脊尾白虾VgR基因克隆及其在卵巢发育过程中的表达分析

为了解卵黄蛋白原受体(vitellogenin receptor,Vg R)在脊尾白虾卵巢发育中的作用,采用同源克隆和RACE技术,克隆了脊尾白虾Vg R基因全长c DNA序列,用实时荧光定量PCR方法分析了Vg R基因在雌虾不同组织、卵巢发育不同时期的表达特征。结果显示,脊尾白虾Vg R基因全长5892 bp,开放阅读框5661 bp,编码1886个氨基酸。脊尾白虾Vg R具有低密度脂蛋白受体(LDLR)家族典型结构特征,属于LDLR家族,进化上与日本沼虾等甲壳动物Vg R亲缘关系最近,然后与昆虫Vg R分支聚为一支,而与LDLR家族中其他成员亲缘关系较远。脊尾白虾Vg R在各组织中均有表达,但主要在卵巢内表达。随着卵巢的发育,卵巢Vg R表达量逐渐升高,在III期达到最大,与肝胰腺Vg表达量、血液Vg浓度变化趋势相一致;而在卵巢成熟时,卵巢Vg R表达量降到了最低,与肝胰腺Vg表达情况截然相反;排卵后的恢复期,血液中Vg浓度仍维持在较高水平,卵巢Vg R表达量又升至III期水平,同时卵巢Vg表达量也升至最高。由此可见,甲壳动物与昆虫Vg R起源于同一祖先,但在进化上已形成独立一支;脊尾白虾卵巢成熟前,卵巢主要通过Vg R介导作用摄取血液中Vg,以供卵巢快速成熟;卵巢成熟期,肝胰腺呈现补偿性合成Vg,以尽快恢复其营养储备功能;卵巢恢复期,卵巢通过Vg R介导摄入外源Vg和内源合成Vg两种途径,为卵巢二次发育提供营养物质。     

克氏原螯虾卵黄蛋白原受体cDNA部分序列的克隆及mRNA表达量的相对定量

从成熟的克氏原螯虾卵巢中提取总RNA,通过同源克隆得到了卵黄蛋白原受体c DNA部分序列,长度为506 bp,在NCBI网站上进行比对后发现,其与斑节对虾、短沟对虾、罗氏沼虾的卵黄蛋白原受体(Vg R)c DNA序列有较高的相似性。采用实时荧光定量PCR方法,测定了卵巢不同发育时期卵巢和肝胰腺两个组织中卵黄蛋白原受体m RNA的相对表达水平。结果显示,卵巢是卵黄蛋白原受体基因表达的主要部位,而肝胰腺只能检测到少量表达,卵黄蛋白原受体基因在卵巢的卵原细胞增殖期相对表达量最高,随后下降,成熟期达到最低值,恢复期开始回升。结合卵巢不同发育阶段卵巢质量的变化计算表达总量,结果发现,卵黄蛋白原受体基因的表达总量在卵原细胞增殖期为最低,随后持续升高并至成熟期达到峰值,恢复期急剧下降,但仍略高于卵原细胞增殖期的表达量。此外,运用所构建的系统进化树比较了克氏原螯虾Vg R m RNA与其他物种间的遗传距离。     

Vitellogenin of the Chilean flounder <Emphasis Type="Italic">Paralichthys adspersus</Emphasis> as a biomarker of endocrine disruption along the marine coast of the South Pacific. Part I: induction,purification, and identification

We sought to provide a useful indicator of the presence of endocrine-disrupting contaminants along the marine coast of the South Pacific using Chilean flounder (Paralichthys adspersus). In light of the lack of information on vitellogenin for this species, we induced, purified, and identified the plasma vitellogenin of Chilean flounder inhabiting the Chilean coast. Vitellogenin (Vg) from Chilean flounder was purified by size exclusion and ion-exchange chromatography using plasma from juvenile males induced by injecting 17β-estradiol. The Vg was detected by SDS–PAGE and Western blot analyses using an antibody against turbot (Scophthalmus maximus) vitellogenin. These analyses revealed a protein band of 205 kDa and three minor bands of 120, 90, and 68 kDa. These proteins were identified as Vg by means of mass spectrometry (LCQ Duo ESI-IT-MS), matching sequences of tryptic peptides to known sequences for several other fish species. The matches showed the presence of vitellogenin (VgI, VgII, Vg A and Vg B) in Chilean flounder, similar to species such as mummichog (Fundulus heteroclitus), Japanese medaka (Oryzias latipes), and white perch (Morone americana). These results are discussed in terms of identifying Vg in Paralichthys adspersus with the antibody to turbot Vg. Moreover, we compare the molecular size of Vg from Chilean flounder (large) with that of other flatfish species. Finally, we discuss the potential use of this molecule as a biomarker for the presence of xeno-estrogenic compounds along the Chilean coastline.     

