Effects of Pluronic F-68 on shoot regeneration from cultured jute cotyledons and on growth of transformed roots

The effects have been studied of the non-ionic surfactant, Pluronic F-68, on the growth in culture of jute (Corchorus capsularis L.) cotyledons with attached petioles, cotyledon explants and transformed roots. Supplementation of culture medium with 0.001–0.5% (w/v) of either commercial grade Pluronic F-68 or a purified fraction prepared by passage through silica gel, stimulated shoot production from the petioles of C. capsularis var. D154 and C134 cotyledons. This effect was most marked in C134, because of the failure of control cotyledons to produce shoots in the absence of Pluronic. Plants regenerated from Pluronic-treated cotyledons were morphologically normal. Growth of transformed roots of C. capsularis var. D154 was stimulated in medium supplemented with commercial grade or purified Pluronic F-68, with maximum increases in both fresh and dry weights with 0.1% (w/v) of the surfactant. Roots cultured in the presence of Pluronic F-68 could be maintained without sub-culture for up to 70 days, whereas roots cultured in the absence of Pluronic required subculture every 7 days, to prevent necrosis. Transformed roots also produced callus in the presence of 0.001–1.0% (w/v) of either commercial grade or purified Pluronic. The biotechnological implications of these results are discussed in relation to the potential value of non-ionic surfactants as growth-stimulating additives to plant culture media.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog (1962)     

Effects of Pluronic F-68 on growth of transformed roots ofSolanum dulcamara

Summary The effects of the non-ionic surfactant, Pluronic F-68, on the growth of transformed roots ofSolanum dulcamara L. have been studied. Growth was stimulated by addition of low concentrations (0.001–0.1% w/v) of freshly-prepared commercial grade Pluronic to liquid culture medium, with maximum increases in root fresh and dry weights at 0.01% (w/v). In contrast, higher concentrations (0.25–1.00% w/v) of freshly-prepared Pluronic inhibited growth. Freshly-prepared purified Pluronic retarded root growth, even at concentrations that were stimulatory with the commercial preparation. Similarly, commercial grade Pluronic solutions stored at 4°C or 22°C for 5 days (aged) were inhibitory to root growth.     

In vitro cellular responses to a non-ionic surfactant,Pluronic F-68

Summary The effects of a non-ionic surfactant, Pluronic F-68, on growth of chick embryonic fibroblasts and hamster melanoma cellsin vitro have been studied. Low concentrations (0.05–0.1% w/v) of commercial grade Pluronic stimulated growth of both cell types whereas low concentrations of purified Pluronic inhibited fibroblast growth but strongly stimulated growth of melanoma cells. These observations suggest that Pluronic may have value for regulating growth of cell cultures.     

Effects of Pluronic F-68 on callus growth and protoplast plating efficiency of Solanum dulcamara

Summary The effects of the non-ionic surfactant, Pluronic F-68, on the growth of callus and protoplasts from Solanum dulcamara L. have been studied. Growth of callus was stimulated by addition of 0.1% (w/v) commercial grade Pluronic to culture medium, whereas lower concentrations (0.01% w/v) had no corresponding effect. In contrast, higher concentrations (1.0% w/v) of Pluronic inhibited callus growth. The mean plating efficiency of protoplasts grown at different densities (15 days after plating) was increased up to 26% following culture with 0.1% (w/v) Pluronic, while 0.01% (w/v) Pluronic was ineffective. Mean protoplast plating efficiency decreased by up to 32% following culture with 1.0% (w/v) Pluronic.     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.     

Effects of Pseudomonas fluorescens strain UTPF5 on the mobility,mortality and hatching of root-knot nematode Meloidogyne javanica

The root-knot nematode Meloidogyne spp. includes important plant pathogens worldwide. This study has considered nematode Meloidogyne javanica second stage larvae activity in the extracts of Pseudomonas fluorescens strains UTPF5 and cytotoxic effect of the strain on the nematode. The movement of second stage larvae of nematodes in water agar medium at four concentrations of bacterial extracts and second stage larvae mortality rate of hatching nematode and bacterial strains in vitro were affected. Different concentrates of the strain UTPF5 effect nematode larvae movement and disposal of the same. Bacterial extraction kills almost 100% of the larvae hatching after 24?h and a complete ban on egg hatch of biocontrol nematodes and nematode indicated that root-knot nematode larvae movement on the right attract the bacteria P. fluorescens to extract in the first place.     

