Precursor structure of egg proteins in the coral Galaxea fascicularis

In the egg of the reef coral Galaxea fascicularis, four proteins (named GfEP-1 to -4) are stored in high abundance. In the present study, a cDNA containing a full-length open reading frame for GfEP-1 was cloned, and the translated protein sequence was compared to the N-terminal sequences of GfEP-2, -3, and -4. GfEP-1 and -2 were shown to be generated by processing of a precursor of 1439 amino acids, and GfEP-3 turned out to be a partial fragment of GfEP-2. The precursor protein contained regions which exhibited similarities to vitellogenins (Vgs) in bilaterian animals (oviparous vertebrates and invertebrates including nematodes, arthropods, and molluscs). This study reports the first cloning and characterization of a full-length cDNA encoding a Vg in a non-bilaterian animal, and argues that the emergence of Vg as a precursor of egg yolk proteins predated the divergence of the cnidarian and bilaterian lineages.     

Germline transformation of the sawfly, Athalia rosae (Hymenoptera: Symphyta), mediated by a piggyBac-derived vector

A piggyBac construct carrying two green fluorescent protein (GFP)-coding sequences one driven by Bombyx mori actin gene promoter and the other by Drosophila melanogaster heat-shock protein 70 (hsp70) promoter were injected together with a nonautonomous helper plasmid containing an active piggyBac transposase gene into the posterior end of mature unfertilized eggs dissected from the ovaries of Athalia rosae (Hymenoptera: Symphyta). These injected eggs, which developed as haploid male embryos upon artificial activation, were cultured to adulthood. Of 278 injected eggs, 61 grew to G(0) haploid adult males. These G(0) haploid males were individually mated to diploid females. The progeny embryos (G(1) generation) were examined for GFP expression. Four GFP-positive embryos (from three independent G(0) matings) were obtained. Two eclosed as diploid adult G(1) females. Mature unfertilized eggs dissected from the GFP-positive G(1) diploid females were activated artificially, and the resultant embryos were examined for GFP expression, separated and cultured to adulthood (G(2) generation). The G(2) haploid embryos segregated to GFP-positive and -negative individuals. By mating the G(2) adult haploid males individually to diploid females, stocks were established in which the piggyBac construct was stably integrated into the genome, as evidenced by GFP expression and Southern blot hybridization. The piggyBac transposition occurred at its canonical target TTAA sequence. These results, which demonstrate the first successful stable transposon-mediated germline transformation in Hymenoptera, will expand the usefulness of the piggyBac vector.     

Both-ways sex change in monogamous coral gobies, Gobiodon spp.

Synopsis We confirmed both-ways sex change in the coral-dwelling gobies Gobiodon micropus, G. oculolineatus, G. quinquestrigatus and G. rivulatus rivulatus by mate-removal experiment in the field and by the aquarium experiment of keeping two consexual fish in a coral. Eight species of Gobiodon were found in Acropora corals on the reef flat of Sesoko Island, Okinawa, southern Japan. The 4 species mentioned above bred in monogamous pairs composed of a male and a female matched by size, and the male took care of eggs deposited on the coral branch. In G. quinquestrigatus and G. rivulatus rivulatus males were larger than females in newly formed pairs, and females grew faster than their mates until breeding. The growth-rate advantage in females seems to be the major factor in the evolution of female to male sex change. The gobies strongly depended on host corals, but they moved between the corals after mate loss or coral death to form new pairs. This provides opportunities for the evolution of male to female sex change; the ability to change sex in both directions reduces the frequency of risky movement between host corals to form new pairs. These conditions are very similar to those reported in the both-ways sex change of another coral-dwelling goby Paragobiodon echinocephalus.     

Laboratory adaptation of Bactrocera tryoni (Diptera: Tephritidae) decreases mating age and increases protein consumption and number of eggs produced per milligram of protein

A significant reduction in age of mating occurred during the first four generations (G1-G4) of laboratory adaptation of wild Bactrocera tryoni (Froggatt) and this was associated with the earlier attainment of peak egg load although no significant differences were detected in the peak egg load itself. A long term laboratory (LTL) strain had a significantly earlier mating age and higher peak egg load than flies of wild origin or those from the first four laboratory generations. The amount of protein consumed by females in the first week of adult life was significantly higher in the LTL strain than in flies of wild origin or G1-G4 but there were no significant changes (or only slight changes) with laboratory adaptation in the amounts of protein consumed up to the ages of mating and peak egg load. Laboratory adaptation resulted in no significant changes in egg size, egg dry weight, puparial fresh weight and the dry weight of newly emerged females. The large increase in fecundity with laboratory adaptation is associated with a 4- to 5-fold increase in the rate of conversion of dietary protein to eggs (i.e. eggs produced per mg of protein consumed).     

