Follistatin 基因在牙鲆胚胎中的表达及其在肌肉发育中作用的研究

卵泡抑素(Follistatin)是1987年由Robertson和Ueno分别从牛和猪的卵泡液中分离出的一种富含半胱氨酸的糖基化单链多肽。FS对不同类型的细胞有广泛的调节作用，具有多方面的生物学作用。本研究克隆到牙鲆Follistatin基因，并利用RT-PCR和原位杂交对其在胚胎及成体中的表达进行了分析，并对其启动子进行了组织特异性分析。 1． Follistatin基因组全长4.3 kb左右，其中启动子区长约1 kb。与cDNA序列的比较显示该基因含有5个外显子和4个内含子，内含子和外显子的交界处严格遵守GT…AG规则。Follistatin基因编码了一个含有323个氨基酸的蛋白质前体，该蛋白质前体含有信号肽区域、N-端结构域、Follistatin结构域Ⅰ、Follistatin结构域Ⅱ、Follistatin结构域Ⅲ和C-端结构域。蛋白比较分析表明Follistatin与其他物种的Follistatin的同源性较高，其结构域保守性更高。 三级预测结果显示：在空间构型上，牙鲆Follistatin基因与斑马鱼Follistatin基因完全一致。在序列上高度保守的半胱氨酸在空间上两两相对，它们可能对维持Follistatin蛋白的空间结构起着重要的作用 2. RT-PCR方法和原位杂交方法研究Follistatin基因在牙鲆胚胎中的表达情况结果显示：Follistatin基因在受精后26 hrs开始在中胚层细胞处表达，其后在受精后28、30、32、34、36、38、40、42 hrs等时期Follistatin基因持续表达，并在体节腹部部位、头部，背部体节等部位表达。以MyoD为对照而进行的双色原位杂交结果显示：Follistatin与MyoD在胚胎的体节处表达有重叠区域，对双色原位杂交样品进行的冰冻切片实验结果显示：两个基因的表达区域交叉，互相混合，表明Follistatin基因也在肌肉前体细胞中表达，但并不在所有的前体细胞中表达，Follistatin可能在肌肉发育早期起一定的作用。成体组织中，Follitatin基因在体肾、肠、心脏、头肾、脾中有表达，而在肌肉中没有表达。 3．启动子序列分析结果表明：牙鲆启动子存在AP-1、C/EBP、SP1、USF、E47、MyoD等转录因子的潜在结合位点。显微注射结果显示：该基因启动子能够使绿色荧光蛋白表达在斑马鱼的胚胎肌肉组织中。

牙鲆rag基因的克隆及表达研究

牙鲆在中国北方沿海地区形成了大规模的工厂化养殖，成为我国海水养殖的一大重要产业。然而疾病的频繁发生严重阻碍了这一产业的发展，为了保证牙鲆产业稳定、健康、持续地发展，开展鱼类免疫系统的基础研究，提高鱼类抵抗病原菌侵害的能力，本研究从牙鲆免疫系统的个体发育过程入手，克隆了其rag（recombination activating gene）基因，在此基础上对其组织特异性表达进行了分析。 研究结果表明牙鲆rag1基因由4个外显子和3个内含子组成，第一个外显子位于5’非翻译区，这一外显子存在两种不同的剪切方式。牙鲆rag1基因编码1068个氨基酸。牙鲆rag2基因的编码区序列长为1602 bp，编码533个氨基酸，没有在牙鲆rag2基因编码区内发现内含子的存在。rag基因间序列长为3128 bp，两个基因共用一段3’非编码区。 本试验通过RT-PCR检测发现，牙鲆的rag1和rag2基因在头肾和体肾中均有表达。整装原位杂交结果表明，牙鲆在受精后第8天开始起有rag1和rag2基因的表达，主要在胸鳍前面，腮背部的部位表达，这个区域与后来胸腺出现的位置相一致。冰冻切片结果显示，rag是在靠近咽部的区域有表达。石蜡切片原位杂交结果显示，rag基因在胸腺中的表达显示出不均一性，着色较深的为皮层区，而着色比较浅的为髓部。 将牙鲆rag1基因的一部分（编码559个氨基酸）和rag2全基因序列（编码533个氨基酸）插入到表达质粒pProEXTM HTa上，在大肠杆菌E. coli BL-21中进行体外表达与分析，结合BD TALONTM Metal Affinity Resins亲和柱纯化了RAG2蛋白。

Effects of temperature and delayed initial feeding on the survival and growth of Japanese flounder larvae