Factors affecting entomophilic activity of Neoaplectana feltiae in mushroom compost

The host-searching ability of Neoaplectana feltiae Filipjev (= S. bibionis Bovien) (Nematoda: Steinernematidae) in response to larvae of a mushroom fly, Lycoriella solani Winn. was examined in a mushroom substrate. Individuals of L. solani were less attractive for the parasite than larvae of Galleria mellonella L. The nematode juveniles penetrated a 22 cm layer of casing mixture within 2–4 days. In the casing alone nematode effectiveness was better than in mushroom compost or in compost and casing together. In the casing mixture parasite dosages of 20 and 100 juveniles per cm² led to 22% and 45% parasitization of L. solani respectively, while all G. mellonella larvae were parasitized at both dosages. The prevalence of nematode infection depended on the content of water in the mushroom substrate. The highest N. feltiae infectivity was observed, when the ratio of the dry casing weight to the weight of water content was 1: 2.5. The practical aspects of the observed phenomena, essential for the use of N. feltiae in the protection of commercial mushroom cultivation are discussed.     

Pluronic F-68 Stimulates Growth of Solanum dulcamara in Culture

The effects have been studied of the non-ionic surfactant, PluronicF-68, on the growth of transformed roots, callus and protoplastsof Solanum dulcamara L. Root growth was stimulated by additionof 0001–005% (w/v) of freshly-prepared, commercial gradePluronic to culture medium, with maximum increases in root freshand dry weights at 001%. Higher concentrations (05–10%w/v) of freshly-prepared Pluronic inhibited growth. A Pluronicfraction, prepared by passage through silica-Amberlite resin,retarded root growth even at concentrations that were stimulatorywith the commercial preparation. Similarly, commercial gradePluronic solutions stored at 4C or 22C for 5 d (‘aged’)also inhibited root growth. Roots grew faster on Pluronic F-68-treatedmembrane rafts compared with growth on commercially-availablerafts; such growth enhancement was comparable to that seen inmedium supplemented with 001% (w/v) freshly-prepared commercialPluronic. Callus growth was also stimulated by the addition of freshly-prepared,commercial grade Pluronic F-68 to medium, with maximum increasesat 01% (w/v); in contrast, 10% (w/v) Pluronic was inhibitoryto callus growth. The mean plating efficiency (15 d after plating)of protoplasts cultured at densities of 01–2010⁵ cm_–3was increased up to 26% by 01% (w/v) Pluronic, while 10% wasinhibitory. Both root and callus soluble carbohydrates and proteinswere increased by exposure to freshly-prepared, commercial Pluronic.Similarly, the specific activities of malate dehydrogenase andacid phosphatase were increased in Pluronic F-68-treated callusand roots. The biotechnological implications of these resultsare discussed in relation to the potential value of non-ionicsurfactants as growth-stimulating additives to plant culturemedia. Key words: Solanum dulcamara, Pluronic F-68, surfactant, transformed roots, callus, protoplasts, malate dehydrogenase, acid phosphatase     

Pathogenicity of Heterorhabditis nematodes isolated from north-western Himalaya against the larvae of Plutella xylostella (L.) (Lepidoptera: Plutellidae)

The efficacy of three entomopathogenic nematodes (Heterorhabditis spp.), from north western Himalaya, India was studied against the diamondback moth, Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae), under laboratory conditions. The larvae were exposed to 10, 20, 30 and 40 infective juveniles (IJs) of each nematode species for different time periods and they were found to be susceptible to all the EPNs tested. However, the susceptibility of larvae to nematode infection varied according to the dosages of IJs and their exposure periods. The efficacy of these indigenous entomopathogenic nematodes was also evaluated against the commercially available entomopathogenic nematode H. indica. An indigenous isolate, H. bacteriophora (HRJ), along with the commercial isolate H. indica recorded 100.0% mortality of insect larvae in 96 h exposure time against third instar larvae of P. xylostella. However, it was noticed that with the advancement of larval stage its mortality rate reduces and vice versa with the exposure period. All the tested nematode species were also found to reproduce within the host and produced infective juveniles. In conclusion, the evidence obtained in this study suggests that all the three indigenous EPN species are virulent enough to produce 100% mortality of larvae of P. xylostella. These EPN species thus have potential for the management of P. xylostella under integrated management practices.     

