S. Patricia Stock

Heterorhabditis, steinernema and their bacterial symbionts : lethal pathogens of insects

The entomopathogenic nematodes (EPN) Heterorhabditis and Steinernema together with their symbiont bacteria Photorhabdus and Xenorhabdus, respectively, are obligate and lethal parasites of insects. EPN can provide effective biological control of some important lepidopteran, dipteran and coleopteran pests of commercial crops and they are amenable to large-scale culture in liquid fermentors. They are unique among rhabditids in having a symbiotic relationship with an enteric bacterium species. The bacterial symbiont is required to kill the insect host and to digest the host tissues, thereby providing suitable nutrient conditions for nematode growth and development. This review describes the general biology of EPN and their symbionts and gives an overview of studies to date on EPN biodiversity, biogeography and phylogeny. The impetus for research in EPN and their symbionts has come about because of their biological control potential, with much of the focus in EPN research having been on applied aspects relating to pest control. However EPN and their symbionts are increasingly being viewed as exciting subjects for basic research in the areas of ecology, biodiversity, evolution, biochemistry, symbiosis and molecular genetics. Much progress has been made over the past 20 years in our understanding of the basic biology and genetics of EPN and their symbionts. We are now entering a new phase in which the tools of molecular genetics are being increasingly used to address a range of biological questions in EPN research. The knowledge gained from this endeavour should ensure that EPN will become even more effective biopesticides and should also ensure that EPN and their symbionts gain prominence as unique and intrinsically interesting biological systems. Les nematodes entomopathogenes (EPN) Heterorhabditis et Steinernema, avec leur bacteries symbiotes Photorhabdus et Xenorhabdus, respectivement, sont des parasites obliges et mortels des insectes. Les EPN peuvent servir a un controle biologique de quelques lepidopteres, dipteres et coleopteres importants pour les cultures commerciales et ils sont elevables a grande echelle dans des fermenteurs liquides. Ils sont uniques chez les rhabditides par leur relation symbiotique avec une espece de bacterie enterique. La bacterie symbiote est necessaire pour tuer l’insecte hote et pour digerer les tissus de l’hote, permettant ainsi des conditons de nutrition favorables a la croissance et au developpement du nematode. La presente revue decrit la biologie generale des EPN et de leur symbiotes et donne un etat des etudes actuelles sur la biodiversite, la biogeographie et la phylogenie des EPN. L’impulsion donnee aux recherches sur les EPN et leur symbiotes provient de leur potentialites pour le controle biologique, une grande partie des recherches sur les EPN ayant trait a des aspects appliques en relation avec ce controle des parasites. Cependant, les EPN et leur symbiotes bacteriens sont de plus en plus consideres comme des sujets interessants pour la recherche fondamentale dans les domaines de l’ecologie, de la biodiversite, de l’evolution, de la biochimie, des processus symbiotiques et de la genetique moleculaire. De nombreux progres ont ete realises ces 20 dernieres annees dans la comprehension de la biologie et de la genetique des EPN et de leur symbiotes. Nous entrons actuellement dans une nouvelle phase ou les moyens de la biologie moleculaire sont utilises de maniere croissante pour formuler une serie de questions biologiques pour la recherche sur les EPN. Les connaissances resultant de ces efforts doivent conduire a verifier que les EPN deviendront des biopesticides toujours plus efficaces et que les EPN et leur symbiotes prendront de l’importance en tant que systemes biologiques uniques et intrinsequement interessants.

