Role of symbiotic and non-symbiotic bacteria in carbon dioxide production from hosts infected with Steinernema riobrave

Entomopathogenic nematodes of the family Steinernematidae and their mutualistic bacteria (Xenorhabdus spp.) are lethal endoparasites of insects. We hypothesized that growth of the nematode’s mutualistic bacteria in the insect host may contribute to the production of cues used by the infective juveniles (IJs) in responding to potential hosts for infection. Specifically, we tested if patterns of bacterial growth could explain differences in CO₂ production over the course of host infection. Growth of Xenorhabdus cabanillasii isolated from Steinernema riobrave exhibited the characteristic exponential and stationary growth phases. Other non-nematode symbiotic bacteria were also found in infected hosts and exhibited similar growth patterns to X. cabanillasii. Galleria mellonella larvae infected with S. riobrave produced two distinct peaks of CO₂ occurring at 25.6–36 h and 105–161 h post-infection, whereas larvae injected with X. cabanillasii alone showed only one peak of CO₂, occurring at 22.8–36.2 h post-injection. Tenebrio molitor larvae infected with S. riobrave or injected with bacteria alone exhibited only one peak of CO₂ production, which occurred later during S. riobrave infection (41.4–64.4 h post-infection compared to 20.4–35.9 h post-injection). These results indicate a relationship between bacterial growth and the first peak of CO₂ in both host species, but not for the second peak exhibited in G. mellonella.     

Expression and activity of a probable toxin from Photorhabdus luminescens

As an insect pathogen, Photorhabdus luminescens possesses an arsenal of toxins. Here we cloned and expressed a probable toxin from P. luminescens subsp. akhurstii YNd185, designated as Photorhabdus insecticidal toxin (Pit). The pit gene shares 94% nucleotide and 98% predicted amino acid sequence identity with plu1537, a predicted ORF from P. luminescens subsp. laumondii TT01 and 30% predicted amino acid sequence similarity to a fragment of a 13.6 kDa insecticidal crystal protein gene of Bacillus thuringiensis (Bt). The pit was expressed as a GST-Pit fusion protein in E. coli, most of which was insoluble and sequestered into inclusion bodies. The inclusion bodies were harvested and dissolved. The resultant protein was purified and the Pit was cleaved from the fusion protein by thrombin and purified from GST then used for bioassay. Pit killed Galleria mellonella (LD₅₀, 30 ng/larva) and Spodoptera litura (LD₅₀, 191 ng/larva) via hemocoel injection. Relative to a control that lacked toxin, Pit did not significantly increase mortality of S. litura and Helicoverpa armigera when introduced orally, but the treatment did inhibit growth of the insects. The present study demonstrated that Pit possessed insecticidal activity.     

Heterorhabditis bacteriophora as a vector for introducing its associated bacterium into the hemocoel of Galleria mellonella larvae

The nematode Heterorhabditis bacteriophora serves as a vector enabling its bacterial associate to reach the hemocoel of its host, the seventh-instar larva of Galleria mellonella. At 28.5°C, the LD₅₀s of the orally introduced nematode-bacterial complex and the intrahemocoelically injected bacteria are three to six nematodes and one to two cells, respectively.     

Biocontrol Potential of Steinernema thermophilum and Its Symbiont Xenorhabdus indica Against Lepidopteran Pests: Virulence to Egg and Larval Stages

Under laboratory conditions, the biocontrol potential of Steinernema thermophilum was tested against eggs and larval stages of two important lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura (polyphagous pests), as well as Galleria mellonella (used as a model host). In terms of host susceptibility of lepidopteran larvae to S. thermophilum, based on the LC₅₀ 36 hr after treatment, G. mellonella (LC₅₀ = 16.28 IJ/larva) was found to be more susceptible than S. litura (LC₅₀ = 85 IJ/larva), whereas neither host was found to be significantly different from H. armigera (LC₅₀ = 54.68 IJ/larva). In addition to virulence to the larval stages, ovicidal activity up to 84% was observed at 200 IJ/50 and 100 eggs of H. armigera and S. litura, respectively. To our knowledge this is the first report of entomopathogenic nematode pathogenicity to lepidopteran eggs. Production of infective juvenile (IJ) nematodes/insect larva was also measured and found to be positively correlated with rate of IJ for H. armigera (r = 0.990), S. litura (r = 0.892), as well as G. mellonella (r = 0.834). Both Phase I and Phase II of symbiotic bacteria Xenorhabdus indica were tested separately against neonates of H. armigera and S. litura by feeding assays and found to be virulent to the target pests; phase variation did not affect the level of virulence. Thus S. thermophilum as well as the nematode’s symbiotic bacteria applied separately have the potential to be developed as biocontrol agents for key lepidopteran pests.     