A novel method for the isolation of nematode larvae using pluronic F-68-treated cellulose strips.

A new method is described for the isolation of cultured nematode larvae. This allows effective separation of larvae from fecal contamination, exsheathed larvae from cast sheaths, and viable larvae from nonviable larvae. The method involves the use of cellulose strips and has been assessed using larvae from 2 hookworm species, Necator americanus and Ancylostoma ceylanicum. Pretreatment of the cellulose strips with 1.0% (w/v) of the nonionic surfactant, Pluronic F-68, significantly increased larval recovery of both species.     

An enzyme from Myrothecium verrucaria that degrades insect cuticles for biocontrol of Aedes aegypti mosquito

Summary Myrothecium verrucaria produced a high activity of extracellular insect cuticle degrading enzymes, chitinases, proteinases and lipases. Both first (I) instar and fourth (IV) instar larvae of a mosquito, Aedes aegypti, a vector of yellow fever and dengue, were susceptible to crude culture filtrate (100% mortality within 48 h at 170 mg/l) . The supplementation of purified M. verrucaria endo-chitinase with commercial lipase decreased the lethal time (LT₅₀) from 48 h to 24 h for I instar and 120 h to 96 h for IV instar. Our results of the larvicidal activity indicate that the cuticle degrading enzyme complex of M. verrucaria has a good potential for the control of mosquitoes.     

Effect of humidity and a superabsorbent polymer formulation on the efficacy of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae) to control codling moth,Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Adequate moisture levels are required for nematode survival and subsequent efficacy as entomopathogens. Formulation of nematodes aimed at aboveground applications may assist in maintaining such moisture levels. In this study, we report the effects of a superabsorbent polymer formulation, Zeba® on the performance of an entomopathogenic nematode, Heterorhabditis zealandica Poinar, for controlling diapausing codling moth, Cydia pomonella (L.) larvae in cryptic habitats on trees. Water activity (a_w-value) on bark was considered to be an indication of moisture levels on trees in cryptic habitats where codling moth larvae are known to occur, thereby influencing nematode efficacy. H. zealandica was only able to infect codling moth larvae at a_w≥0.92, with a_w50=0.94 and a_w90=0.96. Laboratory experiments in which nematode concentration was investigated indicated a positive linear relationship between the concentration of nematodes applied and the level of control obtained, with the highest level of mortality recorded at 80 IJs/larva, requiring at least 4 h of conditions conducive to nematode activity to ensure infectivity and subsequent efficacy. Further experimentation showed that the use of the Zeba formulation, together with the nematodes, improved the level of control obtained at 60% and 80% RH in the laboratory and that it also enhanced the survival and infection-ability of the nematodes in the field. The study conclusively illustrates that the tested formulation assisted in maintaining adequate moisture levels on the application substratum, as required for nematode survival and subsequent efficacy.     

Effect of nematode Panagrellus redivivus density on growth, survival, feed consumption and carcass composition of bighead carp Aristichthys nobilis (Richardson) larvae

