Bacteriophora Infective Juveniles Research Articles

Volatile organic compounds of Metarhizium brunneum influence the efficacy of entomopathogenic nematodes in insect control

The entomopathogenic fungus (EPF) Metarhizium brunneum occupies the same ecological niche as entomopathogenic nematodes (EPN), with both competing for insects as a food source in the rhizosphere. Interactions between these biocontrol agents can be antagonistic or synergistic. To better understand these interactions, this study focussed on investigating the effect of M. brunneum volatile organic compounds (VOCs), 1-octen-3-ol and 3-octanone, on EPN survival and behaviour. These VOCs proved to be highly toxic to the infective juveniles (IJs) of the EPN Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora with mortality being dose dependent. Chemotaxis studies of H. bacteriophora IJs in Pluronic F127 gel revealed significant preference for the VOCs compared with controls for all tested concentrations. The VOCs also impacted on the test insects in a dose-dependent manner with 3-octanone being more toxic to Galleria mellonella, Cydia splendana and Curculio elephas larvae than 1-octen-3-ol. Mortality of C. splendana and G. mellonella larvae was significantly higher when exposed to relatively high doses (>25%) of 3-octanone. Lower doses of 3-octanone and 1-octen-3-ol immobilised test insects, which recovered after exposure to fresh air for 2 hrs. In depth studies on H. bacteriophora showed that exposure of IJs to > 10% concentration of 3-octanone or 1-octen-3-ol negatively affected infectivity whereas exposure to lower doses (0.1%, 0.01%) had no effect. The VOCs affected IJs, reducing penetration efficacy and the number of generations inside G. mellonella but they failed to inhibit the bacterial symbiont, Photorhabdus kayaii. The ecological significance of VOCs and how they could influence EPF-EPN insect interactions is discussed.

Evaluation of different sponge types on the survival and infectivity of stored entomopathogenic nematodes

Sponges are one of the cheapest and most suitable substrates used to formulate and/or store the infective juveniles (IJs) of entomopathogenic nematodes (EPNs). Our study investigated the survival and infectivity of the IJs on five different sponges compared to that in an aqueous suspension (control). The sponges were Oasis® floral, Nanosponge, ScotchbriteTM, or Lysol® and natural sea sponge. EPN species tested were Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae. The recovery efficiency of the IJs from sponges was initially assessed. Subsequently, IJs were stored in the sponges and placed in plastic bags or Falcon tubes and incubated at 10° or 27 °C for 8 months or 11 weeks, respectively. IJ survival and infectivity were monitored monthly for the 10 °C and weekly for 27 °C in these sponge types. The IJs were recovered from the sponges, and their survival was based on observing their movement under a dissecting microscope, and infectivity was based on larval mortality in Galleria mellonella. Recovery efficiency of IJs was best for the Oasis floral sponge for all nematode species ranging between 83 and 91%. The survival and infectivity of stored IJs in all sponge types and control for both 10° and 27 °C gradually decreased over time. IJs stored in Scotchbrite, Lysol, and Nanosponge had the best survival and infectivity, whereas Oasis floral and natural sea sponges showed the poorest results. After 8 months at 10 °C in plastic bags, the survival ratio of all IJs in these three sponges (Scotchbrite, Lysol, and Nanosponge) was approximately 55%. IJs in Scotchbrite and Nanosponge were also able to survive and retain their infectivity at 27 °C for 3 months. IJs stored in Falcon tubes had survival that ranged from 26 to 53% at 27 °C and 55 to 77% at 10 °C. H. bacteriophora IJs lost their infectivity when stored at 27 °C after 10 weeks. However, S. carpocapsae and S. feltiae exhibited 85% infectivity when stored in Scotchbrite and 50% in Nanosponge, respectively. Overall, sponges made from polyurethane (Scotchbrite) followed by melamine (Nanosponge) and cellulose (Lysol) are recommended for long-term nematode storage and transportation of nematode samples. However, Oasis floral sponge may be preferred for short-term IJ formulation for field applications because of easier recovery of IJs.