The effects of the timing of initial feeding (0, 1, 2 3 and 4 days after yolk exhaustion) and temperature (15, 18 and 21degrees C) on the point-of-no-return (PNR), survival and growth of laboratory-reared Japanese flounder Paralichthys olivaceus larvae were studied under controlled conditions. The larvae reached PNR on 7(.)7, 5(.)2 and 4(.)2 days-post-hatching (dph) at 15, 18 and 2 V C, respectively. At each temperature, larval growth did not differ significantly among the delayed initial feedings 1 day before PNR but decreased significantly in larvae first fed after that. In the treatments where initial feeding was equally delayed, larvae grew significantly faster at 18 and 21degrees C than at 15degrees C. The larvae survived apparently better at 15 and 18degrees C than at 21degrees C when initial feeding was equally delayed. At each temperature, survival of the larvae first fed before PNR did not differ noticeably, while delayed initial feeding after that apparently reduced their survival. These results indicated that there existed a negatively temperature-dependent PNR in the Japanese flounder larvae. Survival and growth of the larvae strongly depended on temperature as well as the timing of initial feeding. High temperature accelerated the yolk exhaustion and growth of the larvae and thus reduced their starvation tolerance and survival. To avoid potential starvation mortality and obtain good growth, the Japanese flounder larvae must establish successful initial feeding within 2 days after yolk exhaustion at 15degrees C and within 1 day at both 18 and 21degrees C. (C) 2005 The Fisheries Society of the British Isles.

Feeding resumption, morphological changes and mortality during starvation in Japanese flounder larvae

Japanese flounder Paralichthys olivaceus larvae established first feeding 3 days after hatching (DAH) at c. 17degreesC. Non-fed fish reached irreversible starvation at age 5 DAH. Non-fed fish showed similar feeding rate and feeding intensity as the fed fish when they were provided with prey before 5 DAH, after which the starved larvae did not feed even when prey became available. None of the six morphological measurements examined (total length, body height, eye height, head height, gut height and myotome height) showed significant differences between the non-fed and fed larvae until 5 DAH. Normal development continued only in the fed group, and the non-fed larvae showed reverse growth or body collapse after 5 DAH. Owing to the shrinkage and collapse at the top of head due to starvation, head height could be a sensitive indicator of starvation in Japanese flounder larvae. In the fed treatments, high mortality occurred from first feeding (3 DAH) to irreversible starvation (5 DAH), accounting for about two-thirds to three-quarters of the overall mortality (46-52%) throughout the experiments. This mortality was not prey density or larval density dependent. Mortality during the same period in the non-fed larvae accounted for about a third of the overall mortality (100%). (C) 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.

Feeding behaviour of Japanese flounder larvae under laboratory conditions

Tank-reared Japanese flounder larvae, Paralichthys olivaceus, had a major feeding peak in the morning and a secondary peak in the afternoon throughout the larval development, with light being the primary factor regulating their feeding activity. The larvae consumed rotifers in preference to Artemia for up to 10 days, after which the food preference shifted to Artemia. Feeding rates of the larvae prior to 10 days post-batch depended on prey density, but in the old larvae, feeding rates were independent of prey density. Maximum feeding rate occurred at 19 degrees C. The occurrence of the attack posture, after its onset at first feeding (2 days post-hatch), increased up to 25 days, began to decrease when the larvae prepared to settle down, then disappeared after settlement. The occurrence frequency of the attack posture was positively related to fish density, but inversely related to starvation duration, and occurred most frequently at 19 degrees C. This posture depended on prey density in larvae prior to 10 days post-hatch, but became independent of prey density as the larvae developed. It was obvious that, for flounder larvae, attack posture was a behavioural character closely related to feeding and subject to larval development and environmental factors. (C) 2000 The Fisheries Society of the British Isles.

Food utilization of adult flatfishes co-occurring in the Bohai Sea of China

Stomach contents were examined of 4527 adult individuals of 12 flatfish species collected during the 1982 - 1983 Bohai Sea Fisheries Resources Investigation. Their food habits, diet diversity, similarity of prey taxa, trophic niche breadth and diet overlap were systematically analysed. Ninety-seven prey species belonging to the Coelenterata, Nemertinea, Polychaeta, Mollusca, Crustacea, Echinodermata, Hemichordata and fish were found and five of them were considered to be principal prey for flatfishes: Alpheus japonicus, Oratosquilla oratoria, Alpheus distinguendus, Loligo japonicus and Crangon affinis. Among the flatfishes, Paralichthys olivaceus was piscivorous, whereas Pseodopleuronectes yokohamae and Pseudopleuronectes herzensteini both had polychaetes and molluscs as their main prey groups. Pleuronichthys cornutus was classified as a polychaete-mollusc eater, with a strong preference for crustaceans. Verasper variegatus, Cynoglossus semilaevis, Eopsetta grigorjewi and Cleisthenes herzensteini ate crustaceans. Kareius bicoloratus was classified as a mollusc-crustacean eater: Cynoglossus abbreviatus, Cynoglossus joyneri and Zebrias zebra were grouped as crustacean-fish eaters. However, Z. zebra also took polychaetes and C. abbreviatus and C. joyneri preyed on some molluscs. Trophic relationships among the flatfishes were complicated, but they occupied distinctive microhabitats in different seasons and selected their specific prey items, which was favourable to the stability of the flatfish community in the Bohai Sea.

