Molecular and cytogenetic analysis of the telomeric (TTAGGG)n repetitive sequences in the Nile tilapia,Oreochromis niloticus (Teleostei: Cichlidae)

The majority of chromosomes in Oreochromis niloticus, as with most fish karyotyped to date, cannot be individually identified owing to their small size. As a first step in establishing a physical map for this important aquaculture species of tilapia we have analyzed the location of the vertebrate telomeric repeat sequence, (TTAGGG)n, in O. niloticus. Southern blot hybridization analysis and a Bal31 sensitivity assay confirm that the vertebrate telomeric repeat is indeed present at O. niloticus chromosomal ends with repeat tracts extending for 4-10 kb on chromosomal ends in erythrocytes. Fluorescent in situ hybridization revealed that (TTAGGG)n is found not only at telomeres, but also at two interstitial loci on chromosome 1. These data support the hypothesis that chromosome 1, which is significantly larger than all the other chromosomes in the karyotype, was produced by the fusion of three chromosomes and explain the overall reduction of chromosomal number from the ancestral teleost karyotype of 2n=48 to 2n=44 observed in tilapia.     

Territorial aggression, circulating levels of testosterone, and brain aromatase activity in free-living pied flycatchers

Testosterone (T) is a critical endocrine factor for the activation of many aspects of reproductive behavior in vertebrates. Castration completely eliminates the display of aggressive and sexual behaviors that are restored to intact level by a treatment with exogenous T. There is usually a tight correlation between the temporal changes in plasma T and the frequency of reproductive behaviors during the annual cycle. In contrast, individual levels of behavioral activity are often not related to plasma T concentration at the peak of the reproductive season suggesting that T is available in quantities larger than necessary to activate behavior and that other factors limit the expression of behavior. There is some indication from work in rodents that individual levels of brain aromatase activity (AA) may be a key factor that limits the expression of aggressive behavior, and in agreement with this idea, many studies indicate that estrogens produced in the brain by the aromatization of T may contribute to the activation of reproductive behavior, including aggression. We investigated here in pied flycatcher (Ficedula hypoleuca) the relationships among territorial aggression, plasma T, and brain AA at the peak of the reproductive season. In a first experiment, blood samples were collected from unpaired males holding a primary territory and, 1 or 2 days later, their aggressive behavior was quantified during standardized simulated territorial intrusions. No relationship was found between individual differences in aggressive behavior and plasma T or dihydrotestosterone levels but a significant negative correlation was observed between number of attacks and plasma corticosterone. In a second experiment, aggressive behavior was measured during a simulated territorial intrusion in 22 unpaired males holding primary territories. They were then immediately captured and AA was measured in their anterior and posterior diencephalon and in the entire telencephalon. Five males that had attracted a female (who had started egg-laying) were also studied. The paired males were less aggressive and correlatively had a lower AA in the anterior diencephalon but not in the posterior diencephalon and telencephalon than the 22 birds holding a territory before arrival of a female. In these 22 birds, a significant correlation was observed between number of attacks/min displayed during the simulated territorial intrusion and AA in the anterior diencephalon but no correlation was found between these variables in the two other brain areas. Taken together, these data indicate that the level of aggression displayed by males defending their primary territory may be limited by the activity of the preoptic aromatase, but plasma T is not playing an important role in establishing individual differences in aggression. Alternatively, it is also possible that brain AA is rapidly affected by agonistic interactions and additional work should be carried out to determine whether the correlation observed between brain AA and aggressive behavior is the result of an effect of the enzyme on behavior or vice versa. In any case, the present data show that preoptic AA can change quite rapidly during the reproductive cycle (within a few days after arrival of the female) indicating that this enzymatic activity is able to regulate rapid behavioral transitions during the reproductive cycle in this species.     