Effects of abiotic factors on the osmotic response of alginate-formulated entomopathogenic nematode, Heterorhabditis bacteriophora (Nematoda: Rhabditida)

ABSTRACTLimited shelf life of entomopathogenic nematodes severely restricts their use in biological control programs. In a series of experiments, the dehydration and rehydration response of Heterorhabditis bacteriophora infective juveniles (IJs) was investigated under a range of glycerol concentrations, temperatures and incubation periods. Based on the outcome of these initial studies, nematodes dehydrated using the optimal process were formulated in alginate granules to understand how these nematodes would undergo survival formulation in a model carrier. The highest rate of osmotically arrested IJs occurred in the 22% glycerol solution (98.06%). IJ recovery was considerably improved when dehydration was processed at 10% glycerol solution and 15°C. By trapping IJs in calcium alginate, depending on the adjuvants, the survival rate of IJs differed significantly. IJ state (dehydrated or non-dehydrated) and the addition of formaldehyde had a profound effect on IJ viability, though the severity of the effect varied was dependent on whether the IJs were alginate formulated. Among different formulations, the highest viability (84.18%) was observed where dehydrated IJs were formulated in alginate granules containing formaldehyde. The results showed that the concentration of osmotic solution not only determines the percentage of dehydrated IJs but also affect their subsequent recovery in an aqueous environment. Overall, the results indicate that the shelf life of formulated IJs is significantly affected by combination effects of a broad range of factors. Then understanding the interactive mode of actions of involved factors in formulation play a critical role in developing and introducing more efficient formulations.

Stress avoidance: vertical movement of entomopathogenic nematodes in response to soil moisture gradient

Survival of entomopathogenic nematodes (EPNs) in soils is attributed to their entering a dormant state—anhydrobiosis—as soil moisture decreases, but EPNs with poor desiccation tolerance and low anhydrobiotic capabilities may practice desiccation avoidance. We compared the effect of soil moisture gradient on downward movement of the highly desiccation-tolerant Steinernema carpocapsae and the poorly desiccation-tolerant Heterorhabditis bacteriophora. Infective juveniles (IJs) were applied to the surface of moist (11–13% w/w moisture) sandy soil in buckets. Nematode distribution was monitored at different depths 3, 14 and 28 days after application. In uncovered buckets, soil moisture decreased to 1% in the upper 5-cm layer after 28 days. H. bacteriophora IJs abandoned the upper soil layers as dryness intensified with >80% found in the bottom (20–25 cm) layer. In contrast, >70% S. carpocapsae IJs remained in the upper layer. In covered buckets, with 10% moisture throughout the experiment, heterorhabditid IJs were equally distributed between the 10–15 cm and 20–25 cm layers; only 7% remained in the upper layer. Again, >70% S. carpocapsae IJs remained in the upper layer throughout. Soil type influenced H. bacteriophora IJs' downward migration. In sandy and sandy loam soils, with rapid evaporation, >80% IJs were in the bottom layer 14 and 28 days after application. In the loam soil, with higher moisture retention, >75% IJs remained in the 10–15 cm layer and <20% migrated to the bottom. Results provide initial evidence of a possible stress-avoidance strategy in H. bacteriophora under natural conditions.

Efficacy of the entomopathogenic nematode Heterorhabditis bacteriophora using different application methods in the presence or absence of a natural enemy

Various application methods for the entomopathogenic nematode Heterorhabditis bacteriophora were evaluated under laboratory and field conditions. Four different methods of applying the infective juveniles (IJs) of the nematode to soil were assessed including (1) insect cadavers (referred to as nematode-infected cadavers), (2) subsurface injection, (3) spraying, and (4) drip irrigation. In the laboratory experiment, except for the control with no nematodes, all treatments showed more than 95% insect mortality of the bait insect (Galleria mellonella) with no significant differences among treatments. The same experimental setup was conducted with the introduction of mites, Sancassania polyphyllae (Acari: Acaridae), which are natural enemies of entomopathogenic nematodes. The treatment groups with mites and the control group without mites showed more than 87% insect mortality and no significant differences were observed. The nematode-infected cadaver application method was further evaluated by using cadavers of different ages containing H. bacteriophora IJs in the presence of mites. Larval mortality of the bait insect was significantly lower when 3- or 6-day-old nematode-insect cadavers were used in the presence of mites. Different application methods were also tested in a corn field. No significant differences were observed among the application methods. Our results indicate that the different application methods had no significant effect on larval mortality, but the presence of mites had a negative effect on the cadaver application when the cadavers were 3 days old but had no significant effect when the cadavers were 6 and 9 days old.