Biosynthesis of the Cyclooligomer Depsipeptide Beauvericin, a Virulence Factor of the Entomopathogenic Fungus Beauveria bassiana

Beauvericin, a cyclohexadepsipeptide ionophore from the entomopathogen Beauveria bassiana, shows antibiotic, antifungal, insecticidal, and cancer cell antiproliferative and antihaptotactic (cell motility inhibitory) activity in vitro. The bbBeas gene encoding the BbBEAS nonribosomal peptide synthetase was isolated from B. bassiana and confirmed to be responsible for beauvericin biosynthesis by targeted disruption. BbBEAS utilizes D-2-hydroxyisovalerate (D-Hiv) and L-phenylalanine (Phe) for the iterative synthesis of a predicted N-methyl-dipeptidol intermediate, and forms the cyclic trimeric ester beauvericin from this intermediate in an unusual recursive process. Heterologous expression of the bbBeas gene in Escherichia coli to produce the 3189 amino acid, 351.9 kDa BbBEAS enzyme provided a strain proficient in beauvericin biosynthesis. Comparative infection assays with a BbBEAS knockout B. bassiana strain against three insect hosts revealed that beauvericin plays a highly significant but not indispensable role in virulence.

Phylogeny of Steinernema travassos, 1927 (Cephalobina: Steinernematidae) inferred from ribosomal DNA sequences and morphological characters

Entomopathogenic nematodes in Steinernema, together with their symbiont bacteria Xenorhabdus, are obligate and lethal parasites of insects that can provide effective biological control of some important lepidopteran, dipteran, and coleopteran pests of commercial crops. Phylogenetic relationships among 21 Steinernema species were estimated using 28S ribosomal DNA (rDNA) sequences and morphological characters. Sequences of the rDNA internal transcribed spacers were obtained to provide additional molecular characters to resolve relationships among Steinernema carpocapsae, Steinernema scapterisci, Steinernema siamkayai, and Steinernema monticolum. Four equally parsimonious trees resulted from combined analysis of 28S sequences and 22 morphological characters. Clades inferred from analyses of molecular sequences and combined datasets were primarily reliably supported as assessed by bootstrap resampling, whereas those inferred from morphological data alone were not. Although partially consistent with some traditional expectations and previous phylogenetic studies, the hypotheses inferred from molecular evidence, and those from combined analysis of morphological and molecular data, provide a new and comprehensive framework for evaluating character evolution of steinernematids. Interpretation of morphological character evolution on 6 trees inferred from sequence data and combined evidence suggests that many structural features of these nematodes are highly homoplastic, and that some structures previously used to hypothesize relationships represent ancestral character states.

Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal virulence factor of Beauveria bassiana.

Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana.

An Entomopathogenic Nematode by Any Other Name

Among the diversity of insect-parasitic nematodes, entomopathogenic nematodes (EPNs) are distinct, cooperating with insect-pathogenic bacteria to kill insect hosts. EPNs have adapted specific mechanisms to associate with and transmit bacteria to insect hosts. New discoveries have expanded this guild of nematodes and refine our understanding of the nature and evolution of insect–nematode associations. Here, we clarify the meaning of “entomopathogenic” in nematology and argue that EPNs must rapidly kill their hosts with the aid of bacterial partners and must pass on the associated bacteria to future generations.

Distribution of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in natural habitats in California, USA

A total of 270 soil samples from 30 different habitats in 10 geographic regions of California were evaluated for the presence of rhabditid entomopathogenic nematodes. Nematodes were isolated from 26.3% of the samples. The recovered isolates were identified as Steinernema carpocapsae, S. feltiae, S. kraussei, S. longicaudum, S. oregonense, Heterorhabditis marelatus and H.bacteriophora. Among the steinernematids, S. kraussei and S. feltiae were the most commonly encountered species, generally occurring in acidic soils high in organic matter. Among the heterorhabditids, H. bacteriophora was isolated along the southern coast, whereas H. marelatus was recovered along the northern coast of California. Steinernematids were recovered from coniferous forests, oak woodlands and grasslands whereas heterorhabditids were isolated from coastal marshes.

New Insights into the Colonization and Release Processes of Xenorhabdus nematophila and the Morphology and Ultrastructure of the Bacterial Receptacle of Its Nematode Host, Steinernema carpocapsae

ABSTRACT We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen.