Inhibition of phagocytic activity and nodulation in Galleria mellonella by the entomopathogenic fungus Nomuraea rileyi

Changes in phagocytic activity and nodulation in the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), were examined after treatment with the culture fluid of the entomopathogenic fungus Nomuraea rileyi SH1. When isolated hemocytes of G. mellonella were incubated with the conidia of N. rileyi in vitro, the rates of phagocytosis increased at 4 h after incubation but decreased subsequently. On the contrary, the rates of phagocytosis of isolated hemocytes decreased by 80% after 24 h preincubation with the fungal culture fluid (1/200, 1/100 dilutions). Levels of inhibition of phagocytic activity by the culture fluid depended on dilutions used. Galleria mellonella larvae showed a peak of nodulation at 4 h after injection with conidia. The percentage of nodules in hemolymph did not decrease by preinjection with the culture fluid, whereas the percentage of nodule‐containing conidia decreased, depending on the injected fluid. However, phagocytosis and nodulation in G. mellonella did not change after treatment of the culture fluid with proteinase K, indicating that the culture fluid contained proteinaceous immunosuppressive factors. Electrophoretic analysis of the culture fluid and not the fresh medium without culturing the fungus exhibited protein bands. Therefore, N. rileyi possibly secretes toxic proteins that impair cellular immune responses in G. mellonella larvae.     

Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti

Background  

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H₂O₂ and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria.     

The virulence of protease and cell surface mutants of Pseudomonas aeruginosa for the larvae of Galleria mellonella

Pseudomonas aeruginosa mucoid strains as well as mutants defective in pili, flagella, lipopolysaccharide, and proteases were isolated and tested for their virulence for the larvae of Galleria mellonella. Of all of the mutants, only the lipopolysaccharide-deficient (rough) strain showed a major decrease in virulence when compared to the wild type. The LD₅₀ of the rough strain was about 30,000 bacteria/larva or roughly 10,000-fold higher than the wild type, suggesting an important role for the lipopolysaccharide in P. aeruginosa infections of G. mellonella larvae.     

Use of Galleria mellonella larvae to evaluate the in vivo anti-fungal activity of [Ag2(mal)(phen)3]

Larvae of the insect Galleria mellonella were employed to assess the in vivo antifungal efficacy of ([Ag₂(mal)(phen)₃]), AgNO₃ and 1,10-phenanthroline. Larvae pre-inoculated with these compounds were protected from a subsequent lethal infection by the yeast Candida albicans while larvae inoculated 1 and 4 h post-infection showed significantly increased survival (P < 0.01) compared to control larvae. Administration of these compounds resulted in an increase over 48 h in the density of insect haemocytes (immune cells) but there was no widespread activation of genes for antimicrobial peptides. This work demonstrates that G. mellonella larvae may be employed to ascertain the antifungal efficacy of silver(I) compounds and offers a rapid and effective means of assessing the in vivo activity of inorganic antimicrobial compounds.     

Influences of host size and host species on theinfectivity and development of Heterorhabditismegidis (strain NLH-E87.3)

The infectivity, time to first emergence of infective juveniles (IJs), total number of IJs per insect and IJs body length of the entomopathogenic nematode Heterorhabditis megidis (strain NLH-E87.3) after development in larvae of two insect hosts, Galleria mellonella (greater wax moth) and Otiorhynchus sulcatus (vine weevil) was studied. At a dose of 30 IJs, larvae of G. mellonella show to be significantly more susceptible than O. sulcatus larvae. At a dose of one IJ, vine weevil larvae were more susceptible. The number of invading infective juveniles (IJs) increased with host size while the host mortality at a dose of one IJ decreased with the increase of host size. Time to first emergence was longer at a dose of one IJ per larva and increased with the increase of host size in both insect species. Reproduction of IJs differed between host species, host sizes and doses of nematodes. Generally, the IJs body size increased with an increasing host size. The longest infective juveniles were produced at the lowest IJ doses. Results are discussed in relation to the influence of different host species and their different sizes on the performance of H. megidis (strain NLH-E87.3) as a biological control agent.     

Functional response of Trichogramma chilonis to Galleria mellonella and Chilo sacchariphagus eggs