Effect of Soil Moisture on the Persistence and Efficacy ofHeterorhabditis bacteriophora(Rhabditida: Heterorhabditidae) AgainstAnastrepha ludens(Diptera: Tephritidae) Larvae

The efficacy of Heterorhabditis bacteriophora (Poinar) infective juveniles (IJs) was evaluated against third instar Anastrepha ludens (Loew) (Diptera: Tephritidae) under laboratory conditions in a sandy clay soil at various levels of soil moisture. Three experiments were performed in which the efficacy of the IJs against A. ludens was estimated, i.e., (a) at 6 different levels of soil moisture, (b) in soil that was allowed to lose moisture over a 15 day period, and (c) in soil with an initial moisture content of 16% and in which moisture loss was partially mitigated by adding water at 5-day intervals. In the first experiment, the greatest A. ludens mortality (80%) was observed in soil with 18% moisture (-63.1 bars), although this was not significantly greater than A. ludens mortality at 21% moisture (-20.4 bars). At 24% soil moisture (-7.70 bars), percentage of mortality of A. ludens declined to about 50%. Likewise insect mortality was substantially lower at soil moisture levels of 15% (-240.1 bars) and 12% (-1,232 bars) and very much lower (about 16%) at 9% soil moisture (-10,147 bars). In the second experiment, as soil moisture declined from 16% to less than 10% over a 15 day period, the infectivity of IJs, as indicated by A. ludens larval mortality, progressively declined from more than 55% to less than 10%. In the third experiment, in which moisture loss was partially mitigated by adding water at 5-day intervals, the decline in infectivity of IJs was gradual up to 21 days, but decreased thereafter. We conclude that soil moisture levels must be carefully considered when applying H. bacteriophora IJs to control A. ludens under field conditions, because soil moisture has a marked effect on the efficacy of IJs for the biological control of this pest.

Identification of two down-regulated genes in entomopathogenic nematode Heterorhabditis bacteriophora infective juveniles upon contact with insect hemolymph

Entomopathogenic nematode Heterorhabditis bacteriophora infective juveniles (IJs) employ multiple strategies to combat with insect innate immune system after invasion. We employed suppressive subtractive hybridization (SSH) technique to study the gene expression in the IJs upon contact with the insect hemolymph in vitro. We identified two clones having higher expression levels in the IJs than IJs treated with insect hemolymph. The differential expression levels were confirmed by Northern blot hybridization with reference to the constitutive expression level of Heterorhabditis bacteriophora actin2 gene. Clone HbGPS11.C1G02 encoded a phosphofructokinase (PFK) with a 2.5kb transcript and clone HbGPS11.C4C08 corresponded to a 2.1kb transcript encoding a protein that had weak similarity to trans-sialidase from Trypanosoma cruzi. The differential expression of PFK in H. bacteriophora IJs before and during recovery process represented a switch from active movement in search of insect hosts to a state of combating insect innate immune system. This first report of H. bacteriophora differential gene expression provides a glimpse at the gene expression profile of H. bacteriophora IJ recovery process.

Impact of the host cadaver on survival and infectivity of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) under desiccating conditions.

Entomopathogenic nematode species of Steinernema carpocapsae, Steinernema riobrave, or Heterorhabditis bacteriophora were used to compare survival and infectivity among infective juveniles (IJs) emerging in water from hosts in White traps (treatment a), emerging in sand from hosts placed in sand (treatment c), and emerging from hosts placed on a mesh suspended over sand (treatment m). Nematode survival and infectivity was recorded in sand at three-day intervals during 21 days of storage in desiccators at 75% relative humidity and 25 degrees C. Infectivity was measured by exposing 5 Galleria mellonella for 16 h to IJs. Treatment did not affect percent survival of H. bacteriophora IJs. Percent survival of S. riobrave and S. carpocapsae IJs was lowest in treatment a. Across all treatments, by 10 days after the beginning of the experiments, IJ survival declined to 93, 43, and 28% of levels on day 1 for H. bacteriophora, S. riobrave, and S. carpocapsae, respectively. For the three treatments, infection rate over time was described by a negative exponential function for S. riobrave and S. carpocapsae and by a sigmoid function for H. bacteriophora.

