Role ofCristispira sp. and other bacteria in the chitinase and chitobiase activities of the crystalline style ofCrassostrea virginica (Gmelin)

Activity was found for chitinase and chitobiase in the crystalline styles of American oysters (Crassostrea virginica Gmelin) collected from the Chesapeake Bay (Maryland, USA). The oysters were maintained in tanks on natural food from a constant flow of unfiltered estuarine water. Chitinase and chitobiase specific activities were compared with total, viable, and chitinoclastic bacterial counts andCristispira counts. Regression analyses revealed that one correlation, chitobiase vsCristispira, was significant (P < 0.05). Several oysters were fed chitin in the presence or absence of chloramphenicol. Although no chitinoclasts were present in the antibiotic-treated oysters, the treatment means did not differ significantly (P > 0.05) for either chitinase or chitobiase activity. In several cases with both chitin-fed and naturally fed oysters, enzyme activity was found when noCristispira were present. The results of the investigations suggest that the oyster produces chitinase and chitobiase endogenously.     

First report on bacteria of the family <Emphasis Type="Italic">Spirochaetaceae</Emphasis> from digestive tract of endemic gastropods from Lake Baikal

Detection of bacteria of the family Spirochaetaceae in the crystalline style of 11 species of endemic gastropods from Lake Baikal is reported. Investigation by transmission and scanning electron microscopy showed that these spirochetes belonged to the genus Cristispira.     

Scanning Electron Microscopy of Cristispira Species in Chesapeake Bay Oysters

Scanning electron microscopy was employed to observe the physical interactions between Cristispira spp. and the crystalline style of the Chesapeake Bay oyster (Crassostrea virginica Gmelin 1791). Cristispira organisms were found associated with both the inner and outer layers of the posterior two-thirds of the style. The spirochetes possessed blunt-tipped ends, a cell diameter range of 0.6 to 0.8 μm, and distended spirochetal envelopes which followed the contour of the cells. Transmission electron microscopy showed that the distension of the envelope was probably due to the containment of numerous axial filaments. In addition, they were found to possess two distinct spiral shapes which were dependent on whether their location was inside or on the surface of the style.     

New phylogenetic lineages of the <Emphasis Type="Italic">Spirochaetes</Emphasis> phylum associated with <Emphasis Type="Italic">Clathrina</Emphasis> species (Porifera)

Though spirochetes have been repeatedly found in marine sponges and other invertebrates, little attention has been paid to the specificity of this association. This study demonstrates that different genoand morphotypes of spirochetes can reside within the same sponge individual and develop in considerable numbers. Specimens of the calcareous sponge Clathrina clathrus collected from the Adriatic Sea off Rovinj (Croatia) were found to harbor spirochete-like bacteria, which were characterized by scanning electron microscopy (SEM), 16S rRNA gene analysis, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Two novel spirochete sequence types related to the Brachyspiraceae could be retrieved. By use of specifically designed CARD-FISH probes, the C. clathrus-associated sequences could be assigned to a linear and a helical spirochete morphotype. Both were located within the sponge mesohyl and resembled the spirochete-like cells identified by SEM. In addition, from a Clathrina sp., most likely C. coriacea, that originated from Indonesian coastal waters, four different spirochete type sequences were recovered. Two of these also affiliated with the Brachyspiraceae, the other two were found associated with the Spirochaetaceae, one with the genera Borrelia and Cristispira.     

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protect Acacia food bodies from exploitation

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two‐dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz‐type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin‐like and elastase‐like proteolytic activity in the guts of seed‐feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase‐like and to a lower degree chymotrypsin 1‐like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term ‘exclusive rewards’ can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.     

Co-evolution and symbiont replacement shaped the symbiosis between adelgids (Hemiptera: Adelgidae) and their bacterial symbionts

The Adelgidae (Insecta: Hemiptera), a small group of insects, are known as severe pests on various conifers of the northern hemisphere. Despite of this, little is known about their bacteriocyte‐associated endosymbionts, which are generally important for the biology and ecology of plant sap‐sucking insects. Here, we investigated the adelgid species complexes Adelges laricis/tardus, Adelges abietis/viridis and Adelges cooleyi/coweni, identified based on their coI and ef1alpha genes. Each of these insect groups harboured two phylogenetically different bacteriocyte‐associated symbionts belonging to the Betaproteobacteria and the Gammaproteobacteria, respectively, as inferred from phylogenetic analyses of 16S rRNA gene sequences and demonstrated by fluorescence in situ hybridization. The betaproteobacterial symbionts of all three adelgid complexes (‘Candidatus Vallotia tarda’, ‘Candidatus Vallotia virida’ and ‘Candidatus Vallotia cooleyia’) share a common ancestor and show a phylogeny congruent with that of their respective hosts. Similarly, there is evidence for co‐evolution between the gammaproteobacterial symbionts (‘Candidatus Profftia tarda’, ‘Candidatus Profftia virida’) and A. laricis/tardus and A. abietis/viridis. In contrast, the gammaproteobacterial symbiont of A. cooleyi/coweni (‘Candidatus Gillettellia cooleyia’) is different from that of the other two adelgids but shows a moderate relationship to the symbiont ‘Candidatus Ecksteinia adelgidicola’ of A. nordmannianae/piceae. All symbionts were present in all adelgid populations and life stages analysed, suggesting vertical transmission from mother to offspring. In sharp contrast to their sister group, the aphids, adelgids do not consistently contain a single obligate (primary) symbiont but have acquired phylogenetically different bacterial symbionts during their evolution, which included multiple infections and symbiont replacement.     

Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples

Antibiotic resistance in 40 Staphylococcus aureus clinical isolates from 110 diabetic patients (36%) was evaluated. Of these, 32 (80%) of the isolates showed multidrug-resistance to more than eight antibiotics and 35% isolates were found to be methicillin resistant S. aureus (MRSA). All 40 S. aureus strains (100%) screened from diabetic clinical specimens were resistant to penicillin, 63% to ampicillin, 55% to streptomycin, 50% to tetracycline and 50% to gentamicin. Where as low resistance rate was observed to ciprofloxacin (20%) and rifampicin (8%). In contrast, all (100%) S. aureus strains recorded susceptibility to teicoplanin, which was followed by vancomycin (95%). Genotypical examination revealed that 80% of the aminoglycoside resistant S. aureus (ARSA) have aminoglycoside modifying enzyme (AME) coding genes; however, 20% of ARSA which showed non-AME mediated (adaptive) aminoglycoside resistance lacked these genes in their genome. In contrast all MRSA isolates possessed mecA, femA genetic determinants in their genome.

     

Studies on the antileishmanial properties of the antimicrobial peptides temporin A,B and 1Sa

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector‐borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetoplast DNA permits characterization of pathogenic plant trypanosomes of economic importance

Summary— Twelve Phytomonas isolates were obtained from different plants originating from several countries and cultured in vitro in complex media. The kinetoplast DNA (kDNA) was purified and observed by electron microscopy. The structure of kDNA from all isolates appeared as a large network of interlocked minicircles with some maxicircles extruding from the network, as has often been shown for Trypanosomatidae. Topoisomerase II resolved the kDNA network into free minicircles which were then analyzed by electron microscopy and by electrophoresis in agarose gel. The minicircle sizes varied from 1.3 to 2.8 kilobase pairs according to the Phytomonas isolate. The analysis by restriction endonucleases revealed a base sequence heterogeneity in the minicircles of 10 of these Phytomonas isolates. By contrast, in 2 Phytomonas isolates, more than 90% of their minicircle content was found to be homogeneous. Most interestingly, the minicircle cleavage patterns were found to be different between Phytomonas isolates and thus could be used to distinguish them.     

Direct observation of biopolymers produced from submerged culture of edible mushrooms by atomic force microscopy

Two biopolymers produced from submerged culture of edible mushrooms were directly observed by atomic force microscopy. Biopolymers were deposited on mica from dilute aqueous solution and imaged in air through a thin layer of adsorbed water and their hydrated structures were observed by a tapping mode. A single biopolymer molecule obtained from Cordyceps militaris was typical of a rod-like structure with bending point, which can form intra- and inter-molecular supercoils. In contrast, the image for low molecular weight biopolymer from Paecilomyces sinclarii is typical of a branched structure in which more extensive interaction leads to the formation of network-like matrix.     

Characterization of Salmonella spp. isolated from patients below 3 years old with acute diarrhoea

Fifteen strains of Salmonella were isolated from children with clinically diagnosed diarrhoea aged below 3 years old, who had been admitted to K7 ward, Pediatric Institute, Kuala Lumpur Hospital. The isolates were tested for their susceptibility to a range of antimicrobial agents, and typed by serological tests and randomly amplified polymorphic DNA (RAPD) fingerprinting. All the strains had a similar pattern of antimicrobial susceptibility, where they were susceptible to a wide range of antimicrobial agents. The serological test has typed them into three serovars, which were identified as Salmonella enterica ser. Akanji, Salmonella enterica ser. Hindmarch and Salmonella enterica ser. Richmond. In contrast, the RAPD fingerprinting classed them into two major clusters, cluster 1 consisting of 12 strains of Salmonella and cluster 2 consisting of three strains of Salmonella.     

