Olaf Strauch, Ralf-Udo Ehlers and Ayako Hirao

Abstract

The life cycle and population dynamics of the entomopathogenic nematodes Steinernema carpocapsae and S. feltiae were studied in monoxenic liquid culture with their symbiotic bacteria Xenorhabdus nematophila and X. bovienii. To distinguish between the different juvenile and adult stages, their size was recorded. No differences were observed between the species in the size of the juvenile stages but significant differences were recorded in the length of the F1 adults, pre-dauer (J2d) and dauer juvenile stages (DJ). On average, 90% of inoculated DJ of S. feltiae recovered and 77% of S. carpocapsae. In general, S. feltiae developed from the inoculum DJ to the adult approximately 1 day faster than S. carpocapsae. The sex ratio was female-biased (59.2 ± 2.2% in S. carpocapsae, 66.7 ± 2.6% in S. feltiae) in the parental population but not in the F1 generations. Steinernematid adults, like heterorhaditids, respond to depleting food resources with the cessation of egg laying. Juveniles hatch inside the uterus and develop at cost of the maternal body content causing the death of the adult (endotokia matricida). In contrast to Heterorhabditis spp. and in vivo observations of steinernematids by other authors, who reported that readily developed DJ leave the carcass of the dead adult, in this study J2d emerged 12 h after cessation of egg laying. The density of both bacterial cultures decreased due to the feeding of the parental juveniles. However, X. nematophila continued at very low density, whereas the density of X. bovienii increased again until 15 days post-inoculation. The vast majority of F1 S. carpocapsae offspring developed to DJ, whereas in S. feltiae a significant second and third generation of adults was observed, probably due to the increasing bacterial population. However, second and third generation adults in S. feltiae cultures did not contribute significantly to the DJ yield. Mean yields of 158 × 103 DJ ml–1 were recorded for S. carpocapsae and 106 × 103 DJ ml–1 for S. feltiae. The results provide valuable information for future process improvement.

Olaf Strauch, John Mukuka and Ralf-Udo Ehlers

Abstract

The shelf life of biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora is rather short. In order to prolong shelf life, the metabolism of nematodes during storage must be reduced. This can be achieved by means of desiccation of the infective third-stage dauer juveniles (DJ). The tolerance can be increased by an adaptation to moderate desiccation conditions. Previous investigations indicate that the heritability of the desiccation tolerance is high, justifying a genetic selection for enhanced tolerance. This investigation screened the desiccation tolerance of 43 strains of Heterorhabditis spp. and 18 hybrid/inbred strains of H. bacteriophora. Dehydrating conditions measured as water activity (aw values) were produced by treating DJ with different concentrations of the non-ionic polymer poly(ethylene glycol) 600. Significant inter-specific variation was recorded between nematode strains and species. The mean tolerated aw value (MW50) ranged from 0.90 to 0.95 for non-adapted and 0.67 to 0.99 for adapted nematode populations. For selective breeding, only the 10% most tolerant individuals would be used. The lowest aw value tolerated by 10% of a population (MW10) ranged from of 0.845 to 0.932 for non-adapted nematode populations and 0.603 to 0.950 for adapted nematode populations. Adaptation significantly increased the desiccation tolerance and a weak correlation was recorded for tolerance with and without adaptations. The most tolerant nematode strains will form the basis for the foundation of a parental stock produced by cross-breeding and following genetic selection for enhanced tolerance. Thus, this investigation is another milestone on the road to domestication of H. bacteriophora for commercial use in sustainable pest management.

Farhana Ayub, Olaf Strauch, Laurent Seychelles and Ralf-Udo Ehlers

Life cycle analysis data of the free-living, bacterial-feeding Panagrolaimus sp. strain NFS 24-5 were assessed at different temperatures using a hanging drop method with single male and female individuals and a food density of 3 × 109 Escherichia coli cells ml−1. Lifespan at the moment when the first egg was laid was 5.7 days at 21°C and 4 days at 25, 27 and 29°C. The intrinsic rate of natural increase (rm) was 0.53 at 21°C, 0.81 at 25°C, 0.93 at 27°C and 0.81 at 29°C, corresponding to population doubling times (PDT=ln2rm) of 1.3, 0.9, 0.7 and 0.9 days, respectively. Over 200 offspring per female were produced at 27°C. All other temperatures yielded fewer offspring. When females were kept without males, the life span was 49 days, whereas the last reproductive female (hanging drop with male individual) died after 16.5 days. These data will contribute to the interpretation of nematode population dynamics recorded in liquid culture.