The study aimed to determine the optimum density of free‐living nematodes in feeding bighead carp, Aristichthys nobilis, larvae. In the first experiment, carp stocked at 25 larvae L^?1 were fed varying levels of nematodes (50, 75, 100, 125 and 150 per ml) twice a day for 21 days from the start of exogenous feeding. Final body weight was significantly higher (P < 0.05) in larvae fed 125 and 150 nematodes per ml than in those fed 50 and 75 per ml, but survival was low (61.8 and 63.6%, respectively). Survival rate was highest in larvae fed 100 nematodes ml^?1 (81.3%). Carcass analysis showed that larvae fed 125 and 150 nematodes ml^?1 had significantly lower body protein and higher body lipid than those fed other nematode densities. Carcass ash was similar for larvae fed 50–100 nematodes ml^?1 but it decreased significantly at the higher nematode densities. Carp larvae in a subsequent experiment were given 50, 75 and 100 nematodes ml^?1 per feeding. Newly hatched Artemia was the control feed. Nematode consumption and growth of the larvae were determined. Larvae were sampled at intervals of 2–4 days and the nematodes in the gut were counted and measured. At each nematode density, the number of nematodes present in the gut of the larvae increased significantly with time. At each sampling day, the number of nematodes in the gut did not differ significantly among treatments (P > 0.05) although it tended to increase with nematode density at day 2 and day 4 but decrease at day 7 onward. The carp larvae consumed significantly shorter nematodes on day 2 and day 4 than on the succeeding sampling days regardless of nematode density. However, the length of nematodes in the gut of the larvae did not differ significantly among the nematode densities. The final body weight of larvae increased with increasing nematode density. The body weight of larvae fed 100 nematodes ml^?1 did not differ significantly from that of larvae given Artemia nauplii. Results show that bighead carp larvae should be fed 100 free‐living nematodes per ml at each feeding time.     

Isolation and Characterization of a Serine Protease from the Nematophagous Fungus, <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>, Displaying Nematicidal Activity

Lecanicillium psalliotae produced an extracellular protease (Ver112) which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 32 kDa. The optimum activity of Ver112 was at pH 10 and 70 °C (over 5 min). The purified protease degraded a broad range of substrates including casein, gelatin, and nematode cuticle with 81% of a nematode (Panagrellus redivivus) being degraded after treating with Ver112 for 12 h. The protease was highly sensitive to PMSF (1 mM) indicating it to be a serine protease. The N-terminal amino acid residues of Ver112 shared a high degree of similarity with other cuticle-degrading proteases from nematophagous fungi which suggests a role in nematode infection.     

Entomopathogenic Nematodes for Control of Codling Moth,Cydia pomonella(Lepidoptera: Tortricidae): Effect of Nematode Species, Concentration, Temperature, and Humidity

The susceptibility of codling moth diapausing larvae to three entomopathogenic nematode species was assessed in the laboratory using a bioassay system that employed cocooned larvae within cardboard strips. The LC₅₀values forSteinernema carpocapsae, S. riobrave,andHeterorhabditis bacteriophorawere 4.7, 4.8, and 6.0 infective juveniles/cm², respectively. When a discriminating concentration of 10 infective juveniles/cm²of each of the three nematode species was evaluated at 15, 20, 25, and 30°C,S. carpocapsaewas the most effective nematode with mortalities ranging from 66 to 90%. Mortalities produced byS. riobraveandH. bacteriophoraat the four temperatures were 2–94 and 25–69%, respectively. Studies were also conducted to test infectivity at 10, 35, and 40°C. No mortality was produced by any of the nematode species at 10°C.S. riobravewas the most infective nematode at 35°C producing 68% mortality which was more than twice that observed forS. carpocapsaeorH. bacteriophora.Codling moth larvae treated with 10 infective juveniles/cm²ofS. carpocapsaeand kept in 95+% RH at 25°C for 0–24 h followed by incubation at 25–35% RH indicated that more than 3 h in high humidity was needed to attain 50% mortality. Trials ofS. carpocapsae, S. riobrave,andH. bacteriophoraat 50 infective juveniles/cm²against cocooned larvae on pear and apple logs resulted in reductions of codling moth adult emergence of 83, 31, and 43%, respectively, relative to control emergence. Trials of the three entomopathogenic nematodes at 50 infective juveniles/cm²against cocooned larvae in leaf litter resulted in 99 (S. carpocapsae), 80 (S. riobrave), and 83% (H. bacteriophora) mortality, respectively. Our results indicate good potential of entomopathogenic nematodes, especiallyS. carpocapsae,for codling moth control under a variety of environmental conditions.     