Molecular properties and tissue distribution of 30K proteins as ommin-binding proteins from diapause eggs of the silkworm, Bombyx mori

We previously reported the purification of an ommin-binding protein (OMBP) from an acid-methanol extract of diapause eggs of the silkworm and that OMBP reacted with the anti-30K proteins antiserum. In order to clarify the relationship between OMBP and the 30K proteins, we attempted to determine the sequence of the N-terminal amino acid of OMBP, which was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We observed ten protein spots of various isoelectric points; the spots corresponded with 30 kDa. Based on the sequence of the N-terminal amino acid (20 residues), the spots belonged to two kinds of 30K proteins (6G1 and 19G1), which are known as the major plasma proteins in the larval hemolymph of the silkworm. The proteins are expected to attach to polysaccharide because they reacted with concanavalin A and elderberry bark lectin. Immunohistochemical observations clarified that the proteins were localized in yolk granules and serosa in the diapause egg. These results suggest that OMBP is composed of 30K proteins which were modified with polysaccharides. In addition, the expression of 30K proteins mRNA was observed at early embryonic stage in diapause eggs by RT-PCR analysis. The 30K proteins as OMBP may play an important role in the transport and accumulation of tryptophan metabolites and ommochrome during the formation of serosa.     

丛生盔形珊瑚共附生可培养真菌多样性分析

旨在研究珊瑚共附生可培养真菌多样性。运用稀释平板法和基于ITS-rDNA基因序列的系统发育分析对珊瑚共附生可培养真菌多样性进行研究, 将所得到的基因序列与NCBI数据库GenBank中的序列进行相似性比较并构建系统发育树。实验中从丛生盔形珊瑚表面和内部共分离得到19株菌落形态各异的真菌。ITS-rDNA序列分析及形态学鉴定表明, 丛生盔形珊瑚共附生真菌主要包括曲霉菌Aspergillus sp.、枝孢霉菌Cladosporium sp.、炭角菌Xylariales sp.、青霉菌Penicillium sp.、葡萄穗霉菌Stachybotrys sp.、赤霉菌Gibberella moniliformis、镰刀霉菌Fusarium sp.等。分离得到的菌株中, 4-13与GenBank中已报道的基因序列的相似性仅为89%。结果表明, 与丛生盔形珊瑚共附生的可培养真菌较为丰富, 是潜在的新的微生物菌种资源, 具有进一步研究的价值。     

Gender and sexual behavior modulate the composition of serum lipocalins in Nile tilapia (Oreochromis niloticus)

In tilapia species, plasma lipoproteins with high electrophoretic mobility function in intra- and intergender communication. Blood samples taken at onset and peak of daily sexual activity from dominant and subordinate Oreochromis niloticus males and females were fractionated by native gel electrophoresis and the fast-migrating proteins were subjected to mass spectrometry. Mining the sequence data of the Cichlid Genome Consortium, we identified 11 proteins from the lipocalin super-family and characterized their genes' structures. Phylogenetic and structural analyses subdivided these genes into two classes: (I) 3-coding-exon apolipoproteins and (II) more complex 6-coding-exon sulfide-bond-containing lipocalins. Five apolipoproteins and PTGDSL1, TBTBP, and MSP proteins were modulated by gender and sexual behavior. PTGDSL1 protein was only observed in the plasma serum of dominant males. However, the cysteine residue in the position that is crucial for synthetase activity in mammalian prostaglandin D synthetases was not conserved in PTGDSL1 or PTGDSL2 proteins. In line with previous reports suggesting their involvement in male functions as pheromone transporters, TBTBP and MSP proteins were not detected in females at the onset of daily activity. Their increasing amount in males was concordant with the increase in apolipoproteins AFP4L, APOA4a, APOA4b, APO14kD and APOC2, which were detected exclusively in dominant males, indicating a possible role in mobilization of the energy required to maintain their social hierarchy.     