Purification and development of ELISAs for two forms of vitellogenin in Indian walking catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis> (L.)

Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E₂)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450 kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67 kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100 ng ml^?1 for Vg1 and 40 ng ml^?1 for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E₂ induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.     

Investigation into the possible role of androgens in the induction of hepatic vitellogenesis in the European eel:in vivo andin vitro studies

Changes in the levels of plasma vitellogenin (Vg), estradiol (E₂) and testosterone (T) were examined following gonadal development induced by carp gonadotropin treatment (cGTH) of freshwater female yellow and silver eels (Anguilla anguilla L.). The animals received injections of cGTH (250 μg kg⁻¹ body weight) or saline vehicle three times a week, for 6 to 8 weeks. No effect of vehicle was observed. Steroidogenic activity of the ovary was stimulated by cGTH treatment as shown by the increase in circulating steroid levels in both stages. However, the responses of T, E₂ and Vg differed according to the stage of development of eels. At the yellow stage, the initial steroid plasma levels were undetectable (< 0.01 ng ml⁻¹) before treatment and ovarian steroidogenic activity was slightly stimulated following cGTH treatment; steroid levels reached their highest values after 3 weeks and 6 weeks of treatment for E₂ (0.62 ± 0.13 ng ml⁻¹ and T (0.33 ± 0.30 ng ml⁻¹), respectively. The cGTH treatment slightly increased plasma Vg levels (0.2–0.7 μg ml⁻¹ during the experiment compared with the initial values of the group. At the silver stage, the initial steroid levels were detectable (0.7 ng ml⁻¹ for E₂ and 0.1 ng ml⁻¹ for T); cGTH treatment did not significantly increase plasma E₂ level which remained at initial levels. Nevertheless, plasma T levels dramatically increased from 0.1 to 3 ng ml⁻¹ and peaked after 1 or 2 weeks of cGTH treatment; a rapid increase in plasma Vg levels occurred, reaching its highest value at 5 mg ml⁻¹ after 3 weeks of treatment. Thus, the steroid kinetic profiles in relation to the appearance of Vg in the plasma following cGTH treatment was closely related to androgen levels and there was a strong vitellogenic response induced by chronic cGTH treatment. In order to test if androgens could be implicated in the vitellogenic response, we evaluated the potencies of various androgens (testosterone and 5α-androstane-3β,17β-diol)in vivo andin vitro, associated with E₂ to induce the production of Vg.In vitro experiments showed that Vg synthesis was induced by high doses (10⁻⁶ to 10⁻⁵ M) of androgen in the eel. Tamoxifen totally inhibited the action of androgens suggesting that androgens were acting through binding to the E₂ receptor.In vivo, androgens given alone at 50 μg kg⁻¹ 3 times a week for 1 months had no significant effect on plasma Vg levels. In addition, E₂-androgen cotreatment showed that the presence of androgen did not modify the vitellogenic response induced by E₂.     

Vitellogenesis with special emphasis on Indian fishes

Oocyte growth in most oviparous vertebrates including fish is due to the formation of yolk, and eggshell proteins (zona radiata proteins). Zonagenesis leads to the formation of zona radiata proteins in oocytes, which play an important role during oogenesis, whereas vitellogenesis leads to the formation of yolk in oocytes through a series of events during which the yolk precursor protein vitellogenin (Vg) is synthesized and secreted from liver into blood from where it is sequestered into the developing oocytes and thereafter proteolytically cleaved to form yolk proteins (YPs) and finally deposited in the ooplasm. Much research has been done in many fish species with respect to the number and nature of Vg and YPs and their probable functions during fish reproduction. Recent findings of multiplicity of Vg molecules in fishes reject the earlier view of a single-Vg model and have led scientists to explore the functions of individual Vg and their YP derivatives, lipovitellin, phosvitin, and β′-component. Two distinct types of Vg or Vg genes, containing or encoding the three YPs, have been detected in many teleosts. A third unusual, incomplete, phosvitin-poor Vg has been described recently in many fishes. In comparison to much of the information on vitellogenesis in many fishes very little is known for Indian fishes. In India research has been done in a few species such as the catfish, Heteropneustes fossilis and Clarias batrachus, the murrel, Channa punctatus and the Indian major carps, Labeo rohita and Cirrhinus mrigala. Immunological and biochemical analyses suggest the occurrence of multiple forms of Vg and their YP derivatives. The synthesis and incorporation of Vg are regulated by gonadotropin (GTH) and estradiol-17β (E₂). A differential role between estrone (E₁₎ and estriol (E₃) has been demonstrated for Vg synthesis. Enzyme-linked immunosorbent assays (ELISAs) for Vg have been developed to measure plasma Vg. Finally the different roles of Vg1 (HAI) and Vg2 (HAII) on vitellogenesis have been demonstrated. However, more research remains to be carried out in other fish species with respect to the number and nature of Vg and YPs and their genes in order to describe their reproductive functions.     