Key elements in the successful control of diapausing codling moth,Cydia pomonella (Lepidoptera: Tortricidae) in wooden fruit bins with a South African isolate of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae)

The non-insecticidal control strategies currently being implemented in South African orchards for the control of codling moth, Cydia pomonella (L.) may be hampered by wooden fruit bins being infested with diapausing codling moth larvae, acting as a potential source of re-infestation. Key factors contributing to the success or failure of an entomopathogenic nematode application were investigated using the SF 41 isolate of Heterorhabditis zealandica in laboratory bioassays with wooden minibins. Under operational conditions, an application rate of 100 IJs/mL (LD₉₀₌102 IJs/mL) effectively controlled codling moth larvae in these bins, and for further laboratory bioassays, the LD₅₀ value of 18 IJs/mL (?25 IJs/mL) was identified as the discriminating dosage. Maximum mortality was attained when bins were pre-wet for at least 1 min (>90% RH) and maintained at maximum humidity (>95% RH) post-treatment for at least 3 days (LT₉₀=73 h), to ensure nematode survival and subsequent satisfactory infection of diapausing codling moth larvae. Tarping bins achieved the desired high level of humidity required. Furthermore, adjuvants (specifically Reverseal 10?) also improved an application. The study conclusively illustrated that if all the above-mentioned conditions are met, H. zealandica has the potential to successfully disinfest wooden fruit bins of codling moth.     

Trichostrongylus colubriformis: isolation and characterization of ovicidal activity from Bacillus thuringiensis israelensis

Bioassay of media fractions from cultivation of Bacillus thuringiensis israelensis revealed that ovicidal activity for eggs of the ruminant nematode Trichostrongylus colubriformis was found in microbial crystals, but was not released into culture medium. The purified delta-endotoxin of B. t. israelensis, composed of two 25 kDa proteins, had no effect on nematode eggs. A fraction that had high ovicidal activity for eggs of T. colubriformis was isolated by high performance liquid chromatography from crystals of B. t. israelensis. Retention of the compound(s) on size exclusion columns indicated a mol wt of 1510 when compared to standards. The LD50 of this fraction for nematode eggs was not altered significantly by 5 mM calcium chloride with and without ethylenediaminetetraacetic acid or the enzyme inhibitor phenylmethylsulfonyl fluoride (10(-6) M). The fraction was susceptible to proteolytic hydrolysis by bacterial protease VI whereas the fungal protease XIII slightly decreased the ovicidal effects of the fraction. The ovicidal activity was stable for 2 days at ambient temperature or 2 months at 0 C, but declined after 7 days at ambient temperature. Little activity was lost after heating to 100 C for 60 min. The ovicidal effects were also pH dependent with increased toxicity at alkaline pH values. The fraction, however, had no effect on larvae of the mosquito Aedes aegypti or mice after intraperitoneal injection.     

Evaluation of a Peroxyacetic Acid Disinfectant in Hot-water Treatment for the Control of Basal Rot (Fusarium oxysporum f. sp. narcissi) and Stem Nematode (Ditylenchus dipsaci) in Narcissus

Formaldehyde is used routinely in the hot-water treatment (HWT) of narcissus bulbs for the control of stem nematode (Ditylenchus dipsaci) and basal rot (caused by Fusarium oxysporum f.sp. narcissi). Formaldehyde is unpleasant for operators to use, and does not kill all of the thick-walled chlamydospores of F. oxysporum and so less hazardous but more effective materials are being sought. Peroxyacetic acid (as Jet 5, a commercial disinfectant containing 5% peroxyacetic acid) was evaluated in vitro and in a field trial as a possible alternative to formaldehyde. In laboratory studies, peroxyacetic acid (as 1% Jet 5) was as effective as formaldehyde (as 0.5% commercial formalin containing 38 to 40% formaldehyde) in killing free-swimming stem nematodes and nematodes in the wool stage. Peroxyacetic acid (as 0.5% Jet 5) killed F. oxysporum chlamydospores within 1 h, whereas total kill was not achieved with formaldehyde (concentration as above) after 4 h. In a 2 year field trial, there was no evidence of detrimental effects on a healthy narcissus stock due to using peroxyacetic acid. In an infested, diseased stock, bulbs were virtually destroyed by stem nematode within 2 years when HWT was not given. The greatest reduction in nematode symptoms, and the highest bulb yields, were found when formaldehyde or the higher rates of peroxyacetic acid were used in combination with thiabendazole.     

