Evaluation of the mosquitocidal activity of <i>Photorhabdus</i> and <i>Xenorhabdus</i> extracts against the larvae of <i>Aedes aegypti</i> (Diptera: Culicidae)

Larvicidal activity of Xenorhabdus and Photorhabdus bacteria against Aedes aegypti and Aedes albopictus

Secondary metabolites, bioactive compounds produced by living organisms, can unveil symbiotic relationships in nature. In this study, soilborne entomopathogenic nematodes associated with symbiotic bacteria (Xenorhabdus stockiae and Photorhabdus luminescens) were extracted from solvent supernatant containing secondary metabolites, demonstrating significant inhibitory effects against E. coli, S. aureus, B. subtilus, P. mirabilis, E. faecalis, and P. stutzeri. The characterization of these secondary metabolites by Fourier transforms infrared spectroscopy revealed amine groups of proteins, hydroxyl and carboxyl groups of polyphenols, hydroxyl groups of polysaccharides, and carboxyl groups of organic acids. Furthermore, the obtained crude extracts were analyzed by high-performance liquid chromatography for the basic identification of potential bioactive peptides. Gas chromatography-mass spectrometry analysis of ethyl acetate extracts from Xenorhabdus stockiae identified major compounds including nonanoic acid derivatives, proline, paromycin, octodecanal derivatives, trioxa-5-aza-1-silabicyclo, 4-octadecenal, methyl ester, oleic acid, and 1,2-benzenedicarboxylicacid. Additional extraction from Photorhabdus luminescens yielded functional compounds such as indole-3-acetic acid, phthalic acid, 1-tetradecanol, nemorosonol, 1-eicosanol, and unsaturated fatty acids. These findings support the potential development of novel natural antimicrobial agents for future pathogen suppression.

Photorhabdus and Xenorhabdus bacteria, members of the Morganellaceae family, are sources of novel natural products for the biocontrol of fungal pathogens in soybean production. This study demonstrated the inhibitory effects of metabolites from four Photorhabdus and Xenorhabdus strains (including a local isolate, X. szentirmaii PAM 25), against four key phytopathogenic fungi. Bacterial metabolite efficacy and fungal susceptibility varied. Xenorhabdus szentirmaii DSM 16338, X. szentirmaii PAM 25, and X. doucetiae demonstrated significant inhibition (>90%) against Sclerotinia sclerotiorum, Botrytis cinerea, and Macrophomina phaseolina, exhibiting superior efficacy compared to X. nematophila and Photorhabdus kayaii. Fusarium oxysporum demonstrated greater resistance to the bacterial supernatants. We identified fabclavine, pyrollizixenamide, and szentirazine from X. szentirmaii, and xenocoumacins from X. doucetiae as the antifungal bioactive compounds in the respective easyPACid mutants. Furthermore, we assessed the efficacy of X. szentirmaii PAM 25 and its metabolites in protecting soybean seeds from S. sclerotiorum and investigated the shelf stability of the bacterial metabolites as the fungus suppressors. Cell-free supernatant maintained >80% inhibition of S. sclerotiorum after one year at 5–35 °C. Importantly, the cell-free supernatant, as well as the bacterial culture, effectively inhibited S. sclerotiorum in seed treatments, ensuring ≥80% seed germination, comparable to thiophanate-methyl + fluazinam fungicide. This study demonstrates that the direct seed application of Xenorhabdus and Photorhabdus bacteria offers a practical and innovative biological control method against soil-borne fungal pathogens.

Xenorhabdus and Photorhabdus bacteria are symbionts of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis, respectively. To determine the nutrient potential of these bacteria for a free-living nematode, Panagrellus redivivus, a promising food source for first-feeding fish and crustacean, sterile first-stage juveniles (J1) of P. redivivus were fed on various isolates of Xenorhabdus and Photorhabdus bacteria in liquid cultures. Most of the tested bacterial isolates did not support the growth of P. redivivus. However, four of the Xenorhabdus isolates (X. nematophila All, X. bovienii T319, X. beddingii X-7 and X. poinarii KG) provided nutrients for the production of these nematodes in a liquid medium. Two Xenorhabdus isolates (X. beddingii X-7 and X. poinarii KG) even supported mass production of the nematode in a sponge medium, with yields comparable to those with yeast strains. This is the first report that Xenorhabdus bacteria can function as a nutrient source for mass production of nematodes other than their usual symbiotic partners.

Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture

Summary Entomopathogenic nematodes (EPN) Steinernema and Heterorhabditis with symbionts with Xenorhabdus and Photorhabdus bacteria, respectively, are reported as biocontrol agents for insect control. The objectives of this study were to identify EPN and their symbiotic bacteria in national parks of Phitsanulok Province, Thailand, and to test how Xenorhabdus isolates attack Aedes aegypti larvae. We collected 810 soil samples from four national parks. The juvenile stage of EPN was isolated from soil samples using a baiting technique with Galleria mellonella followed by a White trap. Partial regions of 28S rDNA and internal transcript spacer were sequenced to identify EPN, and recA sequencing was used to discriminate between Xenorhabdus and Photorhabdus. We found that 74 of the 810 soil samples (9.1%) were positive for the EPN. The EPN were molecularly identified as S. surkhetense, S. longicaudum, H. indica and Heterorhabditis sp. SGmg3, and their symbiotic bacteria were identified as X. stockiae, X. griffiniae, X. indica, X. vietnamensis, P. luminescens subsp. akhurstii, and P. temperata subsp. temperata. Xenorhabdus griffiniae showed potential larvicidal activity against A. aegypti (91% mortality at 72 and 96 h after exposure). This study demonstrates the diversity of EPN and symbiotic bacteria in national parks of Thailand and the potential to use X. griffiniae as a biocontrol agent to kill A. aegypti larvae.

Larvicidal activity of Photorhabdus and Xenorhabdus bacteria isolated from insect parasitic nematodes against Aedes aegypti and Aedes albopictus

A taste of a toxin paradise: Xenorhabdus and Photorhabdus bacterial secondary metabolites against Aedes aegypti larvae and eggs

Aedes aegypti is an insect vector that transmits several viruses affecting humans worldwide. Entomopathogenic nematodes (EPNs) and their symbiotic bacteria are organisms with the potential to control many insects. In this study, we did a survey aimed to identify EPNs and their symbiotic bacteria and evaluate the larvicidal activity of bacteria against Ae. aegypti. We collected 540 soil samples from 108 sites in Phitsanulok Province, lower northern Thailand. Baiting techniques and White traps were used to isolate EPNs from soil samples. By sequencing of 28S rDNA and internal transcribed spacer regions, 51 EPN isolates were identified as Steinernema surkhetense (35 isolates), Heterorhabditis indica (14 isolates) and Heterorhabditis sp. SGmg3 (two isolates). Based on sequencing of a partial region of the recA gene, 35 isolates of Xenorhabdus were identified as Xenorhabdus stockiae, and 20 Photorhabdus isolates were identified as Photorhabdus luminescens subsp. akhurstii (10 isolates), P. luminescens subsp. hainanensis (seven isolates) and P. asymbiotica subsp. australis (three isolates). Screening for larvicidal activity of bacteria against Ae. aegypti was performed in the laboratory. Xenorhabdus WB5.4 and Xenorhabdus WB12.5, which were closely related to X. stockiae, resulted in high mortality of Ae. aegypti (99.99% and 70%, respectively) at 96 hr after exposure. Comparing with control groups, mortality of Ae. aegypti larvae was low (1.11%–6.67%) after exposure for 24–96 hr. Our findings showed the potential of X. stockiae for controlling Ae. aegypti. Further studies are needed to elucidate the mechanisms through which these bacteria kill Ae. aegypti larvae.

BackgroundAedes aegypti is a potential vector of West Nile, Japanese encephalitis, chikungunya, dengue and Zika viruses. Alternative control measurements of the vector are needed to overcome the problems of environmental contamination and chemical resistance. Xenorhabdus and Photorhabdus are symbionts in the intestine of entomopathogenic nematodes (EPNs) Steinernema spp. and Heterorhabditis spp. These bacteria are able to produce a broad range of bioactive compounds including antimicrobial, antiparasitic, cytotoxic and insecticidal compounds. The objectives of this study were to identify Xenorhabdus and Photorhabdus isolated from EPNs in upper northern Thailand and to study their larvicidal activity against Ae. aegypti larvae.ResultsA total of 60 isolates of symbiotic bacteria isolated from EPNs consisted of Xenorhabdus (32 isolates) and Photorhabdus (28 isolates). Based on recA gene sequencing, BLASTN and phylogenetic analysis, 27 isolates of Xenorhabdus were identical and closely related to X. stockiae, 4 isolates were identical to X. miraniensis, and one isolate was identical to X. ehlersii. Twenty-seven isolates of Photorhabdus were closely related to P. luminescens akhurstii and P. luminescens hainanensis, and only one isolate was identical and closely related to P. luminescens laumondii. Xenorhabdus and Photorhabdus were lethal to Ae aegypti larvae. Xenorhabdus ehlersii bMH9.2_TH showed 100% efficiency for killing larvae of both fed and unfed conditions, the highest for control of Ae. aegypti larvae and X. stockiae (bLPA18.4_TH) was likely to be effective in killing Ae. aegypti larvae given the mortality rates above 60% at 72 h and 96 h.ConclusionsThe common species in the study area are X. stockiae, P. luminescens akhurstii, and P. luminescens hainanensis. Three symbiotic associations identified included P. luminescens akhurstii-H. gerrardi, P. luminescens hainanensis-H. gerrardi and X. ehlersii-S. Scarabaei which are new observations of importance to our knowledge of the biodiversity of, and relationships between, EPNs and their symbiotic bacteria. Based on the biological assay, X. ehlersii bMH9.2_TH begins to kill Ae. aegypti larvae within 48 h and has the most potential as a pathogen to the larvae. These data indicate that X. ehlersii may be an alternative biological control agent for Ae. aegypti and other mosquitoes.

