Temporal relationship between the daily profiles of gut melatonin,oxidative status and major digestive enzymes in carp Catla catla

The present study demonstrated a temporal relationship between the concentrations of melatonin, oxidative status and digestive physiology in the gut of a tropical carp Catla catla. We measured the levels of gut melatonin, malondialdehyde (MDA) – a faithful marker of intracellular stress, different antioxidants and major digestive enzymes in the carp gut at four different clock hours in a daily cycle under natural photo-thermal conditions. A correlation between the gut variables was sought to point their possible functional relationship. Gut melatonin titers displayed significant diurnal variations with a peak at midday. An identical temporal pattern with the highest value at midday and nadir at midnight was noted in the activities of superoxide dismutase, catalase and glutathione peroxidase. In contrast, levels of MDA and reduced glutathione (GSH) were highest at midnight and lowest at midday. The activity of all the studied digestive enzymes (α-amylase, cellulase, protease and lipase) showed significant daily variations with a peak at midday. Gut melatonin concentrations by showing a positive correlation with the activity of both enzymatic antioxidants and digestive enzymes, and a negative correlation with the levels of GSH and MDA indicated their possible physiological interplay in a daily cycle. Collectively, our study presented the first information on the daily profiles of oxidative stress, different antioxidants and digestive enzymes in the gut tissues of any fish species, and suggested their functional relationship with the concentrations of gut melatonin in carp Catla catla.     

Influence of Altered Photoperiods on Serum Melatonin and Its Receptors (MT1 and MT2) in the Brain,Retina, and Ovary in Carp Catla catla

Daily variation in melatonin receptor (MT1 and MT2) density in three specific tissues—brain, retina, and ovary—and its temporal relationship with serum melatonin were evaluated for the first time in a freshwater teleost, the carp Catla catla, under natural as well as altered photoperiods in different reproductive phases of the annual cycle. Cosinor analysis was used to determine rhythmic features of the serum melatonin and receptors (MT1 and MT2) in different tissues. In each photoperiodic group, irrespective of season, the daily minimum serum melatonin level was noted at midday. However, the daily peak value of melatonin varied in relation to both photo-schedules and reproductive phases. Under natural photoperiods (NPs; duration varied with seasons) and short photoperiods (SPs; light [L]:dark [D] 8:16), it occurred in the late dark phase during the preparatory phase, and at midnight in the remaining parts of the annual cycle. On the other hand, in each reproductive phase, compared to corresponding NP carp, the daily melatonin peak under long photoperiods (LPs; L:D 16:8) exhibited a phase delay of ～2–3?h (occurring during the late dark phase). The melatonin levels at each sampling point were highest during the postspawning phase and lowest during the spawning phase, irrespective of the photoperiodic history of the fish. In each tissue, Western blot analysis revealed a band at ～37?kDa and a band at ～36?kDa corresponding to the molecular weights of native MT1 and MT2 receptor proteins, respectively, with the band intensity of MT1 always being higher than that of a 36-kDa protein. The content of both melatonin receptor proteins varied significantly according to the studied tissue (being highest in the retina, intermediate in the brain, and lowest in the ovary), time in the daily cycle (peak at midnight and fall at midday), and reproductive phase in the annual cycle (highest in the spawning phase and lowest in the postspawning phase). Remarkably, no significant effects of altered photoperiod were detected on any rhythm parameters of either MT1 or MT2 in any of the studied tissues. Collectively, the results of the present study suggest a role of photoperiod in determining daily and seasonal profiles of serum melatonin, but not its receptor proteins, on the ovary or on any nongonad tissues in carp. (Author correspondence: dgp_skmaitra@yahoo.co.in)     

PHOTOPERIODIC REGULATION OF MT1 AND MT2 MELATONIN RECEPTOR EXPRESSION IN SPLEEN AND THYMUS OF A TROPICAL RODENT FUNAMBULUS PENNANTI DURING REPRODUCTIVELY ACTIVE AND INACTIVE PHASES

Photoperiodic regulation of melatonin receptor types on target tissues, such as lymphatic organs, has never been explored for any seasonal breeder. In the present study, we accessed the high affinity membrane melatonin receptors MT1 and MT2 expression dynamics in lymphoid organs (i.e., spleen and thymus) of a seasonally breeding rodent Funambulus pennanti during two major reproductive phases (i.e., active and inactive), when the internal hormonal (melatonin and gonadal steroid) as well as the ecological conditions were entirely different. Photoperiod regulates circulatory melatonin level; hence, we noted the effect of different photoperiodic regimes (long; 16L:8D and short; 10L:14D photoperiod) equivalent to summer and winter daylength on membrane melatonin receptor MT1 and MT2 expression in spleen and thymus. We have correlated the melatonin receptor expression with two major hormones varying seasonally (i.e., melatonin and testosterone) also being responsible for modulation of immunity of a seasonal breeder. Differential immunoreactivity of MT1 and MT2 receptor in spleen and thymus of F. pennanti suggests an involvement of both the receptor types in signal transduction of photoperiod for seasonal immunomodulation, because in the tropical zone, a slight difference (1:45–2?h) in daylength may change reproductive physiology and immunity of animals for adaptation. Our above suggestion receives strong support from the experiment of photoperiodic exposure on MT1 and MT2 expression at the translational level, where long daylength decreased the circulatory melatonin level and melatonin receptor expression in both lymphatic tissues. On the other hand, under short daylength, expression of MT1 and MT2 receptor increased in both spleen and thymus along with concomitant increase in circulatory melatonin level. Differential hormonal level of melatonin and gonadal hormones during reproductively active and inactive phase and its direct relation with melatonin receptor expression dynamics in lymphoid organs could be responsible for seasonal adjustment of immunity and reproduction. (Author correspondence: chaldar2001@yahoo.com)     