澄黄滨珊瑚和丛生盔型珊瑚非培养放线菌多样性

【目的】研究澄黄滨珊瑚(Porites lutea)和丛生盔型珊瑚(Galaxea fascicularis)联合放线菌物种多样性。【方法】实验提取两种珊瑚的总DNA,利用放线菌特异性引物对样品总DNA进行扩增,通过构建16S rRNA基因克隆文库和系统发育分析,对三亚鹿回头岸礁区优势物种澄黄滨珊瑚和丛生盔型珊瑚联合放线菌的多样性和群落结构进行研究。【结果】118个从澄黄滨珊瑚克隆文库中随机挑选的阳性克隆子归为58个OTUs,主要分布于酸微菌亚目、棒状杆菌亚目、微球菌亚目、丙酸杆菌亚目和未知类群。丛生盔型珊瑚克隆文库共获得96个序列,归为31个OTUs,主要分布于酸微菌亚目和未知的放线菌类群。多样性指数和稀疏度曲线分析结果显示澄黄滨珊瑚联合放线菌物种多样性比丛生盔型珊瑚更高。【结论】澄黄滨珊瑚和丛生盔型珊瑚拥有较高水平的放线菌物种多样性和复杂的群落结构,并隐藏着大量的高等级放线菌新分类单元。     

Effects of handling and pair management on reproduction in Japanese quail (Coturnix coturnix )

Three experiments were designed to study the effects of handling, pairing, and frequency of mating opportunities on reproduction in Japanese quail (Coturnix coturnix ) when the sexes were caged separately except during the 90 minute mating periods. In Experiments 1 and 2, 48 females and 48 males were allocated randomly to one of four treatments: 1) continuously paired, 2) paired daily with the same male, 3) paired daily with a different male, and 4) paired every third day with the same male. In Experiment 3, 44 males and 44 females used in Experiments 1 and 2 were again assigned randomly to one of four treatments: 1) continuously paired, 2) paired continuously but the females were handled at the beginning and end of a 90 minute period every third day to simulate the handling associated with moving birds between cages for mating, 3) paired every third day with the same male, and 4) paired every third day with a different male. In Experiments 1 and 3, the females were introduced into the males' cages, while in Experiment 2 males were introduced into the females' cages. In Experiment 1, the females paired every 1 or 3 days with the same males had fewer eggs with embryonic development than continously paired females. In Experiment 2, a reduced number of eggs with embryonic development was observed only in fermales paired every 3 days with the same male. In Experiment 1, more eggs hatched from continuously paired females than from females paired every third day with the same male. In Experiment 3, the females paired every third day with the same males had fewer settable, developing, or hatching eggs. In conclusion, it was found that when the sexes need to be caged separately, it is better to expose the females to different males.     

Identification of nuclear receptors involved in regulation of male reproduction in the red flour beetle, Tribolium castaneum

Nineteen canonical and two Knirps-like family nuclear receptors (NRs) were identified in the genome of Tribolium castaneum. The current study was conducted to identify NRs involved in regulation of male reproduction. RNA interference (RNAi)-aided knockdown in the expression of genes coding for all 21 NRs showed that reduction in the levels of 11 NRs (E75, E78, FTZ-F1, HR38, HR4, Knirps-like, HNF4, Tailless, HR51, Dsf and HR39) in the male beetles caused more than 50% reduction in the eggs laid by the female beetles mated with RNAi male beetles. Among these 11 NRs that are required for male reproduction, knockdown in the expression of genes coding for E78 and HR39 in the male beetles resulted in a reduction in the number of sperm produced and transferred to the female when compared to the sperms produced and transferred by the control male beetles injected with bacterial malE dsRNA. In contrast, knockdown in the expression of genes coding for E75 and HR38 caused a reduction in the size of male accessory glands (MAG), the amount of protein produced by the MAG and the expression of genes coding for accessory gland proteins. These data suggest that NRs such as E78 and HR39 regulate sperm production and their transfer to the females and the other NRs such as E75 and HR38 regulate the development of MAG and the production of accessory gland proteins.     