奥尼罗非鱼及其亲本感染无乳链球菌后生理响应变化

研究通过比较杂交子一代奥尼罗非鱼(Oreochromis spp.)与亲本在腹腔注射无乳链球菌(Streptococcus agalactiae)后累积死亡率差异,血液生理指标、生化指标和脾脏促炎性细胞因子的表达水平变化,以深入了解奥尼罗非鱼抗病性杂种优势的生理和分子基础。结果显示:在无乳链球菌人工感染后,奥尼罗非鱼的累积死亡率显著低于亲本(P<0.05)。感染前奥尼罗非鱼的白细胞数、红细胞数和红细胞压积最高,感染后3种罗非鱼的白细胞都显著性升高(P<0.05),红细胞数、红细胞压积和血红蛋白显著性下降(P<0.05),呼吸暴发受到抑制,奥尼罗非鱼血液生理指标与父本奥利亚罗非鱼更为接近;奥尼罗非鱼在感染细菌后的血糖浓度和溶菌酶含量显著高于亲本,呼吸暴发强于亲本(P<0.05)。定量PCR结果显示感染后3种罗非鱼脾脏TNF-α、IL-1β和IL-6的mRNA表达水平都显著上升(P<0.05), 3种罗非鱼的TNF-α和IL-6都在感染后7h达到峰值,奥尼罗非鱼IL-1β在感染后48h达到峰值,尼罗罗非鱼IL-1β在感染后24—48h达到峰值,奥利亚罗非鱼IL...     

Female Choice Predicts the Best Father in a Biparental Fish,the Midas Cichlid (Cichlasoma citrinellum)

The Midas cichlid is a monogamous, biparental species. It breeds in a highly competitive system where pairs have a low probability of raising fry to independence. Both parents must cooperate to retain the territory and protect the fry from predation. Previous experiments showed that females prefer large, aggressive and sexually experienced males as mates but males do not display any consistent preferences. Here I present the results of two experiments designed to see whether qualities preferred by females correlate with increased success in retaining territories and in providing parental care. Pairs with either large or aggressive males had an advantage in appropriating and holding a breeding territory; reproductive experience conferred no advantage in usurping a territory. Aggressive and reproductively experienced males had an advantage in defending the brood from predators of fry, but size had no effect. Thus, the qualities preferred by females confer advantages both in holding territories and in protecting fry. In contrast, males need not be selective because females, once in possession of a brood, defend it equally well regardless of size, aggressiveness, or reproductive experience. The system is one of mutually enforced monogamy based on female choice; females drive the system because they provide more investment than do males (combining gametes and time) and because this investment is a reliable resource to the male.     

Non-lethal detection of DNA from Cichlidogyrus spp. (Monogenea, Ancyrocephalinae) in gill mucus of the Nile tilapia Oreochromis niloticus

Infection of Nile tilapia Oreochromis niloticus by monogeneans of the genus Cichlidogyrus is harmful. Currently, diagnosis of this infection is based on invasive techniques and the identification of isolated parasites by their morphology. To facilitate diagnosis, we have developed a non-lethal polymerase chain reaction (PCR) test for detection of Cichlidogyrus spp. DNA in the gill mucus of O. niloticus, using 5 pairs of specific primers based on Cichlidogyrus sclerosus 28S rRNA (Cicly 1 to Cicly 5) which generate fragments of approximately 188, 180, 150, 159 and 189 bp, respectively. PCR specificity was tested using genomic DNA extracted individually from 175 isolated Cichlidogyrus spp., 75 Gyrodactylus cichlidarum and 75 endopararasitic Enterogyrus spp., as well as from 75 protozoans Trichodina spp. The Cicly primers were used to detect Cichlidogyrus spp. DNA in mucus from the gills of 23 Nile tilapia confirmed to be infected with the parasite. Negative controls consisted of 45 uninfected Nile tilapia. The limit of sensitivity of the assay was 1.2 ng of purified parasite DNA. The Cicly primers did not amplify DNA from the mucus of non-infected Nile tilapia, G. cichlidarum, Trichodina spp. or Enterogyrus spp. In all cases, the sensitivity and specificity of the test were 100%. The sequences of all the amplified fragments showed a high similarity to that of the 28S rRNA region of C. sclerosus (93 to 100% identical to GenBank Accession No. DQ157660.1). We provide evidence for a safe and non-invasive DNA-based diagnostic method for the presence of Cichlidogyrus in the gill mucus of O. niloticus.     