Physiological ageing and behavioural plasticity of Heterorhabditis bacteriophora infective juveniles

AbstractHeterorhabditis bacteriophora is a cruiser strategist that exhibits long distance ranging searches for insect hosts in soil. We characterised age-based variations in constituent behaviours and total lipid reserves of laboratory-aged H. bacteriophora infective juveniles. Individuals demonstrated extensive behavioural variability within the first 7 days, followed by accentuated ranging behaviour during the next 14 days and, finally, a decline in activity during the last 15 days. Frequency, bout duration and total time investment decreased almost 50% for forward crawling but almost doubled for backward crawling and curling. Both pausing and head waving frequency, a theoretical correlate of searching intensity, varied considerably without relation to age. Lipid content declined steadily, decreasing by 56% after 28 days. Behavioural changes became apparent after a 20% reduction in total lipid, and performance continued to decline with further lipid depletion. These findings emphasise the combined influences of environmental and physiological factors on behavioural plasticity in entomopathogenic nematodes.

Lipids of in Vivo and in Vitro Cultured Heterorhabditis bacteriophora

The lipid, sterol, and fatty acid compositions of Heterorhabditis bacteriophora infective juveniles reared by in vivo and in vitro methods (Galleria mellonella, Popillia japonica, solid and liquid culture) were compared. The greatest accumulation of lipids per dry weight was achieved by growing nematodes in P. japonica and solid culture (54.7 and 60.3%, respectively). Nematodes produced by liquid fermentation accumulated lower levels of sterols (2%) per total lipids compared with other production methods. However, the level of polar lipids was higher in nematodes produced in artificial media. Fractionation of the polar lipids revealed that an increased level of phosphatidylglycerol accompanied production in liquid culture. This was not observed for nematodes produced in solid culture in which the major polar lipid fractions were phosphatidylcholine and phosphatidylethanolamine. Lipids from nematodes produced in artificial media had more linoleic (C18: 2) fatty acids, whereas lipids from in vivo cultures had more stearic (C18: 0) fatty acids. The ratio of saturated to unsaturated fatty acid composition decreased in cultures produced in vitro. Artificial media composition should be adjusted to meet the nutritional composition of a natural host. Media for H. bacteriophora culture should be modified to boost deficiencies in sterols (e.g., liver extract) and oleic acid (e.g., olive oil).

Pathogenicity of New Combinations ofHeterorhabditisspp. andPhotorhabdus luminescensagainstGalleria mellonellaandTipula oleracea

Abstract Photorhabdus luminescens isolates were exchanged between four entomopathogenic nematode strains; two isolates of Heterorhabditis megidis from The Netherlands and two isolates of H. bacteriophora , one from Australia and one from Moldavia. When cultured on H. megidis symbionts only few H. bacteriophora infective juveniles retained the symbiotic bacterium. These infective juveniles, without symbiotic bacteria, were not pathogenic to insects. When P. luminescens was injected into insect larvae it could kill Galleria mellonella (Lepidoptera: Galleridae) larvae but it was not pathogenic to Tipula oleracea (Diptera: Tipulidae). The combination of nematode and bacterium killed T. oleracea , showing that the nematode is more than just a vector for the bacterium. Small differences in pathogenicity between the combinations could only be observed in T. oleracea , not in the highly susceptible G. mellonella . The pathogenicity of a combination depends on the pathogenicity of the bacterium, the pathogenicity of the nematode and the interaction between them. The pathogenicity of H. bacteriophora strains against T. oleracea was low, partly because of the low penetration rate of these strains.