Pelagic spawning of the hawkfish Oxycirrhites typus (Cirrhitidae)

Synopsis Pelagic spawning of the deep slope coral-dwelling cirrhitid Oxycirrhites typus was observed for two social groups at Papua New Guinea. This species was previously reported to be a demersal spawner in an aquarium. Courtship in social groups consisting of a single male and one or two females commenced just prior to or after sunset among the branches of gorgonian or antipatharian corals. Males and females occupied separate corals; males either visited females at their corals or met them at an adjacent coral just prior to courtship. Courtship was sequential and consisted of two or more bouts with each female that culminated in a rapid ascent into the water column and the release of floating eggs. Fertilized eggs, taken from a third social group, were spherical and averaged 0.69 mm in diameter. Spawning pairs sought refuge in their resident corals or in the coral where courtship occurred immediately after spawning was completed.     

VEGF和Flt-1在精索静脉曲张大鼠附睾中的表达变化

目的研究血管内皮生长因子(VEGF)及其受体Flt-1在实验性左侧精索静脉曲张(ELV)大鼠附睾组织中的表达变化,探讨它们与精索静脉曲张(VC)的关系及致男性不育的病理生理学机制。方法建立青春期雄性SD大鼠ELV模型,采用免疫组化SP法检测ELV及对照组附睾中VEGF和Flt-1的表达。结果 VEGF和Flt-1蛋白在大鼠附睾中均有表达,并具有细胞和区域特异性。ELV4周时双侧附睾中VEGF蛋白的表达明显上调(P<0.01),8周时则显著下降(P<0.01);而Flt-1蛋白在ELV4周左侧显著下降(P<0.01),右侧未见明显差异(P>0.05),8周组均显著下降(P<0.01)。结论 ELV引起大鼠附睾中VEGF和Flt-1表达量发生变化,可能影响精子的成熟,是VC引起男性生育力下降甚至不育的原因之一。     

转基因小鼠乳腺表达G-CSF的研究

用PCR法从正常中国人脐带血提取总DNA作为模板,扩增出1.5 kb的人G-CSF基因组基因。序列分析证实其正确性。将其插入小鼠乳清酸蛋白(WAP)基因的起始密码子ATG前的KpnⅠ位点,使其受控于2.6kb的WAP调控序列,构建成乳腺表达载体pWGG。回收经EcoRⅠ酶切后的8.7kb片段用于显微注射。共注射1200枚受精卵,移植34受体母鼠,产仔鼠85只。经PCR检测和DNA印迹分析,证实获得两只整合有人G-CSF基因的雄性鼠,整合率为2.37％。建立的转基因鼠系表明,采用ELASA方法对F1代雌鼠乳汁检测,成功地表达出人G-CSF。表达量为120～250ng/ml。这一结果表明转基因的表达具有乳腺特异性。这为在大动物中实施转基因提供了依据。     

Skeletal muscle protein synthesis and degradation exhibit sexual dimorphism after chronic alcohol consumption but not acute intoxication

Epidemiological evidence suggests alcoholic myopathy is more severe in females than males, but comparable animal studies are lacking that make elucidating the biochemical locus for this defect problematic. The present study determined whether skeletal muscle protein synthesis and markers of degradation exhibit a sexual dimorphic response to either chronic alcohol consumption or acute intoxication. Male and female rats were fed an alcohol-containing diet, pair-fed for 26 wk (chronic), or received an intraperitoneal injection of alcohol (acute). In males, chronic alcohol decreased gastrocnemius protein synthesis by 20%. This reduction was associated with a twofold increase in the inactive eukaryotic initiation factor (eIF) 4E.4E-binding protein 1 (4E-BP1) complex and a 60% reduction in the active eIF4E.eIF4G complex. This redistribution of eIF4E was associated with decreased phosphorylation of both 4E-BP1 and eIF4G (50-55%). The phosphorylation of ribosomal protein S6 was also reduced 60% in alcohol-consuming male rats. In contrast, neither rates of protein synthesis nor indexes of translation initiation in muscle were altered in alcohol-fed female rats despite blood alcohol levels comparable to males. Chronic alcohol ingestion did not alter atrogin-1 or muscle RING finger-1 mRNA content (biomarkers of muscle proteolysis) in males but increased their expression in females 50-100%. Acute alcohol intoxication produced a comparable decrease in muscle protein synthesis and translation initiation in both male and female rats. Our data demonstrate a sexual dimorphism for muscle protein synthesis, translation initiation, and proteolysis in response to chronic, but not acute, alcohol intoxication; however, they do not support evidence indicating females are more sensitive toward the development of alcoholic skeletal muscle myopathy.