Phytochemical and pharmacological investigation of <Emphasis Type="Italic">Spiraea chamaedryfolia</Emphasis>: a contribution to the chemotaxonomy of <Emphasis Type="Italic">Spiraea</Emphasis> genus

Objective

Diterpene alkaloids are secondary plant metabolites and chemotaxonomical markers with a strong biological activity. These compounds are characteristic for the Ranunculaceae family, while their occurrence in other taxa is rare. Several species of the Spiraea genus (Rosaceae) are examples of this rarity. Screening Spiraea species for alkaloid content is a chemotaxonomical approach to clarify the classification and phylogeny of the genus. Novel pharmacological findings make further investigations of Spiraea diterpene alkaloids promising.

Results

Seven Spiraea species were screened for diterpene alkaloids. Phytochemical and pharmacological investigations were performed on Spiraea chamaedryfolia, the species found to contain diterpene alkaloids. Its alkaloid-rich fractions were found to exert a remarkable xanthine-oxidase inhibitory activity and a moderate antibacterial activity. The alkaloid distribution within the root was clarified by microscopic techniques.

     

Photosynthetic response of <Emphasis Type="Italic">Bangia fuscopurpurea</Emphasis> (Bangiales,Rhodophyta) towards dehydration and hyposalinity

Bangia fuscopurpurea, an important farmed species in China, inhabits upper intertidal zones where it suffers periodical desiccation and salinity stress. However, the physiological response and acclimation mechanism of Bangia to abiotic stress is unknown. Here, the photosynthetic response of B. fuscopurpurea to desiccation and hyposalinity was investigated by using chlorophyll fluorescence measurement. The optimum photosynthetic efficiency of photosystem II (Fv/Fm), photochemical quenching (qP) and the non-photochemical quenching (NPQ) of B. fuscopurpurea thalli maintained at basal level when the absolute water content (AWC) was 32%. As AWC decreased from 32% to 9%, Fv/Fm dropped from 0.62 to 0.1 and NPQ increased from 0.2 to 1.2. No significant change occurred in the mean qP but great standard deviation was present as AWC was 9%. Fv/Fm, qP and NPQ of the thalli with 9% AWC fully recovered after rehydration. That B. fuscopurpurea kept high photosystem II photochemical reactions even when AWC was mere 32% enabled this species to survive extreme air drying at low tide. Fv/Fm and qP dropped while NPQ increased with 1 h of varying hyposaline treatment and they regained the basal levels after 6–24 h treatment. Nine days later, Fv/Fm, qP and NPQ levels of the thalli in 100% freshwater was equal to the control level (0.62, 0.9, 0.1, respectively). The present finding suggested that this alga has high photosynthetic capacity to survive during low tide, even during heavy rainfall. We hope this study would facilitate further investigation on the stress acclimation mechanism of B. fuscopurpurea.     

Genome-wide analysis of the terpene synthase gene family in physic nut (<Emphasis Type="Italic">Jatropha curcas</Emphasis> L.) and functional identification of six terpene synthases

Plant terpenes constitute a large class of compounds and have numerous biological roles as either primary or secondary metabolites. Terpene synthases (TPSs) play key roles on catalyzing the formation of different terpenes; they are divided into seven subfamilies based on sequence relatedness (TPSa–h). TPS-a proteins catalyze the formation of sesquiterpenes and diterpenes in plants. Physic nut (Jatropha curcas L.) is an attractive biofuel tree, but its seeds contain diterpene derivatives, which make them inedible for animals. In this study, 59 putative TPS genes (JcTPS01 to JcTPS59) were identified in the physic nut genome, and 26 belong to the TPS-a subfamily. Eight among the 26 TPS-a genes showed expression in developing seeds of physic nut in the present study. After heterologous expression of these eight genes in Escherichia coli and in vitro enzyme assays, six were shown to have TPS activities. Two (JcTPS09 and JcTPS11) catalyzed the production of diterpene casbene, which was consistent with earlier findings. The other four (JcTPS02, 23, 55, and 56) catalyzed the production of sesquiterpenes. These results may facilitate the efforts for identifying TPS genes involving the physic nut terpene synthesize.     

