Bacteria in ovarioles of females from maleless families of ladybird beetles Adalia bipunctata L. (Coleoptera: Coccinellidae) naturally infected with Rickettsia,Wolbachia, and Spiroplasma

Ovarioles were found to be infected with Spiroplasma, Wolbachia, and Rickettsia in Adalia bipunctata females with maleless progeny in different natural populations. Ooplasm was infected with few Wolbachia bacteria. In ooplasm infected by Rickettsia, bacteria were present in small foci. Spiroplasmas were found encapsulated into ooplasm from the wider intercellular spaces between epithelial and oocyte cells. The cytoplasm of follicular epithelia infected with Rickettsia was heavily destroyed, but the nucleus was intact and free from bacteria. The essential feature of follicular epithelium cells from Spiroplasma and Wolbachia infected A. bipunctata females was inclusions of three types: crystalline, filaments, and concentric myelin-like lamellae. Observations of smears prepared from ovaries of A. bipunctata from natural populations revealed a low concentration of bacteria within a microscopy field (less 10 bacteria) in more than 90% of specimens, and only a few ovaries were heavily infected. Two different ways of bacterial invasion of the oocyte are suggested: Spiroplasma-like, through the intercellular spaces in the epithelium and Rickettsia-like, through the cytoplasm of follicular epithelium cells. Bacteria were not found in germarium zones and we suggest that each follicle is infected from haemolymph.     

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected ¹³C- and ¹⁵N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate ¹³C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.     

Isolation of an alpha-methylene-gamma-butyrolactone derivative, a toxin from the plant pathogen Lasiodiplodia theobromae

Lasiodiplodia theobromae is known as a multi-infectious microorganism that causes considerable crop damage, particularly to tropical fruits. When the fruits are infected by L. theobromae, the typical symptom is the appearance of black spots on the surface of the infected fruit. When injected in to the peel of banana, the culture filtrate of L. theobromae induced formation of black spots. The structure of the isolated compound responsible for this effect was determined to be (3S,4R)-3-carboxy-2-methylene-heptan-4-olide on the basis of analysis of MS, IR, and 1H and 13C NMR spectroscopic data, including HMQC, HMBC, and 1H-1H COSY experiments. The active compound was not only isolated from the culture filtrate derived from potato dextrose medium, but also from the extract of infected peels of bananas.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts,propagation in cell culture,and description of ultrastructural and genetic characteristics

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.     

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of na?ve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.     

Spermatogenesis and chromatin condensation in male germ cells of sea cucumber Holothuria leucospilota (Clark, 1920)

Male germ cells in the testis of Holothuria leucospilota can be divided into 12 stages based on ultrastructure and patterns of chromatin condensation. The spermatogonium (Sg) is a spherical-shaped cell with a diameter of about 6.5-7microm. Its nucleus mostly contains euchromatin and small blocks of heterochromatin scattered throughout the nucleus. The nucleolus is prominent. Primary spermatocytes are divided into six stages, i.e., leptotene (LSc), zygotene (ZSc), pachytene (PSc), diplotene (DSc), diakinesis (DiSc) and metaphase (MSc). The early cells are round while in DiSc and in MSc cells are oval in shape. From LSc to MSc, the sizes of cells range from 3.5 to 4microm. LSc contains large blocks of heterochromatin as a result of increasingly condensed 17nm fibers. In ZSc, the nucleus contains prominent synaptonemal complexes but a nucleolus is absent. In PSc, heterochromatin blocks are tightly packed together by 26nm fibers and appeared as large patches in DSc. Heterochromatin patches were enlarged to form chromosomes in DiSc and MSc and then the chromosome are moved to be aligned along equatorial region. The secondary spermatocyte (SSc) is an oval cell about 4.5-5.5microm. Their nuclei contain large clumps of heterochromatin along the nuclear envelope and in the center nuclear region. Spermatids are divided into two stages, i.e., early spermatid (ESt) and late spermatid (LSt). The nuclei decrease in size by a half and become spherical; thus the chromatin fibers condensed into 20nm and are closely packed together leaving only small spaces in LSt. The spermatozoa (Sz), with chromatin tightly packed in the spherical nucleus with a diameter of 2microm and a small acrosome situated at the anterior of the nucleus. The tail consists of a pair of centrioles lying perpendicular to each other and surrounded by a mitochondrial ring, and an axonemal complex, surrounded by a plasma membrane.     

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.     

