Entomopathogenic Nematode Heterorhabditis Indica Research Articles

Heterorhabditis indica (Nematoda: Rhabditida) a possible new biological control agent against the vector of Chagas disease

Chagas disease is a zoonosis caused by the protozoan Trypanosoma cruzi and transmitted through the feces of triatomines, mainly in Latin America. Since the 1950s, chemical insecticides have been the primary method for controlling these triatomines, yet resistance has emerged, prompting the exploration of alternative approaches. The objective of this research was to test the capacity of the entomopathogenic nematodes Heterorhabditis indica and its symbiotic bacteria Photorhabdus luminescens, to produce mortality of Triatoma dimidiata a key vector of T. cruzi in Mexico under laboratory conditions. Two bioassays were conducted. In the first bioassay, the experimental unit was a 250 ml plastic jar with 100 g of sterile soil and three adult T. dimidiata. Three nematode quantities were tested: 2250, 4500, and 9000 nematodes per 100 g of sterile soil (n/100 g) per jar, with 3 replicates for each concentration and 1 control per concentration (1 jar with 100 g of sterile soil and 3 T. dimidiata without nematodes). The experimental unit of the second bioassay was a 500 ml plastic jar with 100 g of sterile soil and 4 adult T. dimidiata. This bioassay included 5, 50, 500, and 5000 n/100 g of sterile soil per jar, with 3 replicates of each quantity and 1 control per quantity. Data were analyzed using Kaplan-Meyer survival analysis. Electron microscopy was used to assess the presence of nematodes and tissue damage in T. dimidiata. The results of the first bioassay demonstrated that the nematode induced an accumulated average mortality ranging from 55.5 % (2250 n/100 g) to 100 % (4500 and 9000 n/100 g) within 144 h. In the second bioassay, the 5000 n/100 g concentration yielded 87.5 % mortality at 86 h, but a concentration as small as 500 n/100 g caused 75 % mortality from 84 h onwards. Survival analysis indicated higher T. dimidiata mortality with increased nematode quantities, with significant differences between the 4500, 5000, and 9000 n/100 g and controls. Electron microscopy revealed the presence of nematodes and its presumably symbiotic bacteria in the digestive system of T. dimidiata. Based on these analyses, we assert that the H. indica and P. luminescens complex causes mortality in adult T. dimidiata under laboratory conditions.

Characterisation and toxins analysis of symbiotic bacteria Photorhabdus luminescens isolated from Heterorhabditis indica ICRI-18 strain

Entomopathogenic nematodes are proven alternatives to pesticides in crop protection as they belong to the Heterorhabditidae and Steinernematidae families. The role of entomopathogenic nematodes has expanded considerably in the recent past for their efficacy as biopesticide which is influenced by nematode species and strain. Members of the genus Heterorhabditis have been found to harbor a symbiotic bacteria Photorhabdus sp. When the nematode is ingested by the insect, Photorhabdus sp. is released into the host haemocoel where they multiply rapidly and kill the insect. The main objective of our research was to examine the insect killing toxin property of symbiotic bacteria isolated from the entomopathogenic nematodes Heterorhabditis indica. The antagonistic potential of insecticidal toxin from Photorhabdus sp. was investigated. The results prove that toxic components produced by these bacteria could kill pests either through ingestion or injection. The broad-spectrum antimicrobial property produced by Photorhabdus sp. was analysed by conducting both antibiogram and antibiosis studies.

Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomics.

