Host Cadavers Research Articles

Investment in multiple defences protects a nematode-bacterium symbiosis from predation

The act of predation often comprises multiple sequential steps whereby prey can employ defences at all or some of these stages to deter predation. However, investment in defences is costly unless they are outweighed by conferring some benefit to the bearer. One system that employs multiple defences is that of the entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus luminescens. This nematode–bacterium complex infects and kills soil-dwelling insect larvae, in which they then reproduce and juveniles emerge 2 weeks later. Predation of the infected host cadaver at any point during infection is fatal for the parasitic colony inside. Infected individuals, however, turn red, produce a chemical defence, bioluminesce and smell strongly at various stages of the infection process. We tested whether these colour and scent cues conferred a benefit to the infecting nematode–bacterium complex, utilizing feeding trials of nematode-infected waxworms, Galleria mellonella, with wild-caught great tits, Parus major. We tested for multimodality, as the cues are in different sensory modalities, and found no overall benefit in terms of initial attack on the first prey item, although this does not rule out the possibility of multimodality within this system. We then examined the first five prey attacked and found that scent overshadowed colour at various stages of infection, in terms of reducing levels of attack, but not when both signals were in concert in terms of consumption of infected individuals.

NprR, a moonlighting quorum sensor shifting from a phosphatase activity to a transcriptional activator.

Regulation of biological functions requires factors (proteins, peptides or chemicals) able to sense and translate environmental conditions or any circumstances in order to modulate the transcription of a gene, the stability of a transcript or the activity of a protein. Quorum sensing is a regulation mechanism connecting cell density to the physiological state of a single cell. In bacteria, quorum sensing coordinates virulence, cell fate and commitment to sporulation and other adaptation properties. The critical role of such regulatory systems was demonstrated in pathogenicity and adaptation of bacteria from the Bacillus cereus group (i.e. B. cereus and Bacillus thuringiensis). Furthermore, using insects as a model of infection, it was shown that sequential activation of several quorum sensing systems allowed bacteria to switch from a virulence state to a necrotrophic lifestyle, allowing their survival in the host cadaver, and ultimately to the commitment into sporulation. The chronological development of these physiological states is directed by quorum sensors forming the RNPP family. Among them, NprR combines two distinct functions connecting sporulation to necrotrophism in B. thuringiensis. In the absence of its cognate signaling peptide (NprX), NprR negatively controls sporulation by acting as a phosphatase. In the presence of NprX, it acts as a transcription factor regulating a set of genes involved in the survival of the bacteria in the insect cadaver.

The mitochondrial genome of the lepidopteran host cadaver (Thitarodes sp.) of Ophiocordyceps sinensis and related phylogenetic analysis

To understand the phylogeny of the host insect (Thitarodes sp.) of the fungus Ophiocordyceps sinensis, we sequenced, annotated and characterized the complete mitochondrial (mt) genome of the host cadaver of a natural O. sinensis. Further, we compared the Thitarodes sp. mt genome with those of the other 7 sequenced Hepialidae and examined the phylogenetic relationships using a constructed Maximum Likelihood (ML) phylogenetic tree and mt genomic features (genetic distances and intergenic spacers). The mt genome is a circular molecule of 16,280bp in length with a high A+T content (81.20%) and contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and an AT-rich region. The gene arrangement is identical to the ancestral arrangement but differs from those of other lepidopteran mt genomes because of the arrangement of tRNA genes. The tRNA region, which is located between the AT-rich region and nad2, is trnI/trnQ/trnM (IQM) in Thitarodes sp., rather than the trnM/trnI/trnQ (MIQ) of the Lepidoptera-specific rearrangement. All PCGs begin with the canonical start codons ATN or NTG, except for cox1, which starts with CGA. Most PCGs terminate with the typical stop codon TAA, although some have an incomplete stop codon (T). The 1473bp AT-rich region is located between the rrnS (12S rRNA) and trnI, which is the longest sequenced in a Thitarodes mt genome to date, containing nine 112bp copies and one partial copy of a 55bp sequence. The results derived from the phylogenetic tree, the genetic distances and the intergenic spacers of the mt genome show that the host insect of O. sinensis belongs to the Thitarodes, while Endoclita signifer and Napialus hunanensis form a relatively distinct lineage from Thitarodes. The sequence and full annotation of this moth mt genome will provide more molecular information about the Exoporia within the Lepidoptera, and the clarification of its phylogeny will improve the management of this insect resource and the conservation and sustainable use of this endangered medicinal species in China.