Life history cycles of flatfish species in the Bohai Sea, China

This paper provides basic information on the general ecology and life history cycles of various flatfish species in the Bohai Sea, China. The species studied are Paralichthys olivaceus (Temminck & Schlegel), Cleisthenes herzensteini (Schmidt), Eopsetta grigorjewi (Herzenstein), Verasper variegatus (Temminck & Schlegel), Pleuronichthys cornutus (Temminck & Schlegel), Pseudopleuronectes yokohamae (Gunther), Pseudopleuronectes herzensteini (Jordan & Snyder), Kareius bicoloratus (Basilewsky), Zebrias zebra (Bloch), Cynoglossus semilaevis Gunther, Cynoglossus abbreviatus (Gray) and Cynoglossus joyneri Gunther. Information on reproduction, eggs and larval distribution, growth and adult abundance is presented. Based on the biology and ecology of these flatfish, artificial enhancement of the commercial species in the Bohai Sea is discussed.

Validations and descriptions of European syntaxa of vegetation dominated by lichens, bryophytes and algae

Fourty-two high-rank syntaxa and seven associations of the thallophyte system of syntaxa are either described as new or validated in this paper. Among those, there are the following nine classes: Aspicilietea candidae, Caulerpetea racemosae, Desmococcetea olivacei, Entophysalidetea deustae, Gloeocapsetea sanguineae, Mesotaenietea berggrenii, Naviculetea gregariae, Porpidietea zeoroidis, Roccelletea phycopsis. Eleven orders and ten alliances as well as three associations are described or validated: the Aspicilietalia verruculosae (incl. Aspicilion mashiginensis and Teloschistion contortuplicati), the Caulerpetalia racemosae (incl. Caulerpion racemosae), the Desmococcetalia olivacei (incl. Desmococcion olivacei), the Dirinetalia massiliensis, the Fucetalia vesiculosi (incl. Ascophyllion nodosi), the Gloeocapsetalia sanguineae, the Lecideetalia confluescentis (incl. Lecideion confluescentis), the Mesotaenietalia berggrenii (incl. Mesotaenion berggrenii, Mesotaenietum berggrenii and Chloromonadetum nivalis), the Naviculetalia gregariae (incl. Oscillatorion limosae and Oscillatorietum limosae), the Porpidietalia zeoroidis (incl. Porpidion zeoroidis), and the Roccelletalia fuciformis (incl. Paralecanographion grumulosae). Further, five orders, seven alliances and four associations, classified in known classes, were described as well. These include: the Bacidinetalia phacodis, the Agonimion octosporae and the Dendrographetalia decolorantis (all in the Arthonio radiatae-Lecidelletea elaeochromae), the Staurothelion solventis (in the Aspicilietea lacustris), the Pediastro duplicis-Scenedesmion quadricaudae and the Pediastro duplicis-Scenedesmetum quadricaudae (both in the Asterionelletea formosae), the Peccanion coralloidis and the Peltuletalia euplocae (both in the Collematetea cristati), the Laminarion hyperboreae, the Saccorhizo polyschidi-Laminarietum and the Alario esculenti-Himanthalietum elongatae (all in the Cystoseiretea crinitae), the Delesserietalia sanguinei, the Delesserion sanguinei and the Delesserietum sanguineae (all in the Lithophylletea soluti), as well as the the Rinodino confragosae-Rusavskietalia elegantis and the Rhizocarpo geographici-Rusavskion elegantis (both in the Rhizocarpetea geographici).

Dieta del guanay leucocarbo bougainvilli, del piquero peruano Sula Variegata y otras aves de la costa peruana. Abril y mayo de 1997.