Predation by Nile perch Lates niloticus (L.) on Oreochromis niloticus (L.), Cyprinus carpio (L.), Mugil sp. and its role in controlling tilapia recruitment in Egypt

Vulnerability of three fish groups (tilapia, common carp, mullet) to predation by Nile perch Lates niloticus was evaluated. The study was conducted in aquaria and in cages placed in concrete ponds. Common carp Cyprinus curpio was the more vulnerable to predation by Nile perch followed in order by mullet ( Mugil cephulus and M. cupito ) and Nile tilapia Oreochromis niloricus . Medium-size Nile perch (30–32 cm t.l. ) were able to consume tilapia up to 14 cm t.l. , while tilapia of 15 cm total length were not eaten. Ratios of 1 : 10 to 1 : 15 between Nile perch and ready-to-spawn females of O. niloticus were found to be adequate to control tilapia reproduction.     

Effects of dietary fibre on the intestinal absorption of dextrin, blood sugar level and growth of tilapia, Oreochromis niloticm × O. aureus

The roles of five different dietary fibres (cellulose, agar, guar gum, carrageenan and carboxy-methylcellulose) each as 10% wt/wt added into the diet, affecting the intestinal sugar absorption, blood sugar level and utilization of dextrin were studied in hybrid tilapia, Oreochromis niloticus × O. aureus ; dextrin and glucose were also included in the study as controls for comparison. There were seven dietary groups: each diet was fed to three aquaria each containing 15 fish. The experiment was carried out in a closed circulation, filtered, rearing system for 2 months. The results indicated that the weight gain percentage and food conversion ratio were significantly (P<0.05) lower in tilapia fed fibre-containing diets than those of tilapia fed dextrin or glucose diet. The intestinal absorption of carbohydrate and the blood sugar content of tilapia were low when diets contained fibre, regardless of the source.     

Comparative susceptibility of two different genetic types of tilapia to Neobenedenia sp. (Monogenea)

Two different genetic types of tilapia, Mozambique tilapia Oreochromis mossambicus (MT), and Pargo-UNAM (PU; a synthetic hybrid whose genetic composition is 50% Florida red tilapia, 25% Rocky Mountain tilapia, and 25% red variant Oreochromis niloticus), were acclimatized to salinity and exposed to seawater from the Gulf of Mexico off the port of Veracruz, Mexico. Both fish types were infected by the monogenean ectoparasite Neobenedenia sp. and were killed within 2 to 3 wk. A crude worm extract was prepared from whole specimens collected during the original outbreak and used to immunize naive hosts of the same 2 types of tilapia. Immunized fish were then exposed to seawater, which resulted in Neobenedenia sp. infection. Immunization did not confer any protection against Neobenedenia sp. infection. However, the experiment enabled detailed analysis of the dynamics of infection and comparison of the effects of the parasite on the 2 host types. Although both tilapia types exhibited similar resistance to infection (as they harbored similar parasite burdens in the early phase of infection), PU is less tolerant to Neobenedenia sp., as a mean parasite abundance of ca. 50 worms fish-' killed all hosts within a fortnight, while 22% of MT survived up to 3 wk, harboring a mean parasite abundance of ca. 900 worms fish-'. Our results suggest that, as reported elsewhere, Neobenedenia sp. could negatively affect mariculture off the Mexican coast of the Gulf of Mexico.     