Laboratory Studies of the Interactions of Environmental Conditions on the Susceptibility of Green June Beetle (Coleoptera: Scarabaeidae) Grubs to Entomopathogenic Nematodes

In the laboratory, four nematode species differed significantly in the percentage mortality that each caused in second-instar green June beetles (grubs), Cotinis nitida (L.). Water suspensions of 1,000 infective juveniles (IJ)/one grub/Petri dish each of Heterorhabditis bacteriophora, Steinernema glaseri, and S. feltiae caused 34, 22 and 18% grub mortality, respectively. These values were significantly (P = 0.05) greater than the grub mortality caused by either S. carpocapsae (12%) or the water check (0%) that were similar. Several factors affected third- and fourth-instar susceptibility in the laboratory when exposed to 10,000 S. carpocapsae or H. bacteriophora IJ in 75 g of soil (133 IJ/cm3 soil)/plastic cup. Soil at 30% moisture by weight resulted in significantly more (P= 0.006) dead grubs (6.6%) than at 10% soil moisture (2.8%). Significantly more (P = 0.03) grubs died when at a grub density of 2 grubs/plastic cup (5.6%) than at 1 grub/plastic cup (3.8%). Significantly more (P = 0.01) grubs were killed by either S. carpocapsae and H. bacteriophora at a soil temperature of 25°C (6.5%) than at 12°C (2.8%). There was also a significant interaction of temperature and nematode species. Heterorhabditis bacteriophora killed significantly more (P= 0.02) grubs at 25°C (9.5%) than at 12°C (1.9%) and more than S. carpocapsae killed at either 12°C or 25°C (&amp;lt;3.9%). More virulent nematode strains or species than these four nematode species must be identified if green June beetles are to be controlled by nematodes.

Infection of Entomopathogenic Nematodes Steinernema glaseri and Heterorhabditis bacteriophora against Popillia japonica (Coleoptera: Scarabaeidae) Larvae

Defense mechanisms of Japanese beetle larvae, Popillia japonica, and their effectiveness against entomopathogenic nematodes were investigated in the laboratory. Behavioral grooming by P. japonica larvae was effective in reducing invasion of both Steinernema glaseri and Heterorhabditis bacteriophora. Forty percent of H. bacteriophora infective juveniles were inactivated or killed in the host gut fluid within 8 hr, whereas 11% of S. glaseri were killed. Host melanotic encapsulation, induced by injection of 20 H. bacteriophora infective juveniles, caused 75% nematode mortality. S. glaseri was less susceptible to the host immune response, which caused only 12% mortality of injected nematodes. In a sand column assay, S. glaseri was found in the host hemocoel 8 hr earlier than H. bacteriophora. However, Photorhabdus luminescens, the bacterium of H. bacteriophora, was detected 16 hr earlier in the host's hemolymph than S. glaseri's bacterium, Xenorhabdus poinarii. Despite the differences in effectiveness of host defenses, S. glaseri and H. bacteriophora caused similar host mortality in the sand column assay. We demonstrated that the two nematode species use different strategies for invasion of P. japonica larvae. S. glaseri, which tolerated P. japonica gut fluid and avoided the host immune response, has a high initial rate of penetration and survival; H. bacteriophora, which exhibited poor tolerance to the host gut fluid and induced a strong host immune response, releases its bacteria during or soon after penetration to kill the host.

Behavioural response of the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to oxamyl

SummaryInfective juveniles of the entomopathogenic nematode Steinernema carpocapsae show a low level of locomotory activity that is presumed to limit their usefulness as biological insecticides. A 30 μg ml‐1 solution of the carbamate pesticide oxamyl reduced the proportion of nonmobile nematodes by nearly two thirds to 35%, while stimulating a 7.5‐fold increase in sinusoidal movement. This increase in activity did not result in a corresponding increase in host‐finding. Oxamyl treatment did not enhance infective juvenile pathogenicity to Galleria mellonella larvae. At higher concentrations, oxamyl caused aberrant nematode movement and partial paralysis. Heterorhabditis bacteriophora infective juveniles maintain a high level of locomotory activity. Treatment with 30 μg ml‐1 oxamyl increased the proportion of sinusoidal over nonsinusoidal movements, but infective juvenile host‐finding and pathogenicity were significantly reduced. Higher rates impaired movement and induced complete paralysis. We conclude that oxamyl is incompatible with S. carpocapsae and H. bacteriophora. The concept of chemically activating infective juveniles to increased locomotory activity and thereby achieving enhanced efficacy is inconsistent with our results.