Temesgen Addis, Shiferaw Demissie, Olaf Strauch and Ralf-Udo Ehlers

Heterorhabditis bacteriophora, associated with Photorhabdus luminescens, is commonly used against insect pests. Dauer juveniles (DJ) develop into self-fertilising hermaphrodites that lay eggs until juveniles hatch inside the uterus and feed on the body content of the mother (endotokia matricida). The life history traits of H. bacteriophora were studied at 2.5 × 109, 5 × 109, 10 × 109 and 20 × 109 cells ml−1 of P. luminescens at 25°C using a hanging drop technique. The number of offspring produced per hermaphrodite increased from 50 at 2.5 × 109 cells ml−1 to 269 at 20 × 109 cells ml−1 of P. luminescens. The bacterial density did not influence the beginning of endotokia matricida, hermaphrodite death, DJ release from the maternal carcass and the percentage of juveniles obtained through endotokia matricida. Mating of automictic females could not increase offspring production and survival. Endotokia matricida is an obligatory developmental step in H. bacteriophora.

Temesgen Addis, Nevena Mijušković, Olaf Strauch and Ralf-Udo Ehlers

Using the hanging drop technique with nematode growth gelrite medium, life history traits of Steinernema yirgalemense (strain Sy 157-C) were investigated at a bacterial density of 10 × 109 cells ml−1 of Xenorhabdus indica at 25°C. With the same technique, the exit of dauer juveniles (DJ) from the arrested stage (recovery) was assessed at 5 × 109, 10 × 109 and 20 × 109 cells ml−1 of X. indica. Additionally, S. yirgalemense was incubated in nematode liquid medium at 25, 27 and 30°C. At each culture temperature, DJ recovery, sex ratio at 3 days post DJ inoculation and DJ yield and DJ as a percentage of non-DJ stages at 15 days post DJ inoculation were assessed. DJ survival in Ringer’s solution stored at 4, 15 and 25°C was assessed for 66 days. Steinernema yirgalemense has a total fertility rate and net reproductive rate of 487 and 314 offspring per female, respectively. The intrinsic rate of natural increase rm was 0.98 day−1, population doubling time PDT = 0.71 days and mean generation time T=5.72 days. The average lifespan of S. yirgalemense females starting from first-stage juveniles was 6.55 days. In liquid culture, DJ recovery ranged from 63-75% at 72 h post DJ inoculation and was not significantly different between the incubation temperatures. Parental male to female ratio was not influenced by incubation temperature and usually was at a ratio of 1:2. The percentage of females that entered into endotokia matricida at 72 h post DJ inoculation was 61% at 25°C, whereas at 27 and 30°C it was 24% and 0.5%, respectively. The highest DJ yield was recorded at 25°C (284 114 DJ ml−1) followed by 27°C (176 932 DJ ml−1) and the lowest at 30°C with 26 298 DJ ml−1. At a storage temperature of 4°C, DJ survival did not exceed 42 days, whereas at 15 and 25°C more than 95% of the DJ survived 66 days. Although S. yirgalemense DJ survived for long periods at both 15 and 25°C in liquid storage, their survival in formulated product and virulence after storage needs further investigation.