Pathology survey of the short-neck clam Ruditapes philippinarum occurring on sandy tidal flats along the coast of Ariake Bay, Kyushu, Japan

The pathological condition of the short-neck clam Ruditapes philippinarum was surveyed along the coast of Kumamoto, Japan, in June 2004. DNA sequences of the non-transcribed spacer region and internal transcribed spacer region flanking 5.8S rRNA identified Perkinsus olseni among the clams. Ray’s fluid thioglycollate medium assay indicated that 96.7% of the clams surveyed from the Kiguchi River tidal flat (native clams, Stn KR-N) and 96.7% of the clams surveyed from the Midori River tidal flat (Stn MR) were infected with P. olseni with an infection intensity of 464,278 and 199,937 Perkinsus cells/gram tissue wet weight (gWW), respectively. In contrast, 66.7% of the clams imported from China and stored along the Kiguchi River tidal flat (Stn KR-I) and 20.2% of clams from the Arao tidal flat (Stn AT) were infected with P. olseni with an infection intensity of 37,547 and 3382 Perkinsus cells/gWW, respectively. Brown ring disease was detected in the clam population from Stn KR-I at a prevalence of 90.0%. Polymerase chain reaction and the 16S rRNA sequence suggested that the agents of brown ring disease observed at Stn KR-I were Vibrio tapetis-like bacteria. Sporocysts and metacercariae of unidentified trematodes were also observed in the gonads and mantle of the clams from Stn KR-I, Stn MR, and Stn AT, at prevalences of 7.1-42.9%. Metacestodes (larval tapeworms) were found in the foot and digestive gland at a prevalence of 52.5%, 30.0%, and 14.3% in clams from Stns MR, AT, and KR-N, respectively. Histology also showed massive hemocyte infiltration and inflammation among clams heavily infected with P. olseni. Castration of the follicle was typical among clams infected with the trematode. The data indicate that most of the clams along the coast of Kumamoto are infected with various pathogens at various rates of infection, and these pathogens could have negative effects on the clam population in the long term.     

Development and morphology of teratocytes in Encarsia berlesei and Encarsia citrina: first record for Chalcidoidea

In several species of hymenopteran parasitoids of the superfamilies of Ichneumonoidea and Platygastroidea, the membrane enveloping the parasitoid embryo dissociates at hatching into a number of cells, called teratocytes, which autonomously develop in the host haemolymph. In this work we report for Encarsia berlesei and Encarsia citrina (Hymenoptera: Chalcidoidea), the dissociation of the extraembryonic membrane into cells whose morphological and embryological features correspond to those of teratocytes. In E. berlesei the membrane dissociated at hatching into 4-9 larger cells (100 microm diameter) and about 10 smaller cells (60 microm), which scarcely doubled their size during maturation. In E. citrina the membrane dissociated into five large cells (250 microm) which did not grow appreciably. Ultrastructural investigation of the dissociated cells in E. berlesei revealed that their surface was covered by microvilli, whose density and length increased from the egg stage to the 12 h following hatching. During the same period, rough endoplasmic reticulum evolved from a parallel profile to that of the cisternal type, while abundant vesicles represented the dominant cytological feature. The ploidy level of these cells ranged between 8c and 140c at hatching, but increased to 40c-350c at maturation. These findings provide the first clear evidences for the presence of teratocytes in the superfamily Chalcidoidea.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocytic phospholipase A2 (PLA2) in tobacco hornworms Manduca sexta

The entomopathogenic bacterium, Xenorhabdus nematophila, induces immunodepression in target insects and finally leads to lethal septicemia of the infected hosts. A hypothesis has been raised that the bacteria inhibit eicosanoid-biosynthesis pathway to interrupt immune signaling of the infected hosts. Here, we show direct evidence that X. nematophila inhibits the activity of phospholipase A2 (PLA2), the initial step in the eicosanoid-biosynthesis pathway. Inhibition of PLA2 was dependent on both incubation time with X. nematophila and the bacterial concentration in in vitro PLA2 preparations of Manduca sexta hemocytes. While living bacteria inhibited PLA2 activity, heat-killed X. nematophila rather increased PLA2 activity. X. nematophila secreted PLA2 inhibitor(s) which were detected in the organic, but not aqueous, extract of the bacterial culture medium. The PLA2 inhibitory activity of the organic extract was lost after heat treatment. These results clearly indicate that X. nematophila inhibits PLA2 activity, and thereby inhibits eicosanoid biosynthesis which leads to immunodepression of the infected hosts.     

Quantitative and qualitative evaluation of iNOS expression in turbot (Psetta maxima) infected with Enteromyxum scophthalmi

Enteromyxum scophthalmi is the causative agent of turbot enteromyxosis, an intestinal parasitisation that produces severe desquamative enteritis leading to a cachectic syndrome and eventually the death. It is well known the importance of the innate immune response against parasites in fish, with the release of antimicrobial substances such as reactive oxygen and nitrogen species, produced by the inducible nitric oxide synthase (iNOS). This enzyme is mainly found in phagocytes, but also in structural cells from the intestinal mucosa. The aim of this study was to characterize iNOS in intestine and lymphohaematopoietic organs (spleen and anterior kidney) of turbot by means of immunohistochemistry in order to assess the possible changes of this enzyme through the infection. The presence of the enzyme was evaluated in control and E. scophthalmi-infected turbot. The results showed immunoreactivity in the apical border of enterocytes and mild staining of goblet cells in both control and infected turbot although it was more evident and widespread in infected turbot compared to control. Moderate numbers of iNOS+ cells were present in the lamina propria-submucosa of fish which presented moderate and severe inflammatory infiltrates at this level. In spleen and kidney, iNOS+ cells were scattered through the parenchyma and, in severely infected fish, tended to be allocated near the vascular structures and melano-macrophage centres. The number of positive cells at the lymphohaematopoietic organs was significantly higher in infected turbot and increased as infection progressed. The increase in the expression of iNOS in the tissues of E. scophthalmi-infected turbot was more evident in individuals with severer lesions. The measurement of the levels of iNOS during turbot enteromyxosis reveals a possibly delayed response that would not able to eliminate the parasites but would exacerbate mucosal injury.     

