Enhancing mass production of Heterorhabditis bacteriophora: influence of different bacterial symbionts (Photorhabdus spp.) and inoculum age on dauer juvenile recovery.

The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, invading host insects. They carry cells of their bacterial symbiont Photorhabdus spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the Photorhabdus cells into the insect's haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called "recovery". For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with Photorhabdus before DJs are added. In absence of the insect's food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from H. bacteriophora were isolated and identified as P. laumondii, P. kayaii and P. thracensis. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.

Life history trait analysis of the entomopathogenic nematode Steinernema feltiae provides the basis for prediction of dauer juvenile yields in monoxenic liquid culture.

Entomopathogenic nematodes (Steinernema spp.) are used in integrated pest management to control insect pests in cryptic environments. The nematodes are mass produced in monoxenic liquid culture with their symbiotic bacteria Xenorhabdus spp. For a better understanding of nematode population dynamics, the life history traits (LHTs) of the entomopathogenic nematode Steinernema feltiae were assessed at 25 °C by observing single pairs of male and female nematodes using a hanging drop technique. To investigate the influence of different food supplies on nematode reproduction, the LHTs were assessed with a daily supply of 5 ×, 10 × and 20 × 10(9) cells ml(-1) of the nematode's bacterial symbiont Xenorhabdus bovienii in semi-solid nematode growth gelrite (NGG) medium. Increasing bacterial density had a significant positive influence on the average number of offspring produced, which ranged from 359 to 813 per female. The intrinsic rate of natural increase r m, which ranges from 1.10 to 1.19 day(-1), was neither influenced by the bacterial density, nor was the mean generation time T (5.12-5.25 days) and population doubling time (PDT) (0.64-0.59 days). The average lifespan of reproductive females, which ranged from 6.7 to 7.3 days, was positively correlated with bacterial density. A positive correlation between female body volume and bacterial density was recorded (R = 0.67) as well as a significant positive correlation between female body size and offspring production (R = 0.89) in hanging drops. Whether these data can be used to predict nematode yields in liquid culture was tested. The total female body volume calculated as the average female body volume × total number of parental females per millilitre 3 days after nematode inoculation was positively correlated (R = 0.72) with nematode yields. The total female body volume on process day 3 is thus a good indicator for the estimation of nematode yield at the end of the process (12-15 days post dauer juvenile (DJ) inoculation) in both Erlenmeyer flasks and bioreactors. With a mean deviation of 9467 DJs ml(-1), the error resembles approximately 5 % of the final DJ yields.

Monoxenic liquid culture with Escherichia coli of the free-living nematode Panagrolaimus sp. (strain NFS 24-5), a potential live food candidate for marine fish and shrimp larvae.

The free-living, bacterial-feeding nematode Panagrolaimus sp. (strain NFS 24-5) has potential for use as live food for marine shrimp and fish larvae. Mass production in liquid culture is a prerequisite for its commercial exploitation. Panagrolaimus sp. was propagated in monoxenic liquid culture on Escherichia coli and parameters, like nematode density, population dynamics and biomass were recorded and compared with life history table data. A mean maximum nematode density of 174,278 mL(-1) and a maximum of 251,000 mL(-1) were recorded on day 17 after inoculation. Highest average biomass was 40 g L(-1) at day 13. The comparison with life history table data indicated that the hypothetical potential of liquid culture is much higher than documented during this investigation. Nematode development is delayed in liquid culture and egg production per female is more than five times lower than reported from life history trait analysis. The latter assessed a nematode generation time of 7.1 days, whereas the process time at maximum nematode density in liquid culture was 16 days indicating that a reduction of the process time can be achieved by further investigating the influence of nematode inoculum density on population development. The results challenge future research to reduce process time and variability and improve population dynamics also during scale-up of the liquid culture process.

Selective breeding for desiccation tolerance in liquid culture provides genetically stable inbred lines of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is used in biological plant protection to control pest insects. In the past, several attempts targeted at an enhancement of the desiccation tolerance of EPN by genetic selection in order to improve their storage stability. The subsequent loss of improved beneficial traits after release of selection pressure has often been reported. In order to stabilize progress of selective breeding, selection during liquid culturing was tested against propagation in host insects. After release of the selection pressure, the tolerance was monitored over additional reproductive cycles in vivo and in vitro to compare the stability of the trait. Furthermore, it was tested whether the virulence of the selected strains would be impaired. Exposure to desiccation stress prior to propagation, in vivo or in vitro, both resulted in increasing desiccation tolerance. When selection pressure was released, the gained tolerance was lost again during in vivo production, whereas the tolerance was maintained at a high level when EPNs were cultured in liquid culture. In Heterorhabditis sp., liquid culture conditions produce highly homozygous, genetically stable inbred lines. The investigation provides easily applicable methods to improve and stabilize beneficial traits of heterorhabditid EPNs through selective breeding in liquid culture. Compared to nematodes from in vivo propagation, production in liquid media yielded EPN of higher virulence.