LC–MS/MS analysis of okadaic acid analogues and other lipophilic toxins in single-cell isolates of several Dinophysis species collected in Hokkaido,Japan

Quantification of diarrhetic shellfish poisoning (DSP) toxins (okadaic acid analogues), and other lipophilic toxins in single-cell isolates of the dinoflagellates Dinophysis fortii, D. acuminata, D. mitra, D. norvegica, D. tripos, D. infundibulus and D. rotundata, collected in coastal waters Hokkaido, Japan in 2005, was carried out by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoyldinophysistoxin-1 (DTX3), pectenotoxin-1 (PTX1), pectenotoxin-11 (PTX11), pectenotoxin-2 (PTX2), pectenotoxin-6 (PTX6), pectenotoxin-2 seco-acid (PTX2sa), yessotoxin (YTX) and 45-hydroxyyessotoxin (45-OHYTX) were quantified by LC–MS/MS. PTX2 was the dominant toxin in D. acuminata, D. norvegica and D. infundibulus whereas both DTX1 and PTX2 were the principal toxins in D. fortii. None of the toxins were detected in D. mitra, D. rotundata and D. tripos. These results suggest that D. fortii is the most important species responsible for DSP contamination of bivalves in Hokkaido. This is the first finding of PTX2 in D. infundibulus, and confirms the presence of PTX2 in Japanese D. acuminata and D. norvegica collected from natural seawater.     

Taxonomic implications of diterpene alkaloids in three toxic tall larkspur species (Delphinium spp.)

The diterpene alkaloid content was used to assess the chemical taxonomic diversity in three larkspur species. Samples (n=163) were collected from 18 different locations in five western states, extracted and analyzed for diterpene alkaloids using electrospray mass spectrometry. The data were statistically analyzed using canonical discriminant analysis and analysis of variance. Delphinium glaucum samples were easily grouped and were significantly different from all other groups (P<0.005) for two compounds. Delphinium barbeyi and Delphinium occidentale were found to be distinct groups, but more closely related. Samples representing a putative hybrid between D. barbeyi and D. occidentale were found to be closely related to D. occidentale, but were significantly different from all other groups. These data support the classification of D. glaucum, D. barbeyi, and D. occidentale as distinct species and suggest that a possible hybrid is more similar to D. occidentale than D. barbeyi.     

A Comparative Study of the Esterase-5 Locus in DROSOPHILA PSEUDOOBSCURA,D. PERSIMILIS and D. MIRANDA

Electrophoretic phenotypes of the esterase-5 locus were examined in the sibling species D. pseudoobscura, D. persimilis and D. miranda. D. persimilis alleles were found to have uniformly higher charge on monomers than corresponding alleles of either D. pseudoobscura or D. miranda. Consequently, D. persimilis shares no alleles in common with either D. pseudoobscura or D. miranda, while the latter two species share a number of alleles. It was discovered that by increasing the concentration of acrylamide gel and increasing the length of migration, more allelic differences could be distinguished. Also more alleles were discovered by examining monomer mobility in addition to dimer mobility. In D. persimilis and D. miranda it was found that the previously known high frequency allelic classes broke down into several allelic classes. A test of goodness-of-fit to the infinite alleles model was done and a rough agreement with the model was found.     

Analysis of courtship behavior in closely related species of Drosophila virilis group: a new approach arises new questions

Courtship behavior of males was studied in four closely related Drosophila species: D. virilis, D. lummei, D. a. americana and D. littoralis. Using a video-computing approach, we compared behavior in males courting conspecific and heterospecific females. In males of all species studied, touching and licking were found to be the most prolonged courtship elements. Touching and licking were typically proceeding together; wing vibration was usually produced against a background of touching and licking. We found only minor interspecific variations in courtship rituals. Heterospecific courtships in D. virilis and D. lummei were almost as active as conspecific ones; however, isolation between D. a. americana and D. littoralis appeared to be much stronger than between D. virilis and D. lummei. Analysis of prolonged touching and licking raises a question about chemical and tactile sensory stimuli exchanged between sexes in the developed courtship of D. virilis group.     

