The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.     

Haemonchus contortus (Nematoda: Trichostrongylidae) is much more sensitive than Caenorhabditis elegans (Nematoda: Rhabditidae) to the ovicidal action of thiabendazole

When eggs of the trichostrongylid nematode Haemonchus contortus were exposed to thiabendazole, the concentration required to prevent hatching in 90% of the eggs (MIC90) was found to be 0.1 microg/ml (using 1% dimethylsulfoxide [DMSO] as solvent). In contrast, eggs of the free-living rhabditid nematode Caenorhabditis elegans hatched at normal rates at a concentration 200 times higher, i.e., 20 microg/ml, and showed only a partial inhibitory effect at a concentration 1,200 times higher, i.e., 120 microg/ml (in 3% DMSO). Because solubility limitations precluded the testing of higher concentrations of thiabendazole, a more soluble derivative, 5-([1-methylethoxy]carbonylamino)-2-(4-thiazloyl)1H-++ +benzimidazolyliminoacetic acid N,N-diethylethanamine salt, was tested against C. elegans eggs. The MIC90 was found to be 400 microg/ml, and although the derivative was not tested against H. contortus eggs, this finding further suggests that C. elegans eggs have an exceptionally low degree of benzimidazole sensitivity.     

蓝尾石龙子杭州和宁德种群繁殖生活史特征的差异

测定处于不同纬度的浙江杭州和福建宁德的蓝尾石龙子(Eumeceselegans)种群的个体大小和繁殖特征。宁德种群的产卵时间为5月27日—6月22日,早于高纬度杭州种群(6月4日—7月12日)。宁德种群最小繁殖雌体及性成熟个体大小均显著小于杭州种群。宁德和杭州两种群的相对窝卵重无显著差异;当统计去除母体体长的影响之后,两地种群的窝卵数和窝卵重也无显著差异,但杭州种群的卵重量显著大于宁德种群。蓝尾石龙子窝卵数和卵重量呈负相关,窝卵数和卵大小的权衡存在种群间差异。特定窝卵数条件下,杭州种群的卵重量显著大于宁德种群。由此可见,蓝尾石龙子种群间的繁殖生活史特征存在显著差异,而且与母体大小的差异密切相关。推测不同纬度地区的蓝尾石龙子种群的繁殖策略存在差异。     

Antinematodal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Caenorhabditis elegans.

The antinematodal activity and mechanism of a 23-mer antimicrobial peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed a strong antinematodal activity against the eggs and worms of Caenorhabditis elegans. To investigate the antinematodal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. C. elegans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide (PI) staining than normal cells. Confocal microscopy showed that the peptide was localized in the egg's shell and cell membrane. The action of the peptide against C. elegans membranes was examined by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w). The result suggests that PMAP-23 may exert its antinematodal activity by disrupting the structure of the cell membrane via pore formation or via direct interaction with the lipid bilayers.     

Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection of Biomphalaria glabrata (Pulmonata: Planorbidae) on a challenge infection with Schistosoma mansoni (Digenea: Schistosomatidae)

Prior exposure of Biomphalaria glabrata to the eggs of an incompatible digenean, Plagiorchis elegans, rendered this snail host less suitable to a compatible species, Schistosoma mansoni. Although P. elegans failed to develop patent infections in B. glabrata, it reduced the production of S. mansoni cercariae by 88%. Concomitantly, host attributes such as reproduction, growth, and survival were compromised. The effect of P. elegans infection was most severe among snails that, in addition, had developed patent schistosome infections. Although few S. mansoni cercariae were produced, egg production by B. glabrata was only 4% of control values. Furthermore, no doubly infected snails survived for more than 3 wk after patency, whereas controls experienced no mortality during the same time period. The above effects were attributable to the establishment and persistence of P. elegans sporocysts in the tissues of the incompatible snail host. Their indirect antagonistic interaction with thelarval stages of S. mansoni may be mediated, in part, through their long-term stimulation of the host's internal defense mechanisms. These findings are discussed with a view to use P. elegans and other plagiorchiid digeneans as agents in the biological control of snails and snail-borne diseases.     

果蝇细胞凋亡核心机制的基因组比较

基因组比较研究是从基因组序列推测调控网络的主要途径。细胞凋亡信号网络是调控网络的一个典型代表。ＥＧＬ１、ＣＥＤ３、ＣＥＤ４和ＣＥＤ９及其同源蛋白质的线虫和哺乳动物构成保守的凋亡核心机制。目前果蝇细胞凋亡核心机制尚不完整,还未找到ＥＧＬ１和ＣＥＤ９类似蛋白质。通过一系列基于生物信息学的基因组比较分析,在果蝇的基因组数据库中发现了两个ＢＣＬ２／ＣＥＤ９和一个ＥＧＬ１的同源蛋白质的编码基因,并重构了果蝇     

Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system

The environmental saphrophyte Burkholderia pseudomallei is the causative agent of melioidosis, a systemic, potentially life-threatening condition endemic to many parts of south-east Asia and northern Australia. We have used the soil nematode Caenorhabditis elegans as a model host to characterize the mechanisms by which this bacterium mounts a successful infection. We find that C. elegans is susceptible to a broad range of Burkholderia species, and that the virulence mechanisms used by this pathogen to kill nematodes may be similar to those used to infect mammals. We also find that the specific dynamics of the C. elegans-B. pseudomallei host-pathogen interaction can be highly influenced by environmental factors, and that nematode killing results at least in part from the presence of a diffusible toxin. Finally, by screening for bacterial mutants attenuated in their ability to kill C. elegans, we genetically identify several new potential virulence factors in B. pseudomallei. The use of C. elegans as a model host should greatly facilitate future investigations into how B. pseudomallei can interact with host organisms.     