The entomopathogenic nematode, Heterorhabditis indica, is a popular biocontrol agent of high commercial significance. It possesses tremendous genetic architecture to survive desiccation stress by undergoing anhydrobiosis to increase its lifespan-an attribute exploited in the formulation technology. The comparative transcriptome of unstressed and anhydrobiotic H. indica revealed several previously concealed metabolic events crucial for adapting towards the moisture stress. During the induction of anhydrobiosis in the infective juveniles (IJ), 1584 transcripts were upregulated and 340 downregulated. As a strategy towards anhydrobiotic survival, the IJ showed activation of several genes critical to antioxidant defense, detoxification pathways, signal transduction, unfolded protein response and molecular chaperones and ubiquitin-proteasome system. Differential expression of several genes involved in gluconeogenesis - β-oxidation of fatty acids, glyoxylate pathway; glyceroneogenesis; fatty acid biosynthesis; amino-acid metabolism - shikimate pathway, sachharopine pathway, kyneurine pathway, lysine biosynthesis; one-carbon metabolism-polyamine pathway, transsulfuration pathway, folate cycle, methionine cycle, nucleotide biosynthesis; mevalonate pathway; and glyceraldehyde-3-phosphate dehydrogenase were also observed. We report the role of shikimate pathway, sachharopine pathway and glyceroneogenesis in anhydrobiotes, and seven classes of repeat proteins, specifically in H. indica for the first time. These results provide insights into anhydrobiotic survival strategies which can be utilized to strengthen the development of novel formulations with enhanced and sustained shelf-life.

Compatibility studies of Heterorhabditis indica with newer insecticides under laboratory condition

Entomopathogenic nematodes (EPNs) have been identified as promising biocontrol agents for controlling economically important insect pests of agricultural and horticultural crops. The compatibility of entomopathogenic nematode Heterorhabditis indica with 7 CIB registered insecticides was investigated under laboratory conditions. The effect of these insecticides on nematode survival at recommended concentrations was observed after 12, 24, 48, 72 hours upon direct exposure. EPN H. indica was compatible with Imidacloprid 17.8% SL as maximum per cent of live H. indica were observed after 72 h of exposure to this insecticide. Similarly, H. indica was compatible with Fipronil 5% SC up to 48 h of exposure whereas, less than 70% live EPN were there in Thiamethoxam 25% WG, Diafenthiuron 50% WP and Cypermethrin 25% EC resulting these insecticides to be least compatible. Emamectin benzoate 5% SG and chlorpyriphos 20% EC were incompatible with H. indica after 48 h of exposure. The result of this experiment will help in reducing the dependence on chemical insecticides and thus slowing down the development of insecticide resistance and preventing adverse effects on public health and the environment.

In vitro study on the nematicidal activity of entomopathogenic bacteria against the root knot nematode Meloidogyne incognita

National loss of Rs. 21,068.73 million has been estimated due to plant-parasitic nematodes in India. Among plant-parasitic nematodes, one of the major nematodes, root-knot nematodes (RKNs), are well-known diseases causing major losses in vegetable crops. An in vitro experiment was conducted to evaluate the nematicidal activities of the cell-free culture filtrate (CFCF) of entomopathogenic bacteria Photorhabdus spp. and Xenorhabdus spp. isolated from entomopathogenic nematodes Heterorhabditis indica (DH3) and Steinernema abbasi (CS-39), respectively. The applied doses were 90, 50, 25 and 10% CFCFs. The experiment was performed on the plant-parasitic nematode Meloidogyne incognita, and the % mortalities were determined at 6, 12, 24 and 48 hr intervals. The results of the present study revealed that 100% mortality was achieved after 48 hrs with a 10% filtrate of H. indica isolate DH3, while no significant result was achieved even after 48 hrs and at 90% CFCF of isolate CS39. Therefore, 10% CFCFs may be recommended for application in root-knot nematode-infected fields for the control.