Effect of granulovirus infection of Spodoptera litura (Lepidoptera: Noctuidae) larvae on development of the endoparasitoid Chelonus inanitus (Hymenoptera: Braconidae)

Infection of parasitized Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae by a granulovirus (GV) deleteriously affected the development of the endoparasitoid Chelonus inanitus (Linnaeus) (Hymenoptera: Braconidae). When second-instar parasitized S. litura larvae were inoculated with GV, the percentages of emergence, pupation, and adult eclosion of C. inanitus were significantly lower from GV-infected hosts than those from uninfected ones. Lower parasitoid emergence was due to the earlier death of parasitized and GV-infected hosts and slower development of parasitoid larvae in GV-infected hosts than in uninfected ones. Some of the parasitoid larvae that emerged from GV-infected hosts died before pupation and adult eclosion as a result of lower external feeding on GV-infected cadavers. Furthermore, the body volume of parasitoids developing in GV-infected hosts was significantly lower than that of parasitoids developing in uninfected hosts. The experiment in which host cadavers and emerged parasitoids were switched revealed that external feeding of parasitoids was affected both by infection of host cadavers with GV and also by parasitoid development within GV-infected hosts. Our data show that GV infection of S. litura larvae has deleterious effects on C. inanitus development.

In Vivo Production of Entomopathogenic Nematodes.

In nature, entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects. The nematodes are used widely as biopesticides for suppression of insect pests. More than a dozen entomopathogenic nematode species have been commercialized for use in biological control. Most nematodes intended for commercial application are produced in artificial media via solid or liquid fermentation. However, for laboratory research and small greenhouse or field trials, in vivo production of entomopathogenic nematodes is the common method of propagation. Additionally, small companies continue to produce nematodes using in vivo methods for application in niche markets. Advances in mechanization and alternative production routes (e.g., production geared toward application of nematodes in infected host cadavers) can improve efficiency and economy of scale. The objective of this chapter is to describe basic and advanced procedures for in vivo production of entomopathogenic nematodes.

Unraveling the intraguild competition between Oscheius spp. nematodes and entomopathogenic nematodes: Implications for their natural distribution in Swiss agricultural soils

Entomopathogenic nematodes (EPN) are excellent biological control agents to fight soil-dwelling insect pests. In a previous survey of agricultural soils of Switzerland, we found mixtures of free-living nematodes (FLN) in the genus Oscheius, which appeared to be in intense competition with EPN. As this may have important implications for the long-term persistence of EPN, we studied this intraguild competition in detail. We hypothesized that (i) Oscheius spp. isolates act as scavengers rather than entomopathogens, and (ii) cadavers with relatively small numbers of EPN are highly suitable resources for Oscheius spp. reproduction. To study this, we identified Oscheius spp. isolated from Swiss soils, quantified the outcome of EPN/Oscheius competition in laboratory experiments, developed species-specific primers and probe for quantitative real-time PCR, and evaluated their relative occurrence in the field in the context of the soil food web. Molecular analysis (ITS/D2D3) identified MG-67/MG-69 as Oscheius onirici and MG-68 as O. tipulae (Dolichura-group). Oscheius spp. indeed behaved as scavengers, reproducing in ∼64% of frozen-killed cadavers from controlled experiments. Mixed infection in the laboratory by Oscheius spp. with low (3 IJs) or high (20 IJs) initial EPN numbers revealed simultaneous reproduction in double-exposed cadavers which resulted in a substantial reduction in the number of EPN progeny from the cadaver. This effect depended on the number of EPN in the initial inoculum and differed by EPN species; Heterorhabditis megidis was better at overcoming competition. This study reveals Oscheius spp. as facultative kleptoparasites that compete with EPN for insect cadavers. Using real-time qPCR, we were able to accurately quantify this strong competition between FLN and EPN in cadavers that were recovered after soil baiting (∼86% cadavers with >50% FLN production). The severe competition within the host cadavers and the intense management of the soils in annual crops readily explain the low EPN numbers in Swiss field samples. The developed molecular tools can be used to elucidate the extent to which the competitive interactions affect EPN populations. This can help to develop strategies to achieve good persistence and natural EPN recycling, in particular in systems where native EPN levels are low, such as annual crops.