The Prevalence of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>, the Causal Agent of Chagas Disease,in Texas Rodent Populations

Rodent species were assessed as potential hosts of Trypanosoma cruzi, the etiologic agent of Chagas disease, from five sites throughout Texas in sylvan and disturbed habitats. A total of 592 rodents were captured, resulting in a wide taxonomic representation of 11 genera and 15 species. Heart samples of 543 individuals were successfully analyzed by SybrGreen-based quantitative PCR (qPCR) targeting a 166 bp fragment of satellite DNA of T. cruzi. Eight rodents representing six species from six genera and two families were infected with T. cruzi. This is the first report of T. cruzi in the pygmy mouse (Baiomys taylori) and the white-footed mouse (Peromyscus leucopus) for the USA. All infected rodents were from the southernmost site (Las Palomas Wildlife Management Area). No differences in pathogen prevalence existed between disturbed habitats (5 of 131 tested; 3.8%) and sylvan habitats (3 of 40 tested; 7.5%). Most positives (n = 6, 16% prevalence) were detected in late winter with single positives in both spring (3% prevalence) and fall (1% prevalence). Additionally, 30 Triatoma insects were collected opportunistically from sites in central Texas. Fifty percent of these insects, i.e., 13 T. gerstaeckeri (68%), and two T. lecticularia (100%) were positive for T. cruzi. Comparative sequence analyses of 18S rRNA of samples provided identical results with respect to detection of the presence or absence of T. cruzi and assigned T. cruzi from rodents collected in late winter to lineage TcI. T. cruzi from Triatoma sp. and rodents from subsequent collections in spring and fall were different, however, and could not be assigned to other lineages with certainty.     

Genetic diversity of free-living <Emphasis Type="Italic">Symbiodinium</Emphasis> in the Caribbean: the importance of habitats and seasons

Although reef corals are dependent of the dinoflagellate Symbiodinium, the large majority of corals spawn gametes that do not contain their vital symbiont. This suggests the existence of a pool of Symbiodinium in the environment, of which surprisingly little is known. Reefs around Curaçao (Caribbean) were sampled for free-living Symbiodinium at three time periods (summer 2009, summer 2010, and winter 2010) to characterize different habitats (water column, coral rubble, sediment, the macroalgae Halimeda spp., Dictyota spp., and Lobophora variegata, and the seagrass Thalassia testudinum) that could serve as environmental sources of symbionts for corals. We detected the common clades of Symbiodinium that engage in symbiosis with Caribbean coral hosts A, B, and C using Symbiodinium-specific primers of the hypervariable region of the chloroplast 23S ribosomal DNA gene. We also discovered clade G and, for the first time in the Caribbean, the presence of free-living Symbiodinium clades F and H. Additionally, this study expands the habitat range of free-living Symbiodinium as environmental Symbiodinium was detected in T. testudinum seagrass beds. The patterns of association between free-living Symbiodinium types and habitats were shown to be complex. An interesting, strong association was seen between some clade A sequence types and sediment, suggesting that sediment could be a niche where clade A radiated from a free-living ancestor. Other interesting relationships were seen between sequence types of Symbiodinium clade C with Halimeda spp. and clades B and F with T. testudinium. These relationships highlight the importance of some macroalgae and seagrasses in hosting free-living Symbiodinium. Finally, studies spanning beyond a 1-yr cycle are needed to further expand on our results in order to better understand the variation of Symbiodinium in the environment through time. All together, results presented here showed that the great diversity of free-living Symbiodinium has a dynamic distribution across habitats and time.     

Drivers of <Emphasis Type="Italic">Bromus tectorum</Emphasis> Abundance in the Western North American Sagebrush Steppe

Bromus tectorum can transform ecosystems causing negative impacts on the ecological and economic values of sagebrush steppe of the western USA. Although our knowledge of the drivers of the regional distribution of B. tectorum has improved, we have yet to determine the relative importance of climate and local factors causing B. tectorum abundance and impact. To address this, we sampled 555 sites distributed geographically and ecologically throughout the sagebrush steppe. We recorded the canopy cover of B. tectorum, as well as local substrate and vegetation characteristics. Boosted regression tree modeling revealed that climate strongly limits the transformative ability of B. tectorum to a portion of the sagebrush steppe with dry summers (that is, July precipitation <10 mm and the driest annual quarter associated with a mean temperature >15°C) and low native grass canopy cover. This portion includes the Bonneville, Columbia, Lahontan, and lower Snake River basins. These areas are likely to require extreme efforts to reverse B. tectorum transformation. Our predictions, using future climate conditions, suggest that the transformative ability of B. tectorum may not expand geographically and could remain within the same climatically suitable basins. We found B. tectorum in locally disturbed areas within or adjacent to all of our sample sites, but not necessarily within sagebrush steppe vegetation. Conversion of the sagebrush steppe by B. tectorum, therefore, is more likely to occur outside the confines of its current climatically optimal region because of site-specific disturbances, including invasive species control efforts and sagebrush steppe mismanagement, rather than climate change.     