罗非鱼胰蛋白酶ⅠmRNA克隆与分析

应用3′/5-′RACE方法对尼罗罗非鱼(Oreochromis niloticus)和奥利亚(Oreochromis aureus)罗非鱼胰蛋白酶ⅠmR-NA进行了克隆和测序,序列分析表明,尼罗罗非鱼和奥利亚罗非鱼胰蛋白酶ⅠmRNA序列全长分别为863bp和858bp,序列中均含有738个核苷酸组成的开放阅读框,编码长度为245个氨基酸残基的胰蛋白酶Ⅰ。胰蛋白酶Ⅰ氨基末端均存在信号肽和激活肽序列,序列中均具有与催化活性必须的高度保守的催化三联体氨基酸残基(His、Asp、Ser)和构成二硫键的12个半胱氨酸残基,以及决定底物特异性的保守性氨基酸残基即天冬氨酸残基和S1结合袋。南极鱼(Patanotothenia magellanica)、大西洋鲑(Salmo salar)、日本鲽(Paralichthys olivaceus)、大西洋鳕(Gadusmorhua)、牛和人类胰蛋白酶mRNA序列以及氨基酸序列与奥利亚罗非鱼相比较同源性分别为58.3%—72.5%和63.3%—76.1%,与尼罗罗非鱼相比较同源性分别为59.1%—73.1%和63.6%—75.6%。奥利亚罗非鱼和尼罗罗非鱼胰蛋白酶mRNA序列以及氨基酸序列同源性分别为96.2%和99.2%。       

The properties of tilapia sperm and its cryopreservation*

The specific differences between the testis, milt and sperm of six species of tilapia including Oreochromis aureus, O. mossambicus, O. niloticus, Tilapia zillii, O. nilolicus×O. aureus hybrid and red tilapia, Oreochromis sp., were studied. The shape of testis is tubular; the gonadosomatic index varied from 0.07 to 2.71. The pH values of individual milts ranged from 6.2 to 8.2 and the osmolarity from 240 to 380 mOsmol kg^?1. The quantity of milt obtained by stripping averaged only about 0.3 ml, and only in the O. niloticus×O. aureus hybrid did it exceed 3 ml. Sperm motility graded from weak to moderate was determined for the stripped tilapia milt. Sperm concentrations ranged from 7.70 × 10⁸ sperms ml^?1 in T. zillii to 2.74 × 10¹⁰ sperms ml^?1 in O. mossambicus. Tilapia sperm was active in various salinity ranges such as 0–5‰ for O. niloticus, and 0–15‰ for O. mossambicus and T. zillii. Extender containing 15% milk and 5% methanol was used to prepare milt mixture before cooling rapidly to ?35° C and then at 5° C min^?1 to ?75° C for storage in liquid nitrogen (– 196° C). Fertility tests on frozen tilapia milt resulted in a fertilization rate of 72.7% (v. control 85.7%) for the 22-day frozen milt of the O. nilolicus×O. aureus hybrid used to fertilize the eggs of O. honorum, and 93.4% (v. control 90%) for the 304-day frozen sperm of red tilapia used to fertilize eggs of red tilapia.     

奥利亚罗非鱼DMRT1,DMO基因片段的克隆及序列分析

根据其他鱼类DMRT基因中的保守序列设计了一对简并引物,利用RT-PCR扩增了雌雄奥利亚罗非鱼的DMRT基因,并对其扩增产物进行了克隆与测序。结果在雌雄奥利亚罗非鱼个体中获得了两个不同的片段,分别命名为DMO,DMRT1基因。序列同源性分析表明,奥利亚罗非鱼DMRT1,DMO cNA系列的同源性为61.78%,氨基酸同源性为86%,说明了DMRT基因存在性别差异。与尼罗罗非鱼、红鳍东方豚、虹鳟、青鱼将等鱼类DM保守区相比,氨基酸序列同源性为86%-100%,这充分显示了DM-RT基因在进化上的高度保守性。     

我国新引进吉富品系尼罗罗非鱼群体的遗传多样性分析

选取罗非鱼(Oreochromis spp.)第二代遗传连锁图谱中的26个微卫星位点,对淡水渔业研究中心引进的、由60个家系组成的吉富品系尼罗罗非鱼(O.niloticus)群体进行遗传结构分析。结果显示,26个微卫星位点在吉富罗非鱼群体中共检测到124个等位基因,各位点的等位基因数为3～7个,平均4.8个。片段长度104～322 bp,平均杂合度观测值为0.622 1,平均杂合度期望值为0.642 3,平均多态信息含量(PIC)为0.633 4。所检测的26个位点中,有25个位点属于高度多态位点(PIC0.5),占所检测位点的96.15%;1个位点属于中度多态位点。结果表明,该吉富罗非鱼群体多态信息含量丰富,遗传多样性水平较高。因而该群体仍然具有较大的选育潜力,可以作为选育的基础群体开展进一步的选育工作。     

