Molecular screening of Entamoeba spp. (E. histolytica, E. dispar, E. coli, and E. hartmanni) and Giardia intestinalis using PCR and sequencing.

A wide range of intestinal protozoan parasites inhabit the human gut. To establish a more comprehensive molecular screening, we designed PCR-sequencing screening methods for Entamoeba spp., including commensal species, and Giardia intestinalis, and performed such methods using 174 stool samples collected from Kenyan children. The prevalences of the target species were as follows: E. histolytica (2/174, 1.1%), E. dispar (20/174, 11.5%), E. coli (107/174, 61.5%), E. hartmanni (77/174, 44.3%), and G. intestinalis (54/174, 31.0%). PCR amplicons specific to G. intestinalis was differentiated to assemblages A (8/174, 4.6%) and B (46/174, 26.4%). PCR specificity for Entamoeba spp. was quite high, except for some cross-reactions between E. hartmanni detection primers and G. intestinalis, although the false-positive amplicons were discernible by the band size. The 18S rRNA PCR primers that was designed by Monis et al. in 1999 for G. intestinalis, have specificity issue, therefore amplicon sequencing was essential not only to determine assemblage classifications but also to confirm the positive results by eliminating potential non-specific reactions. The detection sensitivity of both the Entamoeba universal PCR and the G. intestinalis PCR was more than 100 copies of the target loci, which is sufficient for detecting a single trophozoite or cyst of both species.

Parallel chemical switches underlying pollinator isolation in Asian Mitella.

Floral scents are among the key signals used by pollinators to navigate to specific flowers. Thus, evolutionary changes in scents should have strong impacts on plant diversification, although scent-mediated plant speciation through pollinator shifts has rarely been demonstrated, despite being likely. To examine whether and how scent-mediated plant speciation may have occurred, we investigated the Asimitellaria plant lineage using multidisciplinary approaches including pollinator observations, chemical analyses of the floral scents, electroantennographic analyses and behavioural bioassays with the pollinators. We also performed phylogenetically independent contrast analyses of the pollinator/floral scent associations. First, we confirmed that the pairs of the sympatric, cross-fertile Asimitellaria species in three study sites consistently attract different pollinators, namely long-tongued and short-tongued fungus gnats. We also found that a stereoisomeric set of floral volatiles, the lilac aldehydes, could be responsible for the pollinator specificity. This is because the compounds consistently elicited responses in the antennae of the long-tongued fungus gnats and had contrasting effects on the two pollinators, that is triggering the nectaring behaviour of long-tongued fungus gnats while repelling short-tongued fungus gnats in a laboratory experiment. Moreover, we discovered that volatile composition repeatedly switched in Asimitellaria between species adapted to long-tongued and short-tongued fungus gnats. Collectively, our results support the idea that recurrent scent-mediated speciation has taken place in the Asimitellaria-fungus gnat system.

Purification and identification of major soluble 40-kDa antigenic protein from Entamoeba histolytica: its application for serodiagnosis of asymptomatic amebiasis.

One of the major soluble antigenic proteins of Entamoeba histolytica was purified to homogeneity and identified on a molecular basis. Its recombinant protein was expressed in Escherichia coli as a fusion protein with Shistosoma japonicum glutathione S-transferase. Apparent molecular weight of the purified antigenic protein was estimated to be 40-kDa and molecular-based analysis indicated that the purified protein was NADP+-dependent alcohol dehydrogenase (EhADH1). The application of the purified protein for the serodiagnosis of amebiasis was evaluated using an enzyme-linked immunosorbent assay applied to sera obtained from patients with amebiasis and healthy human controls. The purified protein was well recognized by the sera from asymptomatic amebiasis humans (22/22, 100%), whereas, it was less recognized by the sera from symptomatic amebiasis patients (5/16, 31%) with amebic colitis or liver abscess. To confirm the antigenicity of EhADH1, the recombinant glutathione S-transferase-EhADH1 fusion protein was also evaluated by the enzyme-linked immunosorbent assay using the same sera. The recombinant protein was also recognized by the sera from asymptomatic amebiasis humans (14/22, 64%) and less recognized by the sera from symptomatic amebiasis patients (2/16, 13%). These results suggest that the purified protein is applicable antigen for serodiagnostic screening of asymptomatic amebiasis humans.

Behaviour of pathogenic E. coli and Salmonella enteritidis in small domestic sewage treatment apparatus ("Johkasou").

"Johkasou" is a small sewage treatment apparatus commonly used in Japan which can effectively treat domestic wastewater in places where a public sewage system is difficult to supply. The behaviour of enterohaemorrhagic E. coli O157 and Salmonella enteritidis in a "Johkasou" was studied. Their reduction rates depended significantly on the water temperature in the "Johkasou" with minimal decrease in numbers at 10 degrees C within 48 h. The reduction rates increased at 20 degrees C and 30 degrees C where 4 log reduction could be expected. The reduction rates were influenced by the BOD of the solutions that contained the pathogens with the lower the BOD the higher the reduction rate. The reduction rates were about the same between both pathogens. The result showed that it was necessary to disinfect the effluent as some pathogens can pass through the apparatus when some users of the apparatus excrete pathogens.

Evolving learnable neutral networks under changing environments with various rates of inheritance of acquired characters: comparison between Darwinian and Lamarckian evolution.

