeutic candidate that displays potent and selective inhibition of human plasmin. By using an innovative multi-unit peptide expression cassette, we show that in planta yields reach ~60 µg/g dry weight at 6 days post leaf infiltration. Using nuclear magnetic resonance structural analysis and functional in vitro assays, we demonstrate the equivalence of plant and synthetically derived plasmin inhibitor peptide. The methods and insights gained in this study provide opportunities for the large scale, cost effective production of SFTI-1-based therapeutics.

Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization.

Cyclotides are plant defense peptides that have been extensively investigated for pharmaceutical and agricultural applications, but key details of their posttranslational biosynthesis have remained elusive. Asparaginyl endopeptidases are crucial in the final stage of the head-to-tail cyclization reaction, but the enzyme(s) involved in the prerequisite steps of N-terminal proteolytic release were unknown until now. Here we use activity-guided fractionation to identify specific members of papain-like cysteine proteases involved in the N-terminal cleavage of cyclotide precursors. Through both characterization of recombinantly produced enzymes and in planta peptide cyclization assays, we define the molecular basis of the substrate requirements of these enzymes, including the prototypic member, here termed kalatase A. The findings reported here will pave the way for improving the efficiency of plant biofactory approaches for heterologous production of cyclotide analogs of therapeutic or agricultural value.

External Validity of the New York University Caregiver Intervention: Key Caregiver Outcomes Across Multiple Demonstration Projects.

Purpose of the Study: The Administration on Aging funded six New York University Caregiver Intervention (NYUCI) demonstration projects, a counseling/support intervention targeting dementia caregivers and families. Three sites (Georgia, Utah, Wisconsin) pooled data to inform external validity in nonresearch settings. This study (a) assesses collective changes over time, and (b) compares outcomes across sites on caregiver burden, depressive symptoms, satisfaction with social support, family conflict, and quality of life. Design and Methods: Data included baseline/preintervention (N = 294) and follow-up visits (approximately 4, 8, 12 months). Results: Linear mixed models showed that social support satisfaction increased (p < .05) and family conflict decreased (p < .05; Cohen's d = 0.49 and 0.35, respectively). Marginally significant findings emerged for quality of life increases (p = .05) and burden decreases (p < .10). Depressive symptoms remained stable. Slopes did not differ much by site. Implications: NYUCI demonstrated external validity in nonresearch settings across diverse caregiver samples.

Field Efficacy of Autodissemination and Foliar Sprays of an Entomopathogenic Fungus, Isaria fumosorosea (Hypocreales: Cordycipitaceae), for Control of Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), on Residential Citrus.

Autodissemination and foliar sprays of PFR-97 (Certis Inc., Columbia, MD) microbial insecticide, a blastospore formulation of Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae), were evaluated for control of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), on residential citrus. Seasonal trials on dooryard trees in South Texas evaluated: 1) pathogenicity of I. fumosorosea (Ifr) spores on autodisseminators (dispensers) deployed up to 3 wk on grapefruit trees; 2) psyllid control on several citrus species by dispensers and sprays; 3) infection range of the dispenser. Decline in spore pathogenicity over time was similar among dispensers during fall, winter, or spring and decreased by 30% after 1 d, 59% after 7 d, 81% after 14 d, and 100% after 21 d. Dispensers or sprays were equally effective for psyllid control on heavily infested lime trees from fall to spring and reduced mean reproduction (cumulative eggs) by 90% and mean attack intensity (cumulative psyllid-days) of adults by 76% and nymphs by 82%. Dispensers or sprays were also equally effective for psyllid control on lightly infested lime trees from spring to mid-summer and on orange or grapefruit trees from fall to winter. Very light infestations on grapefruit trees from spring to mid-summer were not significantly reduced by dispensers or sprays. Psyllid control was not improved by combining dispensers and sprays. Adult psyllids infected by Ifr were recovered in trees located 3-4 m away from trees with dispensers but not at greater distances. PFR-97 dispensers could be a treatment option for D. citri in settings where chemical control is problematic.

Healthy publics: enabling cultures and environments for health.

Despite extraordinary advances in biomedicine and associated gains in human health and well-being, a growing number of health and well-being related challenges have remained or emerged in recent years. These challenges are often 'more than biomedical' in complexion, being social, cultural and environmental in terms of their key drivers and determinants, and underline the necessity of a concerted policy focus on generating healthy societies. Despite the apparent agreement on this diagnosis, the means to produce change are seldom clear, even when the turn to health and well-being requires sizable shifts in our understandings of public health and research practices. This paper sets out a platform from which research approaches, methods and translational pathways for enabling health and well-being can be built. The term 'healthy publics' allows us to shift the focus of public health away from 'the public' or individuals as targets for intervention, and away from the view that culture acts as a barrier to efficient biomedical intervention, towards a greater recognition of the public struggles that are involved in raising health issues, questioning what counts as healthy and unhealthy and assembling the evidence and experience to change practices and outcomes. Creating the conditions for health and well-being, we argue, requires an engaged research process in which public experiments in building and repairing social and material relations are staged and sustained even if, and especially when, the fates of those publics remain fragile and buffeted by competing and often more powerful public formations.

Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta.

Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants.

Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays.

The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective for Isaria javanica and Isaria poprawskii when assayed against all members of the genus. In addition, the high resolution melting profile of this amplicon can be used to discriminate between I. javanica, I. poprawskii and a 1:1 mixture of the two species. The practical application of this technique was confirmed using a bioassay on whitefly nymphs (Bemisia tabaci biotype B) inoculated with I. javanica, I. poprawskii or a 1:1 mixture of the two species. This assay provides a simple assay to identify these two species of entomopathogenic fungi.

