Background: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. Results: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. Conclusions: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

Evaluations of tactics for detection and management of raspberry crown borer, RCB, Pennisettia marginata (Harris) on blackberries were performed in Mississippi from January 2005 - July 2006. Randomized split plots with infested blackberries and certified nursery stock of two recommended cultivars, thorny ?Chickasaw? and thornless ?Apache? were planted. Pest management tactics targeting RCB included drenches: chemical insecticide bifenthrin, recently registered for RCB management; experimental pesticide E2Y45; and entomopathogenic nematodes Steinernema feltiae. Insect pheromones (E,Z)3-13-octadecadien-1-ol and (E,Z)3-13-octadecadien-1-yl-acetate were evaluated as RCB lures. Effects of fungicides captan, pyraclostrobin + boscalid, lime-sulfur and insecticide acetamiprid on viability and infectivity of entomopathogenic nematode species, S. feltiae and Heterorhabditis bacteriophora were evaluated in laboratory bioassays with host greater wax moth larvae. RCB larvae and soil pests on and around infested blackberry plants were observed. Bait traps with 5 mg of (E.Z)3-13-octadecadien-1-ol captured an adult RCB on October 19, 2005. Laboratory bioassays indicated that lime-sulfur and pyraclostrobin + boscalid decrease nematode movement and infectivity.

Photorhabdus are motile Gram-negative bacteria that have a mutualistic association with entomopathogenic Heterorhabditis nematodes. Nematodes vector the bacteria from one insect host to another, while the bacterial symbiont produces toxins and secondary metabolites that kill that the insect host. In this study, we characterize the bacterial symbiont of Heterorhabditis sonorensis, recently discovered in the Sonoran desert. Biochemical and molecular methods including sequence data from five genes: 16s rDNA, gyrB, recA, gltX, dnaN were considered. Evolutionary relationships of this new Photorhabdus subsp. were inferred considering maximum parsimony and Bayesian analyses. We also surveyed for secondary metabolites (SM) produced by this microorganism, considering HPLC and mass spectrometry analyses. SM crude extracts showed activity against the corn ear worm Helicoverpa zea, the root-knot nematode (Meloidogyne incognita), the bacterium Pseudomonas syringae, and the fungus Fusarium oxysporum; and were more toxic that those produced by related species. Results from these studies showed that Photorhabdus l. sonorensis' secondary metabolites have potent antagonistic activity against these plant pathogens.

Entomopathogenic nematodes (EPNs) (genera Heterorhabditis and Steinernema) kill their invertebrate hosts with the aid of a mutualistic bacterium. The bacteria (Xenorhabdus spp. for steinernematids and Photorhabdus spp. for heterorhabditids) are primarily responsible for killing the host and providing the nematodes with nutrition and defense against secondary invaders. Photorhabdus is a Gram-negative bacterium in the Enterobacteriaceae family with high virulence towards their insect hosts. To achieve high mortality rates Photorhabdus produces a variety of virulence factors such as toxins, lipases, proteases, secretion systems, and fimbriae. EPNs are amenable to laboratory rearing and mass production for biocontrol applications against insects using in vivo or in vitro methods; however, in vitro liquid culture is considered to be the most efficient. In this method the symbiotic bacteria are cultured prior to the addition of their partner EPN. This can leave the bacteria susceptible to a number of problems such as genetic drift and inadvertent selection. Regardless of the culture method the symbiotic bacteria exhibit trait deterioration or changes due to laboratory rearing. This project had three primary aims: 1) investigate the role of nutrition in trait deterioration, 2) examine virulence evolution using a phylogenetic context, and 3) identify genes that are necessary for survival and virulence inside the insect host. Prior to studying these objectives we first determined the optimal conditions for growing and counting viable cells of Photorhabdus. We discovered that growth is enhanced by the addition of pyruvate to growth media. To determine the role of nutrition in trait deterioration we repeatedly sub-cultured Photorhabdus in three different media types. Throughout this study we found that, in contrast to previous studies, trait deterioration does not always happen and the environment influences trait deterioration. Furthermore, based on our phylogenetic studies we found that Photorhabdus spp. are evolving to an increase in insect virulence. Lastly, using Tn-seq we determined a list of 84 genes that are needed for efficient virulence inside the insect host and provide suggestions for ongoing research efforts.

No description available.

Thesis (MScAgric (Conservation Ecology and Entomology))--Stellenbosch University, 2008. / The codling moth, Cydia pomonella (L.), is a key pest in pome fruit orchards in South Africa. In the past, broad spectrum insecticides were predominantly used for the local control of this moth in orchards. Concerns over human safety, environmental impact, widespread dispersal of resistant populations of codling moth and sustainability of synthetic pesticide use have necessitated the development and use of alternative pest management technologies, products and programmes, such as the use of entomopathogenic nematodes (EPNs) for the control of codling moth. Entomopathogenic nematodes belonging to either Steinernematidae or Heterorhabditidae are ideal candidates for incorporation into the integrated pest management programme currently being developed for pome fruit orchards throughout South Africa with the ultimate aim of producing residuefree fruit. However, these lethal pathogens of insects are not exempted from governmental registration requirements and have therefore not yet been commercialized in South Africa. A nontarget survey was conducted to find suitable isolates of EPNs from local soils and to test their effectiveness as control agents against the codling moth. Soil samples were collected from several habitats and regions throughout South Africa and nematodes were recovered using the insect baiting technique. All EPN isolates obtained were identified to species level using a molecular taxonomic approach. Entomopathogenic nematodes were recovered from 20 of the 200 soil samples (10 %). Of these, eight (40 %) yielded Steinernema spp., identified as three isolates of S. khoisanae and five undescribed Steinernema spp. The other 12 (60 %) of the samples were positive for Heterorhabditis spp. and included six isolates of H. bacteriophora, five H. zealandica and one H. safricana. These isolates were then evaluated in laboratory bioassays for their potential as microbial agents of codling moth under varying conditions. A morphometric study indicated that all natural openings (mouth, anus and spiracles) of final instar codling moth were large enough for the infective juveniles (IJs) of all tested EPN species to gain entry into the larvae.