Lateral Dispersal and Foraging Behavior of Entomopathogenic Nematodes in the Absence and Presence of Mobile and Non-Mobile Hosts.

Entomopathogenic nematodes have been classified into cruisers (active searchers) and ambushers (sit and wait foragers). However, little is known about their dispersal and foraging behavior at population level in soil. We studied lateral dispersal of the ambush foraging Steinernema carpocapsae (ALL strain) and cruise foraging Heterorhabditis bacteriophora (GPS11 strain) from infected host cadavers in microcosms (0.05 m2) containing Wooster silt-loam soil (Oxyaquic fragiudalf) and vegetation in the presence or absence of non-mobile and mobile hosts. Results showed that the presence of a non-mobile host (Galleria mellonella larva in a wire mesh cage) enhanced H. bacteriophora dispersal for up to 24 hr compared with no-host treatment, but had no impact on S. carpocapsae dispersal. In contrast, presence of a mobile host (G. mellonella larvae) increased dispersal of S. carpocapsae compared with no host treatment, but had no effect on H. bacteriophora dispersal. Also H. bacteriophora was better at infecting non-mobile than mobile hosts released into the microcosms and S. carpocapsae was better at infecting mobile than non-mobile hosts, thus affirming the established cruiser-ambusher theory. However, results also revealed that a large proportion of infective juveniles (IJs) of both species stayed near (≤ 3.8 cm) the source cadaver (88-96% S. carpocapsae; 67–79% H. bacteriophora), and the proportion of IJs reaching the farthest distance (11.4 cm) was significantly higher for S. carpocapsae (1.4%) than H. bacteriophora (0.4%) in the presence of mobile hosts. S. carpocapsae also had higher average population displacement than H. bacteriophora in the presence of both the non-mobile (5.07 vs. 3.6 cm/day) and mobile (8.06 vs. 5.3 cm/day) hosts. We conclude that the two species differ in their dispersal and foraging behavior at the population level and this behavior is affected by both the presence and absence of hosts and by their mobility.

Transcriptome of an entomophthoralean fungus (Pandora formicae) shows molecular machinery adjusted for successful host exploitation and transmission

Pandora formicae is an obligate entomopathogenic fungus from the phylum Entomophthoromycota, known to infect only ants from the genus Formica. In the final stages of infection, the fungus induces the so-called summit disease syndrome, manipulating the host to climb up vegetation prior to death and fixing the dead cadaver to the surface, all to increase efficient spore dispersal. To investigate this fascinating pathogen–host interaction, we constructed interaction transcriptome libraries from two final infection stages from the material sampled in the field: (1) when the cadavers were fixed, but the fungus had not grown out through the cuticle and (2) when the fungus was growing out from host cadaver and producing spores. These phases mark the switch from within-host growth to reproduction on the host surface, after fungus outgrowth through host integument. In this first de novo transcriptome of an entomophthoralean fungus, we detected expression of many pathogenicity-related genes, including secreted hydrolytic enzymes and genes related to morphological reorganization and nutrition uptake. Differences in expression of genes in these two infection phases were compared and showed a switch in enzyme expression related to either cuticle breakdown or cell proliferation and cell wall remodeling, particularly in subtilisin-like serine protease and trypsin-like protease transcripts.

