Effects of host desiccation on development, survival, and infectivity of entomopathogenic nematode Steinernema carpocapsae

This study investigates the effect of host desiccation on entomopathogenic nematode (EPN) development, emergence, infectivity, and cross-protection against secondary environmental stress. Galleria mellonella hosts infected with the EPN Steinernema carpocapsae A10 were allowed to dehydrate in an environmental chamber for up to 56 days at 23 degrees C achieving a weight loss of approximately 86% by day 44 post-infection. Host carcasses were rehydrated on water-saturated filter paper in White traps to collect emergent infective juveniles (IJ) at specific time intervals. Populations were counted with an apparent peak coinciding with desiccated hosts rehydrated at 24-day post-infection. Desiccation-stressed IJ populations from each time interval were tested for infectivity, and cross-resistance to secondary temperature and pH stresses and were found to have significant increases in both infectivity and protection from extremes of temperature and pH compared with controls. Total aqueous soluble protein profiles from control and desiccation-stressed IJs were analyzed using 10% SDS Laemmli gels. Several novel proteins were over-expressed in EPN from hosts subjected to desiccation suggesting the induction and expression of stress response genes.     

Susceptibility and behavioral responses of the dampwood termite Zootermopsis angusticollis to the entomopathogenic nematode Steinernema carpocapsae

Termites exploit microbially rich resources such as decayed wood and soil that are colonized by potentially pathogenic and parasitic fungi, bacteria, viruses, and nematodes. In colonies composed of thousands of individuals, the risk of infection among nestmates is significant, and individual and social behavior could involve various adaptations to resist disease and parasitism. Here we show that the dampwood termite Zootermopsis angusticollis (Hagen) exhibits a dosage dependent susceptibility to the soil nematode Steinernema carpocapsae (Weiser) (Mexican strain) and that this social insect significantly alters its behavior in response to this entomopathogenic roundworm. Relative to their baseline behavior, termites exposed to infective juveniles increased the frequency and duration of allogrooming and vibratory displays as well as two other novel behaviors, abdominal tip-raising and self-scratching. Whereas the first two behaviors likely reflect general adaptations to reduce susceptibility to a variety of pathogens and parasites, the latter behaviors might be specific to nematodes because they have never been observed in Z. angusticollis in any other pathogenic context. Our results support the hypotheses that behavioral responses in termites are important in the control of pathogenic and parasitic microorganisms and that termite susceptibility is socially mediated.     

Impacts of fluctuating temperature on the development and infectivity of entomopathogenic nematode Steinernema carpocapsae A10

Three different laboratory conditions were used to examine the impacts of fluctuating temperature on the development and infectivity of entomopathogenic nematode (EPN) Steinernema carpocaposae A10. Set I experiments focused on the impact of cold stress early in the development cycle. In these studies Galleria mellonella hosts were infected and incubated for 2 days at the control temperature of 23 degrees C and then subjected to lower temperatures of -10, 4, 10 or 14 degrees C, respectively, from days 3 to 36 post-infection (PI). Dissections of infected cadavers indicated arrested development at the adult stage at all lower temperatures tested. Set II experiments examined the impacts of cold stress early in the development followed by a return to 23 degrees C. Hosts were infected and incubated as in Set I and subjected to the same temperatures as above for 7 days, followed by incubation at 23 degrees C until 23 days PI. A limited number of EPN populations were able to complete development at 10 and 14 degrees C though emergent population numbers were significantly lower than those of control infections incubated continuously at 23 degrees C. In Set III experiments, infected hosts were subjected to cold stress later during development starting at day 4 post-infection followed by incubation at the control temperature. Population survival past first and second stage juveniles was reduced by at least 95% or more at the lower temperatures compared with controls. Emergent populations from the Set III cold-stressed hosts were not infectious. These studies may provide insights as to how EPN survive seasonal temperature fluctuations under natural environmental conditions.     