Frosty pod rot or moniliasis is a very destructive disease of cocoa plantations in Latin America. The conventional methods to control this disease such as the use of chemical pesticides have failed or are too expensive for smallholders. Nowadays, biological alternatives are being investigated to improve yields and to keep moniliasis controlled. Xenorhabdus and Photorhabdus bacteria and their exudates were evaluated against Moniliophthora roreri. All bacteria demonstrated antifungal action reaching up to 97% after 13 days of exposure, whereas some exudates demonstrated more than 70%. Moniliophthora roreri did not sporulate in bacteria presence and sporulation was reduced by their exudates in 70%. The bacteria and exudates were compared with a commercial fungicide used by Venezuelan farmers; better performance of the bacteria and exudates was found and the fungicide was unable to control M. roreri. These experiments showed promising results of Xenorhabdus and Photorhabdus as biocontrol agents for being included in the integrated disease management programmes in Venezuela.

Fluent genetic manipulation of prokaryote genomes is still limited to only a few commonly used hosts. Ideally the advanced technologies available for cloning into recombinant Escherichia coli should also be applicable in other prokaryotes. In particular, 'recombineering' is mediated by the lambda Red operon that permits fluent and precise engineering of the E. coli genome and associated recombinant DNA. The major limitation is that host-specific phage-derived recombination systems are also required in more distant species. Recently, an endogenous Red-like operon Pluγβα has been reported to be effective in both Photorhabdus and Xenorhabdus bacteria. The Pluγβα recombineering system is based on three host-specific phage proteins from Photorhabdus luminescens, Plu2935, Plu2936, and Plu2934, which are functional analogs of Redβ, Redα, and Redγ, respectively. In this chapter, we provide a comprehensive and up-to-date method for P. luminescens and Xenorhabdus stockiae genome engineering via the Pluγβα recombineering system. In order to facilitate the rapid construction of knock-in vectors, recET-mediated recombineering is incorporated in the pipeline. Concerted recET system in E. coli with Pluγβα system in Photorhabdus and Xenorhabdus could promote reverse genetics, functional genomics, and bioprospecting research for these two genera.

Dengue, Chikungunya, and Zika are important vector-borne diseases, and Aedes aegypti L. is their main transmitter. As the disease management is mainly based on mosquito control strategies, the search for alternative and cost-effective approaches is ongoing. The Gram-negative bacteria Xenorhabdus nematophila and Photorhabdus luminescens are symbiotically associated with entomopathogenic nematodes and are highly pathogenic for insect larvae. After we have recently confirmed the toxicity of these bacteria in Ae. aegypti larvae, we here evaluated the toxic activity of culture fluids on the development of this mosquito species. Larval susceptibility was assessed by exposing larvae to different concentrations of P. luminescens or X. nematophila culture fluids to confirm whether secondary metabolites might cause the mosquitos' death. Xenorhabdus nematophila culture fluid was more effective and stable during the mosquito pathogenicity bioassays compared to that of P. luminescens. Larval mortality started a few hours after exposure of the insects to the fluids. Furthermore, the residual effect of larvicidal activity of X. nematophila fluid persisted at full efficiency for 4 d. Particularly, larval mortality was still higher than 50% for up to 8 d. Exposure of larvae to a sublethal dose of X. nematophila fluid delayed pupation as well as emergence of adult mosquitoes and caused cumulative larval mortality higher than 90% by day 14. Here, we describe for the first time the use of stable culture fluids and therefore secondary metabolites of P. luminescens and X. nematophila as a promising basis for the use as biopesticide for control of Ae. aegypti in the future.

Dengue fever is an important vector-borne disease, mainly transmitted by Aedes aegypti. To date, there are no vaccines or effective drugs available against this arboviral disease. As mosquito control is practically the only method available to control dengue fever, alternative and cost-effective pest control strategies need to be explored. The gram-negative enteric bacteria Xenorhabdus and Photorhabdus are symbiotically associated with nematode parasites, which themselves are highly pathogenic for insect larvae. Here, we evaluate the oral toxicity of these entomopathogenic bacteria in A. aegypti larvae. The susceptibility of larvae (third late or fourth early instars) was assessed by exposing them to suspensions containing Photorhabdus luminescens or Xenorhabdus nematophila, respectively. Two diet treatments were tested with larvae fed on pet food and unfed larvae. After 24 h, larvae began to die when exposed to the bacteria. Exposure to P. luminescens killed 73% of the fed and 83% of the unfed larvae, respectively. In comparison, X. nematophila was less pathogenic, killing 52% of the larvae in the fed and 42% in the unfed treatment. Remarkably, cannibalism was observed in all bioassays after exposing larvae to either of the bacterial species. To our knowledge, this is the first report demonstrating the efficiency of these entomopathogenic bacteria for oral A. aegypti killing. Our results provide a promising basis for using these bacteria as bioinsecticides for mosquito control in the future.

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.