Identification of growth hormone receptor in the ovary of tilapia, Oreochromis mossambicus

Growth hormone receptor (GH-R) cDNA was isolated and characterized in the tilapia, Oreochromis mossambicus. GH-R mRNA was expressed in the nucleus and cytoplasm of the young oocytes and in the follicles surrounding vitellogenic oocytes showing a correlation with ovarian IGF-I mRNA levels. However, no change was seen in liver GH-R/IGF-I mRNA levels or plasma GH/IGF-I levels during ovarian development, suggesting that GH/IGF-I axis may be involved in the ovarian development in paracrine or autocrine manner, independent of liver derived IGF-I.     

Isolation and expression of rainbow trout (Oncorhynchus mykiss) ovarian cDNA encoding 3β-hydroxysteroid dehydrogenase/Δ5−4-isomerase

A distinct shift in steroidogenesis from testosterone to 17α-hydroxyprogesterone occurs in the salmonid ovarian thecal cell layers immediately prior to oocyte maturation, and is a prerequisite for the production of 17α,20β-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of salmonid fishes) by granulosa cells during oocyte maturation. 17α-Hydroxylase/17,20-lyase cytochrome P-450 (P-450_17α) and 3β-hydroxysteroid dehydrogenase/Δ^5?4-isomerase (3β-HSD) are the two major steroidogenic enzymes involved in the production of 17α-hydroxyprogesterone and testosterone. Using mammalian cDNA probes, we isolated and characterized full-length cDNAs encoding these two enzymes from a rainbow trout (Oncorhynchus mykiss) ovarian thecal cell cDNA library. The cloning of 2.4-kilobase cDNA encoding P-450_17α and transient expression of this clone in nonsteroidogenic monkey kidney tumor COS-1 cells have recently been reported (Sakai et al. 1992). We have isolated a 1.4-kilobase cDNA which is hybridized to the mammalian 3β-HSD cDNAs. Expression of this cDNA in COS-1 cells led to the production of an enzyme which is capable of converting dehydroepiandrosterone to androstenedione. In this study, enzymatic activities and expression of rainbow trout ovarian P-450_17α and 3β-HSD are discussed in relation of the steroidogenic shift occurring in the ovarian follicle layers.     

A wide difference in susceptibility to nitrite between Eurasian perch (Perca fluviatilis L.) and largemouth bass (Micropterus salmoides Lac.)

Influence of nitrite on two fish species, Eurasian perch (Perca fluviatilis L.) and largemouth bass (Micropterus salmoides Lacépède), was assessed in two acute toxicity tests. In the first one, lethal concentrations (48hLC50) of nitrite were estimated at 11 mg l^?1 NO₂ ^? for perch and 882 mg l^?1 NO₂ ^? for bass. In the second test, fishes were exposed for 48 h to concentrations representing ¼ and ½ value of 48hLC50 for each species. This test showed that the higher nitrite concentration in the water the higher methaemoglobin content in the blood, and nitrite levels in the blood plasma were observed in both species. On the other hand, leucocyte count showed opposite trend. Activity of NADH-methaemoglobin reductase was markedly lower in largemouth bass compared to Eurasian perch and was stimulated by nitrite exposure in neither of the species.     