Decrease of larval and subsequent adult Anopheles sergentii populations following feeding of adult mosquitoes from Bacillus sphaericus-containing attractive sugar baits.

BackgroundBacillus sphaericus is a mosquito-larvae pathogen which proliferates in the host cadavers, spreading and preserving the infection within the larval habitats for prolonged periods. In this pilot field study, we presented B. sphaericus-containing attractive sugar baits (ASB) to wild Anopheles sergentii adults, with the assumption that bait-fed, B. sphaericus-carrying mosquitoes are able to efficiently transmit the pathogen to the larval habitats, causing larval mortality and leading to a decrease in the subsequent adult population.MethodsThe experiment was conducted over 75 days in two desert-surrounded streamlets. Blooming Ochradenus baccatus bushes were sprayed with bait solutions consisting of sugar and food dye marker solutions, with or without B. sphaericus at the experimental and control streamlets, respectively. Adult mosquito and larvae numbers were monitored before and after the treatment application, and the proportion of bait-fed adults was determined by visual inspection for dye presence.ResultsPresence of food dye confirmed a large fraction of the adult mosquito population (70%-75%) readily ingested Bacillus sphaericus- containing bait. By the end of the study period, the larval population at the experimental site was six-fold smaller than the concurrent larval population at the control site. The ensuing adult An. sergentii population was also reduced to about 60% at the experimental site, while the adult mosquito population at the control site had increased 2.4 fold over the same time-frame. The reduction in adult mosquito numbers became apparent after a lag time (10 days), suggesting the treatment had minimal effect on adult longevity, also indicated by the post-treatment increase in the proportion of old mosquitoes and concomitant decrease in the proportion of young mosquitoes.ConclusionsPresentation of B. sphaericus-containing ASB substantially impacts the larval population, which in turn leads to a significant reduction of the ensuing numbers of adult mosquitoes. Although such outcomes may be the result of other causes, these preliminary results raise the possibility that adult mosquitoes can efficiently transmit the pathogen into the larval habitats. The transfer of B. sphaericus via contaminated adult mosquito carriers may allow introduction of pathogens to breeding places which are dispersed, hard to find, or difficult to access.

