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Ralf-Udo Ehlers and Richou Han

Abstract

Mixed culture filtrates of Photorhabdus luminescens isolated from Heterorhabditis bacteriophora H06 and from H. indica LN2 had a toxic effect on axenic H. bacteriophora H06 dauer juveniles. Single culture filtrates had no effect. When one filtrate originated from a secondary phase culture, the toxic effect was lost. Heat treatment of one of the filtrates to 80°C destroyed the effect. The toxin is probably synthesized de novo after mixing the culture supernatants of these two P. luminescens symbionts. Dilution of the filtrate reduced the effect, and it was lost when P. luminescens was cultured for more than two days. Steinernema carpocapsae A24 was not affected by the mixture, and was affected only by the filtrate of primary phase P. luminescens H06. The toxic effect was recorded also when axenic dauer juveniles of H. bacteriophora were inoculated into a mixed bacterial culture of H06 and LN2. Inoculating monoxenic dauer juveniles of H. bacteriophora H06 into P. luminescens LN2 or into mixtures containing LN2 bacteria resulted in significant dauer juvenile mortality. These manifestations of the interaction of bacteria to produce toxic effects on the non-symbiotic nematode (trans-specific activity) may have an impact on competitive interactions when one insect host is infected by different nematode species. Eine transspezifische nematizide Aktivitat von Photorhabdus luminescens - Eine Mischung der Kulturfiltrate des Bakteriums Photorhabdus luminescens, isoliert aus Heterorhabditis bacteriophora H06 mit dem Filtrat des Symbionten aus H. indica LN2 wirkt toxisch auf axenische Dauerlarven von H. bacteriophora H06. Die einzelnen Kulturfiltrate zeigten keine Wirkung. Sofern ein Filtrat einer Sekundarformkultur entnommen wurde, konnte kein Effekt erzielt werden. Eine Hitzebehandlung bei 80°C zerstorte die Wirkung. Das Toxin wird wahrscheinlich de novo synthetisiert in dem Moment, in dem die Kulturfiltrate dieser P. luminescens Symbionten gemischt werden. Eine Verdunnung der Filtrate reduzierte die Wirkung. Sie ging ganz verloren, wenn P. luminescens langer als zwei Tage kultiviert wurde. Die Mischung hatte keine Wirkung auf axenische Steinernema carpocapsae. Dieser Nematode wurde nur durch Filtrate der Primarform von P. luminescens H06 abgetotet. Die toxischen Effekte wurden auch festgestellt, wenn axenische Dauerlarven von H. bacteriophora in Gemische von H06 und LN2 Bakterienkulturen inokuliert wurde. Bei Inokulation von monoxenischen Dauerlarven von H. bacteriophora H06 in P. luminescens LN2 oder in Gemische, die LN2 enthielten, wurde ebenfalls eine gesteigerte Mortalitat festgestellt. Dieses Deutlichwerden von Wechselbeziehungen zwischen den Bakterien bei Erzeugung toxischer Effekte auf den nicht-symbiontischen Nematoden (trans-spezifische Aktivitat) konnte eine Auswirkung auf die Konkurrenz bei Koinfektion eines Wirtsinsekts mit zwei Nematodenarten haben.

Ralf-Udo Ehlers and Jens Aumann

Abstract

Recovery in entomopathogenic nematodes is the exit from the dauer juvenile stage. It is a response to environmental queues signalling the presence of food sources (e.g., insect haemolymph). The bacterium Photorhabdus luminescens excretes a signal which also induces recovery of its symbiotic Heterorhabditis bacteriophora dauer juveniles. This bacterial signal is composed of at least two compounds with different polarity. The symbiotic bacteria also secrete an antagonistic signal which inhibits nematode recovery. The recovery-inducing signal compounds have a molecular mass of less than 20 kDa and are negatively charged. The data indicate that at least one compound is smaller than 5 kDa. The bacterial signal triggers by receptor binding, the first step in a recovery-inducing muscarinic signalling pathway.

Hilke Honnens and Ralf-Udo Ehlers

Free-living nematodes have potential to be used as live food for early life stages of several species in marine aquaculture. Panagrolaimus sp. displays several favourable characteristics for this application. The present study proved the feasibility of propagation in monoxenic liquid culture on Saccharomyces cerevisiae. The development of yeast cell density, nematode numbers and size distribution was assessed daily for 15 days. After a lag phase of 4 days the inoculated first-stage juveniles started development to adults. Yields in terms of nematode number as well as biomass were highly variable. The maximum number of nematodes varied from 45 000 to 238 000 ml−1 and maximum biomass from 49 to 143 g l−1. Information on size, dry and wet weight of the nematodes is provided. The size spectrum of Panagrolaimus sp. individuals ranged from 176 × 8 μm to 1377 × 61 μm and 8.15 to 3202.39 ng wet weight. Water content of the nematodes was 71.7 ± 2.5%, so dry weight per individual was 2.31-905.95 ng. Differentiation of juvenile stages by body length was not possible. Based on comparison of dry weight per individual the Panagrolaimus sp. might be used as a substitute for rotifers, a commonly used live food organism.