Involvement of androgens and growth hormone in the synthesis of vitellogenin in Japanese eel (Anguilla japonica)

Immature eels positively responded to estradiol-17β (E₂) injection in terms of vitellogenin (Vg) synthesis but not to growth hormone (GH) or 17α-methyltestosterone (MT) injection. However, injection of MT or GH combined with E₂ strongly increased Vg synthesis. In in vitro experiments, eel hepatocytes treated with E₂, GH, or MT alone did not produce detectable amount of Vg, whereas the combination of E₂ with GH or MT, or both greatly increased Vg synthesis in the hepatocytes.     

Induction of fertilizable eggs by conspecific vitellogenin implantation in captive female walking catfish,Clarias batrachus (Linn.)

The synthesis of vitellogenin (Vg) is induced by conspecific Vg (Vg1 and Vg2) and estradiol‐17β (E₂) as demonstrated by the pattern of ³H‐serine incorporation in the liver and plasma proteins. The incorporation studies indicated that the label was first incorporated into the liver after which it appeared in the blood in both E₂‐ and Vg‐treated male catfish. Since Vg was capable of inducing its own synthesis, experiments were conducted in females during preparatory–prespawning period (March–May) to make them gravid by implanting Vg pellets. Two implantations of 4 mg Vg1 pellets into female catfish with an interval of 15 days, followed by laboratory maintenance for 45 days of initial implantation showed a significant increment in ovarian weight with concomitant formation of yolky oocytes through synthesis and incorporation of Vg, whereas Vg2 implantation was not effective in this regard. Histological observation of yolky oocytes in Vg1‐treated group showed the peripheral migration of germinal vesicle (eccentric germinal vesicle), which indicates the onset of maturation. On 45th day, third implantation with 2 mg Vg pellets was performed and after 15 days, fish were hormonally induced with a single injection of hCG (2,000 IU/kg fish). Six groups were considered such as initial control, BSA‐implanted control, Vg1‐implanted, Vg2‐implanted, catfish collected from the field on the last day of the experiment and catfish collected during spawning period in this experiment with 3–7 fish in each group. Each of the experimental fish was sexually mature and the body weight was between 100 and 125 g. The percentage of ovulation and fertilization in the eggs of Vg1‐implanted group was 91% and 78%, respectively, which was almost similar to that of gravid female catfish collected during breeding period (July). The breeding performance in BSA‐ and Vg2‐treated females was very poor. The fertilized eggs were hatched in the laboratory conditions. Thus, in the female catfish, Vg1 not only induces vitellogenesis but also makes the oocytes viable for fertilization.     

Identification, purification and classification of multiple forms of vitellogenin from white perch (Morone americana)

Three forms of female-specific plasma protein (FSPP 1-3) were purified from blood plasma of estrogen-treated white perch (Morone americana) by combining several types of ion-exchange chromatography including a novel, fast flow, strong anion exchanger (POROS media), followed by gel filtration. Native FSPP 1, FSPP 2 and FSPP 3 had molecular masses of 532 kDa, 532 kDa and 426 kDa, respectively. The apparent mass of purified FSPP 1 and FSPP 2 after SDS-PAGE under reducing conditions was ∼ 180 kDa, while FSPP 3 appeared as a major ∼ 148 kDa band. All of the FSPPs resembled one another with respect to amino acid composition but each appeared to be immunologically distinct. In double immunodiffusion using anti-total FSPP (antiserum raised against vitellogenic female plasma pre-absorbed by male plasma), each FSPP formed one precipitin line that crossed those produced by both others. A rabbit antiserum was raised against each FSPP and absorbed with combinations of the other two FSPPs to ensure specificity. Using the antisera, each FSPP was detected by immuno-electrophoresis in plasma from vitellogenic females or estrogen-treated male or immature fish, but no FSPP was detected in normal male plasma. Endoprotease (Asp-N) digests of the FSPPs were subjected to HPLC separation for N-terminal sequencing and mapping of isolated peptides to published vitellogenin (Vg) sequences. Results of these analyses indicate that white perch FSPP 1, FSPP 2, and FSPP 3 can be classified into three Vg groups identified in previous studies: VgA, VgB, and VgC-like protein, respectively. This is the first report, of which we are aware, on isolation of more than two Vg proteins from any species of vertebrate except the chicken. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gill ATPase activities of silver perch, Bidyanus bidyanus (Mitchell), and golden perch, Macquaria ambigua (Richardson): Effects of environmental salt and ammonia

