Efficacy of plants extracts from the Cerrado against adult female of <Emphasis Type="Italic">Dermacentor nitens</Emphasis> (Acari: Ixodidae)

Dermacentor nitens tick is commonly found in the equine auditory canal, where it causes economic losses due to its direct damage, causing blood spoliation, stress, transmission of pathogens, and predisposition to myasis and secondary bacterial infection in its hosts. In this study we evaluated the effect of ethanolic extracts of Cerrado plants on biological parameters of engorged females of D. nitens. Ethanolic extracts were prepared from the leaves of Schinopsis brasiliensis, Piptadenia viridiflora, Ximenia americana, and Serjania lethalis at 25–150 mg mL^?1. Groups of 10 engorged adult females were treated with these extracts and compared with a control containing distilled water and another control with organophosphate, using five replicates for each group. Compared with the control with water, S. lethalis and X. americana extracts at 100 and 150 mg mL^?1 significantly inhibited the posture ability. Differently, extracts of S. brasiliensis and P. viridiflora were the most effective in inhibiting larval hatching. Extracts of X. americana and P. viridiflora showed effective inhibition of reproductive parameters of the tick, presenting dose-dependent effect with IC₉₀ 78.86 and 78.94 mg mL^?1, respectively. Theses effective extracts contained low condensed tannin levels and their HPLC chromatograms revealed the presence of flavonoids. The efficacies of P. viridiflora and X. americana extracts were higher than 90% indicating that these extracts are promising as alternative agents for D. nitens control.     

Interactions of the manganese hyperaccumulator <Emphasis Type="Italic">Phytolacca americana</Emphasis> L. with soil pH and phosphate

Hyperaccumulators are plants that store exceptionally high concentrations of heavy metals or metalloids in their leaves. Phytolacca americana is one of the few species known to hyperaccumulate manganese (Mn); however, it is a common weedy species and has no specific association with high-Mn soils. Neither the mechanism by which P. americana hyperaccumulates Mn nor the ecological significance of this trait are well understood. It has recently been suggested that P. americana secretes acids into the rhizosphere as a means of acquiring phosphate, which might coincidentally increase Mn uptake. To determine whether P. americana acidifies the surrounding soil, plants were grown in rhizoboxes providing access to living roots. A thin layer of agar containing bromocresol green pH indicator dye was placed on the roots to observe color changes indicating acidification. Comparative studies showed that P. americana acidifies the rhizosphere significantly more than the non-accumulating plant Acalypha rhomboidea. A second experiment studied whether adjustment of soil pH and phosphate affect foliar Mn concentrations of P. americana. Concentrations of Mn in leaves were highest when plants were grown in acidified soils but were significantly lower in soils that were alkaline and/or enriched with phosphate. These results suggest that Mn hyperaccumulation may be a side effect of rhizosphere acidification as a phosphorus-acquisition mechanism, rather than an adaptation in its own right. The findings provide fundamental information about hyperaccumulator physiology and evolution, and may be relevant to attempts to utilize P. americana for phytoremediation.     

Variability,stability, and resilience of fecal microbiota in dairy cows fed whole crop corn silage

The microbiota of whole crop corn silage and feces of silage-fed dairy cows were examined. A total of 18 dairy cow feces were collected from six farms in Japan and China, and high-throughput Illumina sequencing of the V4 hypervariable region of 16S rRNA genes was performed. Lactobacillaceae were dominant in all silages, followed by Acetobacteraceae, Bacillaceae, and Enterobacteriaceae. In feces, the predominant families were Ruminococcaceae, Bacteroidaceae, Clostridiaceae, Lachnospiraceae, Rikenellaceae, and Paraprevotellaceae. Therefore, Lactobacillaceae of corn silage appeared to be eliminated in the gastrointestinal tract. Although fecal microbiota composition was similar in most samples, relative abundances of several families, such as Ruminococcaceae, Christensenellaceae, Turicibacteraceae, and Succinivibrionaceae, varied between farms and countries. In addition to the geographical location, differences in feeding management between total mixed ration feeding and separate feeding appeared to be involved in the variations. Moreover, a cow-to-cow variation for concentrate-associated families was demonstrated at the same farm; two cows showed high abundance of Succinivibrionaceae and Prevotellaceae, whereas another had a high abundance of Porphyromonadaceae. There was a negative correlation between forage-associated Ruminococcaceae and concentrate-associated Succinivibrionaceae and Prevotellaceae in 18 feces samples. Succinivibrionaceae, Prevotellaceae, p-2534-18B5, and Spirochaetaceae were regarded as highly variable taxa in this study. These findings help to improve our understanding of variation and similarity of the fecal microbiota of dairy cows with regard to individuals, farms, and countries. Microbiota of naturally fermented corn silage had no influence on the fecal microbiota of dairy cows.     