Storage and incubation of Echinostoma revolutum eggs recovered from wild Branta canadensis, and their infectivity to Lymnaea tomentosa snails

Echinostoma revolutum eggs recovered from naturally infected wild Canada geese (Branta canadensis) were cold stored (4-6 degrees C) for up to 72 weeks. Successful hatching followed incubation for from 6 to 8 days at an optimum temperature of between 25 and 30 degrees C. A partial life cycle from adult worm to metacercarial encystment in Lymnaea tomentosa snails was completed in the laboratory. Snails were infected both by free miracidia and by ingestment of unhatched embryonated eggs. Infection was equally successful in environmental temperature ranges from 10 to 25 degrees C, and at challenge levels of 2, 5 or 10 embryonated eggs per snail. Exposure to 10 eggs was lethal. Ingestion by snails of embryonated eggs with successful infection at 10 degrees C suggests that embryonated eggs may be used to infect wild snails when the environmental water temperature has reached 10 degrees C.     

Developmental control of oocyte maturation and egg activation in metazoan models

Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.     

New DAPI and FISH findings on egg maturation processes in related hybridogenetic and parthenogenetic Bacillus hybrids (Insecta, Phasmatodea)

Bacillus stick insects have proved adequate for studying a wide array of reproductive modes: sexual, parthenogenetic, hybridogenetic, androgenetic. Hybridogenetic strains (B. rossius-grandii) were thought to discard the paternal "grandii" haploset during first meiotic division and keep the "rossius" hemiclone, whereas the clonal B. whitei (=rossius/grandii) would maintain its hybrid structure by fusing back two nonsister nuclei-each derived from previously segregated heterospecific complements-by the end of the 2(nd) meiotic division. New investigations on laid eggs and ovariole squashes, either DAPI stained or FISH labeled, revealed that in hybridogens the "grandii" set is excluded from the germ line prior to meiosis and that a DNA extra-synthesis should occur to produce hemiclonal eggs after two cytologically normal meiotic divisions. On the other hand, in B. whitei eggs no genome segregation appears to occur and an intrameiotic DNA extra-synthesis must take place to produce 2n tetrachromatidic oocytes I; these divide twice and give unreduced clonal eggs. The new findings bring hybridogenetic oogenesis of Bacillus to be coincident with that of the known hemiclonal organisms and point to an independent onset of B. whitei from hemiclonal strains. In addition, B. whitei gains a closer resemblance to B. lynceorum owing to the sharing of a cytologically identical egg maturation mechanism, of the same maternal ancestor and of peculiar chromosomal features. It is here suggested that B. lynceorum originated from the incorporation of an "atticus" genome into a B. whitei egg, according to a pathway of repeated hybridization often occurred with other polyploid hybrids.     

Schistosomiasis mansoni in areas of low transmission: epidemiological characterization of Venezuelan foci

Severe schistosomiasis is a rare event in Venezuela nowadays, after a successful national campaign by the Schistosomiasis Control Program. Unfortunately, this program has practically disappeared, and snail surveillance in field is not a priority, anymore. Thus, schistosomiasis has become a neglected disease in this country. However, surveys in different populations from the endemic area have shown particular epidemiological features described herein. In five communities we evaluated 2,175 persons and searched for the presence of Biomphalaria glabrata snails. Some markers were used for classifying schistosomiasis foci: mean age of the persons with Schistosoma mansoni eggs in the stools, serological tests, presence of B. glabrata snails, and intensity of infection. Places without B. glabrata snails and with few schistosomiasis cases were defined as "past transmission sites"; a site with abundant snails but few cases was defined as "potential risk"; "new transmission" foci were characterized by the presence of infected snails and young people passing eggs in the stools. A "re-emergent" focus has shared these last features, showing in addition a place where schistosomiasis had been reported before. Recent evidences of active transmission with the increasing dispersion of B. glabrata snails, point out the necessity for the re-establishment of the Schistosomiasis Control Program in Venezuela.     