Stefan-Andreas Johnigk and Ralf-Udo Ehlers

Abstract

Heterorhabditis bacteriophora and H. indica were cultured in vitro in monoxenic liquid cultures. Nematodes were examined for morphological characters which can be used to determine the sexes in all juvenile stages. When hatching from the egg no sex-specific characters could be distinguished. Twelve hours later sex determination resulted in male phenotypes which were identified by the asymmetric shape of the primordial testis. Female phenotypes, with symmetrical primordial gonads, were observed 12 h later. Of the eggs laid by the parental hermaphrodite 57% developed to amphimictic adults. The other individuals were determined to become automictic dauer juveniles which further develop to hermaphrodites. The pre-dauer J1 was the last of the first juvenile stages to be determined. They moult to the pre-dauer second stage juvenile (J2D) which is distinguished from the other J2 stages by a thin and spindle-like shape, elongated pharynx and needle-like tip of the tail. The late JD2 stage is immobile, pharyngeal pumping ceases and intercalated fat reserves make it appear darker then the amphimictic J2. The further development to adults is described and the occurrence of the different stages in liquid culture documented. Developpement larvaire et cycle biologique d'Heterorhabditis bacteriophora et H. indica (Nematoda: Heterorhabditidae) - Heterorhabditis bacteriophora and H. indica ont ete eleves in vitro sur milieux liquides monoxeniques. Les nematodes ont ete etudies pour les caracteres morphologiques qui pourraient etre utilises dans la determination des sexes dans tous les stades juveniles. A l'eclosion, aucun caractere sexuel specifique n'a pu etre identifie. Douze heures plus tard, le dimorphisme sexuel se revele avec des phenotypes males caracterises par la forme asymetrique du testicule. Les phenotypes femelles avec des primordium symetriques ont ete observes 12 h plus tard. 57% des individus issus des oeufs de parent hermaphrodite se sont developpes en adulte amphimictique. Les autres individus se sont developpes en dauerlarva automictiques qui se sont plus tard developpes en hermaphrodites. La pre-dauerlarvae (J1) a ete le dernier des stades juveniles a etre identifie. Ils ont mue en stade pre-dauer J2 qui peut etre differencie des autres stades J2 par le corps mince et fusiforme, un pharynx allonge et l'extemite de la queue tres effilee. Le dernier stade DJ2 est immobile, le pompage pharyngien cesse et des reserves lipidiques interstitielles le rendent plus sombre que chez le stade amphimictique J2. Le developpement jusqu'a l'adulte est decrit et l'apparition de differents stades en milieu de culture liquide est detaillee.

Hilke Honnens, Thomas Assheuer and Ralf-Udo Ehlers

Panagrolaimus sp. strain NFS-24-5 has potential to be used as live food for early stages of fish and crustacean species in marine aquaculture. One constraint to its commercialisation is the lack of a method that enables storage of nematodes over a longer time span. The objective of this study was to develop a procedure to transfer nematodes into a dormant state by desiccation. The nematodes were concentrated at densities of 25, 50, 100 and 200 × 103 individials cm−2 on nylon net or cellulose paper, preconditioned for 72 h at 97.3% relative humidity (RH) and then stored at 52.9 or 32.8% RH for 1 week. Cellulose was a better carrier for the nematodes. Survival of the nematodes was reduced only at the highest nematode density on both materials. The water activity of desiccated nematodes was 0.44 and 0.33 at 52.9% and 32.8% RH, respectively, well beyond a point to prevent microbial growth. After storage over a period of 10 weeks at 25 × 103 nematodes cm−2 at 52.9 and 32.8% RH, 92% of the nematodes were still alive. Monitoring the size distribution revealed no changes at 52.9% RH, but there were more of the larger nematodes dying at 32.8% RH in two out of three experiments. The method can be used to store quiescent Panagrolaimus sp. (strain NFS-24-5) for transportation and use in small scale feeding experiments for marine fish and crustacean larvae.

