Development of Eighteen Microsatellite Markers in Anemone amurensis (Ranunculaceae) and Cross-Amplification in Congeneric Species

Polyploidy plays an important role in the evolution of plant genomes. To enable the investigation of the polyploidy events within the genus Anemone, we developed eighteen microsatellite markers from the hexaploid species A. amurensis (Ranunculaceae), and tested their transferability in five closely related species. The number of total alleles (N(A)) for each resulting locus varied from one to eight. The polymorphism information content (PIC) and Nei's genetic diversity (N(GD)) for these microsatellites ranged from 0.00 to 0.71 and 0.00 to 0.91, respectively. For each population, the N(A) was one to seven, and the values of PIC and N(GD) varied from 0.00 to 0.84 and 0.00 to 0.95, respectively. In addition, most of these microsatellites can be amplified successfully in the congeneric species. These microsatellite primers provide us an opportunity to study the polyploid evolution in the genus Anemone.     

Population Genetics of the Endemic Hawaiian Species Chrysodracon hawaiiensis and Chrysodracon auwahiensis (Asparagaceae): Insights from RAPD and ISSR Variation

The genus Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon’s index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested.     

Molecular Characterization of Sec2 Loci in Wheat—Secale africanum Derivatives Demonstrates Genomic Divergence of Secale Species

The unique 75 K γ-secalins encoded by Sec2 loci in Secale species is composed of almost half rye storage proteins. The chromosomal location of Sec2 loci in wild Secale species, Secale africanum, was carried out by the wheat—S. africanum derivatives, which were identified by genomic in situ hybridization and multi-color fluorescence in situ hybridization. The Sec2 gene-specific PCR analysis indicated that the S. cereale Sec2 was located onchromosome 2R, while the S. africanum Sec2 was localized on chromosome 6R^afr of S. africanum. A total of 38 Sec2 gene sequences were isolated from S. africanum, S. cereale and S. sylvestre by PCR-based cloning. Phylogenetic analysis showed that S. africanum Sec2 diverged from S. cereale Sec2 approximately 2–3 million years ago. The illegitimate recombination of chromosome 2R–6R involving the Sec2 loci region may accelerate sequence variation during evolutionary process from wild to cultivated Secale species.     

Inhibitory Effects of Amorphigenin on the Mitochondrial Complex I of Culex pipiens pallens Coquillett (Diptera: Culicidae)

Previous studies in our laboratory found that the extract from seeds of Amorpha fruticosa in the Leguminosae family had lethal effects against mosquito larvae, and an insecticidal compound amorphigenin was isolated. In this study, the inhibitory effects of amorphigenin against the mitochondrial complex I of Culex pipiens pallens (Diptera: Culicidae) were investigated and compared with that of rotenone. The results showed that amorphigenin and rotenone can decrease the mitochondrial complex I activity both in vivo and in vitro as the in vivo IC₅₀ values (the inhibitor concentrations leading to 50% of the enzyme activity lost) were determined to be 2.4329 and 2.5232 μmol/L, respectively, while the in vitro IC₅₀ values were 2.8592 and 3.1375 μmol/L, respectively. Both amorphigenin and rotenone were shown to be reversible and mixed-I type inhibitors of the mitochondrial complex I of Cx. pipiens pallens, indicating that amorphigenin and rotenone inhibited the enzyme activity not only by binding with the free enzyme but also with the enzyme-substrate complex, and the values of K_I and K_IS for amorphigenin were determined to be 20.58 and 87.55 μM, respectively, while the values for rotenone were 14.04 and 69.23 μM, respectively.     

Homogeneous Nature of Malaysian Marine Fish Epinephelus fuscoguttatus (Perciformes; Serranidae): Evidence Based on Molecular Markers,Morphology and Fourier Transform Infrared Analysis

Taxonomic confusion exists within the genus Epinephelus due to the lack of morphological specializations and the overwhelming number of species reported in several studies. The homogenous nature of the morphology has created confusion in the Malaysian Marine fish species Epinephelus fuscoguttatus and Epinephelus hexagonatus. In this study, the partial DNA sequence of the 16S gene and mitochondrial nucleotide sequences of two gene regions, Cytochrome Oxidase Subunit I and III were used to investigate the phylogenetic relationship between them. In the phylogenetic trees, E. fuscoguttatus was monophyletic with E. hexagonatus species and morphology examination shows that no significant differences were found in the morphometric features between these two taxa. This suggests that E. fuscoguttatus is not distinguishable from E. hexagonatus species, and that E. fuscoguttatus have been identified to be E. hexagonatus species is likely attributed to differences in environment and ability to camouflage themselves under certain conditions. Interestingly, this finding was also supported by Principal Component Analysis on Attenuated Total Reflectance–Fourier-transform Infrared (ATR-FTIR) data analysis. Molecular, morphological and meristic characteristics were combined with ATR-FTIR analysis used in this study offer new perspectives in fish species identification. To our knowledge, this is the first report of an extensive genetic population study of E. fuscoguttatus in Malaysia and this understanding will play an important role in informing genetic stock-specific strategies for the management and conservation of this highly valued fish.     