温度对蚜科专化菌暗孢耳霉休眠孢子形成的影响

PDF HTML阅读 XML下载 导出引用 引用提醒 温度对蚜科专化菌暗孢耳霉休眠孢子形成的影响 DOI: 10.5846/stxb201401070048 作者: 作者单位: 浙江农林大学 林业与生物技术学院,浙江农林大学 林业与生物技术学院,浙江农林大学 林业与生物技术学院,浙江农林大学 林业与生物技术学院,浙江农林大学 林业与生物技术学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金(31300550); 国际科学基金(IFS, C/5146-1) The effect of temperature on in vivo resting spore formation of the aphid-specific pathogen Conidiobolus obscurus Author: Affiliation: Zhejiang Agricultural and Forestry University Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为探究虫霉休眠孢子形成的关键影响因素,通过孢子浴接种和多浓度生物测定实验,观察蚜科专化菌暗孢耳霉(Conidiobolus obscurus)在5个温度处理(10,15,20,24,28 ℃)和3个接种浓度梯度下感染寄主桃蚜(Myzus persicae)的情况,并观测不同处理下蚜尸内产休眠孢子的比例。暗孢耳霉对桃蚜的毒力随温度和接种浓度升高而提高。同时,感病致死的蚜尸镜检结果表明:温度显著影响休眠孢子形成,温度越高,形成几率越大;接种浓度的影响次之,在15-24 ℃间,形成几率随接种浓度提高而增大。这一现象可能的解释:高温环境将使寄主种群增长停滞或消退,暗孢耳霉通过感知环境温度情况,倾向于在较高温时于寄主体内形成休眠孢子来规避接下来可能出现的寄主匮乏期。 Abstract:Fungi belonging to the order of Entomophthorales are found to induce host epizootics under natural conditions, restraining many agroforestry pest populations below economic damage level. For decades, researchers have focused on exploring these valuable resources with respect to their application in pest biocontrol programs, but the process has proven to be difficult. A poor understanding of the fungal epizootic mechanism and survival strategies in the nature, including the mechanism of in vivo resting spore formation, is one of the main obstacles hindering this effort. Entomophthorales can actively eject numerous conidia to infect nearby hosts, thus transmitting within the host populations. When the host population decreases dramatically, the fungi tend to form thick-walled spores (resting spores, RS) within the bodies of cadavers. Once the conditions improve, RS germinate to restart the infection cycle and regulate the host population. Thus, the RS formation is an important step for fungal survival and the subsequent initiation of the epizootic. Previous reports indicated that many biotic and abiotic factors influence in vivo RS formation, including temperature, humidity, light duration, host age, nutritive conditions, and inoculum levels. However, no consistent conclusion has been formed regarding the vital factors, as contradictory results have been reported for different pathogen-host systems. Temperature and inoculum levels were recently found to exert dominant effects on the formation of Pandora nouryi RS in Myzus persicae inhabiting fields in cold climates. To investigate the main factors influencing in vivo RS formation of the aphid-specific pathogen Conidiobolus obscurus, we performed a series of multi-concentration bioassays of C. obscurus against the host M. persicae under five different temperature treatments (10 ℃, 15 ℃, 20 ℃, 24 ℃, and 28 ℃). The results of the bioassays showed that aphid mortality increased with increasing conidial concentrations used to inoculate the aphids (inoculum level) under each temperature condition. The data were well fitted to the time-concentration-mortality model, and the lowest calculated median lethal concentration (LC50) with 95% confidence intervals was estimated to be 13.6 (11-16.8) conidia/mm2 on day 4 at 28 ℃. Within the crushed cadaver bodies, proportion of the RS formation significantly increased with increased temperature, reaching a peak of 0.66 at 28 ℃. The proportion also increased with inoculum levels at temperatures from 15-24 ℃. Thus, C. obscurus tends to form RS more readily at higher temperatures and inoculum levels. We explain this phenomenon as follows: the rise of temperature to high levels signals the upcoming variation in the host population to C. obscurus, because high temperatures always cause host diapause and population collapse in the fields. For survival in the successive period of host shortage, the fungi tend to form thick-walled RS in host cadavers. Further observation of other entomophthoralean fungi and their hosts is required to verify this prediction. The present results also showed that C. obscurus possesses high infectivity under higher temperature conditions and is thus suitable for aphid control in thefields. 参考文献 相似文献 引证文献

A putative methyltransferase, mtrA, contributes to development, spore viability, protein secretion and virulence in the entomopathogenic fungus Beauveria bassiana

The filamentous fungus, Beauveria bassiana, is a ubiquitously distributed insect pathogen, currently used as an alternative to chemical pesticides for pest control. Conidiospores are the means by which the fungus disseminates in the environment, and these cells also represent the infectious agent most commonly used in field applications. Little, however, is known concerning the molecular basis for maintenance of spore viability, a critical feature for survival and persistence. Here, we report on the role of a putative methyltransferase, BbmtrA, in conidial viability, normal fungal growth and development, and virulence, via characterization of a targeted gene knockout strain. Loss of BbmtrA resulted in pleiotropic effects including reduced germination, growth and conidiation, with growing mycelia displaying greater branching than the WT parent. Conidial viability dramatically decreased over time, with <5 % of the cells remaining viable after 30 days as compared with >80 % of the WT. Reduced production of extracellular proteins was also observed for the ΔBbmtrA mutant, including protease/peptidases, glycoside hydrolases and the hyd1 hydrophobin. The latter was further confirmed by hyd1 gene expression analysis. Insect bioassays using the greater wax moth, Galleria mellonella, further revealed that the ΔBbmtrA strain was attenuated in virulence and failed to sporulate on host cadavers. These data support a global role for mtrA in fungal physiological processes.