Effects of the novel anthelmintic emodepside on the locomotion, egg-laying behaviour and development of Caenorhabditis elegans

Emodepside, a cyclooctadepsipeptide, is a broad-spectrum anthelmintic previously shown to paralyse body wall muscle and pharyngeal muscle in the model nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans L4 are less sensitive than adults to emodepside in two independent assays of locomotor behaviour: body bend generation on agar (adult IC(50) 3.7 nM, L4 IC(50) 13.4 nM) and thrashing behaviour in liquid (thrashing behaviour as a % of controls after 1h in 10 microM emodepside: adults 16%, L4 worms 48%). We also show that continuous exposure of wild-type C. elegans to emodepside throughout the life-cycle from egg onwards, slows worm development, an effect that is emodepside concentration-dependent. The rate of worm-hatching from eggs on agar plates containing emodepside was not significantly different from controls, suggesting that it is development post-hatching rather than hatching itself that is affected by the drug. Emodepside also inhibits wild-type C. elegans egg-laying, with acute exposure to the drug at 500 nM resulting in an almost total inhibition within the first hour. However, the rate of egg production was not inhibited and therefore emodepside-treated worms became bloated with eggs, eventually rupturing. This suggests that the effect of emodepside on reproduction is not due to an inhibition of egg production but rather a paralytic effect on the egg-laying muscles. These results, when coupled with previous research, suggest that emodepside interferes with signalling at the neuromuscular junction on the body-wall muscles (Willson et al., 2003), pharynx (Willson et al., 2004) and egg-laying muscles and thus inhibits three important physiological functions: locomotion, feeding and reproduction.     

Activation at the germinal vesicle stage of starfish oocytes produces parthenogenetic development through the failure of polar body extrusion

Starfish oocytes can be fertilized after germinal vesicle breakdown (GVBD) and artificial parthenogenesis can be induced by activating the oocytes after GVBD (post-GVBD activation). In the present study, parthenogenotes were obtained by the activation of immature oocytes with caffeine before treatment with 1-methyladenine (1-MeAde) to induce oocyte maturation. Most of the caffeine-treated eggs developed as tetraploids, as parthenogenotes produced by the post-GVBD activation. The parthenogenotes were derived only from eggs that failed to extrude polar bodies, mostly from eggs failing to extrude a second polar body. Eggs derived from immature oocytes activated by A23187, treated with 1-MeAde and post-treated with cytochalasin B failed to extrude polar bodies, and eventually developed into parthenogenetic embryos. These results indicate that the present parthenogenesis mechanism shares the same characteristics as that achieved by post-GVBD activation in the suppression of polar body formation as a key means for successful starfish parthenogenesis.     

Oocyte signals derived from polyunsaturated fatty acids control sperm recruitment in vivo

A fundamental question in animal development is how motile cells find their correct target destinations. During mating in the nematode Caenorhabditis elegans, males inject sperm through the hermaphrodite vulva into the uterus. Amoeboid sperm crawl around fertilized eggs to the spermatheca--a convoluted tube where fertilization occurs. Here, we show that polyunsaturated fatty acids (PUFAs), the precursors of eicosanoid signalling molecules, function in oocytes to control directional sperm motility within the uterus. PUFAs are transported from the intestine, the site of fat metabolism, to the oocytes yolk, which is a lipoprotein complex. Loss of the RME-2 low-density lipoprotein (LDL) receptor, which mediates yolk endocytosis and fatty acid transport into oocytes, causes severe defects in sperm targeting. We used an RNAi screen to identify lipid regulators required for directional sperm motility. Our results support the hypothesis that PUFAs function in oocytes as precursors of signals that control sperm recruitment to the spermatheca. A common property of PUFAs in mammals and C. elegans is that these fats control local recruitment of motile cells to their target tissues.     

Cyclin is a component of maturation-promoting factor from Xenopus

Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies. Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates. Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity. These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.     

A novel macrocyclic lactone with insecticidal bioactivity from Streptomyces microflavus neau3

A novel macrocyclic lactone (1) was isolated from the fermentation broth of Streptomycesmicroflavus neau3, and the structure was elucidated by extensive spectroscopic analysis. Compound 1 showed high acaricidal activity against adult mites (IC(50)=11.1 μg mL(-1)), and nematocidal activity against Caenorhabditis elegans (IC(50)=17.4 μg mL(-1)), especially the acaricidal activity against mite eggs with an IC(50) of 37.1 μg mL(-1), which was relative higher than that of the commercial acaricide and nematocide milbemycins A(3)/A(4).     

The influence of larval density, food stress, and parasitism on the bionomics of the dengue vector Aedes aegypti (Diptera: Culicidae): implications for integrated vector management

New larval control strategies for integrated vector management of Aedes aegypti are in high demand, including the use of biological control agents. Exposure of Aedes aegypti to parasites, starvation, and overcrowded conditions during larval development reduces the probability of survival to eclosion, can directly affect fitness parameters such as adult size and fecundity, and can affect the size, provisioning, and viability of eggs produced by females. We compared these parameters after exposing larvae to 1) abundant food at low larval densities, 2) food deprivation and high larval density, and 2) infection with the endoparasite Plagiorchis elegans, an entomopathogenic digenean trematode. Female mosquitoes that eclosed from larval conditions of starvation and overcrowding were smaller and laid fewer and smaller eggs than controls. The proportion of females to complete an oviposition cycle was reduced in the P. elegans-infected treatment group. Parasite load was negatively correlated with wing length and egg size. Infection of Ae. aegypti with P. elegans has sublethal effects and may reduce population-level reproductive output, but one-time low-density P. elegans exposure does not have sufficient effect on Ae. aegypti fitness parameters to be considered a viable biocontrol option.