Antibiotic-Resistant Genes and Pathogens Shed by Wild Deer Correlate with Land Application of Residuals

The purpose of this study was to investigate genetic biomarkers of zoonotic enteric pathogens and antibiotic-resistant genes (ARGs) in the feces of white-tailed deer (Odocoileus virginianus) as related to proximity of deer to land that receives livestock manure or human waste biosolid fertilizers. Deer feces were collected in the St. Lawrence River Valley and Adirondack State Park of New York. Campylobacter spp. 16S rDNA was detected in 12 of 232 fecal samples (8 of 33 sites). Salmonellae were cultivated from 2 of 182 fecal samples (2 of 29 sites). Genetic virulence markers for Shiga-like toxin I (stx₁) and enterohemolysin (hylA) were each detected in one isolate of Escherichia coli; E. coli O157 was not detected in any of 295 fecal samples. ARGs detected in deer feces included ermB (erythromycin-resistant gene; 9 of 295 fecal samples, 5 of 38 sites), vanA (vancomycin-resistant gene; 93 of 284 samples, 33 of 38 sites), tetQ (tetracycline-resistant gene; 93 of 295 samples, 25 of 38 sites), and sul(I) (sulfonamide-resistant gene; 113 of 292 samples, 28 of 38 sites). Genetic markers of pathogens and ARGs in deer feces were spatially associated with collection near concentrated animal feeding operations (CAFOs; Campylobacter spp., tetQ, and ermB) and land-applied biosolids (tetQ). These results indicate that contact with human waste biosolids or animal manure may be an important method of pathogen and ARG transmission and that deer in proximity to land-applied manure and human waste biosolids pose increased risk to nearby produce and water quality.     

Investigation of the midgut structure and ultrastructure in <Emphasis Type="Italic">Cimex lectularius</Emphasis> and <Emphasis Type="Italic">Cimex pipistrelli</Emphasis> (Hemiptera: Cimicidae)

Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions—the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.     

Degradation capacities of bacteria and yeasts isolated from the gut of <Emphasis Type="Italic">Dendroctonus rhizophagus</Emphasis> (Curculionidae: Scolytinae)

Bark beetles (Curculionidae: Scolytinae) feed on the xylem and phloem of their host, which are composed of structural carbohydrates and organic compounds that are not easily degraded by the insects. Some of these compounds might be hydrolyzed by digestive enzymes produced by microbes present in the gut of these insects. In this study, we evaluated the enzymatic capacity of bacteria (Acinetobacter lwoffii, Arthrobacter sp., Pseudomonas putida, Pseudomonas azotoformans, and Rahnella sp.) and yeasts (Candida piceae, Candida oregonensis, Cyberlindnera americana, Zygoascus sp., and Rhodotorula mucilaginosa) isolated from the Dendroctonus rhizophagus gut to hydrolyze cellulose, xylan, pectin, starch, lipids, and esters. All isolates, with the exception of C. piceae, showed lipolytic activity. Furthermore, P. putida, P. azotoformans, C. americana, C. piceae, and R. mucilaginosa presented amylolytic activity. Esterase activity was shown by A. lwoffii, P. azotoformans, and Rahnella sp. Cellulolytic and xylanolytic activities were present only in Arthrobacter sp. and P. azotoformans. The pectinolytic activity was not recorded in any isolate. This is the first study to provide evidence on the capacity of microbes associated with the D. rhizophagus gut to hydrolyze specific substrates, which might cover part of the nutritional requirements for the development, fitness, and survival of these insects.     