Transfer of Brassica tournefartii (TT) genes to allotetraploid oilseed Brassica species (B. juncea AABB, B. napus AACC, B. carinata BBCC): homoeologous pairing is more pronounced in the three-genome hybrids (TACC, TBAA, TCAA, TCBB) as compared to allodiploids (TA, TB, TC)

For the transfer of genes from B. tournefortii (TT) to the allotetraploid oilseed brassicas, B. juncea AABB, B. carinata BBCC and B. napus AACC, B. tournefortii was first crossed with the three basic diploid species, B. campestris (AA), B. nigra (BE) and B. oleracea (CC), to produce the allodiploids TA, TB and TC. These were tetraploidized by colchicine treatment to produce the allotetraploids TTAA, TTBB and TTCC, which were further crossed with B. juncea and B. napus to produce three-genome hybrids with substitution-type genomic configurations: TACC, TBAA and TCAA. These hybrids along with another hybrid TCBB produced earlier, the three allodiploids, their allotetraploids and the four diploid parent species were studied for their male meiotic behaviour. The diploid parent and the allotetraploids (TTAA, TTBB and TTCC) showed regular meiosis although the pollen viability was generally low in the allotetraploids. In the allodiploids (TA, TB and TC) only some end-to-end associations were observed without any clearly discernible chiasmata or exchange points. Chromosomes involved in end-to-end associations were randomly distributed at the metaphase/anaphase-I stages. In contrast, the three-genome hybrids (TACC, TBAA, TCAA and TCBB) showed normal bivalents whose number exceeded the expected bivalent values. Bivalents arising out of homoeologous pairing were indistinguishable from normal pairs by their disjunction pattern but could be distinguished on the basis of the heteromorphy of the homoeologous chromosomes. The three-genome hybrids could be backcrossed to allotetraploid oilseed brassicas as they had some fertility. In contrast, the allodiploids could neither be selfed nor back-crossed. On the basis of their meiotic stability, in terms of more pronounced homoeologous pairing and fertility for backcrossing, the three-genome configurations provide the best possible situation for the introgression of alien genes from the secondary gene pool to the allotetraploid oilseed crops B. juncea, B. napus and B. carinata.     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

The influence of the developing bacterial spore on the mother cell

Mutants unable to develop a completely engulfed forespore do not lose their viability, i.e., their ability to resume cell division, for at least 10 hr after the end of exponential growth. In contrast, mutants, which are blocked at later stages in development and which are able to produce completely engulfed forespores, lose their ability to divide. The time course of this decrease in viability coincides with the time course for the appearance of completely enclosed forespores. Experiments with the sporulating standard strains of Bacillus subtilis and B. megaterium suggest that the mother cells also lose their viability at about the time of forespore enclosure. These results indicate that the forespore, as soon as it is completely engulfed and thus committed to continue differentiation, somehow prevents the mother cell (sporangium) from resumption of growth.     

Resource exploitation of larvae ofGastrophysa atrocyanea Motschulsky andgalerucella vittaticollis Baly (Coleoptera: Chrisomelidae) under a limited resource condition

Resource exploitation by and intraspecific competition in larvae of Gastrophysa atrocyanea and Galerucella vittaticollis were investigated in field and laboratory experiments. Larvae of both species frequently suffered from food shortages in the field. When G. atrocyanea larvae suffered from a food shortage, severe intraspecific competition occurred because of lack of predation and parasitism. This exploitive competition was caused by a local food shortage of the host plant. Individuals survived by fast exploitation when food became abundant (contest type competition). the G. atrocyanea larvae were wasteful of the food resource, and no mechanism by which to economize on the utilization of the resource was acquired because of their exploitation of the abundant resource. In contrast, the G. vittaticollis population probably is regulated by extrinsic factors such as predation and parasitism. Those larvae grew into smaller adults than those of G. atrocyanea under a food shortage, so that their wasted food consumption was lower than that of G. atrocyanea. Although intraspecific competition was similar to that for G. atrocyanea, it was not as severe. The food for G. vittaticollis was apt to be appropriated by other wasteful exploitators such as G. atrocyanea, which was superior in resource exploitation; therefore G. vittaticollis frequently suffered a food shortage. Consequently selection in relation to tolerance to starvation became more acute for G. vittaticollis than for G. atrocyanea, and individuals of G. vittaticollis that could endure starvation better may have been selected.     

Germination and seedling growth under anaerobic conditions in Echinochloa crus-galli (barnyard grass)*

Abstract Although rice has long been recognized to be uniquely adapted for growth in low oxygen environments of flooded rice fields, rice weeds of the Echinochloa crus-galli complex appear to be at least as well specialized for germination and growth under such unusual biological conditions. Seeds of two varieties of E. crus-galli germinate and grow for prolonged periods in a totally oxygen-free environment. E. crus-galli germinates as well as rice (Oryza sativa) under a total nitrogen atmosphere and produces as large a seedling in spite of its much smaller seed size. Like rice, the seedlings of E. crus-galli are unpigmented, the primary leaves do not emerge from the coleoptile and no root growth occurs without oxygen. Of particular interest is the ultrastructure of mitochondria from anaerobically-grown seedlings. Mitochondrial profiles from the primary leaf of seedlings grown continuously in nitrogen are very similar to those grown aerobically. The size and shape of the mitochondria are similar and the cristae are numerous and normal in appearance. This is in sharp contrast to previous studies of other species which have reported that mitochondria were vesiculate and tended to lose their normal fine-structure after similar periods without oxygen. Finally, based on ultrastructure and ¹⁴C labeling studies, anaerobically-grown seedlings are highly active metabolically, which may explain, at least for E. crus-galli var. oryzicola, its ability to germinate and emerge from flooded rice fields.     