Complete sequencing and expression of three complement components,C1r,C4 and C1 inhibitor,of the classical activation pathway of the complement system in rainbow trout<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>

Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M _r of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M _r of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a -chain of 773 aa, a -chain of 635 aa and a -chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M _r of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.The nucleotide sequence data will appear in the EMBL/DDBJ/GenBank nucleotide sequence database under the following accession numbers: AJ519929 (trout C1r), AJ519930 (trout C1 inhibitor), AJ544262 (trout C4) and BN000290 (flounder C1 inhibitor)     

Melatonin in the Regulation of Annual Testicular Events in Carp Catla catla: Evidence from the Studies on the Effects of Exogenous Melatonin,Continuous Light,and Continuous Darkness

The physiological significance of melatonin in the regulation of annual testicular events in a major carp Catla catla was evaluated through studies on the effects of graded dose (25, 50, or 100 µg/100 g body wt.) of melatonin exogenously administered for different durations (1, 15, or 30 days) and manipulation of the endogenous melatonin system by exposing the fish to constant darkness (DD) or constant light (LL) for 30 days. An identical experimental schedule was followed during the preparatory (February–March), pre‐spawning (April–May), spawning (July–August), and post‐spawning (September–October) phases of the annual cycle. Irrespective of the reproductive status of the carp, LL suppressed while DD increased the mid‐day and mid‐night values of melatonin compared to respective controls. Influences of exogenous melatonin varied in relation to the dose and duration of treatment and the reproductive status of the carp. However, testicular response to exogenous melatonin (at 100 µg, for 30 days) and DD in each reproductive phase was almost identical. Notably, precocious testicular maturation occurred in both DD and melatonin‐injected fish during the preparatory phase and in LL carps during the pre‐spawning phase. In contrast, testicular functions in both the melatonin‐treated and DD fish were inhibited during the pre–spawning and spawning phases, while the testes did not respond to any treatment during the post‐spawning phase. In conclusion, this study provided the first experimental evidence that melatonin plays a significant role in the regulation of annual testicular events in a sub‐tropical surface‐dwelling carp Catla catla, but the influence of this pineal hormone on the seasonal activity of testis varies in relation to the reproductive status of the concerned fish.     

The genome structure of the <Emphasis Type="Italic">1-FEH</Emphasis> genes in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.): new markers to track stem carbohydrates and grain filling QTLs in breeding

Terminal drought tolerance of wheat is a major target in many areas in the world and is a particular focus in Western Australia. It is widely considered to relate to water soluble carbohydrate (WSC) levels such as fructan in the stem, as the head is maturing. Fructan exohydrolases are key enzymes during both fructan biosynthesis and mobilization. The wheat genome sequences of three fructan 1-exohydrolase (1-FEH) genes with seven exons and six introns were isolated by using the available 1-FEH w2 cDNA sequence. The major size differences among the three genes were located in intron 1 and intron 4. The three 1-FEH genes were mapped to Chinese Spring chromosome 6A, 6B and 6D based on polymerase chain reaction (PCR) polymorphisms and Southern hybridization. 1-FEH-6A, -6B and -6D corresponded to published cDNA sequences 1-FEH w1, w3 and w2, respectively. The overall correlation of the mRNA accumulation profile for the 1-FEH genes in stem and sheath leaf tissue in relation to the profile of soluble carbohydrate accumulation was consistent with their postulated role in stem soluble carbohydrate accumulation. The accumulation of the 1-FEH-6B (1-FEH w3) mRNA was 300 fold greater than that of 1-FEH-6A and -6D. The mRNA accumulation continued after the stem water soluble carbohydrate concentrations reached a peak, consistent with a role of 1-FEH-6B in the breakdown of soluble carbohydrate. The relationship between the 1-FEH genes and soluble carbohydrate accumulation is discussed and the 1-FEH-6B gene in particular is suggested to provide a new class of molecular marker for this trait. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Influence of altered photoperiods on serum melatonin and its receptors (MT1 and MT2) in the brain, retina, and ovary in carp Catla catla