Variation of 15 autosomal microsatellite DNA loci in the Russian population

The allele frequencies of 15 autosomal STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820, D16S539, D2S1338, D8S1179, D21S11, D18S51, and D19S433) used in forensic medicine were determined for the Russian population of European Russia (N = 176). The power of discrimination (PD) and power of exclusion (PE) of the system of the 15 STR loci were 0.999 999 999 999 999 986 and 0.999 999 331 310 171 000, respectively. The allele and genotype frequency distributions in the Russian population corresponded to the Hardy-Weinberg equilibrium. The D2S1338, D18S51, D21S11, and FGA loci were identified as the most informative markers for the Russian population and proposed as a reference for forensic studies in the Russian Federation.     

Characterization of bacteria of the genus Dietzia: an updated review

The aim of this study was to characterize several aspects of species of the genus Dietzia, such as current taxonomic placement, morphological and growth characteristics, biochemical reactions, cellular lipid and fatty acid composition, the amino acids and sugars of whole-cell hydrolysates and the respiratory quinone system, and genomic guanine and cytosine (G + C) content. The species chosen for study were D. aerolata, D. alimentaria, D. aurantiaca, D. cerdiciphylli, D. cinnamea, D. kunjamensis, D. lutea, D. maris, D. natronolimnaea, D. papillomatosis, D. psychralcaliphila, D. schimae, and D. timorensis. The colony morphology study revealed that the colonies were small, smooth, circular and convex. Nitrate reduction, H₂S production, hydrolysis of urea, starch, and Tween 80, and the Voges–Proskauer and methyl red tests were performed for biochemical differentiation of the various Dietzia strains. Optimum growth temperature and pH for the different strains were 25–30 °C and 7–8, respectively. Among the strains studied, D. timorensis ID05-A0528^T had the lowest tolerance level to NaCl (7 %). This strain was also able to utilize a wide range of compounds as the sole carbon source. Short-chain mycolic acids were present in these bacteria. The cell wall contained meso-diaminopimelic acid, arabinose, and galactose; the glycan moiety of the cell wall contained acetyl residues. The major menaquinone was MK-8 (H₂). The G + C contents of the DNA ranged from 64.7 (D. alimentaria 72^T) to 73 mol?% (D. maris DSM 43672^T). The most important phospholipids in these strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, and phosphatidylethanolamine.     

Single nucleotide polymorphisms in the FADS gene cluster are associated with delta-5 and delta-6 desaturase activities estimated by serum fatty acid ratios

Genetic variability in the FADS1-FADS2 gene cluster [encoding delta-5 (D5D) and delta-6 (D6D) desaturases] has been associated with plasma long-chain PUFA (LCPUFA) and lipid levels in adults. To better understand these relationships, we further characterized the association between FADS1-FADS2 genetic variability and D5D and D6D activities in adolescents. Thirteen single nucleotide polymorphisms (SNPs) were genotyped in 1,144 European adolescents (mean ± SD age: 14.7 ± 1.4 y). Serum phospholipid fatty acid levels were analyzed using gas chromatography. D5D and D6D activities were estimated from the C20:4n-6/C20:3n-6 and C20:3n-6/C18:2n-6 ratios, respectively. Minor alleles of nine SNPs were associated with higher 18:2n-6 levels (1.9E-18 ≤ P ≤ 6.1E-5), lower C20:4n-6 levels (7.1E-69 ≤ P ≤ 1.2E-12), and lower D5D activity (7.2E-44 ≤ P ≤ 4.4E-5). All haplotypes carrying the rs174546 minor allele were associated with lower D5D activity, suggesting that this SNP is in linkage disequilibrium with a functional SNP within FADS1. In contrast, only the rs968567 minor allele was associated with higher D6D activity (P = 1.5E-6). This finding agrees with an earlier in vitro study showing that the minor allele of rs968567 is associated with a higher FADS2 promoter activity. These results suggest that rare alleles of several SNPs in the FADS gene cluster are associated with higher D6D activity and lower D5D activity in European adolescents.     

Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture

We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture.     

Divergence in the neural control of oviposition in Drosophila

Of several species of Drosophila tested, only D. melanogaster was capable of ovipositing after decapitation. Two other species (D. tripunctata and D. pseudoobscura) were also capable of reflex oviposition during the operation but D. virilis and D. palustris were not. This divergence of neural control mechanisms suggests the existence of at least two alternate circuits for the control of insect oviposition.     

Next-generation sequencing based genotyping,cytometry and phenotyping for understanding diversity and evolution of guinea yams

Key message

Genotyping by sequencing (GBS) is used to understand the origin and domestication of guinea yams, including the contribution of wild relatives and polyploidy events to the cultivated guinea yams.

Abstract

Patterns of genetic diversity within and between two cultivated guinea yams (Dioscorea rotundata and D. cayenensis) and five wild relatives (D. praehensilis, D. mangenotiana, D. abyssinica, D. togoensis and D. burkilliana) were investigated using next-generation sequencing (genotyping by sequencing, GBS). Additionally, the two cultivated species were assessed for intra-specific morphological and ploidy variation. In guinea yams, ploidy level is correlated with species identity. Using flow cytometry a single ploidy level was inferred across D. cayenensis (3x, N = 21), D. praehensilis (2x, N = 7), and D. mangenotiana (3x, N = 5) accessions, whereas both diploid and triploid (or aneuploid) accessions were present in D. rotundata (N = 11 and N = 32, respectively). Multi-dimensional scaling and maximum parsimony analyses of 2,215 SNPs revealed that wild guinea yam populations form discrete genetic groupings according to species. D. togoensis and D. burkilliana were most distant from the two cultivated yam species, whereas D. abyssinica, D. mangenotiana, and D. praehensilis were closest to cultivated yams. In contrast, cultivated species were genetically less clearly defined at the intra-specific level. While D. cayenensis formed a single genetic group, D. rotundata comprised three separate groups consisting of; (1) a set of diploid individuals genetically similar to D. praehensilis, (2) a set of diploid individuals genetically similar to D. cayenensis, and (3) a set of triploid individuals. The current study demonstrates the utility of GBS for assessing yam genomic diversity. Combined with morphological and biological data, GBS provides a powerful tool for testing hypotheses regarding the evolution, domestication and breeding of guinea yams.     

Carbohydrates of the seaweeds,Desmarestia ligulata and D. firma

Crystalline mannitol and some oligosaccharides were separated from ethanolic extracts of Desmarestia ligulata and D. firma. Laminaran, ‘fucans’ and alginic acid were also isolated from both species. The laminaran from D. ligulata comprised both M- and G-chains but no M-chains were found in the laminaran from D. firma. In both species the amount of ‘fucan’ was small, particularly in D. firma. Both ‘fucans’ contained glucuronic acid, galactose, xylose and fucose and that from D. ligulata also contained mannose. After sequential extraction of D. ligulata with water, acid and alkali evidence was obtained for the presence of cellulose, a uronan, and protein in the residual material.     

Number of primary spermatocytes in the Drosophila immigrans (Sturtevant) group (Diptera : Drosophilidae)

Nine species of the Drosophila immigrans (Sturtevant) group (Diptera Drosophilidae) were examined for the number of primary spermatocytes per cyst. Seven species (D. formosana, D. immigrans, D. kohkoa, D. nasuta, D. neohypocausta, D. quadrilineata and D. sulfurigaster albostrigata) showed nearly 16 primary spermatocytes per cyst, but 2 species (D. annulipes and D. curviceps) had mean numbers of 20.41 and 24.02 primary spermatocytes per cyst, respectively. The frequency distribution patterns of the numbers of primary spermatocytes suggest that the multiplication divisions do not occur synchronously in these species. The systematic positions of D. annulipes and D. curviceps are also discussed.     