Polymorphisms in DNA Repair Genes (APEX1, XPD,XRCC1 and XRCC3) and Risk of Preeclampsia in a Mexican Mestizo Population

Variations in genes involved in DNA repair systems have been proposed as risk factors for the development of preeclampsia (PE). We conducted a case-control study to investigate the association of Human apurinic/apyrimidinic (AP) endonuclease (APEX1) Asp148Glu (rs1130409), Xeroderma Pigmentosum group D (XPD) Lys751Gln (rs13181), X-ray repair cross-complementing group 1 (XRCC) Arg399Gln (rs25487) and X-ray repair cross-complementing group 3 (XRCC3) Thr241Met (rs861539) polymorphisms with PE in a Mexican population. Samples of 202 cases and 350 controls were genotyped using RTPCR. Association analyses based on a χ² test and binary logistic regression were performed to determine the odds ratio (OR) and a 95% confidence interval (95% CI) for each polymorphism. The allelic frequencies of APEX1 Asp148Glu polymorphism showed statistical significant differences between preeclamptic and normal women (p = 0.036). Although neither of the polymorphisms proved to be a risk factor for the disease, the APEX1 Asp148Glu polymorphism showed a tendency of association (OR: 1.74, 95% CI = 0.96–3.14) and a significant trend (p for trend = 0.048). A subgroup analyses revealed differences in the allelic frequencies of APEX1 Asp148Glu polymorphism between women with mild preeclampsia and severe preeclampsia (p = 0.035). In conclusion, our results reveal no association between XPD Lys751Gln, XRCC Arg399Gln and XRCC3 Thr241Met polymorphisms and the risk of PE in a Mexican mestizo population; however, the results in the APEX1 Asp148Glu polymorphism suggest the need for future studies using a larger sample size.     

Passive Immune-Protection of Litopenaeus vannamei against Vibrio harveyi and Vibrio parahaemolyticus Infections with Anti-Vibrio Egg Yolk (IgY)-Encapsulated Feed

Vibrio spp. are major causes of mortality in white shrimp (Litopenaeus vannamei) which is lacking adaptive immunity. Passive immunization with a specific egg yolk antibody (IgY) is a potential method for the protection of shrimp against vibriosis. In this study, immune effects of the specific egg yolk powders (IgY) against both V. harveyi and V. parahaemolyticus on white shrimp were evaluated. The egg yolk powders against V. harveyi and V. parahaemolyticus for passive immunization of white shrimp were prepared, while a tube agglutination assay and an indirect enzyme-linked immunosorbent assay (ELISA) were used for detection of IgY titer. Anti-Vibrio egg yolk was encapsulated by β-cyclodextrin, which could keep the activity of the antibody in the gastrointestinal tract of shrimp. The results showed that the anti-Vibrio egg powders had an inhibiting effect on V. harveyi and V. parahaemolyticus in vitro. Lower mortality of infected zoeae, mysis, and postlarva was observed in groups fed with anti-Vibrio egg powders, compared with those fed with normal egg powders. The bacterial load in postlarva fed with specific egg powders in seeding ponds was significantly lower than those fed with normal egg powders in seeding ponds. These results show that passive immunization by oral administration with specific egg yolk powders (IgY) may provide a valuable protection of vibrio infections in white shrimp.     

Low Diversity Bacterial Community and the Trapping Activity of Metabolites from Cultivable Bacteria Species in the Female Reproductive System of the Oriental Fruit Fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae)

Our goal was to identify the bacteria inhabiting the reproductive system of the female oriental fruit fly, Bactrocera dorsalis (Hendel), and evaluate the chemotaxis of B. dorsalis to the metabolites produced by the bacteria. Based on 16S rRNA-based polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 18 operational taxonomic units (OTUs) were assigned to the five bacterial classes Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria. Nine OTUs were assigned to Gammaproteobacteria, which was the most highly represented class. Enterobacteriaceae constituted the dominant family, and within this family, three genera and five species were identified, including Enterobacter sakazakii, Klebsiella oxytoca, Klebsiella pneumoniae, Raoultella terrigena and Enterobacter amnigenus. In this set, the first two species were the dominant components, and the latter three species were the minor ones. Finally, we found that the metabolites produced by R. terrigena, K. oxytoca and K. pneumoniae were attractive to the B. dorsalis adults, and in field studies, B. dorsalis adults were most attracted to K. oxytoca. Collectively, our results suggest that the female reproductive system plays an important role in the transfer of enterobacteria from the gut to fruit. Our data may prompt the development of a female-targeted population control strategy for this fly.     