Bbmsn2 acts as a pH-dependent negative regulator of secondary metabolite production in the entomopathogenic fungus Beauveria bassiana.

Fungal secondary metabolites are chemical compounds important for development, environmental adaptation and for potential biotechnological and pharmaceutical applications. Oosporein, a red-pigmented benzoquinone, produced by many fungal insect pathogenic Beauveria spp., shows remarkable functional diversity, displaying antimicrobial, antiviral and even anti-proliferative activities. A homologue of the msn2/seb1 transcription factor was identified in a Beauveria bassiana random T-DNA insertion library. Targeted gene-knockout of Bbmsn2 resulted in reduced growth and increased sensitivity to Calcofluor White, H2 O2 and Congo Red. However, when normalized to growth at 26°C, the ΔBbmsn2 mutant was more tolerant to high temperature (32°C) than the wild type parent. The ΔBbmsn2 mutant also displayed a pH-dependent growth phenotype, with little growth seen at pH < 5.0 but, better growth at alkaline conditions (pH > 8.0). Unexpectedly, a pH-dependent deregulation of a red pigment, identified as oosporein, was seen in the ΔBbmsn2 mutant. The ΔBbmsn2 strain was impaired in virulence in both topical and intrahaemocoel injection bioassays against Galleria mellonella. ΔBbmsn2 proliferation in the host haemolymph and conidiation on the host cadaver was reduced. These data indicate that Bbmsn2 acts as a negative regulator of oosporein production and contributes to virulence and growth in response to external pH in B. bassiana.

Natural enemies of natural enemies: the potential top‐down impact of predators on entomopathogenic nematode populations

Top‐down population regulation can influence the success of biological control agents when they are released into the field. Entomopathogenic nematodes (EPNs) are used commonly in biological control programmes, but their efficacy suffers from poor persistence. Although abiotic soil conditions have been shown to reduceEPNpersistence, consumption of infected insects by scavengers and of infective juvenile (IJ) nematodes by predators may also regulate these populations. In the present study, the effects of different soil arthropods onEPNs in laboratory conditions were measured. It was hypothesised that arthropods commonly found in soil communities whereEPNs are applied would consume cadavers of insects parasitised by the nematodes and theIJs themselves.Some species of scavengers consumeEPN‐infected insects. Crickets (Gryllus bimaculatusDe Geer),American cockroaches [Periplaneta Americana(Linnaeus)], ants [Tetramorium chefketiForel andPheidole pallidula(Nylander)], earwigs (Labidura ripariaPallas), mites (Sancassania polyphyllaeZachvatkin), and springtails (Sinella curvisetaBrook andFolsomia candidaWillem) have different responses to nematode‐killed insects. Results suggested that ants (T. chefketi), cockroaches, mites, and earwigs fed onSteinernema‐killed insects whereas neither crickets nor springtails consumed them.In the second part of the study, experiments were conducted to determine whether mites and springtails consumedIJ EPNs. Results showed thatS. polyphyllaemites do not consume infective juveniles in soil, whereas both springtail species consumed significant numbers of theIJs.Top‐down regulatory processes can be a limiting factor forEPNpopulations under laboratory conditions. Both host cadavers andIJs are consumed (albeit by different arthropods), so these results may help explain the difficulties associated with the persistence ofEPNapplication to soil.