Genetic analysis of the traits introgressed from <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch. to bread wheat and determined by chromosome 5A genes

A winter bread wheat accession from the Arsenal collection was genetically examined to study the results of introgression, which substantially changed the physiological and morphological traits of the original spring cultivar Rodina. Apart from its winter habit, the accession was characterized by awned speltoid spikes, suggesting introgression into chromosome 5A, which carries marker genes in the order Vrn-A1-Q-B1. Genetic analysis showed that the chromosome fragment introgressed from Aegilops speltoides recombined well with the homeologous region of bread wheat chromosome 5A in the region between the Vrn-A1 and Q genes. Recombination between the Vrn-A1 and B1 genes was not detected, and it was assumed that the order of the marker genes of chromosome 5A was inverted to produce Q-Vrn-A1-B1. When the winter introgression line was crossed with Triticum spelta L., an interaction of two dominant genes determining the spike character was for the first time detected in F₁, increasing the spike length and the number of spikelets, and followed with transgression in F₂. It was assumed that Ae. speltoides had a homeoallelic speltoid gene, which was designated as Q ^S .     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Frequency of <Emphasis Type="Italic">Aedes sp</Emphasis>. Linnaeus (Diptera: Culicidae) and Associated Entomofauna in Bromeliads from a Forest Patch within a densely Urbanized Area

Little is known about the relationship between the presence of Aedes, abiotic factors and the entomofauna existing in phytotelmatas. The aim of this study was to identify biotic and abiotic factors associated with the presence of Aedes in bromeliads sites located in a forest fragment in Rio de Janeiro, Brazil. In the study area, eight bromeliads from the Aechmea genus were chosen and kept in landscape form. Physical and chemical variables were measured. Collected aquatic insects were identified according to the literature. A total of 3102 immature insects were collected. The presence of Aedes aegypti (Linnaeus) (2.29%) was rare. Few specimens were found concentrated in urban-adjacent areas during summer. On the other hand, Aedes albopictus (Skuse) (17.57%) was found throughout the year in 0%–80% of the sites, averaging 1.0 mosq/bromelia. Aedes albopictus was found predominantly in central sites of the forest fragment. The Canonical Correspondence Analysis indicates that most taxa had a moderate association with temperature, dissolved oxygen, and pH. The abiotic variables, such as temperature and dissolved oxygen, affect the distribution of the genus Aedes vectors, while most of the other variables did not.     

New species of unicellular obligate parasite, <Emphasis Type="Italic">Olpidiopsis pyropiae</Emphasis> sp. nov., that plagues <Emphasis Type="Italic">Pyropia</Emphasis> sea farms in Korea

Pyropia (Porphyra) sea farms are plagued with many diseases, similar to a land crop field. Olpidiopsis disease has been one of the most serious diseases causing multimillion dollars of economic loss every year. From 3 years of epidemiological studies in Pyropia sea farms, we found that the pathogen of Olpidiopsis disease in Korea is different from the oomycete, Olpidiopsis porphyrae, which is known to infect the commercially cultivated Pyropia yezoensis in Japan. The Korean species showed a clearly different small subunit (SSU) 18S rRNA sequence (90.4 % identity) and lacked four intron-like insertions, which are present in O. porphyrae. We therefore established a new species named Olpidiopsis pyropiae. The infection process and asexual life history of O. pyropiae were similar to O. porphyrae. Infection started when zoospores attached to the surface of Pyropia blade, lost flagella, and produced thin germ tubes that penetrated the cell walls of the host. Spherical multinucleate thalli developed within the cytoplasm of its algal host, which grew into mature sporangia within the next 2 days. The shape and size of sporangium was similar to that of the Japanese species, except for longer discharge tubes in O. pyropiae. Transmission electron microscopy revealed the presence of K-body-like organelles with tubular inclusions located close to the nucleus, which is one of the key characters of the genus. However, phylogenetic analysis based on SSU ribosomal RNA (rRNA) gene data showed a loose affinity of the Korean species with the other marine Olpidiopsis spp.     

Taxonomic and functional diversity in <Emphasis Type="Italic">Calogaya</Emphasis> (lichenised Ascomycota) in dry continental Asia

The genus Calogaya (Teloschistaceae, Xanthorioideae) was established to accommodate mainly epilithic lichens with lobate thalli, previously regarded as the “Caloplaca saxicola group.” Data supporting the recognition of this new genus came from European lichens, and although the genus is soundly based, we have found in Asia numerous epiphytic lineages and lineages with reduced, non-lobate thallus in dry continental areas. The taxonomic and functional diversity of Calogaya is distinctly higher in steppe and desert areas of Asia than in the less arid regions of Europe. We sampled 238 specimens, mostly from arid regions of north-western China, Iran, southern Siberia and Turkey. Three nuclear DNA loci were analysed separately and jointly by Bayesian inference, maximum likelihood and *BEAST approaches. Delimitations of 28 putative species were tested by BP&P multispecies coalescent model with joint analysis of species delimitation and species-tree estimation. Finally, we recognised 22 taxonomic units: 16 are at species rank, 3 are treated as subspecies and 3 are complexes, treated here as a single entity, but in reality probably including more than one species. Calogaya altynis, C. biatorina subsp. asiatica, C. decipiens subsp. esorediata, C. haloxylonis, C. orientalis, C. xanthoriella and C. xinjiangis are newly described. Caloplaca zoroasteriorum is combined into Calogaya, and Calogaya persica is reduced to a subspecies. The taxonomic status of Calogaya saxicola is unclear, and the name is employed here “sensu lato” for several non-monophyletic epilithic lineages with short-lobed thalli. Calogaya biatorina and C. ferrugineoides are the two other heterogeneous taxonomic units probably including more species.     

Prevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in questing <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks in relation to the density of wild cervids

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum have been considered as pathogens in animals and humans. The role of wild cervids in the epidemiology is not clear. We analyzed questing Ixodes ricinus ticks collected in spring for these pathogens from sites with high (Fjelløyvær and Strøm) and low density (Tjore, Hinnebu and Jomfruland) of wild cervids to study the spread of the pathogens in questing ticks.

Methods

For detection of Anaplasma phagocytophilum a 77-bp fragment in the msp 2 gene was used. Detection of Borrelia burgdorferi sensu lato was performed using the FL6 and FL7 primers according to sequences of conserved regions of the fla gene. The Osp A gene located on the linear 49-kb plasmid was used as target in multiplex PCR for genotyping. Genospecies-specific primers were used in the PCR for Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii.

Results

Infection rates with Borrelia spp. were significantly lower at Fjelløyvær and Strøm compared to Tjore and Hinnebu; Fjelløyvær vs. Tjore (χ² = 20.27, p < 0.0001); Fjelløyvær vs. Hinnebu (χ² = 24.04, p < 0.0001); Strøm vs. Tjore (χ² = 11.47, p = 0.0007) and Strøm vs. Hinnebu (χ² = 16.63, p < 0.0001). The Borrelia genospecies were dominated by. B. afzelii (82%) followed by B. garinii (9.7%) and B. burgdorferi sensu stricto (6.9%). B. burgdorferi s.s. was only found on the island of Jomfruland. The infection rate of Anaplasma phagocytophilum showed the following figures; Fjelløyvær vs Hinnebu (χ² = 16.27, p = 0.0001); Strøm vs. Tjore (χ² = 13.16, p = 0.0003); Strøm vs. Hinnebu (χ² = 34.71, p < 0.0001); Fjelløyvær vs. Tjore (χ² = 3.19, p = 0.0742) and Fjelløyvær vs. Støm (χ² = 5.06, p = 0.0245). Wild cervids may serve as a reservoir for A. phagocytophilum. Jomfruland, with no wild cervids but high levels of migrating birds and rodents, harboured both B. burgdorferi s.l. and A. phagocytophilum in questing I. ricinus ticks. Birds and rodents may play an important role in maintaining the pathogens on Jomfruland.

Conclusion

The high abundance of roe deer and red deer on the Norwegian islands of Fjelløyvær and Strøm may reduce the infection rate of Borrelia burgdorferi sensu lato in host seeking Ixodes ricinus, in contrast to mainland sites at Hinnebu and Tjore with moderate abundance of wild cervids. The infection rate of Anaplasma phagocytophilum showed the opposite result with a high prevalence in questing ticks in localities with a high density of wild cervids compared to localities with lower density.

     

The influences of temperature and irradiance on thallus length of <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Phaeophyta) during the early stages of cultivation

Korean Saccharina japonica is highly valued, both for human consumption and abalone feed. For the stable production of abalone feed, fresh seaweed biomass is required throughout the year. However, currently, the production of farmed Saccharina is limited by environmental conditions such as temperature, irradiance, and nutrient availability between August and November. Due to shortages experienced in supply, the production of early-season biomass can be highly profitable and, therefore, some famers attempt to start their cultivation activities before prevailing, surface seawater temperatures (SST) are optimal. However, attempting to cultivate too early, can lead to total crop failure. Young kelp sporophytes are easily destroyed between 18 and 22 °C SST, which can occur during the early nursery period when the materials are confined to tanks. This study investigated the growth of S. japonica thalli and photosynthetic quantum yield (F_v/F_m) under five temperatures (i.e., 18–26 °C, at 2° increments) and five irradiances (i.e., 5, 10, 20, 40, and 80 μmol photons m^?2 s^?1). This was undertaken for four different size groups of sporophyte thalli (i.e., 0.25, 1, 5, 10 mm). There were different responses of the initial groups of S. japonica showing different tolerances to temperature and irradiance. In general, the smaller plants (1 mm) were more tolerant of sub-optimal conditions than their larger cohorts. These results indicated the optimum temperature and irradiance ranges for different size groups of S. japonica thalli which, if adopted in management protocols, could contribute to enhanced profitability and a more stable and evenly distributed production of Saccharina raw materials over an entire annual basis.