Enzymatic Degradation of Colistin Isolation and Identification of α-N-Acyl α,γ-Diaminobutyric Acid and Colistin Nonapeptide

Colistin, a fatty acyl peptide antibiotic, was attacked by proteolytic enzymes such as papain, ficin and bromelain, and as degradation product, a peptide portion retaining the ring structure of colistin was liberated. In contrast, an analogous antibiotic polymyxin B showed a characteristic resistance to the catalytic activity of papain.

Colistin nonapeptide and α-N-fatty acyl α,γ-diaminobutyric acid were obtained as products from the above enzymatic hydrolyzates of colistin and their chemical and physicochemical properties were investigated.

Contrary to colistin, this colistin nonapeptide was inactive to Escherichia coli. NIHJ and to many other strains even at a concentration of 800 mcg/ml by the agar dilution method. As α-N-fatty acyl α,γ-diaminobutyric acid which is rest part of colistin was added to colistin nonapeptide, antimicrobial activity of colistin nonapeptide did not increase.     

Partner recognition in a perennially monogamous pipefish,Corythoichthys haematopterus

The pipefish Corythoichthys haematopterus, which forms lifelong pair bonds and exhibits strict monogamy, performs a daily ritualized intrapair interaction, called the greeting. Cross-pairing experiments were performed to examine the capacity for partner recognition in C. haematopterus during the greeting. When pair members were released into an artificially constructed greeting arena, they exhibited typical greeting behavior immediately after contact. In contrast, when males and females from different pairs met, almost no displays were exchanged. These results strongly suggest that paired C. haematopterus can recognize their own partners, irrespective of the meeting location.     

Characterisation of a new thermoalkaliphilic bacterium for the production of high-quality hemp fibres, <Emphasis Type="Italic">Geobacillus thermoglucosidasius</Emphasis> strain PB94A

Novel thermophilic and alkaliphilic bacteria for the processing of bast fibres were isolated using hemp pectin as substrate. The strain PB94A, which showed the highest growth rate (μ = 0.5/h) was identified as Geobacillus thermoglucosidasius (DSM 21625). The strain grew optimally at 60°C and pH 8.5. During growth on citrus pectin, the strain produced pectinolytic lyases, which were excreted into the medium. In contrast to the commercially available pectinase Bioprep 3000 L, the enzymes from G. thermoglucosidasius PB94A converted pectin isolated from hemp fibres. In addition to hemp pectin, the culture supernatant also degraded citrus, sugar beet and apple pectin and polygalacturonic acid. When hemp fibres were incubated with the cell-free fermentation broth of G. thermoglucosidasius PB94A, the fineness of the fibres increased. The strain did not produce any cellulases, which is important in order to avoid damaging the fibres during incubation. Therefore, these bacteria or their enzymes can be used to produce fine high-quality hemp fibres.     

Effects of carotenoid sequestration on a caterpillar's cryptic coloration and susceptibility to predation

Carotenoids are used for many functions by animals, including combining with other pigments to produce aposematic and cryptic coloration. Carotenoids in combination with blue pigments are responsible for green coloration in many caterpillars, and thus carotenoid sequestration may reduce their contrast against a green foliage background. We tested the hypothesis that carotenoid sequestration reduces contrast and enhances survival by rearing Trichoplusia ni Hübner (Lepidoptera: Noctuidae) on Brassica oleracea L. var. Acephala (Brassicaceae) leaves and exposing them to predators. We found that carotenoids derived from the host plant are partially excreted, along with chlorophyll, but also sequestered in hemolymph. Larvae that were given plants that provided carotenoids showed less contrast against their host plants within 1 day compared to larvae that were not provided with carotenoids. Last, both short‐term field observations and laboratory trials of larvae caged with predatory Podisus maculiventris Say (Hemiptera: Pentatomidae) nymphs showed that survival of carotenoid‐sequestering larvae was higher compared to larvae that did not sequester. These results suggest that carotenoid sequestration may be an important adaptive strategy that reduces susceptibility to natural enemies that hunt by sight. Further research that examines the mechanisms by which carotenoids are absorbed and modified will lend insights into the evolution of carotenoids functioning as passive defensive compounds.