Notes on paternal care and sibling cannibalism in the giant water bug, Lethocerus deyrolli (Heteroptera: Belostomatidae)

Males of the giant water bug, Lethocerus deyrolli, care for egg masses on vegetation above the water surface. They supply the developing eggs with water and guard them against predators. In the present study, mechanisms by which paternal care is extended were found. Males were found situated just below the water on the natal substrate (usually a stick), and the first instar nymphs were aggregated around the substrate. When disturbed, the males showed aggressive behavior, threatening the intruder with their forelegs. Nymphs up to 12 h old did not attack the offered sibling nymphs or anuran larvae, which are common prey in the field. The 24 h‐old nymphs attacked both prey animals; however, they preferred anuran larvae. Cannibalistic behavior in the nymphs was well developed 72 h after hatching, when the nymphs had already dispersed from the natal substrate. The suppression of sibling cannibalism in younger nymphs would promote the maintenance of tight nymphal aggregations and consequently extend male care in this predatory species.     

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma

BackgroundFew driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes.MethodsWe searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort.ResultsWe found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC.ConclusionOur integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.     

Ultraconserved Elements Sequencing as a Low-Cost Source of Complete Mitochondrial Genomes and Microsatellite Markers in Non-Model Amniotes

Sequence capture of ultraconserved elements (UCEs) associated with massively parallel sequencing has become a common source of nuclear data for studies of animal systematics and phylogeography. However, mitochondrial and microsatellite variation are still commonly used in various kinds of molecular studies, and probably will complement genomic data in years to come. Here we show that besides providing abundant genomic data, UCE sequencing is an excellent source of both sequences for microsatellite loci design and complete mitochondrial genomes with high sequencing depth. Identification of dozens of microsatellite loci and assembly of complete mitogenomes is exemplified here using three species of Poospiza warbling finches from southern and southeastern Brazil. This strategy opens exciting opportunities to simultaneously analyze genome-wide nuclear datasets and traditionally used mtDNA and microsatellite markers in non-model amniotes at no additional cost.     

Estimating the population structure of brown bears in eastern Hokkaido based on microsatellite analysis

Estimating the genetic structure of a population is important for the conservation and management of wildlife. In the present study, our aim was to estimate the genetic structure of the brown bear (Ursus arctos) population in eastern Hokkaido by performing a Bayesian clustering analysis. To accomplish this goal, we used 15 microsatellites to generate genotypic data from tissue samples collected from 646 bears between 1996 and 2008. Using this genotypic data and the geographic locations where the bears were captured, GENELAND analysis detected six subpopulations. Based on the genotypic data, the STRUCTURE analysis revealed three subpopulations. As inferred from the GENELAND analysis, the core zones of the subpopulations (G-a through G-f) were located in the Shiranuka Hills (G-a), the northern area of the Shiranuka Hills (G-b), the eastern slope of the Daisetsuzan Mountains (G-c), the northern slope of the Akan Mountain Range (G-d), the Shiretoko Peninsula (G-e), and Akkeshi District (G-f). The STRUCTURE analysis indicated that G-b and G-d were influenced by gene flow from other subpopulations. National routes, towns, and farm fields were considered to have formed the distribution boundaries among the subpopulations. A high level of genetic differentiation was not observed among the six subpopulations, with the exception of G-f (F _st?=?1.35–0.176, D _s?=?0.246–0.349), which showed a geographically discontinuous distribution. We suggest that the loss of forest areas through future regional development and road building should be avoided to facilitate gene flow in brown bears in Hokkaido.     

Effect of Pepsin Inhibitor (S-PI) on the Growth of Rhodotorula glutinis No. K-24

A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N,N′-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS–polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0–9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Repression of maleate cis-trans isomerase(maleate isomerase) by carbon sources and its reversal were investigated by using Alcaligenes faecalis IB-14.

The formation of maleate isomerase was induced by malonate favorably in a poor medium, whereas it was repressed in a rich medium by carbon sources such as intermediates of TCA cycle. The repression provoked by dl-malate was accompanied with remarkable promotion of the cell growth and with accumulation of a large amount of pyruvate. The enzyme levels of TCA cycle were elevated several times in the dl-malate repressed cells. It was probable to assume that the formation of maleate isomerase was subject to catabolite repression when a rapid and surplus metabolism of dl-malate via TCA cycle was conducted.

So, as an approach to reveal the chemical nature of the catabolite moiety, reversal of the catabolite repression was studied. It was demonstrated that the repression provoked by dl-malate was reversed by various cultural conditions as follows; addition of higher concentrations of malonate, divided supply of dl-malate, “anaerobic” incubation and addition of higher concentrations of ammonium ion. From physiological significances of these events, it was revealed that catabolite repression of maleate isomerase was reversed by minimizing the functioning of TCA cycle.     