Molecular-cytogenetic analysis reveals sequence differences between the sex chromosomes of Oreochromis niloticus: evidence for an early stage of sex-chromosome differentiation

Sex determination in the Nile tilapia, Oreochromis niloticus, is primarily genetic, with XX females and XY males. A candidate sex-determining region in the terminal region of the largest chromosome pair has been identified by analysis of meiotic chromosomes. This region shows an inhibition of pairing and synapsis in the XY genotype, but not in XX or YY genotypes, suggesting that recombination is inhibited. Here we show that chromosome microdissection and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR) can be used to produce in situ hybridization probes to this largest pair of O. niloticus chromosomes. Furthermore, analysis of the comparative hybridization of X and Y chromosome-derived probes to different genotypes provides the first demonstration that sequence differences exist between the sex chromosomes of O. niloticus. This provides further support for the theory that this chromosome pair is related to sex determination and further suggests that the sex chromosomes are at a very early stage of divergence.     

Masculinization of genetic female nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation

A series of experiments was carried out in which genetically female Nile tilapia (Oreochromis niloticus) fry were treated with Fadrozole, a nonsteroidal aromatase inhibitor (AI), in the diet during the period of sexual differentiation. Batches of tilapia fry treated with AI during the first 30 days following yolk-sac resorption (7-37 days post hatch, dph) showed a dose-dependent increase in the percentage of males from 0 to 200 mg. kg(-1). The percentage of males remained approximately constant (92.5-96.0%) from 200 to 500 mg. kg(-1). Any continuous 2- or 3-week treatment with 500 mg. kg(-1) AI in this 4-week period successfully masculinized the majority of the treated fish (>80%). Treatments of 1 week duration revealed that the most sensitive time to AI lies in the first week (between 7 and 14 dph). Progeny testing of males from AI-treated groups gave results indicating that these were XX males, as expected. These experiments strongly implicate aromatase activity as a key factor in sexual differentiation in the Nile tilapia.     

Reproductive season onset and time invested in territory defense in Colinus leucopogon males (Galliformes: Odontophoridae)

The factors that determine the onset of the reproductive season and the relationship between territory defense and mating success of Colinus leucopogon males are unknown. Here I report on climatic variables influencing the time of permanence on the territory, and how this affects the species mating success. I also analyze the relationship between the time devoted by males on territory defense and the relationship of song and territorial characteristics. The onset of the reproductive season was determined by an amount of rain greater than 14.3 mm during March, favouring the food availability and nesting places abundance, and also allowed an increase in the reproductive success of Colinus leucopogon. The time invested in territory defense by males was not related with their mating success. Moreover, the duration in territory defense was similar for males that paired, compared with those that did not. In addition, song and territory characteristics were not related with males invested time in their territory defense. Therefore, this could be another reason explaining the lack of a relationship between the duration in the territories by males and pair formation, and suggests that song characteristics strongly influence the formation of pairs in this species.     

四种杂交组合奥尼罗非鱼及其亲本的生长对比研究

通过RFID电子标记注射、跟踪和生长性状测量,对2个奥利亚罗非鱼品系(ZZ、AA)、2个尼罗罗非鱼品系(XX、EE)自交群体及其杂交奥尼罗非鱼4个群体(XZ、XA、EZ、EA)进行生长对比研究,罗非鱼亲本来自国家罗非鱼产业技术体系研发中心。结果表明:XZ、XA、EZ、EA四个奥尼罗非鱼群体雄性率均达94%以上,全长、体长、头长、体高等性状特征值显著高于亲本品系(P<0.05),奥尼罗非鱼的生长速率相对其亲本品系具有一定的优势,同时4个奥尼群体间生长性状特征值也存在显著差异(P<0.05);生长性状相关分析显示罗非鱼各配组群体的体质量与尾柄长相关性较低(0.690—0.846),与全长、体长、体高、体厚相关性较高;对罗非鱼各配组群体的比例性状进行主成分分析,第1主成分主要代表BS、HS、CDS、BWS性状特征,第2主成分主要代表TS、HS、CLS性状特征,通过第1、第2主成分作图可将AA与其他6个罗非鱼配组群体有效区分。     