Biology,Pest Status,Microbiome and Control of Kudzu Bug (Hemiptera: Heteroptera: Plataspidae): A New Invasive Pest in the U.S.

Soybean is an important food crop, and insect integrated pest management (IPM) is critical to the sustainability of this production system. In recent years, the introduction into the United States of the kudzu bug currently identified as Megacopta cribraria (F.), poses a threat to soybean production. The kudzu bug was first discovered in the state of Georgia, U.S. in 2009 and since then has spread to most of the southeastern states. Because it was not found in the North American subcontinent before this time, much of our knowledge of this insect comes from research done in its native habitat. However, since the U.S. introduction, studies have been undertaken to improve our understanding of the kudzu bug basic biology, microbiome, migration patterns, host selection and management in its expanding new range. Researchers are not only looking at developing IPM strategies for the kudzu bug in soybean, but also at its unique relationship with symbiotic bacteria. Adult females deposit bacterial packets with their eggs, and the neonates feed on these packets to acquire the bacteria, Candidatus Ishikawaella capsulata. The kudzu bug should be an informative model to study the co-evolution of insect function and behavior with that of a single bacteria species. We review kudzu bug trapping and survey methods, the development of bioassays for insecticide susceptibility, insecticide efficacy, host preferences, impact of the pest on urban environments, population expansion, and the occurrence of natural enemies. The identity of the kudzu bug in the U.S. is not clear. We propose that the kudzu bug currently accepted as M. cribraria in the U.S. is actually Megacoptapunctatissima, with more work needed to confirm this hypothesis.     

Effects of the Bradyrhizobium japonicum waaL (rfaL) Gene on Hydrophobicity,Motility, Stress Tolerance,and Symbiotic Relationship with Soybeans

We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.     

Genetic Diversity and Conservation of the Prespa Trout in the Balkans

The Balkans are known to have a high level of biodiversity and endemism. No less than 15 taxa have been recorded in salmonids of the Salmo genus. Among them, the Prespa trout is found in only four river systems flowing into Lake Macro Prespa, three in the Former Yugoslav Republic of Macedonia and one in Greece. This is the first comprehensive survey of all streams located within the Macro Prespa Basin, encompassing the whole taxon range. A large genetic sample of 536 Prespa trout was collected mainly between 2005 and 2007. The sampling included 59 individuals from the Golema river system, 93 from the Kranska, 260 from the Brajcinska, 119 from the Agios Germanos, and five individuals from the lake itself. These specimens were analyzed with six microsatellite markers and by sequencing the mitochondrial control region. Nuclear data were examined through multidimensional analysis and assignment tests. Five clusters were detected by assignment: Golema, Kranska, Brajcinska upstream, Rzanska Brajcinska tributary and Brajcinska downstream. Most of these river systems thus hosted differentiated Prespa trout populations (with past gene flows likely dating before the construction of dams), except Agios Germanos, which was found to be composed of 5% to 32% of each cluster. Among the five trout individuals from the lake, four originated from Kranska River and one was admixed. Supported parsimonious hypotheses are proposed to explain these specificities. Conservation of this endemic taxon should take these results into account. No translocation should be performed between different tributaries of the lake and preservation of the Brajcinska populations should address the upstream-downstream differentiation described.     

Isolation and Identification of Fourteen Microsatellite Markers in Clivia miniata and Clivia nobilis (Amaryllidaceae)

Clivia is a genus of great horticultural importance and has been widely cultivated as ornamental plants in all over the world. In order to assess the phylogenetic relationships and genetic diversity of the wild Clivia species and cultivars, we isolated AC-enriched repeats using FIASCO from a single clone each of C. miniata Regel. and Clivia nobilis Lindl. Of the fourteen repeats, 10 were polymorphic and 4 were monomorphic. The polymorphic marker loci were characterized using 61 Clivia accessions. The number of alleles ranged from two to six, observed heterozygosity ranged from 0.04 to 1.00 and expected heterozygosity ranged from 0.04 to 0.83. These microsatellite marker loci provide tools for future studies of Clivia species and cultivars.     