This study investigated a system comprised of entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae), their symbiotic bacteria (Xenorhabdus, Photorhabdus) and an insect host, M. sexta. The focus of this investigation was to examine the effects insect host condition on the nematode-bacterium mutualistic partnership as a whole. The EPN used in the study had varying host-searching strategies: an ambusher, Steinernema carpocapsae, and a cruiser, Heterorhabditis sonorensis. Insect host age and diet were evaluated as factors that affect insect host condition. Wandering 5th instar M. sexta were much less susceptible to EPN infection compared to non-wandering 5th instar larvae, regardless of EPN species. Insect host diet had a significant impact on H. sonorensis susceptibility to EPN infection, and a non-significant trend was observed for S. carpocapsae. EPN and bacterial symbiont fitness were unaffected by insect host condition. This is the first record of the effect of insect host condition on both EPN and their symbiotic bacteria.

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae, and their bacterial symbionts, have been studied intensively because of their role as natural mortality factor for soil-dwelling arthropods, and their potential as biological control agents for belowground insect pests. Moreover, EPN are recognized as key players in regulating soil food webs and triggering trophic cascades. However, most studies of interactions with EPN have been conducted under laboratory setting and simplified conditions, without consider the dynamic of the EPN and their interactions with other soil components in a wider context. In this respect, knowledge of the effect that other soil organisms or human induced factor may have on EPN dynamic and life cycle in the soil may contribute to improve tactics for their implementation and success as natural regulators of herbivores. The present investigation focused on the interactions of EPN with a selection of insecticides, and biotic (saprobic fungus and plant parasitic nematodes) elements that may be present in the soil, and may potentially interact with EPN. Specifically, I investigated how these factors may affect the life cycle (host search behavior, virulence and reproduction) of EPN. Appendix A shows the effect that a group of selected synthetic and biological insecticides have on EPN virulence and reproduction. The results obtained from this study revealed that most combinations of EPN and insecticides under study increased the mortality of the insect host. However, it was also found that some of these combinations reduced the nematode progeny production and emergence of IJs from the insect cadaver. In contrast in Appendix B, when examining the effect of the saprobic fungus Fusarium oxysporum in the life cycle of the EPN Heterorhabditis sonorensis, it was found that this fungus negatively affected the virulence and reproduction of the EPN in the insect host. In the third study of this dissertation (Appendix C) the interactions studied considered the effect of two EPN on an organism of a different trophic guild, the plant parasitic nematode Tylenchulus semipenetrans. This plant parasitic nematode causes serious diseases in citrus plants by infecting their roots and defoliating their branches. Previous studies have shown that some EPN species may negatively affect the life cycle of plant parasitic nematodes by reducing the damage produced by this plant parasite. Results from this study confirm the antagonistic effect between the selected EPN and the citrus nematode. Specifically, it was found that the presence in the soil of both EPN reduced the survival of infective juveniles of the citrus nematode and their penetration to the root. Moreover, the presence of EPN had an antagonistic effect in the production of eggs of T. semipenetrans females.

During the evolution some groups of organisms have become coevolutionary associated with other groups, as is the case of host symbiont systems. To explore coevolutionary history of hosts and their associated symbionts, phylogenetic reconstruction of symbionts and phylogenetic reconstruction of hosts are usually compared. Coevolution is described by coevolutionary events (cospeciation, host switch, duplication, failure to diverge events and linage sorting events). The aim of this work was to test the suitability of MLST method for the complex of entomopathogenic nematodes from the genus Steinernema (with detailed analysis of Steinernema feltiae) and their symbiotic bacteria Xenorhabdus bovienii and subsequently use cophylogenetic comparative analysis to determine their level of cospeciation.

It is known that entomopathogenic nematodes rarely live in fields where pesticides and fertilizers are used. Because they are organisms with a high potential for biological and integrated pest control the thesis tried to determine the influence of pesticides on the infectivity and mortality of selected nematodes: Steinernema feltiae, S. arenarium and S. kraussei in water solution of some pesticides. The results are that preparations Vydate (a. i. oxamyl) and Sulka (a. i. sulphur) have the highest negative influence on both characteristics. These pesticides cause a mortality of 94.3 - 99.9 % and reduce infectivity by about 50 {--} 75 % (level 3). The following preparations may be identified as having higher potential for infectivity reduction: above all, Starane 250 EC (fluroxypyr), Sumithion super (fenithrothion), and Novozir MN 80 (mancozeb); on the other hand, Treflan 48 EC (trifluralin) significantly increases mortality. Another important observation is a fact that infectivity is significantly influenced only by concentration of pesticides, not by exposure time. During an experiment with enhacement of resistance to oxamyl (Vydate) I falling mortality and increasing infectivity of entomopathogenic nematodes were observed after five rounds.