Influence of nematode age and culture conditions on morphological and physiological parameters in the bacterial vesicle of Steinernema carpocapsae (Nematoda: Steinernematidae)

Steinernema spp. third-stage infective juveniles (IJs) play a key role in the symbiotic partnership between these entomopathogenic nematodes and Xenorhabdus bacteria. Recent studies suggest that Steinernema carpocapsae IJs contribute to the nutrition and growth of their symbionts in the colonization site (vesicle) [Martens, E.C. and Goodrich-Blair, H., 2005. The S. carpocapsae intestinal vesicle contains a sub-cellular structure with which Xenorhabdus nematophila associates during colonization initiation. Cellular Microbiol. 7, 1723-1735.]. However, the morphological and physiological interactions between Xenorhabdus symbionts and Steinernema IJs are not understood in depth. This study was undertaken to assess the influence of culture conditions and IJ age on the structure, nutrition, and symbiont load (colonization level) of S. carpocapsae vesicles. Our observations indicate the vesicles of axenic IJs are shorter and wider than those of colonized IJs. Moreover, as colonized IJs age the vesicle becomes shorter and narrower and bacterial load declines. The colonization proficiency of several bacterial metabolic mutants was compared between two cultivation conditions: in vitro on lipid agar and in vivo in Galleria mellonella insects. Colonization defects were generally less severe in IJs cultivated in vivo versus those cultivated in vitro. However, IJs from both cultivation conditions exhibited similar declining bacterial load over time. These results suggest that although the vesicle forms in the absence of bacteria, the presence of symbionts within the vesicle may influence its fine structure. Moreover, these studies provide further evidence in support of the concept that the conditions under which steinernematid nematodes are cultivated and stored affect the nutritive content of the vesicle and the bacterial load, and therefore have an impact on the quality of the nematodes for their application as biological control agents.     

Storage temperature influences desiccation and ultra violet radiation tolerance of entomopathogenic nematodes

1. The effect of cold (5 °C) and warm (35 °C) storage on desiccation tolerance of cold-adapted Steinernema feltiae, intermediate S. carpocapsae and warm-adapted S. riobrave was evaluated at 5 and 35 °C.     

Susceptibility of the peachtree borer, Synanthedon exitiosa, to Steinernema carpocapsae and Steinernema riobrave in laboratory and field trials

The nematode Steinernema carpocapsae (All) strain was significantly more effective against peachtree borer larvae (Synanthedon exitiosa [Lepidoptera: Sesiidae]) than Steinernema riobrave (7-12) strain in field and laboratory experiments. Eighty-eight percent control of peachtree borer larvae was obtained with S. carpocapsae in the field trial when applied at 3 x 10(5) infective juveniles per tree, and 92% mortality was obtained in the lab assay using 50 infective juveniles per larva.     

Characteristics of protectant synthesis of infective juveniles of Steinernema carpocapsae and importance of glycerol as a protectant for survival of the nematodes during osmotic dehydration

Two hypotheses on the synthesis of the protectants glycerol and trehalose of the infective juveniles (IJs) of Steinernema carpocapsae during osmotic dehydration were tested and utilised to evaluate the function and importance of glycerol on survival of the nematodes during osmotic dehydration. This was achieved by comparing the changes in survival, morphology, behaviour and levels of glycerol, trehalose and permeated compounds of the IJs dehydrated in seven hypertonic solutions at two temperature regimes: (1) 5 °C for 15 days; and (2) 23 °C for 1 day followed by 5 °C for another 14 days. The results substantiate both hypotheses tested: (1) the permeability of the IJs to various compounds, such as sucrose or ethylene glycol, when they are dehydrated in hypertonic solutions of these compounds; and (2) suppression of the synthesis of protectant glycerol but not trehalose when IJs are dehydrated at low temperature. The results also showed that: (1) although trehalose was the preferred dehydration protectant, glycerol played an important role in rapidly balancing the osmotic pressure when IJs were exposed in hypertonic solutions; (2) the presence of glycerol was essential for the IJs to survive and function properly even under moderate osmotic dehydration, especially when IJs were dehydrated in salt solutions; and (3) some exogenous compounds permeated into IJs during osmotic dehydration such as ethylene glycol, may function in the same way as glycerol and significantly improve the survival and function of the IJs. The results indicate that each of the protectants glycerol and trehalose has a specific function and neither is replaceable by the other.     