Preferential infectivity of entomopathogenic nematodes in an envenomed host

Entomopathogenic nematodes and parasitoid wasps are used as biological control agents for management of insect pests such as the Indian meal moth, Plodia interpunctella. The parasitoid wasp Habrobracon hebetor injects a paralytic venom into P. interpunctella larvae before laying eggs. A previous study reported that the entomopathogenic nematode Heterorhabditis indica preferentially infects P. interpunctella that have been envenomed by H. hebetor while results in this study showed a similar preference by the entomopathogenic nematode, Steinernema glaseri. We therefore tested four hypotheses for why nematode infection rates are higher in envenomed hosts: (1) elevated CO2 emission from envenomed hosts attracts nematodes, (2) paralysis prevents hosts from escaping nematodes, (3) volatile chemicals emitted from envenomed hosts attract nematodes and increase infection, and (4) reduced immune defenses in envenomed hosts increase nematode survival. Results showed that envenomed P. interpunctella larvae emitted lower amounts of CO2 than non-envenomed larvae. Physical immobilization of P. interpunctella larvae did not increase infection rates by S. glaseri but did increase infection rates by H. indica. Emissions from envenomed hosts were collected and analyzed by thermal desorption gas chromatography/mass spectrometry. The most abundant compound, 3-methyl-3-buten-1-ol, was found to be an effective cue for S. glaseri attraction and infection but was not an effective stimulus for H. indica. Envenomed P. interpunctella exhibited a stronger immune response toward nematodes than non-envenomed hosts. Altogether, we conclude that different mechanisms underlie preferential infection in the two nematode species: host immobilization for H. indica and chemical cues for S. glaseri.

Assessing the Pathogenicity of Two Bacteria Isolated from the Entomopathogenic Nematode Heterorhabditis indica against Galleria mellonella and Some Pest Insects.

The entomopathogenic nematodes Heterorhabditis are parasites of insects and are associated with mutualist symbiosis enterobacteria of the genus Photorhabdus; these bacteria are lethal to their host insects. Heterorhabditis indica MOR03 was isolated from sugarcane soil in Morelos state, Mexico. The molecular identification of the nematode was confirmed using sequences of the ITS1-5.8S-ITS2 region and the D2/D3 expansion segment of the 28S rRNA gene. In addition, two bacteria HIM3 and NA04 strains were isolated from the entomopathogenic nematode. The genomes of both bacteria were sequenced and assembled de novo. Phylogenetic analysis was confirmed by concatenated gene sequence datasets as Photorhabdus luminescens HIM3 (16S rRNA, 23S rRNA, dnaN, gyrA, and gyrB genes) and Pseudomonas aeruginosa NA04 (16S rRNA, 23S rRNA and gyrB genes). H. indica MOR03 infects Galleria mellonella, Tenebrio molitor, Heliothis subflexa, and Diatraea magnifactella larvae with LC50 values of 1.4, 23.5, 13.7, and 21.7 IJs/cm2, respectively, at 48 h. These bacteria are pathogenic to various insects and have high injectable insecticide activity at 24 h.

Parasitic potential of entomopathogenic nematode Heterorhabditis indica against two Lepidopteran insect pests of cotton, Helicoverpa armigera (Hubner) and Spodoptera litura (Fabricious)

Ten isolates of entomopathogenic nematode (EPN) Heterorhabditis indica were evaluated for their differential infectivity against two major cotton insect pests viz., Helicoverpa armigera and Spodoptera litura. The results of bioassay revealed that the third instar larvae of H. armigera were relatively more susceptible to the EPN isolates compared to the larvae of S. litura. All the ten EPN isolates could reproduce successfully on H. armigera larvae however, the isolates viz., CICR-Su and CICR-SUB exhibited relatively higher virulence. On the other hand, on S. litura larvae only one isolate- CICR-BBFNS2 was able to produce infective juveniles and the rest failed to do so. Analysis of haemolymph composition of EPN infected H. armigera and S. litura larvae indicated the phase change of bacterial symbionts as a primary reason for the failure of EPN to reproduce and multiply in S. litura larvae. Higher percentage of primary phase colonies of bacterial symbionts were recorded in H. armigera larvae which resulted in successful progeny production of EPN while secondary phase colonies were predominant in the larvae of S. litura which resulted in failure of EPN isolates to reproduce. Phylogenetic tree made using internal transcribed spacer (ITS) region of ribosomal DNA of nematodes and 16sRNA of bacterial symbionts revealed the distinctness and uniqueness of the EPN isolate CICR-BBFNS2 which was able to multiply on the larvae of both H. armigera and S. litura from rest of the nine isolates which could multiply only in H. armigera. The results indicate that isolate CICR-BBF-NS2 has potential for deployment in IPM programme for management of H. armigera and S. litura.