Mudassir Iqbal, Ralf-Udo Ehlers and Lieven Waeyenberge

Entomopathogenic nematodes belong to the families of Steinernematidae and Heterorhabditidae. They are obligate and lethal parasites of insects that can provide effective control of some important pests of commercial crops. A total of 53 isolates of EPN were molecularly characterised (ITS region-based) in the present study. Most of the studied isolates belong to the Steinernema genus and only few isolates belong to the Heterorhabditis genus. The phylogenetic relations of Steinernema and Heterorhabditis species were analysed by utilising the maximum likelihood method. In the Steinernema phylogenetic tree, 99 isolates formed five major, moderately or highly reinforced clades: clade I: affine-intermedium group; clade II: carpocapsae-siamkayai-tami-scapterisci; clade III: bicornutum-riobrave-thermophilum; clade IV: glaseri-arenarium-karii-longicaudum; and clade V: feltiae-schliemanni-kushidai-kraussei-oregonense. The BLAST analysis of the ITS region of the rDNA of the steinernematid isolate PAL10 showed a rather low similarity of 93% with S. vulcanicum (accession number: GU929442), supporting the possible designation of a new species. In the Heterorhabditis phylogenetic tree, 25 isolates formed three main clades: clade I: bacteriophora-argentinensis-hepialius; clade II: baujardi-sonorensis-amazonensis; and clade III: indica-brevicaudis-hawaiiensis. All five studied isolates of Heterorhabditis were identified as H. indica and H. bacteriophora. In both phylogenetic trees, the intra-specific variability level was different among clades for some species. The description of the new species (PAL10 isolate) would need further morphometric characterisation, morphologically identification and cross-breeding studies.

Stefan-Andreas Johnigk and Ralf-Udo Ehlers

Abstract

Intra-uterine birth causing maternal death (endotokia matricida) is relatively common in rhabditid nematodes and typical for entomopathogenic nematodes of the genus Heterorhabditis. A detailed description of this phenomenon is given, including a summary of embryo development, egg-laying, juvenile hatching and development. It is demonstrated that low food supply significantly promotes the beginning of endotokia matricida but has no influence on the time scale of the process. The biological function of endotokia matricida and the intra-uterine induction of the dauer formation is discussed. Endotokia matricida in Heterorhabditis spp. is a well synchronised process of the destruction of the maternal tissues correlated with the juvenile development including the uptake of the symbiotic bacteria by the late pre-dauer stage. It secures the development of dauer juveniles at a moment when the external food supply is reducing and provides offspring which are well equipped with energy reserves and symbiotic bacteria for long term survival and subsequent infection of insects in the soil. Endotokia matricida bei Hermaphroditen von Heterorhabditis spp. und der Einfluss der Nahrungsversorgung . - Der Schlupf juveniler Nematoden im mutterlichen Uterus mit anschliessender Totung des Muttertieres (endotokia matricida) ist relativ weit verbreitet bei rhabditiden Nematoden und typisch fur entomopathogene Nematoden der Gattung Heterorhabditis. Die Endotokia matricida wird beschrieben sowie die Embryonalentwicklung, das Eilegeverhalten, der Schlupf und die Entwicklung der Juvenilen. Der Beginn der endotokia matricida wird durch eine niedrige Nahrungsverfugbarkeit in der Kultur signifikant gefordert, diese hat jedoch keinen Einfluss auf den zeitlichen Ablauf der endotokia matricida. Die biologische Funktion der endotokia matricida und die intra-uterine Induktion der Dauerlarvenbildung werden diskutiert. Die endotokia matricida bei Heterorhabditis spp. ist ein gut synchronisierter Prozess, bei dem die Zerstorung des Muttertiers korreliert mit der Entwicklung der Juvenilstadien, einschliesslich der Aufnahme der symbiotischen Bakterien durch das Pra-Dauer Stadium. Sie sichert die Entwicklung von Dauerlarven zu einem Zeitpunkt, wenn die externe Nahrungsversorgung sich verschlechtert und bringt so Nachkommen hervor, die gut mit Energiereserven und symbiotischen Bakterien ausgestattet sind, um im Boden lange zu uberdauern und Insekten zu befallen.

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.

Sibylle Schroer, Ralf-Udo Ehlers and Xiaoli Yi

Abstract

Increasing resistance to chemical insecticides in field populations of the diamondback moth (Plutella xylostella) has stimulated research on alternative control measures. The entomopathogenic nematode Steinernema carpocapsae may be one such alternative, particularly against the third larval instar of P. xylostella. The LC50 for the second instar is 38, the third 13 and the fourth 22 nematodes/larva. Plutella xylostella pupae were not affected by the nematodes, although mortality in leaf disk bioassays after application of nematodes in water seldom surpassed 50%. Therefore, additives were tested to improve nematode performance. Only Triton X-100 (0.3%) caused phytotoxic effects. The addition of xanthan gum or potassium alginate resulted in a two-fold increase of insect mortality at 80% relative humidity (RH) and a five-fold increase at 60% RH. Mixtures of 0.3% xanthan or alginate with 0.3% surfactants further improved efficacy. In water the LT50 for S. carpocapsae against P. xylostella larvae was > 40 h. Using a mixture of 0.3% xanthan or 0.3% alginate with 0.3% surfactant, the LT50 was reduced to < 25 h.