牡竹属三竹种的秆形质量与材性比较

对牡竹属(Dendrocalamus) 3个竹笋品质佳的竹种［勃氏甜龙竹(D. brandisii)、马来麻竹(D. asper)、花吊丝竹(D. minor var. amoenus)］的竹材形态质量及材性进行比较研究。结果表明:种间的立竹枝下高、相对全高的差异显著或极显著,勃氏甜龙竹相对枝下高最小,立竹胸径、全高、枝下高、尖削度值、壁厚率均为最大;竹秆含水率随立竹年龄增大而下降,3年生立竹的竹秆含水率为花吊丝竹>勃氏甜龙竹>马来麻竹,种间差异极显著;相对材积为勃氏甜龙竹(1771.35 cm³ cm^-1)>马来麻竹(1166.66 cm³ cm^-1)>花吊丝竹(659.78 cm³ cm^-1),种间差异极显著;竹材密度随立竹年龄增大而提高,3年立竹的竹材密度为花吊丝竹(0.914 g cm^-3)>勃氏甜龙竹(0.812 g cm^-3)>马来麻竹(0.749 g cm^-3),种间差异显著。因此,勃氏甜龙竹、马来麻竹可作为优良的笋材兼用竹种,而花吊丝竹宜作为笋用、观赏竹种推广应用。     

Oviposition pattern of several Drosophila species under various light environments

The oviposition pattern over two continuous days under constant light (LL) and light-and-dark (LD) conditions was studied by means of a special apparatus; four Drosophila species (D. lutescens, D. melanogaster, D. hydei, and D. virilis) were used. The results showed that: (1) Under the LL condition, the oviposition pattern was characteristic for each species. The number of eggs laid by D. lutescens was smaller than that laid by the other species, and the oviposition pattern of this species was rhythmical. Females of D. melanogaster laid eggs continuously, but at a low rate. In contrast, D. hydei and D. virilis laid eggs in clusters, the total numbers of eggs being larger than those of D. lutescens and D. melanogaster. (2) Females of three species (D. lutescens, D. melanogaster, and D. virilis) laid more eggs in the light phase than in the dark phase under the LD condition. However, no consistent trend was obtained with D. hydei, suggesting that the oviposition pattern of this species is indifferent to the light condition (i.e., it is independent of it). (3) No effects of pre-experimental light condition on the oviposition were found for any of the species. The light-dependency of the oviposition pattern is discussed from the viewpoint of adaptation.     

Identification of the Daphnia species (Crustacea: Cladocera) in the lakes of the Ob and Yenisei River basins: morphological and molecular phylogenetic approaches

The morphological and genetic variability of Daphnia species from 16 lakes in the upper watersheds of the Ob and Yenisei Rivers were investigated. Four species, namely D. galeata Sars 1863, D. cristata Sars 1862, D. curvirostris Eylmann, 1887 and D. longispina O.F. Müller, 1776 inhabit these lakes. In addition, longispina-like individuals in Lake Borzu-Khol (the Bolshoi Yenisei River basin) were found which were, however, clearly distinguishable from the other species by morphometric and mitochondrial DNA analyses and could belong to D. umbra based on the 12S sequences.     

Studies on the Ribosomal RNA Cistrons in Interspecific Drosophila Hybrids. II. Heterochromatic Regions Mediating Nucleolar Dominance

Interspecific hybrids of D. melanogaster and D. simulans normally exhibit a secondary constriction only at the D. melanogaster nucleolus organizer (NO). This phenomenon, termed nucleolar dominance, occurs only when the NO-bearing sex chromosomes of both species are present in conjunction. Experiments were initiated to localize regions on the sex chromosomes of D. melanogaster involved in mediating this suppression. Sex chromosome heterochromatic rearrangements and deficiencies were introduced into F₁ hybrids and their corresponding effect on simulans NO constriction formation was examined in hybrid mitotic neuroblast tissue. Sex chromosomes deficient for both the D. melanogaster NO and adjacent heterochromatin were unable to restrict the formation of a constriction at the D. simulans NO. The presence of a D. melanogaster NO, however, was not sufficient for the establishment of nucleolar dominance. Results from an array of NO-bearing X and Y chromosome rearrangements and deficiencies indicate that at least one heterochromatic region, proximal to the NO on the D. melanogaster X and distal to the NO on the D. melanogaster Y, affects the induction of this interchromosomal phenomenon.     