Ultrastructural study on a novel microsporidian, Endoreticulatus eriocheir sp. nov. (Microsporidia, Encephalitozoonidae), parasite of Chinese mitten crab, Eriocheir sinensis (Crustacea, Decapoda)

A microsporidian pathogen, infecting the epithelial cells of the hepatopancreas of Chinese mitten crab, Eriocheir sinensis, was studied by electron microscopy. The detailed ultrastructure of life cycle of the pathogen including proliferative and sporogonic developmental stages are provided. All stages of the parasite are haplokaryotic and develop in a vacuole bounded by a single membrane in contact with host cell cytoplasm. Sporogenesis is synchronous with the same developmental stage in one vacuole. Sporogony shows a characteristic of multinucleate sporogonial plasmodia divided by rosette-like division, producing 4 or 8 sporoblasts. The mature spore is ellipsoidal, length (mean) 1.7 microm, width 1.0 microm, with a uninucleate in the center of the sporoplasm, 7 turns of the polar filament, a bell-like polaroplast of compact membranes and obliquely positioned posterior vacuole. The morphological characteristics of this novel microsporidian pathogen have led us to assign the parasite to a new species of Endoreticulatus, E. eriocheir sp. nov., that has not been reported previously from crab.     

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.     

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P ≤ 0.001), either alone or in combination with either bacterium. However, treatment with bacteria did not affect P. nicotianae development in roots. The results suggest different mechanisms by which T. semipenetrans, B. cepacia, and B. megaterium may mitigate virulence of P. nicotianae.     

To complete their life cycle, pathogenic nematode-bacteria complexes deter scavengers from feeding on their host cadaver

The life cycle of commercially used molluscicidal rhabditid nematodes Phasmarhabditis hermaphrodita and entomopathogenic steinernematid nematodes is similar: infective stages carry symbiotic bacteria, which kill their host. Nematodes complete their life cycle feeding on the proliferating symbiont and the host tissue. After 1-2 weeks, new infective stages carrying the bacteria leave the host cadaver in search of new hosts. The removal of invertebrate cadavers by scavengers is extremely fast and represents a severe threat to the developing nematodes.Two-choice trials were used to assess prey choice of the generalist predator/scavenger Pterostichus melanarius (Coleoptera: Carabidae) between Deroceras reticulatum (Mollusca: Agriolimacidae) slugs or wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae killed by infection of P. hermaphrodita/Steinernema affine and control killed by freezing. We demonstrate that the presence of either of the two nematodes tested deters the beetles from consuming infected cadavers. As P. hermaprodita cannot infect an insect host, we hypothesise the deterrent effect being an evolutionary adaptation of the nematode/bacteria complex rather than the ability of the beetles to avoid potentially infective cadavers.     

Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia)

In this report, an experimental infection of Apis mellifera by Nosema ceranae, a newly reported microsporidian in this host is described. Nosema free honeybees were inoculated with 125,000 N. ceranae spores, isolated from heavily infected bees. The parasite species was identified by amplification and sequencing the SSUrRNA gene of the administered spores. Three replicate cages of 20 honeybees each were prepared, along with one control cage (n=20) supplied with sugar syrup only. The infection rate was 100% at the dosage administered. The presence of Nosema inside ventricular cells was confirmed in the samples using ultrathin sectioning and transmission electron microscopy. By day 3 p.i. a few cells (4.4%+/-1.2) were observed to be parasitized, whereas by 6 days p.i. more than half of the counted cells (66.4%+/-6) showed different parasite stages, this value increasing on day 7 p.i. (81.5%+/-14.8). Only one control bee died on day 7 p.i. In the infected groups, mortality was not observed until day 6 p.i. (66.7%+/-5.6). Total mortality on day 7 p.i. was 94.1% in the three infected replicates and by day 8 p.i. no infected bee was alive. After the infection, the parasites invaded both the tip of folds and the basal cells of the epithelium and the autoinfective capacity of the spores seemed to spread the infection rapidly between epithelial cells. On day 3 p.i., mature spores could be seen inside host cell tissue implying that the developmental cycle had been completed. The large number of parasitized cells, even the regenerative ones, the presence of autoinfective spores and the high mortality rate demonstrate that N. ceranae is highly pathogenic to Apis mellifera. Possible relation with bee depopulation syndrome is discussed by authors.