Se estudió la dieta del guanay Leucocarbo bougainvillii y del piquero peruano Sula variegata en siete áreas guaneras del litoral peruano en abril y mayo de 1997. Además se presenta información sobre la dieta del camanay Sula nebouxii, piquero enmascarado Sula dactylatra y por primera vez, del cushuri Hypoleucos olivaceus. El pejerrey Odontesthes regia fue la presa más consumida por el guanay representando el 42,41% de los otolitos hallados. La anchoveta ocupó el segundo lugar, con el 39,40% de los otolitos (33,70% adultos y 5,70% juveniles). La presa principal del guanay en las islas Lobos de Tierra, Macabí y Guañape Sur fue la anchoveta, en las islas Mazorcas y Chincha Norte y Punta Coles fue el pejerrey, en tanto que en San Juan fue el camotillo Normanichthys crockeri. La dieta del guanay refleja poca disponibilidad de anchoveta en el zona norte, siendo esta aún menor en las zonas centro y sur. En general, se ha observado una disminución en el número de presas consumidas por el guanay en todas las zonas muestreadas, con respecto a una evaluación realizada en febrero de 1997.

Discrimination of millipedes by the opossum Didelphis albiventris (Marsupialia, Didelphidae)

Millipedes are toxic preys for many vertebrates. Two individuals of the opossum species D. albiventris were videorecorded when feeding on the millipedes G. olivaceus and L. dentellus. Data were compared with feeding behavior on Tenebrio larvae. The opossums are resistant to intoxication by millipedes. Although chemical defenses of the millipedes were not effective against the opossum predation, they make them less attractive.

Corticolous green algae from tropical forest remnants in the northwest region of São Paulo State, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Systematics and phylogeny of the Neotropical Pachymenes de Saussure and Santamenes Giordani Soika (Hymenoptera, Vespidae, Eumeninae)

The genera Pachymenes de Saussure and Santamenes Giordani Soika arerevised and the phylogenetic relationships among their species, based on external mor-phology and male genitalia, are presented. The cladistics analysis, using 22 terminalspecies (19 ingroup and 3 outgroup species) and 44 characters, produced a single clado-gram under implied weighting. Both genera were recovered as paraphyletic, althoughttwo major clades were formed and were well supported by the re-sampling analysis.We propose the synonymy of Pachymenes with Santamenes, and the description of twonew species: P. saussurei Grandinete n.sp. and P. riograndensis Grandinete n.sp..Newcombinations are: Pachymenes novarae (de Saussure) n.comb., P. olympicus (Zavattari)n.comb., P. peregrinus (Zavattari) n.comb. and P. santanna (de Saussure) revised combi-nation. We state the synonymy of P. obscurus orellanoides under P. obscurus consuetus,reviewing the status of the latter and raising P. consuetus to species level. Pachymenesorellanae vardyi is synonymized under P. orellanae; P. ghilianii olivaceus, P. ghilianiiavissimus and P. peruanus are proposed as synonyms of P. ghilianii; P. picturatusobscuratus is synonymized under P. laeviventris; P. picturatus nigromaculatus andP. picturatus var . intermedia are synonymized under P. picturatus and P. a t ra var . ornatis-sima get its lectotype designated and proposed as synonym of P. ater.

Host Specificity of Calyptospora funduli (Apicomplexa: Calyptosporidae) in Atheriniform Fishes

Calyptospora funduli has a broad host specificity, infecting at least 7 natural and 10 additional experimental definitive hosts, all atheriniform fishes within 5 families, but most in the genus Fundulus. Barriers, apparently innate ones, prevent any development of C. funduli in perciform fishes but allow incomplete or abnormal development of the parasite in a few unnatural atheriniform hosts. In the freshwater species Fundulus olivaceus and Fundulus notti, these abnormalities consisted of asynchronous development, degeneration of the parasite in early stages of development, and the formation of numerous macrophage aggregates. Rivulus marmoratus has the ability to eliminate infections with a granulomatous inflammatory response. Additional barriers that limit natural infections of C. funduli in other hosts include feeding behavior, environmental conditions, and geographic isolation.

Experimental Infection of Contracaecum tipapillatum (Nematoda: Anisakinae) from Mexico in the Domestic Cat

Juveniles of Contracaecum multipapillatum infected the Mayan cichlid (Cichlasoma urophthalmus) and adults infected the olivaceous cormorant (Phalacrocorax olivaceus) and the great egret (Casmerodius albus) in the coastal lagoon at Celestun, State of Yucatan, Mexico. All are new host records, and, even though the geographic locality record of Mexico for the species has not been published, unidentified but presumably conspecific specimens have been reported from there. When juveniles of C. multipapillatum were fed to a kitten, but not rats, ducks, or chickens, they developed into adults. Measurements and morphological data are provided on the specimens from the kitten. Development of an avian ascaridoid in the intestine of a mammal increases the potential of this widespread species to infect other mammals, including humans.

Vitrification of primordial germ cells using whole embryos for gene-banking in loach, Misgurnus anguillicaudatus

In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3'UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.