Leaf Extracts of Selected Gardening Trees Can Attenuate Quorum Sensing and Pathogenicity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

An increasing concern on resistance to multiple-antibiotics has led to the discovery of novel agents and the establishment of new precaution strategy. Numerous plant sources have been widely studied to reduce virulence of pathogenic bacteria by interfering cell-to-cell based communication called quorum sensing (QS). Leaf extracts of 17 gardening trees were collected and investigated for their anti-QS effects using a sensor strain Chromobacterium violaceum CV026. Methanolic extracts of K4 (Acer palmatum), K9 (Acer pseudosieboldianum) and K13 (Cercis chinensis) leaves were selected for further experiments based on their antagonism effect on QS without inhibiting C. violaceum CV026 growth. Subsequently, the leaf extracts on QS-mediated virulence of Pseudomonas aeruginosa PAO1 involved in biofilm formation, motility, bioluminescence, pyocyanin production, QS molecules production, and Caenorhabditis elegans killing activity were evaluated. The biofilm formation ability and swarming motility of P. aeruginosa PAO1 were decreased approximately 50% in the presence of these leaf extracts at a concentration of 1 mg/mL. The expression level of lecA::lux of P. aeruginosa PAO1 and pyocyanin production were also reduced. The three leaf extracts also decreased autoinducer (AI) production in P. aeruginosa PAO1 without direct degradation, suggesting that AI synthesis might have been suppressed by these extracts. The three leaf extracts also showed anti-infection activity in C. elegans model. Taken together, these results suggest that methanolic leaf extracts of K4, K9 and K13 have the potential to attenuate the virulence of P. aeruginosa PAO1.     

Identification and field testing of floral odorants that attract the rove beetle <Emphasis Type="Italic">Pelecomalium testaceum</Emphasis> (Mannerheim) to skunk cabbage, <Emphasis Type="Italic">Lysichiton americanus</Emphasis> (L.)

Western skunk cabbage, Lysichiton americanus (Araceae), is pollinated mainly by the rove beetle Pelecomalium testaceum (Staphylinidae). Our objective was to determine the floral semiochemical(s) of L. americanus that attract(s) P. testaceum. Porapak Q headspace volatile extracts of L. americanus inflorescences were analyzed by gas chromatographic–electroantennographic detection (GC–EAD) and GC–mass spectrometry. In GC–EAD analyses, three floral odorants [(E)-4 nonene, (E)-5-undecene, indole] elicited consistent responses from the antennae of female P. testaceum. In field experiments, traps baited with a blend of these three components (“3-CB”) captured significantly more P. testaceum than unbaited control traps. Traps baited with the 3-CB, the two hydrocarbons, or indole, each captured significantly more beetles than unbaited control traps, indicating redundancy in the semiochemical blend. Moreover, traps baited with indole captured significantly more beetles than traps baited with either the 3-CB, or the hydrocarbons, indicating that indole is a key floral attractant for P. testaceum. Many necrophilous and coprophilous insects respond to indole in search of carrion or feces, but P. testaceum has never been associated with these types of resources. Both electrophysiological and behavioral responses of P. testaceum to two hydrocarbon semiochemicals, which are not signature odorants of carrion or feces, may indicate that the beetles recognize the odor of L. americanus as an honest signal, seek and pollinate its inflorescences, and get rewarded with pollen and on-plant mating opportunities.     

Composition and diversity of the bacterial community in snow leopard (<Emphasis Type="Italic">Uncia uncia</Emphasis>) distal gut

Intestinal microflora influences many essential metabolic functions, and is receiving increasing attention from the scientific community. However, information on intestinal microbiota, especially for large wild carnivores, is insufficient. In the present study, the bacterial community in the feces of snow leopards (Uncia uncia) was described based on 16S rRNA gene sequence analysis. A total of 339 near-full-length 16S rRNA gene sequences representing 46 non-redundant bacterial phylotypes (operational taxonomical units, OTUs) were identified in fecal samples from four healthy snow leopards. Four different bacterial phyla were identified: Firmicutes (56.5 %), Actinobacteria (17.5 %), Bacteroidetes (13 %), and Proteobacteria (13 %). The phylum Actinobacteria was the most abundant lineage, with 40.4 % of all identified clones, but Clostridiales, with 50 % of all OTUs, was the most diverse bacterial order. The order Clostridiales was affiliated with four families: Clostridiaceae I, Lachnospiraceae, Peptostreptococcaceae, and Ruminococcaceae. Lachnospiraceae was the most diverse family with 17 OTUs identified. These findings were basically consistent with previous reports on the bacterial diversity in feces from other mammals.     