Treatment with an aromatase inhibitor suppresses high-temperature feminization of genetic male (YY) Nile tilapia

High temperature (36° C) treatment during sexual differentiation caused significant changes in sex ratio in YY male Nile tilapia Oreochromis niloticus fry (64.5% males compared to 100.0% males at 28° C), while dietary treatment with a chemical aromatase inhibitor (AI: Fadrozole™ CGS16949A) during this period suppressed the high temperature feminization (98.9% males). This implies that cytochrome P450 aromatase is mechanistically associated with temperature-dependent sex determination (TSD) in this species. XY male fry did not show significant sex reversal at 36° C. In XX female fry, high temperature treatment resulted in significant masculinization (62.5% males compared with 21.9% males at 28° C), while treatment with AI at either temperature resulted in very high proportions of males (100.0% males at 36° C; 99.0% males at 28° C). These results confirm the importance of aromatase in sexual differentiation in the Nile tilapia below the TSD threshold and suggest that it also plays a role in TSD, at least in the YY genotype.     

Effects of behavioural time budgets and nest-building efficiency on male reproductive performance in red bishops ( Euplectes orix )

In this study we have investigated the effect of nest-building behaviour, courtship behaviour, and male–male interactions on male reproductive performance of the red bishop (Euplectes orix), a highly polygynous, colonially breeding weaverbird species. Previous studies on red bishops have revealed that male reproductive success is mainly determined by the number of nests built in a territory, and that reproductive success and the number of nests both vary substantially among males. Here we test whether the high variation observed in reproductive performance is related to male behavioural patterns (as assessed by time-budget analysis) and/or nest-building efficiency (as assessed by the number of fibres incorporated in the nest in unit time). We collected data on male time budgets and analysed behavioural patterns for three breeding seasons. A greater number of nests built was positively related to both the amount of time allocated to nest-building behaviour and the efficiency of nest building. Neither the amount of time spent in courtship behaviour nor the amount of time spent in male–male interactions was related to reproductive success. Male reproductive success, irrespective of the number of nests built, was partly determined by the number of fibres incorporated, suggesting some importance of nest quality in terms of nest chamber density to male reproductive success. There were no trade-offs with regard to time allocated to different behaviour. Instead, males differed with regard to their territory attendance and activity levels, which might be because of differences in energy resources and may thus reflect inherent differences in male body condition and, ultimately, male genetic quality.     

罗非鱼颗粒蛋白前体cDNA序列与表达分析

颗粒蛋白前体(Progranulin,PGRN)在先天免疫反应调控及个体生长发育过程中均有重要作用。通过对罗非鱼外周血白细胞全长cDNA文库筛选得到的序列进行生物信息学分析,获得罗非鱼Pgrn全长cDNA序列(GenBank登录号为GQ241348)。该cDNA克隆总长843bp,包含一个完整的开放阅读框,编码206个氨基酸,其中推定信号肽20个氨基酸,两个GRN重复单位均56个氨基酸。研究采用实时定量PCR(Real-time RT-PCR)方法对感染海豚链球菌后奥尼罗非鱼、尼罗罗非鱼、奥利亚罗非鱼和吉富罗非鱼4种组织(脑、肝脏、脾脏和头肾)Pgrn mRNA表达情况进行分析。结果显示,Pgrn mRNA在攻毒后4种罗非鱼4种组织中表达均有上调趋势,并且在脾中的表达量最高,提示PGRN在鱼类先天免疫反应调控中起重要作用。另外,奥尼罗非鱼在感染海豚链球菌后6h的脑和肝脏、6h和12h的脾脏和头肾中Pgrn mRNA表达均下调,然后表达升高,这种表达变化在其他三种鱼中不明显,这也许是奥尼罗非鱼抗病力较强的一个原因。研究为从分子水平探讨PGRN在罗非鱼先天免疫反应中的作用机制提供了数据,也为罗非鱼的抗病选育提供了参考分子标记。