Interactions of two idiobiont parasitoids (Hymenoptera: Ichneumonidae) of codling moth (Lepidoptera: Tortricidae) with the entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae)

Simultaneous use of parasitoids and entomopathogenic nematodes for codling moth (CM) control could produce an antagonistic interaction between the two groups resulting in death of the parasitoid larvae. Two ectoparasitic ichneumonid species, Mastrus ridibundus and Liotryphon caudatus, imported for classical biological control of cocooned CM larvae were studied regarding their interactions with Steinernema carpocapsae. Exposure of M. ridibundus and L. caudatus developing larvae to infective juveniles (IJs) of S. carpocapsae (10 IJs/cm2; approximately LC(80-90) for CM larvae) within CM cocoons resulted in 70.7 and 85.2% mortality, respectively. However, diapausing full grown parasitoid larvae were almost completely protected from nematode penetration within their own tightly woven cocoons. M. ridibundus and L. caudatus females were able to detect and avoid ovipositing on nematode-infected cocooned CM moth larvae as early as 12h after treatment of the host with IJs. When given the choice between cardboard substrates containing untreated cocooned CM larvae and those treated with an approximate LC95 of S. carpocapsae IJs (25 IJs/cm2) 12, 24, or 48h earlier, ovipositing parasitoids demonstrated a significant preference for untreated larvae. The ability of these parasitoids to avoid nematode-treated larvae and to seek out and kill cocooned CM larvae that survive nematode treatments enhances the complementarity of entomopathogenic nematodes and M. ridibundus and L. caudatus.     

Phoresy of the entomopathogenic nematode Steinernema feltiae by the earthworm Eisenia fetida

The free-living stage of entomopathogenic nematodes occurs in soil, and is an environmental-friendly alternative for biological control. However, their dispersal capability is limited. Earthworms improve soil characteristics, changing soil structure and influencing many edaphic organisms. Thus, earthworms could be used as vectors to introduce/disperse beneficial organisms. Nevertheless this interaction has not been studied in detail. This study presents the infectivity results of Steinernema feltiae after passing through the Eisenia fetida gut. Although entomopathogenic nematodes have no deleterious effects on earthworms, their passage through E. fetida gut seriously affected their mobility and virulence.     

Outcrossing and crossbreeding recovers deteriorated traits in laboratory cultured Steinernema carpocapsae nematodes

The nematode Steinernema carpocapsae infects and kills many pest insects in agro-ecosystems and is commonly used in biocontrol of these pests. Growth of the nematodes prior to distribution for biocontrol commonly results in deterioration of traits that are essential for nematode persistence in field applications. To better understand the mechanisms underlying trait deterioration of the efficacy of natural parasitism in entomopathogenic nematodes, we explored the maintenance of fitness related traits including reproductive capacity, heat tolerance, virulence to insects and ‘tail standing’ (formerly called nictation) among laboratory-cultured lines derived from natural, randomly mating populations of S. carpocapsae. Laboratory cultured nematode lines with fitness-related trait values below wild-type levels regained wild-type levels of reproductive and heat tolerance traits when outcrossed with a non-deteriorated line, while virulence and ‘tail standing’ did not deteriorate in our experiments. Crossbreeding two trait-deteriorated lines with each other also resulted in restoration of trait means to wild-type levels in most crossbred lines. Our results implicate inbreeding depression as the primary cause of trait deterioration in the laboratory cultured S. carpocapsae. We further suggest the possibility of creating inbred lines purged of deleterious alleles as founders in commercial nematode growth.     