Female Responses to Synthetic Pheromone and Plant Compounds in <Emphasis Type="Italic">Agriotes brevis</Emphasis> Candeze (Coleoptera: Elateridae)

Traps baited with synthetic pheromone components of Agriotes brevis [geranyl butanoate + (E,E)-farnesyl butanoate] captured significantly higher numbers of not only male, but also female beetles, compared to unbaited controls. Catches of both sexes showed a clear positive relationship with increasing doses. In electroantennogram tests, antennal responses of females and males to a number of known Agriotes pheromone components, identified from pheromone glands, showed a similar trend, with geranyl butanoate eliciting the strongest responses. This suggests that the female and male A. brevis antennae are similar with respect to the perception of pheromone compounds, and female beetles have the sensory capabilities to perceive the pheromone components which they produce. Addition of the plant-derived compounds (Z)-3-hexenyl acetate, methyl benzoate, (Z)-3-hexenol and methyl salicylate (identified earlier from foliage as attractive for A. brevis) to the synthetic pheromone significantly increased catches. All the above results suggest that geranyl butanoate and (E,E)-farnesyl butanoate are constituents of an aggregation pheromone of A. brevis, in contrast to the general view of click beetle pheromones being “classical” sex pheromones. These findings could be useful for more precise monitoring and forecasting of damage, based on female catches.     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

The unusual gynoecium structure and extragynoecial pollen-tube pathway in <Emphasis Type="Italic">Phytolacca</Emphasis> (Phytolaccaceae)

The fusion of carpels into a unified compound gynoecium is considered a dominant feature of angiosperm evolution and it also occurs by postgenital fusion during the gynoecium development in some apocarpous species. However, we found the reverse process, the separation of carpels from combined carpel primordia, during the development of the gynoecium in Phytolacca. Semithin sectioning and scanning electron microscopy were utilised to observe the structure and development of the gynoecia in Phytolacca acinosa and Phytolacca americana, fluorescence microscopy was utilised to observe the pollen tube growth in the gynoecia of the two species, and the topology method was applied to analyze the relationship between the gynoecium structure and pollen tube pathway. Although the gynoecia of P. acinosa and P. americana are both syncarpous, the degree of carpel fusion in the mature gynoecia of the two syncarpous species is different as a result of variant developmental processes. However, change in the degree of carpel fusion during the development of gynoecia in Phytolacca does not affect pollen tube growth because of the existence of the extragynoecial pollen-tube pathway. Thus, the change in the degree of carpel fusion in Phytolacca is primarily the result of diversification of developmental processes related to selection pressure.     

Potential application of genetically identical somatic cell nuclear transfer-cloned dogs for gastrointestinal microbiota analysis

This study investigated the gastrointestinal microbiota in three genetically identical cloned dogs (A, B and C) by somatic cell nuclear transfer. We collected feces from three cloned dogs and their feed to investigate gastrointestinal microbiota using both culture-dependent and culture-independent methods. A total of 962 strains from the feces of cloned dogs were isolated using aerobic and anaerobic culture methods. The dominant microorganisms were Enterococcus faecalis and Enterococcus faecium in all fecal samples. In particular, the fecal sample from cloned dog C had similar proportions of three species (E. faecalis, E. faecium and Lactobacillus murinus). In all, 29 DNA fragments were identified by PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The highest DGGE band intensities were for E. faecalis from cloned dogs A and C and for Clostridium sordellii from cloned dog B, with relative intensities of 15.2, 17.7 and 14.4%, respectively. The other strains identified from the cloned dogs were Chryseobacterium soldanellicola, Escherichia coli, L. murinus, Streptococcus alactolyticus, Weissella confusa and uncultured bacterium. Some microbes isolated from the fecal samples, including C. soldanellicola and W. confuse, were derived from the feed. Overall, gastrointestinal microbiota of all genetically identical cloned dogs, maintained under the same environmental and feeding conditions, showed similar profiles in terms of species diversity analyzed by PCR-DGGE, although there were proportional differences in the amounts of bacterial species. To our knowledge, this is the first report to investigate and compare gastrointestinal microbiota of three genetically identical dogs.     