Natural Occurrence ofHeterorhabditis indicaPoinar, Karunakar & David on Sugarcane Weevil,Sphenophorus incurrensGyllenhal, at Morelos, Mexico

Abstract. The entomopathogenic nematode Heterorhabditis indica Poinar, Karunakar & David (Rhabditida: Heterorhabditidae) was reported for the first time as a natural parasite of the sugarcane weevil, Sphenophorus incurrens Gyllenhal (Coleoptera: Curculionidae), in sugarcane (Saccharum officinarum L.) fields in Mexico.

Genetic and proteomic characterization of rpoB mutations and their effect on nematicidal activity in Photorhabdus luminescens LN2.

Rifampin resistant (RifR) mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The RifR mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs) of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 RifR mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing RifR mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 RifR mutants. The rpoB (P564L) mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s). The non-nematicidal-producing RifR mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and RifR mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS), and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively) were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregualted dsbA). These results indicate that the rpoB mutations greatly influence the symbiotic association between the symbionts and their entomopathogenic nematode hosts.

BIOEFFICACY OF PHOTORHABDUS LUMINESCENS, A SYMBIOTIC BACTERIUM AGAINST THRIPS PALMI KARNY (THRIPIDAE: THYSANOPTERA)

ABSTRACT The primary and secondary forms of Photorhabdus luminescens a symbitotic bacterium of an entomopathogenic nematode Heterorhabditis indica (RCR) was evaluated against Thrips palmi Karny a serious pest on cotton. The treatment included different concentration (101 to 1010 cells/ CFS/ml) of cells and cells free supernatant which was obtained by centrifugation (4000 rpm for 20 min) of broth culture. The treatment was imposed through potter’s tower on aphid colony containing (20 per leaf/replication plants). Observation of aphid mortality was recorded at 24 and 48 hr. The results revealed that both the forms were pathogenic to T. palmi but the cells free supernatant (CFS) of both the forms was more virulent than cells causing high mortality within 24 to 48 hr. The cells of primary and secondary form recorded 67.50 and 72.50 per cent mortality whereas corresponding CFS recorded 85.00 and 80.00 per cent mortality, respectively at the concentration of 1010 cells/ ml. The LC50 of both primary 2.38× 107 cells/ml and secondary form cells 2.35 × 107 cells / ml and CFS of P. luminescens decreased with increase in exposure time from 24 to 48 hr. These results indicate the possibility of developing CFS as a potential biocontrol component against T. palmi.

Antibiotic and antibacterial activity of a symbiotic bacterium, Photorhabdus luminescens.

Antibiotic and antagonistic effects of primary and secondary forms of Photorhabdus luminescens , a symbiotic bacterium of an entomopathogenic nematode Heterorhabditis indica (RCR) were investigated in vitro against plant pathogens and beneficial microorganisms. An inhibition zone assay on potato dextrose agar (PDA) medium revealed that the primary form completely inhibited all plant pathogenic fungi, viz. , Aspergillus flavus , Rhizoctonia solani and Fusarium solani and tested secondary form completely inhibited the growth of F. solani and partially inhibited A. flavus and failed to inhibit R. solani . However, dual culture assay on nutrient agar (NA) in both forms completely inhibited the growth of the bacterial pathogen, Xanthomonas punicae . The forms of P. luminescens did not inhibit the mycelial growth of entomopathogens viz., Metarhizium anisopliae , Verticillium lecanii and Nomuraea rileyi but inhibited the growth of bacterial bioagents Rhizobium sp. and Pseudomonas fluorescens . The present study indicates that P. luminescens can be effectively used in the management of these plant pathogens.