Efficacy of Topical Application, Leaf Residue or Soil Drench of Blastospores of Isaria fumosorosea for Citrus Root Weevil Management: Laboratory and Greenhouse Investigations.

The efficacy of topical, leaf residue, and soil drench applications with Isaria fumosorosea blastospores (Ifr strain 3581) was assessed for the management of the citrus root weevil, Diaprepes abbreviatus (L.). Blastospores of Ifr were applied topically at a rate of 107 blastospores mL-1 on both the larvae and adults, and each insect stage was incubated in rearing cups with artificial diet at 25 °C, either in the dark or in a growth chamber under a 16 h photophase for 2 weeks, respectively. Percent larval and adult mortality due to the infection of Ifr was assessed after 14 days as compared to untreated controls. Leaf residue assays were assessed by feeding the adults detached citrus leaves previously sprayed with Ifr (107 blastospores mL-1) in Petri dish chambers and then incubating them at 25 °C for 2-3 weeks. Efficacy of the soil drench applications was assessed on five larvae feeding on the roots of a Carrizo hybrid citrus seedling ~8.5-10.5 cm below the sterile sand surface in a single 16 cm × 15.5 cm pot inside a second pot lined with plastic mesh to prevent escapees. Drench treatments per pot consisted of 100 mL of Ifr suspension (107 blastospores mL-1), flushed with 400, 900, or 1400 mL of water compared to 500, 1000, and 1500 mL of water only for controls. The mean concentration of Ifr propagules as colony forming units per gram (CFUs g-1) that leached to different depths in the sand profile per treatment drench rate was also determined. Two weeks post-drenching of Ifr treatments, larvae were assessed for percent mortality, size differences, and effect of treatments in reducing feeding damage to the plant root biomass compared to the controls. Topical spray applications caused 13 and 19% mortality in larvae and adults after 7 days compared to none in the control after 14 days, respectively. Adults feeding on a single Ifr treated leaf for 24 h consumed less than the control, and resulted in 100% mortality 35 days post-treatment compared to 33% in the untreated control. Although offered fresh, untreated leaves after 24 h, only adults in the control group consumed them. Ifr CFUs g-1 were isolated 8.5-10.5 cm below the sand surface for the 1000 and 1500 mL drench rates only, resulting in 2%-4% larval mortality. For all the Ifr drench treatments, no differences were observed in percent larval mortality and size or the effect of treatments in reducing feeding damage to the plant root biomass compared to the controls. These results suggest that the foliar application of Ifr may be an efficient biocontrol strategy for managing adult populations of D. abbreviatus; potential alternative larval management strategies are discussed.

Liquid Culture Production of Fungal Microsclerotia.

Fungal microsclerotia ("small" sclerotia) are compact hyphal aggregates, typically 50-600 µm in diameter, that are formed under unfavorable nutritional and/or environmental conditions. These structures are often melanized and desiccated to some degree containing endogenous nutritional reserves for use when favorable conditions return. Many fungi, mostly plant pathogens, produce microsclerotia as a survival structure. Liquid culture methods have been developed for producing microsclerotia of the Ascomycota Metarhizium spp, Colletotrichum truncatum, Mycoleptodiscus terrestris, and Trichoderma spp. While these fungi have varying culture conditions that optimize microsclerotia production, all share common nutritional and environmental requirements for microsclerotia formation. Described are the general liquid culture techniques, media components, and harvesting and drying methods necessary to produce stable microsclerotial granules of these fungi.

The Effects of Nutrient Concentration, Addition of Thickeners, and Agitation Speed on Liquid Fermentation of Steinernema feltiae.

Entomopathogenic nematode production in liquid fermentation still requires improvements to maximize efficiency, yield, and nematode quality. Therefore, this study was aimed at developing a more suitable liquid medium for mass production of Steinernema feltiae, by assessing the effects of nutrient concentration, thickeners (primarily agar), and agitation speed on infective juvenile (IJ) yield. Base medium (BM) contained yeast extract (2.3%), egg yolk (1.25%), NaCl (0.5%), and corn oil (4%). All media were inoculated with Xenorhabdus bovienii, and 2 d later, with 2-d-old S. feltiae juveniles. For the nutrient concentration experiment, we evaluated the base medium versus a modified base medium containing all the components, but with 3× concentrations of yeast extract (6.9%), egg yolk (3.75%), and corn oil (12%). The nematodes and bacteria were cultured in 150-ml Erlenmeyer flasks containing 50 ml of liquid medium at (25°C) and 180 rpm on a rotary shaker incubator. To assess the effect of thickeners, IJs were inoculated in BM with agar (0.2%), carrageen (0.2%), and carboxymethyl cellulose (0.2% and 0.5%). The addition of 3× more nutrients relative to the BM resulted in a significantly lower yield of nematodes. For agar and agitation speed experiments, five levels of agar in the BM (0%, 0.2%, 0.4%, 0.6%, and 0.8% agar) and two agitation speeds (180 and 280 rpm) were evaluated for production. Increasing agitation speed from 180 to 280 rpm and higher levels of agar in the medium (> 0.2%) significantly increased the yield of bacteria. At the lower agitation speed, media amended with 0.4% and 0.6% agar produced higher nematode yields compared to media without agar. Media with 0.2% and 0.8% agar resulted in intermediate levels of nematode production. At the higher agitation speed, media supplemented with 0.8% agar resulted in the lowest yield of nematodes when compared to the other media tested. Results indicated that increasing nutrient concentration levels was detrimental to nematode production. Also, media containing agar (0.4% and 0.6%) increased nematode yields when cultures were grown at low agitation speed. When IJs were used as the inoculum, 0.2% agar also enhanced recovery and nematode yield at the higher agitation speed.