The Australian freshwater fish, silver and golden perch, are increasingly being used for aquaculture. Addition of salt to water is commonly used in commercial aquaculture to reduce stress attributed to high ammonia concentrations. The activities in gill homogenates of ouabain-sensitive Na⁺/K⁺-ATPase and NEM-sensitive ATPases (as a measure of H⁺-ATPases) of silver and golden perch were measured after maintaining the fish in water containing different salt and ammonia concentrations. Six treatments were applied in a 2 × 3 factorial design: two salt treatments, low salt (LS) of 2.5 g l^− 1 and high salt (HS) 5 g l^− 1, and three ammonia treatments, no added ammonia (NA), low ammonia (LA), 3 mg total ammonia nitrogen (TAN) l^− 1 and high ammonia (HA), 5 mg TAN l^− 1. In both species, activity of Na⁺/K⁺-ATPase was lowest in fish kept in the LSNA treatment (7.4 ± 0.4 μmol Pi mg protein^− 1 h^− 1 for silver perch and 3.1 ± 0.6 for golden perch) and highest in the HSHA treatment (15.2 ± 1.0 μmol Pi mg^− 1 protein h^− 1 for silver and 8.4 ± 1.2 for golden perch). In both species there was a significant increase (P < 0.001) in Na⁺/K⁺-ATPase activity with increase in salt concentration and with an increase in ammonia concentrations. A significant interaction (P < 0.036) between salt and ammonia on Na⁺/K⁺-ATPase activity was observed in silver but not in golden perch. In contrast, the lowest activity for NEM-sensitive ATPase was observed in the HSNA treatment (1.0 ± 0.2 μmol Pi mg^− 1 protein h^− 1 for silver and 1.5 ± 0.4 for golden perch) and highest in LSHA treatment (2.9 ± 0.4 μmol Pi mg^− 1 protein h^− 1 for silver and 3.6 ± 1.2 for golden perch). In both species there was a significant decrease in NEM-sensitive ATPase activity with increase in salt concentration and an increase in activity with increase in ammonia (P < 0.003). In silver perch, a significant interaction between the treatments was observed (P < 0.02). The results suggest that in these species of freshwater fish the Na⁺/K⁺-ATPase has a role in salt and ammonia homeostasis and that the NEM-sensitive ATPases are more active in fish kept in water with a lower salt content. It is possible that the increase in ammonia resistance when salt is added to the environmental water in commercial aquaculture systems may be due to the effects of salt on gill Na⁺/K⁺-ATPase activity rather than the NEM-sensitive ATPases.     

Vitellogenin genes in fish: differential expression on exposure to estradiol

Three types of vitellogenins (Vgs) namely vitellogenin A (VgA), vitellogenin B (VgB) and vitellogenin C (VgC) have been identified in fishes. The existence of VgA and VgB is reported in the Indian freshwater murrel Channa punctatus. Gene-specific primers were designed using available nucleotide sequences in National Centre for Biotechnology Information (NCBI), for amplification of VgA and VgB cDNA. Differential processing of Vgs is evident in many fishes. Adult male murrel expressed both the VgA and VgB genes when estradiol-17β (E₂) is injected in vivo and Vg levels in blood quantified by Enzyme linked immunosorbent assay (ELISA) showed a dose-related response in such treatments. Cultured hepatocytes on treatment with E₂, however, expressed only VgB as detected by RT-PCR, suggesting different regulatory mechanism for the VgA and VgB genes.     

Evaluation of plankton surface pushnets and oblique tows for comparing the catch of diadromous larval fish

A bow-mounted surface pushnet and an obliquely towed plankton net were compared to evaluate gear efficiency and effectiveness in collecting larval fishes under daytime and nighttime conditions. The diadromous species targeted were striped bass Morone saxatilis, white perch, Morone americana, and river herring Alosa sp. We sampled the lower Roanoke River, North Carolina, from March through June of 2002 and 2003. Striped bass, white perch and river herring represented over 90% of the larvae collected during the study period. Mean larval densities (number/100 m³) were 63.4 for striped bass, 26.4 for river herring, and 17.7 for white perch. Striped bass larval densities were significantly higher in the surface pushnet for both years (P ≤ 0.05). In 2002, white perch mean larval density was significantly higher at night in the surface pushnet samples, but in 2003 there were no differences between day and night samples. River herring mean densities were significantly higher in the surface pushnets for both years, but showed no clear patterns between day and night samples. Larger larvae were consistently collected in the surface pushnets for all species. Overall, the surface pushnet was easier to operate. The pushnet was mounted on the bow of a small jon boat and required less specialized gear and fewer personnel than oblique sampling. The method also allows for sampling in shallow water or vegetated habitats. Because larvae were significantly larger in the surface samples, using surface pushnets may not allow for detection of the smaller-sized larvae therefore underestimating the abundance of smaller fish. Depending on the question being asked, we recommend that sampling programs should use both gear types to reduce any gear biases.     