Ecological characterization of entomopathogenic nematodes isolated in stone fruit orchard soils of Mediterranean areas

Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae were isolated from stone-fruit orchards in two Mediterranean regions of Spain. A total of 630 soil samples (210 sites) from Catalonia and 90 soil samples (30 sites) from Murcia were evaluated resulting in 5.2% and 20% of the soils testing positive for nematodes, respectively. Ten steinernematid isolates and three heterorhabditid isolates were recovered using the Galleria mellonella baiting method. Based on morphometric data, molecular data, and cross-breeding experiments the nematode species were identified as Steinernemafeltiae and Heterorhabditis bacteriophora. Environmental tolerance to heat, desiccation and hypoxia, the effect of temperature on infectivity and reproduction and nematode migration in sand columns were compared among isolates and one Steinernema carpocapsae strain. Results showed differences among species and a great variability within species. Beneficial traits for each strain were added up to identify a superior candidate to control Mediterranean flat-headed rootborer, Capnodis tenebrionis. When all analyzed factors were considered, three S. feltiae isolates (Bpa, Sor and M116) obtained the best scores, and when hypoxia was removed, two of the strains (Bpa and Sor) continued ranking superior to other strains.     

Infection of the Entomopathogenic Nematode,Steinernema carpocapsae,as Affected by the Presence of Steinernema glaseri

The infection behavior of Steinernema carpocapsae infective juveniles (IJ) was investigated in the presence and absence of S. glaseri. Mixed inoculation of S. carpocapsae with S. glaseri IJ significantly raised the nictation rates of S. carpocapsae IJ. Significantly more S. carpocapsae IJ migrated to the host insect in the mixed inoculation with S. glaseri IJ on agar plates. More S. carpocapsae IJ penetrated into the host insect placed 2 cm below the surface in the mixed inoculation with S. glaseri IJ. More S. glaseri than S. carpocapsae IJ penetrated into hosts placed 7 cm deep. Irrespective of host location, the male ratio of S. carpocapsae IJ established in the host body was always higher in the mixed inoculation with S. glaseri IJ.     

Acclimation of entomopathogenic nematodes to novel temperatures: trehalose accumulation and the acquisition of thermotolerance

The effect of thermal acclimation on trehalose accumulation and the acquisition of thermotolerance was studied in three species of entomopathogenic nematodes adapted to either cold or warm temperatures. All three Steinernema species accumulated trehalose when acclimated at either 5 or 35 degrees C, but the amount of trehalose accumulation differed by species and temperature. The trehalose content of the cold adapted Steinernema feltiae increased by 350 and 182%, of intermediate Steinernema carpocapsae by 146 and 122% and of warm adapted Steinernema riobrave by 30 and 87% over the initial level (18.25, 27.24 and 23.97 microg trehalose/mg dry weight, respectively) during acclimation at 5 and 35 degrees C, respectively. Warm and cold acclimation enhanced heat (40 degrees C for 8h) and freezing (-20 degrees C for 4h) tolerance of S. carpocapsae and the enhanced tolerance was positively correlated with the increased trehalose levels. Warm and cold acclimation also enhanced heat but not freezing tolerance of S. feltiae and the enhanced heat tolerance was positively correlated with the increased trehalose levels. In contrast, warm and cold acclimation enhanced the freezing but not heat tolerance of S. riobrave, and increased freezing tolerance of only warm acclimated S. riobrave was positively correlated with the increased trehalose levels. The effect of acclimation on maintenance of original virulence by either heat or freeze stressed nematodes against the wax moth Galleria mellonella larvae was temperature dependent and differed among species. During freezing stress, both cold and warm acclimated S. carpocapsae (84%) and during heat stress, only warm acclimated S. carpocapsae (95%) maintained significantly higher original virulence than the non-acclimated (36 and 47%, respectively) nematodes. Both cold and warm acclimated S. feltiae maintained significantly higher original virulence (69%) than the non-acclimated S. feltiae (0%) during heat but not freezing stress. In contrast, both warm and cold acclimated S. riobrave maintained significantly higher virulence (41%) than the non-acclimated (14%) nematodes during freezing, but not during heat stress. Our data indicate that trehalose accumulation is not only a cold associated phenomenon but is a general response of nematodes to thermal stress. However, the extent of enhanced thermal stress tolerance conferred by the accumulated trehalose differs with nematode species.     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.     

Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.     