Characterization of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Goose Feces from State Parks in Northeast Ohio

Staphylococcus aureus can colonize a range of species. Although numerous studies have isolated pathogenic bacteria from wild birds, very little is known regarding S. aureus and their potential to spread methicillin-resistant (MRSA) strains. The objective of this study was to determine the presence and molecular characteristics of S. aureus in geese fecal samples collected from ten state parks across Northeast Ohio (NEO). A total of 182 fecal samples from Canada geese (Branta canadensis) were collected in April 2015. Isolates were characterized using multi-locus sequence (MLST) and spa typing, as well as PCR to detect the presence of Panton–Valentine leukocidin (PVL), mecA, and scn genes. Antibiotic susceptibility testing was done via Vitek-2 system. The overall contamination by S. aureus in fecal samples was 7.1% (13/182); 7/182 (3.8%) were MRSA and 6/182 (3.3%) were methicillin-susceptible S. aureus (MSSA). One isolate was positive for PVL. A total of eight different spa types were observed. MLST included ST5, ST8, ST291, ST298, and ST2111. One (7.7%) MSSA isolate was multi-drug resistant. The S. aureus contamination in NEO state parks ranged from 0% (park 1, 4, 8, 9) to 35% (7/20) (park 5). Parks 2, 3, 6, and 7 had 5% (1/20) positive. The results of this study indicate that the feces of geese collected at various state parks in NEO may harbor S. aureus.     

Efficacy of botanical extracts from Brazilian savannah against <Emphasis Type="Italic">Diabrotica speciosa</Emphasis> and associated bacteria

Botanical extracts are a plentiful resource of molecules with different biological activities, such as insecticides and antimicrobial pesticides. In this context, the aim of this work was to evaluate the efficacy of botanical extracts from the Brazilian savannah against Diabrotica speciosa and bacterial strains isolated from the gut of this insect under aseptic conditions. The bacterial isolates were identified by genomic and proteomic approaches, and bioassayed against eighteen botanical extracts in vitro. The best results of bacterial inhibitions were obtained for the extracts of Casearia sylvestris and Psidium laruotteanum. Fractions of C. sylvestris and P. laruotteanum, quantitatively evaluated by chromatographic analyses, showed a relationship between the bactericidal activity and phytochemical profile. In vivo assays showed that P. laruotteanum was also effective for the control of D. speciosa. Those results show that selected natural products can have both antimicrobial and insecticidal activities.     

Evaluation of the Diversity of Probiotic <Emphasis Type="Italic">Bacillus</Emphasis>, <Emphasis Type="Italic">Clostridium</Emphasis>, and <Emphasis Type="Italic">Bifidobacterium</Emphasis> Using the Illumina-Based Sequencing Method

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.     

Cultivation and utility of <Emphasis Type="Italic">Piptoporus betulinus</Emphasis> fruiting bodies as a source of anticancer agents

Piptoporus betulinus is a wood-rotting basidiomycete used in medicine and biotechnology. However, to date, no indoor method for cultivation of this mushroom fruiting bodies has been developed. Here we present the first report of successful production of P. betulinus mature fruiting bodies in artificial conditions. Four P. betulinus strains were isolated from natural habitats and their mycelia were inoculated into birch sawdust substrate supplemented with organic additives. All the strains effectively colonized the medium but only one of them produced fruiting bodies. Moisture and organic supplementation of the substrate significantly determined the fruiting process. The biological efficiency of the P. betulinus PB01 strain cultivated on optimal substrate (moisture and organic substance content of 55 and 65 and 25 or 35 %, respectively) ranged from 12 to 16 %. The mature fruiting bodies reached weight in the range from 50 to 120 g. Anticancer properties of water and ethanol extracts isolated from both cultured and nature-derived fruiting bodies of P. betulinus were examined in human colon adenocarcinoma, human lung carcinoma and human breast cancer cell lines. The studies revealed antiproliferative and antimigrative properties of all the investigated extracts. Nevertheless the most pronounced effects demonstrated the ethanol extracts, obtained from fruiting bodies of cultured P. betulinus. Summarizing, our studies proved that P. betulinus can be induced to fruit in indoor artificial culture and the cultured fruiting bodies can be used as a source of potential anticancer agents. In this respect, they are at least as valuable as those sourced from nature.     