A biological control programme using inundative releases of Trichogramma chilonis Ischii (Hymenoptera: Trichogrammatidae) reared on Galleria mellonella L. (Lepidoptera: Pyralidae) is currently underway to reduce infestations of Chilo sacchariphagus Bojer (Lepidoptera: Crambidae) in sugarcane, Saccharum spp., on Réunion Island. To assess the potential of the parasitoid as an inundative biocontrol agent, the functional response of three T. chilonis strains was tested with G. mellonella and one strain with C. sacchariphagus host eggs in glass tubes in the laboratory. The shape of the functional response (type II or III) was determined using logistic regression, and attack coefficients and handling times (T_h) were determined using non‐linear least‐square regression. The behaviour of all three strains with G. mellonella host eggs corresponded to a type III response. The St Benoît T. chilonis strain had a significantly shorter estimate of T_h than the St Pierre strain (P<0.05) and may, therefore, be more appropriate as a biocontrol agent. The functional response with C. sacchariphagus host eggs was a type II with the St Benoît T. chilonis strain. More T. chilonis wasps developed per host egg from the larger C. sacchariphagus host eggs (2.9) relative to G. mellonella (1.1). Superparasitism at low host egg densities was, therefore, likely to have been less frequent with C. sacchariphagus. Black eggs were chosen as an estimate of number of eggs parasitized, although they represent the number of eggs where parasitism led to complete pupal development. The low rate of detected parasitism at low host densities with G. mellonella eggs may be due to incomplete pupal development due to superparasitism rather than lack of parasitism, thus explaining the type III functional response.     

Effects of Palpita unionalis and Galleria mellonella larval densities on functional response,egg dispersion and progeny sex ratio of Habrobracon hebetor

To assess the potential of the hymenopteran ectoparasitoid, Habrobracon hebetor Say as a biological control agent, we evaluated its response to different larval densities of two pyralid hosts, Palpita unionalis Hbn. and Galleria mellonella L. The former host is a serious pest of olive trees, whereas the latter is used as a factitious host in parasitoid mass rearing. In order to study the functional response of the parasitoid, five host densities (1, 2, 3, 5, 7) of either late instar larva per Petri dish were used. The shape of the functional response curve was determined using logistic regression and could be described as a type II response for both hosts, characterised by a monotonic decelerating increase in the number of hosts attacked with increasing host density. Female parasitoids allocated more eggs to the first larva attacked than all the remaining larvae. Aggregated dispersion patterns for parasitoid egg distribution at different host densities were estimated using the Green index. Multiple visits and ovipositions by females did not significantly affect the total number of progeny produced or their sex ratio. This study has generated novel information on egg laying, egg distribution and sex ratio of H. hebetor when reared on G. mellonella and has the potential to be used in the development of sustainable biological control programmes aimed at P. unionalis in olive orchards.     

Ecological characterisation of <Emphasis Type="Italic">Steinernema siamkayai</Emphasis> (Rhabditida: Steinernematidae), a warm-adapted entomopathogenic nematode isolate from India

Our study describes basic ecological properties of Steinernema siamkayai Tiruchirappalli strain from India. The effect of temperature on nematode infectivity and development, laboratory host range and foraging behaviour were determined. The data showed that S. siamkayai is a warm-adapted nematode species with larval mortality observed between 15°C and 37.5°C and nematode reproduction occurring between 20°C and 35°C. All insect species used in this study were susceptible to S. siamkayai under laboratory conditions. Sixty infective juveniles (IJs) per insect were used and the lepidopterans, Galleria mellonella (100%) and Spodoptera exigua (85%), were the most susceptible species followed by the dipteran, Ceratitis capitata (60%), and lepidopteran, Cydia splendana (55%), and the coleopteran, Tenebrio molitor (45%), whereas the coleopteran, Curculio elephas (25%), was the least susceptible species. S. siamkayai infective juveniles (IJs) stood on their tails and jumped and could also attach to a mobile host at a rate of 27 IJs larvae⁻¹ out of 1000 IJs in 10 min. Larval mortality of G. mellonella by S. siamkayai on different substrates (sand, filter paper, filter paper sprinkled with sand) was 100% on all substrates. Number of IJs out of 100 IJs that penetrated into a G. mellonella host at different soil depths was the highest at the surface (44 IJs larva⁻¹) and the lowest at 5 cm depth (13 IJs larva⁻¹) with no larval mortality observed at 10 cm depth. In addition, the symbiotic bacterium of S. siamkayai was identified as Xenorhabdus stockiae based on genotypic and phenotypic characterisation. Bacterial growth was observed between 15°C and 41°C.     

Anastrepha ludens (Loew) (Diptera: Tephritidae) larvae irradiated at higher doses improve the rearing of two species of native parasitoids