Effect of Entomopathogenic Nematodes on the Fitness Cost of Resistance to Bt Toxin Cry1Ac in Pink Bollworm (Lepidoptera: Gelechiidae)

The widespread use of crop plants genetically engineered to produce toxins from the bacterium Bacillus thuringiensis (Bt) imposes selection on insect populations to evolve resistance. The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major pest of cotton in the southwestern United States that is currently controlled with transgenic cotton that produces Bt toxin Cry1Ac. Previously reported theoretical work suggests that, in conjunction with a high dose/refuge strategy, fitness costs of Bt resistance can slow or prevent the evolution of resistance. We report here that the entomopathogenic nematode Steinernema riobrave (Rhabditida: Steinernematidae) increased the fitness cost of resistance to Cry1Ac in P. gossypiella. Mortality of P. gossypiella from fourth instar to adult eclosion was significantly higher for a Bt-resistant strain than a susceptible strain in tests with two to 14 infective juveniles of S. riobrave per larva, but it did not differ between strains when nematodes were absent. Nematodes established in P. gossypiella larvae at all concentrations tested, and nematode reproduction in infected P. gossypiella larvae occurred at nematode concentrations of four to 14 infective juveniles per larva. Our results suggest that incorporation of entomopathogenic nematodes into an integrated resistance management strategy could help to delay pest resistance to Bt toxins.

Nematode parasites, pathogens and associates of insects and invertebrates of economic importance

Nematodes are an ubiquitous group of invertebrates that occupy numerous niches and exist in nearly every available habitat on every continent. They also utilize many trophic strategies and lifestyles. Many species are associated with invertebrates, and these relationships may span from fortuitous to antagonistic. In this chapter we focus on the techniques used for identifying, isolating, propagating, bioassaying, and preserving nematodes that are parasitic in or pathogenic to insects and invertebrates of economic importance. Particular emphasis is placed on entomopathogenic nematodes and their bacterial symbionts, because they are the most widely studied group at this time and have the most relevance in insect pest management. Moreover, this nematode–bacterium complex represents a model system for the study of eukaryote–prokaryote interactions.

Survival of entomopathogenic nematodes within host cadavers in dry soil

Abstract Our objectives were to determine whether entomopathogenic nematode emergence from host cadavers is influenced by soil moisture, whether the nematodes can survive adverse desiccating conditions in the soil by remaining within the host cadaver, and whether differences in such an adaptation occur among species. In the first experiment, wax moth larvae killed by Steinernema glaseri, Steinernema carpocapsae, Steinernema riobravis , or Heterorhabditis bacteriophora were placed in soil water potentials ranging from −500 MPa (very dry) to −0.006 MPa (moist). No infective juveniles (Us) emerged from cadavers at −500 MPa, and only few S. glaseri and S. carpocapsae emerged at −40 MPa. Large numbers of IJs emerged at ≥ −5 MPa from cadavers containing S. carpocapsae, S. glaseri , or H. bacteriophora. S. riobrauis emerged only at ≥ −0.3 MPa. In the second experiment, cadavers were left in dry soil (−40 MPa) for various periods of time before being rehydrated. The number of Us emerging per cadaver and the infectivity of the emerged IJs were determined. IJ emergence declined with the time that the cadavers were left in dry soil. Regression analysis predicted that IJ emergence from cadavers with S. glaseri, S. carpocapsae, H. bacteriophora , or S. riobravis would stop after 27, 62, 80, and 111 days, respectively, in dry soil. We hypothesize that S. carpocapsae , a sit-and-wait forager, survives longer than S. glaseri because it is adapted to infect insects near the soil surface, whereas S. glaseri , an actively searching forager, is adapted to infect insects deeper in the soil profile. Cadavers colonized by S. carpocapsae , therefore, are more likely to be exposed to dehydrating conditions. H. bacteriophora , an actively searching forager, may survive longer within cadavers because the gummous consistency of its host cadavers retains moisture very efficiently. S. riobrauis may survive for considerable lengths of time within cadavers in adaptation to the subtropical, semiarid climate of its geographic area of origin.