Simulated roots and host feeding enhance infection of subterranean insects by the entomopathogenic nematode Steinernema carpocapsae

Steinernema carpocapsae can be effective against root-feeding insects despite its reputation as a sedentary ambusher. In pot experiments, using twigs as surrogate roots and pine weevil larvae as targets, we tested the hypothesis that roots serve as physical routeways and conduits of feeding-associated stimuli, thus enhancing the success of S. carpocapsae applied at the surface against subterranean hosts. Insect mortality was lowest (25%) in the absence of plant material, increased to 48% when twigs linked nematodes and insects, and further increased to 69% when the insects were allowed feed on the twigs. This is the first experimental support for the root-routeway hypothesis.     

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode,Steinernema riobrave (Rhabditida: Steinernematidae)

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.     

The Complete Mitochondrial Genome of the Entomopathogenic Nematode Steinernema carpocapsae: Insights into Nematode Mitochondrial DNA Evolution and Phylogeny

We determined the complete sequence of the mitochondrial DNA of the entomopathogenic nematode Steinernema carpocapsae and analyzed its structure and composition as well as the secondary structures predicted for its tRNAs and rRNAs. Almost the complete genome has been amplified in one fragment with long PCR and sequenced using a shotgun strategy. The 13,925-bp genome contains genes for 2 rRNAs, 22 tRNAs, and 12 proteins and lacks an ORF encoding ATPase subunit 8. Four initiation codons were inferred, TTT, TTA, ATA, and ATT, most of the genes ended with TAA or TAG, and only two had a T as an incomplete stop codon. All predicted tRNAs showed the nonconventional secondary structure typical of Secernentea. Although we were able to fold the sequences of trnN, trnD, and trnC into more conventional cloverleaf structures after adding adjacent nucleotides, northern blot experiments showed that the nonstandard tRNAs are actually expressed. Phylogenetic and comparative analyses showed that the mitochondrial genome of S. carpocapsae is more closely related to the genomes of A. suum and C. elegans than to that of Strongyloides stercoralis. This finding does not support the phylogeny based on nuclear small subunit ribosomal DNA sequences previously published. This discrepancy may result from differential reproductive strategies and/or differential selective pressure acting on nuclear and mitochondrial genes. The distinctive characteristics observed among mitochondrial genomes of Secernentea may have arisen to counteract the deleterious effects of Muller’s ratchet, which is probably enhanced by the reproductive strategies and selective pressures referred to above. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Abscisic acid and the induction of desiccation tolerance in the extremely xerophilic liverwortExormotheca holstii

Abscisic acid (ABA) induces formation of a set of proteins in the xerophilic liverwortExormotheca holstii. Some of them have immunological properties similar to the dehydrins of desiccated corn embryos and the desiccation-related proteins ofCraterostigma plantagineum. The fluctuations of endogenous ABA during cycles of desiccation and rehydration seem to be sufficiently high to indicate a role for ABA as a stress hormone and there by as an endogenous inductor of stress-related protein synthesis. Desiccation tolerance disappears when thalli are cultivated for a longer period under well-watered conditions; such thalli are not able to increase stress-dependent ABA biosynthesis sufficiently, or to form the desiccation-related proteins unless they are treated with external ABA. The rehydrated thalli cannot recover from a rapid water loss, while ABA-treated, non-hardened thalli regain their photosynthetic activity within two hours.Abbreviations ABA abscisic acid - F_o initial fluorescence yield - F_m maximum fluorescence yield - Q_A primary quinone receptor of PSII We are grateful to Deutsche Forschungsgemeinschaft for financial support (SFB 251, TP3, Graduiertenkolleg Ka 456/5-1), to Prof. E.W. Weiler (Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität, Bochum, Germany), Dr. D. Bartels (Max-Planck-Institut für Züchtungsforschung, Köln, Germany) and Dr. T.J. Close (Department of Botany and Plant Science, University of California, Riverside, Calif., USA) for generous gifts of immunochemicals for ABA assay and antibodies 6–19, 37-31 and Rb-2b, to Miss. B. Dierich for skilful technical assistance and to Mrs. E.M. Arnold, Omaruru, Namibia for the generous supply ofExormotheca thalli.