Efficacy of entomopathogenic nematodes against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory conditions

The susceptibility of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) to native and commercial strains of entomopathogenic nematodes (EPNs) was studied under laboratory conditions. Native strains of EPNs were collected from northeastern Iran and characterised as Steinernema feltiae and Heterorhabditis bacteriophora (FUM 7) using classic methods as well as analysis of internal transcribed spacer (ITS) and D2/D3 sequences of 28S genes. Plate assays were performed to evaluate the efficiency of five EPN strains belonging to four species including Steinernema carpocapsae (commercial strain), S. feltiae, Steinernem glaseri and H. bacteriophora (FUM 7 and commercial strains). This initial assessment with 0, 75, 150, 250, 375 and 500 IJs/ml concentrations showed that S. carpocapsae and H. bacteriophora caused the highest mortality in both larval and prepupal stages of P. operculella, PTM. Thereafter, these three strains (i.e. S. carpocapsae, H. bacteriophora FUM 7 and the commercial strains) were selected for complementary assays to determine the effects of soil type (loamy, loamy–sandy and sandy) on the virulence of EPNs against the second (L2) and fourth instar (L4) larvae as well as prepupa. A soil column assay was conducted using 500 and 2000 IJs in 2-ml distilled water. Mortality in the L2 larvae was not affected by the EPN strain or soil type, while there was a significant interactive effect of nematode strains and soil type on larval mortality. The results also showed that EPN strains have higher efficiency in lighter soils and caused higher mortality on early larvae than that in loamy soil. In L4 larvae, mortality of PTM was significantly influenced by nematode strain and applied concentrations of infective juveniles. The larval mortality induced by S. carpocapsae was higher than those caused either by a commercial or the FUM 7 strain of H. bacteriophora. Prepupa were the most susceptible stage.     

Secretion of anti-parasite substances and leukotrienes from ovine gastrointestinal tissues and isolated mucosal mast cells

The presence of larval migration inhibitory (LMI) compounds in the gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucosal mast cells (MMC) and globule leukocytes (GL). This experiment was designed to determine if LMI compounds were secreted by MMC/GL in response to nematode antigenic challenge and if so, could secretion account for levels observed in mucus. Rommey sheep were immunized by repeated cycles of infection with Trichostrongylus colubriformis or Haemonchus contortus larvae and anthelmintic treatment. After slaughter, gastrointestinal tissue was taken for examination of histology and mucus anti-parasite activity. Segments of small intestine were ligatured to form sacs which were incubated with exsheathed nematode larvae or larval excretory/secretory antigens. Tissue slices from small intestine or abomasum were also incubated with nematode larvae or antigens. After homologous challenge, levels of leukotrienes secreted into small intestinal tissue sacs were significantly higher than levels in heterologously challenged sacs or unimmunized sheep intestinal sacs challenged with larvae of any nematode species (279.4±33.7, 141.0±27.8 and 39.5±15.2 ng h⁻¹ respectively). Tissue slices gave a similar pattern of leukotriene secretion. LMI activity was also significantly elevated in intestinal sacs from immunized sheep challenged homologously with nematode larvae or antigen (64±10 and 68±14% respectively cf. heterologous challenge 32±10% and unimmunized sheep sacs 15±6%). Histological examination of abomasal and small intestinal sections showed that immunized sheep had significantly greater numbers of MMC/GL than unimmunized sheep. MMC/GL isolated and purified from immunized sheep secreted leukotrienes and compounds having LMI activity when cultured with homologous nematode larvae or antigens. Secretion of leukotrienes and molecules having LMI activity from MMC/GL could account for the levels of these substances observed in small intestinal mucus.