The seaweed <Emphasis Type="Italic">Prasiola crispa</Emphasis> (Chlorophyta) neutralizes toxic effects of <Emphasis Type="Italic">Bothrops jararacussu</Emphasis> snake venom

In Brazil, the snake genus Bothrops has many venomous species with 90 % of cases of accidents. Snake bites by Bothrops jararacussu result in moderate to severe envenoming, characterized by hemorrhage, coagulation disorders, tissue necrosis, and death. Antivenom has been regularly used for more than a century but poorly neutralizes myonecrosis. And, as a consequence, victims may have their affected limbs amputated. Thus, the production of antivenom must be improved as well as alternative treatments investigated. Thus, the ability of four extracts of the green alga Prasiola crispa to neutralize some toxic effects of B. jararacussu venom was tested. P. crispa was collected in Antarctica, then extracted using four solvents, dichloromethane (DCM), ethyl acetate (ETA), n-hexane (HEX), or methanol (MET). The extracts were incubated with B. jararacussu venom, and in vivo (hemorrhagic, lethal, and edematogenic) or in vitro (coagulating and proteolytic) activities were performed. Moreover, B. jararacussu venom was injected into mice before or after the injection of alga extracts. Overall, extracts inhibited all activities. The MET extract inhibited less and HEX, DCM and ETA inhibited more efficiently the activities. These latter extracts fully protected mice from B. jararacussu-induced hemorrhage and delayed death of mice. Edema was partially inhibited (20 %) by all extracts. Neutralization of hemorrhage was also observed when the extracts of P. crispa were administered after or before the venom injection. These results indicate that the extracts of P. crispa have potential to treat or to prevent some toxic effects of B. jararacussu venom, thus aiding in the antivenom therapy.     

Temporal Response of Foragers and Guards of Two Stingless Bee Species to Cephalic Compounds of the Robber Bee <Emphasis Type="Italic">Lestrimelitta niitkib</Emphasis> (Ayala) (Hymenoptera,Apidae)

Lestrimelitta spp. are stingless bees that steal food and nesting materials from other highly social bees to survive. Though most of their victim species respond, either aggressively or submissively, to cephalic components of Lestrimelitta, little is known about if such response changes at some point during extended periods of exposure. Moreover, potential synergistic effects due to a mixture of victim’s alarm/defense pheromones and Lestrimelitta mandibular pheromones, like in an actual attack, have not been examined so far. In this paper, we investigated the response of two species of non-robber stingless bees, Scaptotrigona mexicana (Guérin) and Tetragonisca angustula (Latreille), to (a) cephalic compounds from crushed heads of nestmates, (b) cephalic compounds of Lestrimelitta niitkib (Ayala), and (c) a mixture of (a) and (b). We found that even though T. angustula did not react to nestmates’ crushed head, its response towards L. niitkib cephalic compounds was stronger and lasted longer than that of S. mexicana. Interestingly, the addition of crushed heads of the non-robber species to L. niitkib crushed heads caused no significant increase in the alarm response of both species. It may be that the absence of an alarm pheromone in T. angustula made this species more receptive to extraneous odors, which is not the case for S. mexicana; however, more species must be studied to elucidate any pattern regarding the absence/presence of alarm pheromones and the corresponding response to intruders’ pheromones.     

Detection of <Emphasis Type="Italic">Helicobacter</Emphasis> DNA in different water sources and penguin feces from Greenwich,Dee and Barrientos Islands,Antarctica

Helicobacter spp. colonize the gastrointestinal tract of humans and animals and have been associated with gastrointestinal diseases. Antarctic habitats are considered pristine ecosystems, but the increase in human activity could be introducing human bacteria hosted into waters and wildlife. However, Helicobacter spp. occurrence has not been studied in Antarctica. The aim of our study was to detect the Helicobacter DNA in different water sources and penguin feces from Greenwich, Dee and Barrientos Islands during summer of 2012 and 2013. High Helicobacter proportion was observed in water sources amplifying the 16S rRNA (33/40) and 23S rRNA genes (37/40) by semi-nested PCR. Similar results were observed in feces from Gentoo penguins (16S rRNA: 32/39, and 23S rRNA: 28/39) and Chinstrap penguins (16S rRNA: 16/17, and 23S rRNA: 15/17) by PCR. The phylogenetic relationship of 16S rRNA and 23S rRNA sequences from penguin feces was closely related to Helicobacter brantae. Analyses of 16S rRNA sequences showed that the majority of water samples are related to penguin (3/6) and Helicobacter pylori (2/6) sequences, but the 23S rRNA sequences matched with Campylobacter and Arcobacter. These results show for the first time the presence of the genus Helicobacter in different Antarctica water sources and in Gentoo and Chinstrap penguin feces. A few 16S rRNA sequences are very closely related to H. pylori, but specific glmM and ureA H. pylori genes were not detected. More studies are needed to determine the Helicobacter species present in this ecosystem and to establish the human impact in these Antarctic Islands.