The processes of adaptation in natural organisms consist of two complementary phases: learning, occurring within each individual's lifetime, and evolution, occurring over successive generations of the population. In this article, we study the relationship between learning and evolution in a simple abstract model, where neural networks capable of learning are evolved using genetic algorithms (GAs). Individuals try to maximize their life energy by learning certain rules that distinguish between two groups of materials: food and poison. The connective weights of individuals' neural networks undergo modification, that is, certain characters will be acquired, through their lifetime learning. By setting various rates for the heritability of acquired characters, which is a motive force of Lamarckian evolution, we observe adaptational processes of populations over successive generations. Paying particular attention to behaviors under changing environments, we show the following results. Populations with lower rates of heritability not only show more stable behavior against environmental changes, but also maintain greater adaptability with respect to such changing environments. Consequently, the population with zero heritability, that is, the Darwinian population, attains the highest level of adaptation to dynamic environments.

(Z,E,E)-dodecatrien-1-ol: A minor component of trail pheromone of termite,Coptotermes formosanus Shiraki.

In the course of the elucidation of the primary structure of an isolated trail pheromone fromC. formosanus, a minor component that had the same molecular weight as the major trail pheromone, (Z,Z,E)-3,6,8-dodecatrien-1-ol [(Z,Z,E)-DTE-OH], was detected in the mass chromatogram ofm/z 180 of capillary GC-MS. The mass spectrum of the minor component showed a prominent pattern of dodecatrien-1-ol. Chemical analysis demonstrated that the complete structure was (Z,E,E)-DTE-OH. Furthermore, capillary GC-MS-HR-SIM analysis indicated that the component existed only in the workers ofCoptotermes formosanus Shiraki and was not present in workers ofReticulitermes speratus (Kolbe). This minor component may be a species-specific factor ofC. formosanus, although this was not suggested by a two-choice bioassay.

Identification of trail pheromone precursors from Subterranean termite,Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae).

Whole-body extracts of the termite,Coptotermes formosanus Shiraki served for examining the presence of trail pheromone precursor(s). Three trail pheromone precursor candidates, identified as dodecatrienyl stearate, dodecatrienyl oleate, and dodecatrienyl linoleate, were isolated using various chromatographic methods in conjunction with bioassay and by capillary GC-MS analyses.

[An improved differential medium, CA medium, for differentiating Shigella].

We devised a Citrate-Acetate (CA) medium for rapidly differentiating Shigella. The medium consisted of 3.0 g of sodium citrate, 2.0 g of sodium acetate, 0.2 g of glucose, 1.0 g of dipotassium phosphate, 1.0 g of mono ammonium phosphate, 0.2 g of magnesium sulfate, 5.0 g of sodium chloride, 0.08 g of brom thymol blue, 15.0 g of agar, and 1000 ml of distilled water. An evaluation was made of the CA medium, for the rapid differentiation of 23 Shigella strains, 129 Escherichia coli strains and 130 isolates, that formed colourless colonies suspected to be Shigella on SS agar plate, from feces of healthy people. The results obtained were as follows 1) On the CA medium, all Shigella strains did not grow and there was no change in colour. 2) Positive growth rates of E. coli strains after incubation for 24 hr at 37 degrees C on CA medium, sodium acetate medium (Acet) and Christensen citrate medium (C-Cit) were 96.0%, 95.2% and 28.0%, respectively. Therefore, the positive growth rate of E. coli strains after incubation for 24 hr on CA medium was significantly higher (p less than 0.01) than that on C-Cit medium. 3) Positive growth rates of isolates after incubation for 24 hr at 37 degrees C on CA medium, Acet medium and C-Cit medium were 95.4%, 83.1% and 71.5%, respectively. Therefore, the positive growth rates of isolates after incubation for 24 hr on CA medium was significantly higher (p less than 0.01) than that on Acet medium and C-Cit medium.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fundamental studies on serological diagnosis of amoebiasis. 1. Application of amoebic antigen for CF test to ELISA].

We studied the establishment of enzyme-linked immunosorbent assay (ELISA) using amoebic antigen for complement fixation (CF) test. Optimal dilution for ELISA of sera from patients was 1:100, and that of CF-antigen was 1:400. The upper limit of the 99% critical range of the reaction of negative sera was 0.068 (cut-off level). Absorbance of sera from patients diluted 1:100 to antigen and antibody titers of ELISA were strongly correlated, so it was possible to estimate antibody titers from absorbance of serum diluted 1:100. ELISA and CF test were done to compare sensitivity of the tests using 63 sera from patients with invasive amoebic disease. The sensitivity of ELISA compared well with CF test (62 sera were positive by ELISA and 61 by CF test). Only one sample was both positive by ELISA and negative by CF test. This sample had low ELISA titers, so this discrepancy was mainly due to the sensitivity of CF test in detecting lower levels of antibody. These results suggested that the amoebic antigen for CF test can be applicable to ELISA, and this method was so sensitive and specific.

Evidence for trail-pheromone precursor in termiteReticulitermes speratus (Kolbe) (Rhinotermitidae: Isoptera).

Whole body extracts of the termite,Reticulitermes speratus, were subjected to various chemical operations and bioassays to examine the presence of trail-pheromone precursor. Fractions that mainly contained fatty acid esters were obtained from hexane extracts by means of silica gel column chromatography. Trail-following activity of the fractions was activated by alkaline hydrolysis, while the original fractions did not show any conspicuous activity. Bioassay showed that the activity of hydrolyzed product was approximately 20 times as high as the original hexane extract. This suggests that the precursor candidate could be stored in termite bodies as an esterified form. Chemical analyses revealed that the complete structure of the hydrolyzed product was coincident with that of the native pheromone ((Z,Z,E)-3,6,8-dodecatrien-1-ol).