Efeitos Sinérgicos de Combinações entre Nematóides Entomopatogênicos (Nemata: Rhabditida) e Inseticidas Químicos na Mortalidade de Sphenophorus levis (Vaurie) (Coleoptera: Curculionidae)

The effect of combinations of the entomopathogenic nematodes Heterorhabditis indica Poinar, Karunakar & David, 1992 (strain IBCB-n5) and Steinernema sp. (strain IBCB-n6) at doses of 2.4;12 and 60 IJ/cm2, with sub-doses of the chemical insecticides fipronil, thiamethoxam and imidacloprid were tested in laboratory conditions against adults and larvae of the sugarcane billbug, Sphenophorus levis Vaurie, 1978. Firstly, the effect of fipronil (200 g A.I./ha), thiamethoxam (250 g A.I./ha) and imidacloprid (700 g A.I./ha) were evaluated on the nematode viability. The insecticides were compatible with the nematodes, not affecting the viability of the infective juvenile after 24 hours. For the experiments with adults, the treatments containing nematodes and insecticides tested separately showed mortality below 25%, except for the two highest doses of Steinernema sp. (at 12 and 60 IJ/cm2), with mortality levels of 33.3% and 50% respectively. For the mixtures of the nematodes with sub-doses of thiamethoxam (62.5 g A.I./ha), the mortality levels increased significantly, reaching up to 66.7%, with the mixture of H. indica at 60 IJ/cm2, and 70% to 83.3% at all doses of Steinernema sp., showing synergistic effect at these combinations. The combination of H. indica and Steinernema sp. (12 IJ/cm2) with thiamethoxam (62.5 g A.I./ha) against larva, the nematodes tested alone or in mixture with the insecticides showed mortality levels between 75% and 87.5%, indicating no advantage of the mixture for improving the insect mortality.

Effect on Heterorhabditis indica of substances occurring in decomposing palm tissues infested by Rhynchophorus ferrugineus

AbstractEthanol, acetic acid and ethyl acetate are generated during the degeneration of palm tissues infested by the red palm weevil, Rhynchophorus ferrugineus Oliv. This in vitro study determined the influences of concentrations of 0.05-2% v/v of these substances in distilled water on the behaviour, survival and infectivity of the entomopathogenic nematode Heterorhabditis indica, a candidate species for control of R. ferrugineus. The observed responses of the nematode included normal movement, reversible partial paralysis and death. The estimated LC50 were 1.48, 0.25 and 0.14% for ethanol, acetic acid and ethyl acetate, respectively, but only 0.18% for the equal mixture of all three. Nematodes that survived treatment at the LC50 of each substance showed infectivity and penetration activity comparable to those of control nematodes. We conclude that it will be difficult to control R. ferrugineus using H. indica in the presence of these substances in palm trunks.

Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida).

Bacteria naturally associated with the symbiont Photorhabdus luminescens subsp. akhurstii were isolated from the entomopathogenic nematode Heterorhabditis indica. Bacterial isolates distinct from P. luminescens subsp. akhurstii were obtained from 33% of the samples. Fourteen bacterial isolates, from nematodes collected from three different Caribbean islands, were characterized by conventional phenotypic tests, restriction fragment length polymorphism and sequence analyses of PCR-amplified 16S rRNA genes (16S rDNAs). Isolates were grouped into three genotypes, each one being associated with one Caribbean island. Phenotypic characteristics and 16S rDNA analysis showed that the Photorhabdus-associated bacteria were closely related to Ochrobactrum anthropi for the group from Guadeloupe, and to Ochrobactrum intermedium for the two groups from the Dominican Republic and Puerto Rico. No pathogenicity of the Ochrobactrum spp. to the insects Galleria mellonella and Spodoptera littoralis (Lepidoptera) was detected. Since Ochrobactrum spp. are considered as human opportunist pathogens, the mass production of entomopathogenic nematodes for biological control requires strict vigilance.