Effect of 17α-hydroxy-progesterone on vitellogenin secretion in kuruma prawn,Penaeus japonicus

The secretion of vitellogenin (Vg) in kuruma prawn, Penaeus japonicus, by 17α-hydroxy-progesterone was investigated under tank-culture conditions. A significant (P<0.1) increase in Vg concentration in the sera (9.6-fold over initial value) was observed after 48 h in early vitellogenic prawns injected with 17α-hydroxy-progesterone (0.01 μg/g body weight) compared to control I (no injection, 3.0-fold over initial value) and control II (0.1 μl pure ethanol/g body weight, 2.4-fold over initial value). These results indicate that 17α-hydroxy-progesterone stimulates Vg synthesis and/or release into the haemolymph in prawns.     

Estuarine fish responses to strobe light,bubble curtains and strobe light/bubble-curtain combinations as influenced by water flow rate and flash frequencies

This study examined the possible use of strobe lights and strobe light/bubble-curtaincombinations as behavioral guidance systems for estuarine fish. White perch (Morone americana), spot (Leiostomus xanthurus) and Atlantic menhaden (Brevoortia tyrannus) were tested in a behavior experimental tank for avoidance to strobe lights, bubble curtains and strobe light/bubble-curtain combinations at different water flow rates, strobe flash frequencies and light acclimations.Percentage avoidance of strobe light ranged from 8 to 36% for white perch, from 8 to 100% for spot and from 8 to 68% for menhaden, depending on the conditions tested. χ² analyses indicated significant (P < 0.05) avoidance by white perch at most flash rates with a 0.2 ms⁻¹ flow rate, and at 120 and 300 flashes min⁻¹ with 0.3 and 0.5 ms⁻¹ flows. Spot had significant avoidance for all flash rates at the 0.2 ms⁻¹ flow, and for 300 and 600 flashes min⁻¹ at water velocities of 0.3 and 0.5 ms⁻¹. All species showed little avoidance of bubble curtains. Avoidances of 3–58% for white perch, 21–85% for spot and 9–81% for menhaden were obtained with the strobe light/bubble-curtain combinations. χ² analyses indicated significant avoidance for most conditions at 0.2 and 0.5 ms⁻¹ flows for spot and menhaden. White perch had significant avoidance for most conditions at the 0.2 ms⁻¹ flow, but no avoidance at 0.5 ms⁻¹.The strobe light and strobe light/bubble-curtain combinations elicited best avoidance results at flash rates of 300 min⁻¹ or greater and low flow rates. Strobe lights show promise as a guidance system for estuarine fish.     

Effects of fasting,temperature, and photoperiod on <Emphasis Type="Italic">preproghrelin</Emphasis> mRNA expression in Chinese perch

Preproghrelin, a gut/brain peptide, plays an important role in the regulation of food intake and energy homeostasis in teleost and mammals. In the present study, we obtained the full-length preproghrelin cDNA in Chinese perch. The preproghrelin messenger RNA (mRNA) tissue expression showed that level was much higher in stomach and pituitary than in other tissues. The fasting study showed, after gastric emptying (3–6 h), short-term fasting (6–12 h) increased preproghrelin expression in the stomach. While in the pituitary, fasting reduced preproghrelin expression at 1, 3, 12, and 48 h, presenting state fluctuation of self-adjustment. The temperature study showed that the mRNA expression of preproghrelin was the highest in the brain at 26 °C and highest in the stomach at 32 °C, respectively, with different optimum temperature in these two tissues, reflecting spatiotemporal differences of regulation by central nervous system and peripheral organs. The photoperiod study showed that normal light (11 h of lightness and 13 h of darkness) led to highest preproghrelin expression, both in the brain and in the stomach, than continuous light or continuous dark, proving food intake is adapted to natural photoperiod or normal light in this study. These results all indicated that tissue-specific preproghrelin expression of Chinese perch could be significantly affected by environmental factors. Short-term fasting of 6 h after gastric emptying, 26 °C, and normal light led to higher preproghrelin expression, which indicated potential appetite increase in Chinese perch.