Irradiation doses 40, 80, 120 and 160 Gy were used on 6-, 7-, 8- and 9-days-old Anastrepha ludens (Loew) (Diptera: Tephritidae) larvae. The larvae from the different age groups irradiated with different doses were exposed separately to Neotropical-native Doryctobracon crawfordi (Viereck), Utetes anastrephae (Viereck) and the Asian introduced Diachasmimorpha longicaudata (Ashmead) parasitoids (Hymenoptera: Braconidae). A significant increment in parasitoid emergence directly related to radiation increment on parasitoids D. crawfordi and U. anastrephae was observed. This effect was higher on irradiated young larvae; in D. longicaudata, emergence increment was related to age of larva but not with radiation doses. Similar results were obtained for sex ratio. Host larva weight increased with age, and irradiation had no effect on gaining weight of growing larvae. The host larval mortality 72 hr after exposition had an inverse relationship regarding to its age, although it did not have a negative effect as regards radiation doses. Immature stages of parasitoid/larva (>1 = superparasitism) were higher in 8-days-old larvae parasitized by D. Longicaudata, while in D. crawfordi and U. Anastrephae, superparasitism was absent (<1 immature stage per host larva). Superparasitism was not affected by increased radiation dose. No levels of melanin were detected as indicators of immunological reactions. However, these results are considered to be closely related to the lessening of these reactions due to irradiation. The positive relationship between emergence and irradiation dose to host larvae was present in native species D. crawfordi and U. anastrephae, but absent for exotic D. longicaudata. The data presented here are of great relevance for cost effective mass rearing of these parasitoids.     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.     

Bacterial α2-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome?

Background  

Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor α₂-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion.     

Establishment of an EC50 database of pesticides using a Vibrio fischeri bioluminescence method

An EC₅₀ database was established to assess the acute toxicity of 16 PESTANAL pesticide standards and of seven pesticide commercial formulations using a Vibrio fischeri bioluminescence method. Half maximal effective concentration ( EC₅₀) is defined as the concentration of pollutant (in this case, pesticide) destroying 50% of the bacteria population and causing 50% bioluminescence inhibition, after a specified exposure time. Linear curves of bioluminescence inhibition versus pesticide concentration and EC₅₀ values were obtained for exposure times (t) of 5 or 15 min for these pesticides. The EC₅₀ values ranged from 6.90 × 10^?4 to 0.83 mg/ml (t = 5 min), and from 9.00 × 10^?4 to 0.37 mg/ml (t = 15 min) for pesticide standards, plus from 0.0077 to 0.74 mg/ml (t = 5 min), and from 0.0076 and 0.57 mg/ml (t = 15 min) for pesticide commercial formulations. The EC₅₀ database allowed classification of the pesticides under study into three categories according to their toxicity: very toxic, toxic and moderately toxic. These results demonstrated that the establishment of an EC₅₀ database and of linear curves of bioluminescence inhibition versus the pesticide concentration resulted in very important and irreplaceable tools to estimate the global and individual toxicity of pesticides present in environmental samples.     

The main features of Bacillus thuringiensis δ-endotoxin molecular structure

The crystal-forming proteins (-endotoxins) produced by various serotypes of Bacillus thuringiensis and toxic for Lepidoptera reveal the same pattern of molecular organisation. These proteins (M _r of ca. 145,000–130,000) contain an N-terminal domain (M _r of 85,000–65,000) resistant to proteolysis whereas their C-terminal moieties (M _r of 65,000) undergo an extensive degradation by trypsin that leads to stepwise cleavage off the fragments with M _r of 15,000–35,000.The N-terminal domain from serotype V -endotoxin is active when introduced into the hemocoel of Galleria mellonella larvae. Hence, it correponds to the true toxin normally formed by larva proteases action on the crystalforming protein (protoxin). Some differences were found in the properties of the N-terminal domains isolated from the crystal-forming proteins of III, V and IX serotypes, e.g., in their solubility, digestion by subtilisin, molecular weights and the distribution of methionine residues along the polypeptide chains.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacryl amide gel electrophoresis - CFP crystal-forming protein - DNS 5-dimethylamino-1-naphthalene-sulphonyl     

Galleria mellonella apolipophorin III – an apolipoprotein with anti-Legionella pneumophila activity

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.     

Host cell binding of the flagellar tip protein of Campylobacter jejuni

Flagella are nanofibers that drive bacterial movement. The filaments are generally composed of thousands of tightly packed flagellin subunits with a terminal cap protein, named FliD. Here, we report that the FliD protein of the bacterial pathogen Campylobacter jejuni binds to host cells. Live‐cell imaging and confocal microscopy showed initial contact of the bacteria with epithelial cells via the flagella tip. Recombinant FliD protein bound to the surface of intestinal epithelial cells in a dose‐dependent fashion. Search for the FliD binding site on the host cell using cells with defined glycosylation defects indicated glycosaminoglycans as a putative target. Heparinase treatment of wild type cells and an excess of soluble heparin abolished FliD binding. Binding assays showed direct and specific binding of FliD to heparin. Addition of an excess of purified FliD or heparin reduced the attachment of viable C. jejuni to the host cells. The host cell binding domain of FliD was mapped to the central region of the protein. Overall, our results indicate that the C. jejuni flagellar tip protein FliD acts as an attachment factor that interacts with cell surface heparan sulfate glycosaminoglycan receptors.