Farhana Ayub, Olaf Strauch, Laurent Seychelles and Ralf-Udo Ehlers

The nematode Panagrolaimus sp. NFS 24-5 has potential for use as living food for larval shrimps and fish in marine aquaculture. The nematodes are usually produced on bacterial or yeast cells. Nematodes cannot synthesise the long chain fatty acid docosahexaenoic acid (DHA) which is essential for feeding marine aquaculture organisms. The eukaryotic, heterotrophic dinoflagellate Crypthecodinium cohnii consists of approximately 20% DHA. To culture the nematodes and simultaneously enrich them with DHA, single adult male and female individuals were cultured in hanging drops with variable cell density of C. cohnii. Life history traits, such as net reproductive rate (R0), population doubling time (PDT) and intrinsic rate of natural increase (rm), were assessed and compared with data obtained from cultures on Escherichia coli. A maximum R0 was recorded at a cell density of 4 × 106 C. cohnii cells ml−1, corresponding to 2478.82 μg dry mass ml−1. The same was achieved with 7× lower biomass of E. coli at a cell density of 3 × 109 cells ml−1, corresponding to 335.63 μg dry mass ml−1. The results exclude the use of the dinoflagellate culture from application in mass production of the nematode for aquaculture food and limit the use to post-harvest enrichment of the nematodes with essential fatty acids. At a density of 3 × 109 E. coli cells ml−1 the PDT was lowest and the rm was highest, indicating that this cell density might be closest to optimum conditions for nematode reproduction. Exceeding this cell density yielded fewer offspring within a longer time period. Implications for mass production in monoxenic liquid cultures are discussed.

Prakaijan Nimkingrat, Felix Uhlmann, Olaf Strauch and Ralf-Udo Ehlers

For transport of entomopathogenic nematodes to the user, developmentally arrested dauer juveniles (DJ) are mixed with inert carriers at high density. If quiescence is not induced, DJ will quickly lose energy reserves and die. To induce quiescence DJ can be moderately desiccated. This study investigated the desiccation tolerance by measurement of water activity (aw-value) tolerated by 50% of populations (WA50) of different Steinernema species and strains. DJ were tested with or without prior adaptation to desiccation stress. Stress conditions were produced by exposure to various concentrations of poly(ethylene glycol) 600. Significant differences in desiccation tolerance were recorded between strains and species, but, except for Steinernema abbasi, not within strains of one species. Without adaptation to stress conditions, the most tolerant species was S. carpocapsae (WA50 = 0.836) followed by S. abbasi (0.86). Adaption to stress by exposure to an aw-value of 0.95 for 48 h increased tolerance to 0.68 and 0.66, respectively. The least tolerant species were S. kraussei, S. glaseri and S. ethiopiense. Tolerance recorded would allow storage at a water activity that would inhibit growth of bacteria but not of fungi. Analysis of water activity tolerated by only 10% of the population indicated potential for genetic improvement by selective breeding for S. carpocapsae, S. abbasi and S. arenarium to reach water activity levels that would also inhibit fungal growth.

Temesgen Addis, Asmamaw Teshome, Olaf Strauch and Ralf-Udo Ehlers

Life history traits (LHT) of Steinernema riobrave strain Sr 7-12 and Sr HYB19 were assessed at 25°C in monoxenic culture using a hanging drop technique. The LHT were studied with 5 × 109, 10 × 109 and 20 × 109 cells ml−1 of Xenorhabdus cabanillasii in semi-solid Nematode Growth Gelrite. Increased X. cabanillasii densities had a significant positive influence on offspring production on both Sr 7-12 and the hybrid Sr HYB19. At the higher bacterial food density, the total fertility rate (TFR) per female of Sr 7-12 was 2022 offspring and the net reproductive rate (R0) reached 1904 offspring. Similarly, for Sr HYB19, the TFR per female was 2434 and R0 was 1903. The percentage offspring produced via intra-uterine development (endotokia matricida) was relatively higher at 5 × 109 bacterial cells ml−1 than at higher bacterial density, with 64% and 66% of the total offspring produced by Sr 7-12 and Sr HYB19, respectively. A positive correlation (R=0.648) was recorded for offspring production and bacterial food density and for female body volume with bacterial density (R=0.610). Female nematodes survived longer at higher bacterial food density; however, females of both strains could not survive longer than 7.8 days, beginning from hatching and always ending 1 day after initiation of endotokia matricida. Based on LHT results, there is a potential to maximise yield of dauer juveniles in liquid culture through increasing bacterial food density.