Ambush Foraging Entomopathogenic Nematodes Employ ‘Sprinters' for Long-Distance Dispersal in the Absence of Hosts

Ambush foragers must employ a long-distance dispersal strategy to maximize reproductive success in the absence of hosts. This hypothesis was tested by comparing lateral dispersal of the ambusher, Steinernema carpocapsae , and the cruiser, Heterorhabditis bacteriophora , nematodes from infected host cadavers in autoclaved, silt-loam soil in large microcosms (0.05-1.5 m(2)) with or without vegetation in the absence of hosts. Dispersal was estimated by taking soil cores (5 × 2 cm in diameter) from the microcosms at different intervals (6-240 hr) and distances (3.8-61 cm) from the infected host cadavers and baiting with Galleria mellonella larvae. The numbers of baited larvae killed and the numbers of infective juveniles (IJs) penetrated in dead baits were counted to compute the percentage of IJs dispersed from the source cadavers, based on the emergence potential and penetration efficiency of the 2 species, and analyzed. Vegetation enhanced dispersal of both species but more so for H. bacteriophora . Although the pattern of dispersal differed spatio-temporally for the 2 species, average population displacement was similar (∼6 cm/day). A majority of the S. carpocapsae population ambushed close to the source cadaver (<3.8 cm), whereas a majority of H. bacteriophora population dispersed between 7-12 cm away from the source cadaver. About 4% of the S. carpocapsae population dispersed faster than the fastest H. bacteriophora , reaching 30-61 cm, compared to only 2% of the H. bacteriophora population dispersing this far. This use of 'sprinters' for long-distance dispersal may represent an adaptive dispersal strategy by the otherwise ambush forager S. carpocapsae in the absence of hosts.

Lethal fighting in nematodes is dependent on developmental pathway: male-male fighting in the entomopathogenic nematode Steinernema longicaudum.

Aggressive encounters occur between competitors (particularly males) throughout the animal kingdom, and in some species can result in severe injury and death. Here we describe for the first time lethal interactions between male nematodes and provide evidence that the expression of this behaviour is developmentally controlled. Males of the entomopathogenic nematode Steinernema longicaudum coil around each other, resulting in injuries, paralysis and frequently death. The probability of death occurring between pairs of males was affected by the developmental pathway followed, being much greater among males that had passed through the infective juvenile (IJ, or dauer) stage than among males that had not. Post-IJ males are found only in newly colonised hosts, typically with few competing males present. Killing those few competitors may secure valuable resources (both females and a host cadaver for nourishment of offspring). Non-IJ males develop in subsequent generations within a host cadaver, where the presence of many closely related male competitors increases the risk:benefit ratio of fighting. Thus, passage through the IJ stage primes males for enhanced aggression in circumstances where this is more likely to result in increased reproductive success. Fighting occurred between males developing in mixed-sex social groups, indicating that it is an evolved trait and not an abnormal response to absence of females. This is supported by finding high mortality of males, but not of females, across a range of population densities in insect cadavers. We propose that these nematodes, with their relatively simple organization, may be a useful model for studies of aggression.

SinR Controls Enterotoxin Expression in Bacillus thuringiensis Biofilms

The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

Ablation of the creA regulator results in amino acid toxicity, temperature sensitivity, pleiotropic effects on cellular development and loss of virulence in the filamentous fungus Beauveria bassiana

For most organisms, carbon and nitrogen uptake are essential for growth, development and, where applicable, pathogenesis. The role of the carbon catabolite repressor transcription factor homologue BbcreA in the entomopathogenic fungus Beauveria bassiana was investigated. Deletion of BbcreA resulted in pleiotropic effects, including nutrient toxicity, leading to a novel cell lytic phenotype. Fungal growth in rich media and minimal media containing select amino acids/peptides was severely compromised, with microscopic examination revealing conidial-base germ tube degeneration and cell lysis occurring during growth, a phenomenon exacerbated at higher temperatures (32°C). Depending upon nutrient conditions, growth, pigment and aerial mycelium production, sporulation and dimorphic transition to blastospore production were also impaired in the ΔBbcreA strain. Although loss of BbcreA resulted in de-repression of secreted protease and lipase, enzymes critical in mediating pathogenesis, insect bioassays indicated severe defects in virulence using both topical and intra-haemocoel injection assays, with eruption and subsequent sporulation on host cadavers greatly reduced in the mutant. These data suggest that BbcreA functions as more than a carbon repressor and plays important roles in nutrient utilization, cell homeostasis and virulence. In particular, BbcreA is required for proper assimilation of select amino acids and peptides, including asparagine, arginine and proline.