Purification of the Early Sporulation Gene spo0F Product of Bacillus subtilis

The RsaI fragment (750 base pairs) containing the entire early sporulation gene spo0F of Bacillus subtilis was inserted in the downstream region of the P_R promoter of the expression vector, pEBR-151. The recombinant plasmid thus obtained was introduced into Escherichia coli HB101 and the synthesis of the spo0F gene product was induced by a temperature shift up. After induction for 7 hr, a protein of molecular weight 14,000 (14 K protein) was overproduced to about 9% of the total cellular protein. The 14 K protein was purified to 94% purity by four steps of column chromatography. Deletion analysis and the sequence determination of the NH₂-terminal amino acid residues of the purified 14 K protein confirmed that the 14 K protein is the spo0F gene product.     

Isopullulanase from Arthrobacter globiformis T6

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10^-7 and 6.82 × 10^-7 cm²/sec, and 51 and 53 Å, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabinose and d-ribose. The smaller Km values and the larger molecular activities for d-xylose and d-xyluIose indicated that both enzymes are essentially d-xylose isomerases. The optimum temperature was 80°C for both enzymes. The optimum pH was 8 to 10 for the Streptomyces enzymes and 7.5 to 8.0 for the Bacillus enzyme. The Bacillus enzyme was more thermostable than the Streptomyces enzyme, but required cobalt ions in addition to magnesium ions for the full expression of its activity.     

Chemical structures of oligosaccharides in milks of the American black bear (Ursus americanus americanus) and cheetah (Acinonyx jubatus)

The milk oligosaccharides were studied for two species of the Carnivora: the American black bear (Ursus americanus, family Ursidae, Caniformia), and the cheetah, (Acinonyx jubatus, family Felidae, Feliformia). Lactose was the most dominant saccharide in cheetah milk, while this was a minor saccharide and milk oligosaccharides predominated over lactose in American black bear milk. The structures of 8 neutral saccharides from American black bear milk were found to be Gal(β1–4)Glc (lactose), Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)Glc (B-tetrasaccharide), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]Glc (B-pentasaccharide), Fuc(α1–2)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (difucosyl lacto-N-neotetraose), Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (monogalactosyl monofucosyl lacto-N-neotetraose) and Gal(α1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (Galili pentasaccharide). Structures of 5 acidic saccharides were also identified in black bear milk: Neu5Ac(α2–3)Gal(β1–4)Glc (3′-sialyllactose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monofucosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Gal(α1–3)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monogalactosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl monofucosyl lacto-N-neohexaose), and Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl difucosyl lacto-N-neohexaose). A notable feature of some of these milk oligosaccharides is the presence of B-antigen (Gal(α1–3)[Fuc(α1–2)]Gal), α-Gal epitope (Gal(α1–3)Gal(β1–4)Glc(NAc)) and Lewis x (Gal(β1–4)[Fuc(α1–3)]GlcNAc) structures within oligosaccharides. By comparison to American black bear milk, cheetah milk had a much smaller array of oligosaccharides. Two cheetah milks contained Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), while another cheetah milk did not, but contained Gal(β1–6)Gal(β1–4)Glc (6′-galactosyllactose) and Gal(β1–3)Gal(β1–4)Glc (3′-galactosyllactose). Two cheetah milks contained Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (lacto-N-neohexaose), and one cheetah milk contained Gal(β1–4)Glc-3’-O-sulfate. Neu5Ac(α2–8)Neu5Ac(α2–3)Gal(β1–4)Glc (disialyllactose) was the only sialyl oligosaccharide identified in cheetah milk. The heterogeneity of milk oligosaccharides was found between both species with respect of the presence/absence of B-antigen and Lewis x. The variety of milk oligosaccharides was much greater in the American black bear than in the cheetah. The ratio of milk oligosaccharides-to-lactose was lower in cheetah (1:1–1:2) than American black bear (21:1) which is likely a reflection of the requirement for a dietary supply of N-acetyl neuraminic acid (sialic acid), in altricial ursids compared to more precocial felids, given the role of these oligosaccharides in the synthesis of brain gangliosides and the polysialic chains on neural cell adhesion.

     

High altitude adaptation of the schizothoracine fishes (Cyprinidae) revealed by the mitochondrial genome analyses

The schizothoracine fishes, also known as “mountain carps” are widely distributed in the Qinghai-Tibetan Plateau and its peripheral regions. Although they provide a prime example of high altitude adaptation, the phylogenetic relationships and the divergence times among these carp lineages are still controversial. Moreover, the genetic basis for high altitude adaptation is also poorly understood. In this study, we determined the mitochondrial genomes from two species of the schizothoracine fishes, representing a “morphologically primitive” clade and “morphologically specialized” clade, respectively. The phylogenetic tree and the divergence times were estimated within the evolutionary framework of the entire order Cypriniformes. Our results indicate a polyphylyetic relationship of the schizothoracine fishes and suggest two independent migration events into the Qinghai-Tibetan Plateau: one by the “morphologically primitive” clade in the Late Miocene and another by the “morphologically specialized” clade in the Eocene. Rapid speciation events of each clade from the Late Miocene to the Pliocene correspond to the timing of the geologic acceleration of the Qinghai-Tibetan Plateau. Interestingly, we found evidence for positive selection acting on the protein coding genes in the mitochondrial genomes of the “morphologically specialized” clade, implying a possible genetic basis for high altitude adaptation in this derived lineage of cypriniform fishes.