Peter Jessen, Reiner Luttmann, Ralf-Udo Ehlers, Olaf Strauch and Urs Wyss

Abstract

Heterorhabditis spp. (Rhabditida: Nematoda) live in a close symbiosis with the bacterium Photorhabdus luminescens. For biocontrol purposes the nematodes are produced in liquid culture pre-incubated with P. luminescens. The bacteria produce a food signal, inducing dauer juveniles (DJ) to initiate development. In rhabditid nematodes the exit from this developmentally arrested third stage DJ is called recovery. Attempts to produce Heterorhabditis spp. in liquid culture have often failed due to low and delayed recovery of the inoculated DJ. The influence of carbon dioxide as a recovery co-factor was investigated. Increasing concentrations of CO2 enhanced DJ recovery in the presence of the bacterial food signal. The effect could not be related to a decline of the pH caused by increasing CO2 concentrations. On the contrary, at lower pH the DJ recovery decreased. In one experiment a considerable spontaneous recovery was observed in the absence of a food signal. This phenomenon and a variable threshold response of the DJ to CO2 lead to the assumption that they are differently pre-disposed to respond to recovery inducing signals. Providing the results can be confirmed in laboratory scale bioreactors, the control of carbon dioxide during nematode liquid culture can help to improve the bioreactor process technology.Heterorhabditis spp. (Rhabditida: Nematoda) leben in enger Symbiose mit dem Bakterium Photorhabdus luminescens. Für die biologische Bekämpfung werden die Nematoden in Flüssigkulturen vermehrt, die vorher mit P. luminescens inkubiert wurden. Die Bakterien produzieren ein Nahrungssignal, das die Dauerlarven (DJ) veranlasst, ihre Entwicklung wieder aufzunehmen. Bei rhabditiden Nematoden wird das Verlassen des entwicklungsphysiologisch gehemmten Dauerlarvenstadiums als “recovery” bezeichnet. Versuche, Heterorhabditis spp. in Flüssigkultur zu produzieren sind oft aufgrund einer niedrigen oder verspäteten “recovery” gescheitert. Der Einfluß von Kohlendioxid als Einflussfaktor auf die “recovery” wurde untersucht. Zunehmende CO2 Konzentrationen förderten die “recovery” bei Anwesenheit des Nahrungssignals. Einem mit zunehmender CO2-Konzentration fallenden pH-Wert konnte die Wirkung nicht zugeschrieben werden. Im Gegenteil, bei niedrigen pH-Werten nahm die “recovery” ab. In einem Experiment wurde eine spontane “recovery” beobachtet, ohne dass ein Nahrungssignal vorhanden war. Dieses Phänomen und die variable Antwort der Dauerlarven auf gleiche CO2-Konzentrationen lassen den Schluss zu, dass die Dauerlarven unterschiedlich prädisponiert sind in ihrer Reaktion auf die “recovery” induzierenden Signale. Vorausgesetzt die Ergebnisse können in LaborBioreaktoren bestätigt werden, ist die Regelung des Kohlendioxidgehalts während der Nematoden-Flüssigkultur eine Hilfe die Prozesstechnik zu optimieren.

Alper Susurluk, Urs Wyss, Ralf-Udo Ehlers, Olaf Strauch, Erko Stackebrandt and Ilona Dix

Abstract

Two heterorhabditid nematode strains (TUR-H1 and TUR-H2), and their bacterial symbionts isolated from soil samples taken at the campus of the Agriculture Faculty of the University of Ankara, Turkey, were identified by molecular methods and by cross-breeding with Heterorhabditis bacteriophora. The bacterial symbionts shared >99% similarity in the 16S rDNA sequence with Photorhabdus luminescens subsp. laumondii. Results of the restriction fragment length analysis of the ITS region assigned both nematode strains to the species H. bacteriophora. Cross-breeding confirmed the species designation for strain TUR-H2. Crosses of TUR-H1 with a hybrid strain of H. bacteriophora or with TUR-H2 resulted in infertile offspring. Both strains reproduced in monoxenic cultures of the symbionts P.luminescens isolated from H. bacteriophora and H. megidis and the resulting dauer juveniles retained cells of the bacteria. Reproduction on the symbionts isolated from H. indica failed. Infectivity at variable soil moisture and heat tolerance of the two heterorhabditid strains was compared with a Turkish isolate of Steinernema feltiae. Significantly more nematodes invaded the insect Galleria mellonella in a sandy soil assay at 10% water content than at lower values. A higher water content significantly reduced the invasion rate. Steinernema feltiae was better adapted to a temperature of 32°C than the heterorhabditid strains. Hardly any nematodes of all strains survived for longer than 4 h at 36°C.