Desiccation and cold storage of Galleria mellonella cadavers and effects on in vivo production of Steinernema carpocapsae

Direct application of insect cadavers infected with entomopathogenic nematodes (EPN) can successfully control target pest insects. Little is known about the effects of environmental factors (desiccation and temperature) on the production process for infective juveniles (IJ) in insects. We examined the effects of desiccation time and cold storage (6.7 °C) on IJ production of the nematode Steinernema carpocapsae in Galleria mellonella cadavers at 30.8 and 57% humidity. Under desiccation, the IJ yield in cadavers increased gradually and reached a maximum on day 5. IJ yield gradually declined from day 6 onwards and was almost zero by day 15. In general, cold storage at 6.7 °C caused negative effects on IJ production in desiccated cadavers. Approximately 56 h post infection was the time at which nematodes were most sensitive to low temperatures during development in cadavers. Five-day desiccated cadavers generated higher mortality and more rapid death of Galleria mellonella larvae than using newly (day 0) desiccated cadavers. This study describe methods of optimizing rearing techniques such as desiccation and cold storage to promote the mass production and application of EPN- infected host cadavers for the field control of insect pests.

Aggregative group behavior in insect parasitic nematode dispersal

Movement behavior of foraging animals is critical to the determination of their spatial ecology and success in exploiting resources. Individuals sometimes gain advantages by foraging in groups to increase their efficiency in garnering these resources. Group movement behavior has been studied in various vertebrates. In this study we explored the propensity for innate group movement behavior among insect parasitic nematodes. Given that entomopathogenic nematodes benefit from group attack and infection, we hypothesised that the populations would tend to move in aggregate in the absence of extrinsic cues. Movement patterns of entomopathogenic nematodes in sand were investigated when nematodes were applied to a specific locus or when the nematodes emerged naturally from infected insect hosts; six nematode species in two genera were tested (Heterorhabditis bacteriophora, Heterorhabditis indica, Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema riobrave). Nematodes were applied in aqueous suspension via filter paper discs or in infected insect host cadavers (to mimic emergence in nature). We discovered that nematode dispersal resulted in an aggregated pattern rather than a random or uniform distribution; the only exception was S. glaseri when emerging directly from infected hosts. The group movement may have been continuous from the point of origin, or it may have been triggered by a propensity to aggregate after a short period of random movement. To our knowledge, this is the first report of group movement behavior in parasitic nematodes in the absence of external stimuli (e.g., without an insect or other apparent biotic or abiotic cue). These findings have implications for nematode spatial distribution and suggest that group behavior is involved in nematode foraging.

Trade-offs shape the evolution of the vector-borne insect pathogen Xenorhabdus nematophila

Our current understanding on how pathogens evolve relies on the hypothesis that pathogens' transmission is traded off against host exploitation. In this study, we surveyed the possibility that trade-offs determine the evolution of the bacterial insect pathogen, Xenorhabdus nematophila. This bacterium rapidly kills the hosts it infects and is transmitted from host cadavers to new insects by a nematode vector, Steinernema carpocapsae. In order to detect trade-offs in this biological system, we produced 20 bacterial lineages using an experimental evolution protocol. These lineages differ, among other things, in their virulence towards the insect host. We found that nematode parasitic success increases with bacteria virulence, but their survival during dispersal decreases with the number of bacteria they carry. Other bacterial traits, such as production of the haemolytic protein XaxAB, have a strong impact on nematode reproduction. We then combined the result of our measurements with an estimate of bacteria fitness, which was divided into a parasitic component and a dispersal component. Contrary to what was expected in the trade-off hypothesis, we found no significant negative correlation between the two components of bacteria fitness. Still, we found that bacteria fitness is maximized when nematodes carry an intermediate number of cells. Our results therefore demonstrate the existence of a trade-off in X. nematophila, which is caused, in part, by the reduction in survival this bacterium causes to its nematode vectors.