Interactions between Lactobacillus crispatus and Bacterial Vaginosis (BV)-Associated Bacterial Species in Initial Attachment and Biofilm Formation

Certain anaerobic bacterial species tend to predominate the vaginal flora during bacterial vaginosis (BV), with Gardnerella vaginalis being the most common. However, the exact role of G. vaginalis in BV has not yet been determined. The main goal of this study was to test the hypothesis that G. vaginalis is an early colonizer, paving the way for intermediate (e.g., Fusobacterium nucleatum) and late colonizers (e.g., Prevotella bivia). Theoretically, in order to function as an early colonizer, species would need to be able to adhere to vaginal epithelium, even in the presence of vaginal lactobacilli. Therefore, we quantified adherence of G. vaginalis and other BV-associated bacteria to an inert surface pre-coated with Lactobacillus crispatus using a new Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) methodology. We found that G. vaginalis had the greatest capacity to adhere in the presence of L. crispatus. Theoretically, an early colonizer would contribute to the adherence and/or growth of additional species, so we next quantified the effect of G. vaginalis biofilms on the adherence and growth of other BV-associated species by quantitative Polymerase Chain Reaction (qPCR) technique. Interestingly, G. vaginalis derived a growth benefit from the addition of a second species, regardless of the species. Conversely, G. vaginalis biofilms enhanced the growth of P. bivia, and to a minor extent of F. nucleatum. These results contribute to our understanding of BV biofilm formation and the progression of the disorder.     

Molecular Systematics of Genus Atractylodes (Compositae,Cardueae): Evidence from Internal Transcribed Spacer (ITS) and trnL-F Sequences

To determine the evolutionary relationships among all members of the genus Atractylodes (Compositae, Cardueae), we conducted molecular phylogenetic analyses of one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS) and one chloroplast DNA (cpDNA) region (intergenic spacer region of trnL-F). In ITS and ITS + trnL-F trees, all members of Atractylodes form a monophyletic clade. Atractylodes is a sister group of the Carlina and Atractylis branch. Atractylodes species were distributed among three clades: (1) A. carlinoides (located in the lowest base of the Atractylodes phylogenetic tree), (2) A. macrocephala, and (3) the A. lancea complex, including A. japonica, A. coreana, A. lancea, A. lancea subsp. luotianensis, and A. chinensis. The taxonomic controversy over the classification of species of Atractylodes is mainly concentrated in the A. lancea complex. In base on molecular results, the intraspecific division of Atractylodes lancea is not supported, and A. coreana should be treated as a synonym A. chinensis.     

Vaccination of Silver Sea Bream (Sparus sarba) against Vibrio alginolyticus: Protective Evaluation of Different Vaccinating Modalities

In order to develop more effective immunological strategies to prevent vibriosis of farmed marine fish in Hong Kong and southern China, various vaccine preparations including formalin-, phenol-, chloroform- and heat-killed whole cell bacterins and subcellular lipopolysaccharides (LPS), as well as different administration routes, were investigated. Fish immunized with the subcellular LPS exhibited the best protection [Relative Percent of Survival (RPS) = 100], while fish immunized with whole cell bacterins displayed varying degrees of protection (RPS ranged from 28 to 80), in descending order: formalin-killed > phenol-killed > heat-killed > chloroform-killed bacterins. Regarding various administration routes, fish immunized with two intraperitoneal (i.p.) injections exhibited the best protection, and the RPS values were 100 or 85 upon higher or lower doses of pathogenic V. alginolyticus challenges. Both oral vaccination and a combination of injection/immersion trial were also effective, which achieved relatively high protection (the RPS values ranged from 45 to 64.3). However, two hyperosmotic immersions could not confer satisfactory protection, especially when fish were exposed to the severe pathogenic bacteria challenge. Marked elevations of serum agglutinating antibody titer were detected in all immunized fish. Macrophage phagocytosis was enhanced significantly, especially in the fish immunized by formalin- and phenol-killed bacterins through various administration routes. Both adaptive (specific antibody) and innate (phagocytic activity) immunity elicited by different immunization strategies were in parallel with the degree of protection offered by each of them. Although all vaccination trials had no significant effect on the serum hematocrit and hemoglobin levels, the circulating lymphocyte counts were significantly elevated in the fish immunized with LPS, formalin- and phenol-killed bacterins. Serum cortisol levels appeared to be reduced in all immunized fish except the trial of hyperosmotic immersion, which indicated the stressful impact on vaccinated fish.     

Reduced power consumption compared to a traditional stirred tank reactor (STR) for enzymatic saccharification of alpha-cellulose using oscillatory baffled reactor (OBR) technology

Enzymatic saccharification of pure α-cellulose was conducted in oscillatory baffled (OBR) and stirred tank (STR) reactors over a range of mixing intensities requiring power densities (P/V) from 0 to 250 Watts per cubic metre (W/m³). Both reactor designs produced similar saccharification conversion rates at zero mixing. Conversion increased with increasing mixing intensity. The maximum conversion rate occurred at an oscillatory Reynolds number (Re_o) of 600 in the OBR and at an impeller speed of between 185 and 350 rpm in the STR. The OBR was able to achieve a maximum conversion rate at a much lower power density (2.36 W/m³) than the STR (37.2–250 W/m³). The OBR demonstrated a 94–99% decrease in the required power density to achieve maximum conversion rates and showed a 12% increase in glucose production after 24 h at 2.36 W/m³.