Three Cases of Diphyllobothrium nihonkaiense Infection in Korea

Until 2012, a total of 48 cases of diphyllobothriasis had been reported in Korea, all of which were morphologically identified as Diphyllobothrium latum. However, some of these specimens were analyzed by nucleotide sequencing of the mitochondrial cox1 gene, which showed that all were D. nihonkaiense, not D. latum. After that, 3 further cases of diphyllobothriasis were confirmed as D. nihonkaiense. In the present study, 3 new cases of D. nihonkaiense were detected from 2011 through 2013. The hosts were infected through consumption of salmonid fishes, such as the trout or salmon, and 2 of them experienced severe diarrhea prior to proglottid passage. All of the tapeworms were confirmed to be D. nihonkaiense by genetic identification. This proved again that most diphyllobothriasis in Korea have been caused by D. nihonkaiense.     

Molecular genetic identification and phylogeny of Daphnia species (Crustacea, Cladocera) from water bodies of the Lake Chany basin

The data on the molecular genetic identification of Daphnia species from the water bodies of the Lake Chany basin are presented. Phylogenetic relationships between these species have been established. The fragments of the mitochondrial DNA genes were used as genetic markers. According to the data obtained, the water bodies examined were inhabited by five Daphnia species, including Daphnia (Daphnia) galeata Sars, D. (D.) longispina O. F. Müller, D. (D.) curvirostris Eylmann, D. (D.) pulex Leydig, and D. (Ctenodaphnia) magna Straus. In addition, longispina a-like individuals that form a separate phylogenetic lineage was identified.     

Mating strategies in two species of dart-poison frogs: a comparative study

Comparative field studies of species of dart-poison frogs in the genus Dendrobates were carried out to test predictions from two hypotheses that attempt to explain female-female competition for mates in species of Dendrobates with male parental care. The sex role reversal hypothesis proposes that males invest so much time and energy in parental care that receptive males are rare relative to receptive females, and females compete to find and mate with receptive males. The parental quality hypothesis proposes that females compete to monopolize the parental effort of particular males, because they potentially suffer a cost when their mates care for the offspring of other females. Comparisons between species with male parental care (Dendrobates leucomelas) and female parental care (Dendrobates histrionicus) contradicted prediction of the sex role reversal hypothesis, but were consistent with predictions of the parental quality hypothesis. Male D. histrionicus did not compete for mates more aggressively than male D. leucomelas, and male D. leucomelas were not more selective about mating than male D. histrionicus. Female D. leucomelas and D. histrionicus were both selective about mating; female D. leucomelas associated with and competed for particular males, whereas female D. histrionicus did not.     

Reticulate evolution in the apogamous Dryopteris varia complex (Dryopteridaceae,subg. Erythrovariae,sect. Variae) and its related sexual species in Japan

Apogamous fern species are often difficult to distinguish from related species because of their continuous morphological variations. To clarify the genetic relationships among the members of the Dryopteris varia complex, we analyzed the nucleotide sequences of the plastid gene rbcL and the nuclear gene PgiC. We also analyzed the diploid sexual species D. caudipinna and D. chinensis, which have not been included in the complex, but were recently shown to be closely related to the complex in a molecular phylogenetic study. The PgiC sequences of the diploid sexual species, D. varia, D. saxifraga, D. sp. ‘protobissetiana’ (undescribed diploid sexual species), D. caudipinna, and D. chinensis, were well differentiated and hence designated A, B, C, D, and E, respectively. Thus, the PgiC constitution of apogamous species in the complex was as follows: D. bissetiana, B + C; D. kobayashii, B + C + E); D. pacifica, A + C, A + B + C, or A + C + D; D. sacrosancta, A + C + E; and D. saxifragivaria, B + C. These results suggest that these apogamous species are formed by hybridizations of species including not only the three diploid sexual species of the D. varia complex (A, B, and C) but also the two diploid sexual species D. caudipinna (D) and D. chinensis (E), which do not belong to the complex.