Daily variation in melatonin receptor (MT1 and MT2) density in three specific tissues-brain, retina, and ovary-and its temporal relationship with serum melatonin were evaluated for the first time in a freshwater teleost, the carp Catla catla, under natural as well as altered photoperiods in different reproductive phases of the annual cycle. Cosinor analysis was used to determine rhythmic features of the serum melatonin and receptors (MT1 and MT2) in different tissues. In each photoperiodic group, irrespective of season, the daily minimum serum melatonin level was noted at midday. However, the daily peak value of melatonin varied in relation to both photo-schedules and reproductive phases. Under natural photoperiods (NPs; duration varied with seasons) and short photoperiods (SPs; light [L]:dark [D] 8:16), it occurred in the late dark phase during the preparatory phase, and at midnight in the remaining parts of the annual cycle. On the other hand, in each reproductive phase, compared to corresponding NP carp, the daily melatonin peak under long photoperiods (LPs; L:D 16:8) exhibited a phase delay of ～2-3?h (occurring during the late dark phase). The melatonin levels at each sampling point were highest during the postspawning phase and lowest during the spawning phase, irrespective of the photoperiodic history of the fish. In each tissue, Western blot analysis revealed a band at ～37?kDa and a band at ～36?kDa corresponding to the molecular weights of native MT1 and MT2 receptor proteins, respectively, with the band intensity of MT1 always being higher than that of a 36-kDa protein. The content of both melatonin receptor proteins varied significantly according to the studied tissue (being highest in the retina, intermediate in the brain, and lowest in the ovary), time in the daily cycle (peak at midnight and fall at midday), and reproductive phase in the annual cycle (highest in the spawning phase and lowest in the postspawning phase). Remarkably, no significant effects of altered photoperiod were detected on any rhythm parameters of either MT1 or MT2 in any of the studied tissues. Collectively, the results of the present study suggest a role of photoperiod in determining daily and seasonal profiles of serum melatonin, but not its receptor proteins, on the ovary or on any nongonad tissues in carp.     

Daily profiles of serum and gastrointestinal melatonin in response to daytime or night-time supply of tryptophan-rich diet in carp (Catla catla)

Influences of daytime (~10:00 h) or night-time (~22:00 h) supply of L-tryptophan (Trp)-rich diet on daily rhythm features of melatonin and arylalkylamine-N-acetyltransferase (AANAT) protein (key regulator of melatonin biosynthesis) in gastrointestinal (gut) tissue extracts, and melatonin in serum were studied in carp (Catla catla). Analysis of obtained data revealed that the mesor and amplitude values of both melatonin and AANAT in gut tissue-extracts were higher in daytime-fed fish than those supplied with food at night, and their acrophase varied from ~2 h in the daytime-fed carp to ~10 h in night-time-fed fish. Notably, initiation of stimulatory response of melatonin and AANAT in gut to Trp-rich diet varied from ~2 h (following food supply in day) to ~6 h (after food supply during night). However, in either case, their elevated levels were maintained for ~12 h. Trp-rich diet also caused increase in serum melatonin levels, and the duration of such response varied with the time of food supply. Collectively, present study not only demonstrates the role of Trp-rich diet as a potential inducer of gut melatoninergic system and modulator of daily serum melatonin profiles, but underlines the importance of the time of food supply as a determining factor of its influence as well.     

Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish

 The closely linked recombination activating genes, rag1 and rag2, encode components of the recombinase involved in V(D)J recombination of the immunoglobulin and T-cell receptor genes. These genes are expressed together exclusively in immature lymphocytes and are useful markers for following the development of lymphoid tissues. We cloned the rag locus of the zebrafish Danio rerio and sequenced the open reading frames of the rag1 and rag2 genes. Although the gene organization is similar to that in other species, the rag1 gene is unusual in possessing two introns within the coding region. In another teleost, the rainbow trout, the rag1 gene is interrupted by a single intron. Introns are not present in the rag1 gene of any other species examined to date. Expression of both rag1 and rag2 begins late in embryonic development, on day 4, by northern RNA blot analysis. Expression of rag1 was detected in the adult zebrafish thymus, pronephros, mesonephros, and ovary. This pattern of expression is consistent with previous histological studies of adult teleosts, which implicate the kidney as the major site of hematopoiesis and the thymus as the major lymphocyte-containing organ. Received: 16 October 1996 / Revised: 28 November 1996     

Genotyping selection for resistance against tomato yellow leaf curl virus (TYLCV) conferred by Ty-1 and Ty-3 genes in tomato

The tomato yellow leaf curl virus (TYLCV), transmitted by whitefly, causes major disease losses to tomato crops in tropical and subtropical regions of the world. Several genes conferring resistance to TYLCV, mainly Ty-1 and Ty-3 genes, have been introgressed to cultivated tomato (Solanum lycopersicum) from the wild relative species Solanum chilense. By combining bulked segregant analysis and amplified fragment length polymorphisms (AFLP), several AFLP markers closely linked to Ty-1 and Ty-3 genes were identified from the resistant breeding line TZ841-4. Cloning and sequencing of the selected AFLP fragments allowed us to develop codominant cleaved amplified polymorphic sequence and dominant sequence characterized amplified region markers closely linked to Ty-1. In addition, Ty-3-linked allelic-specific markers have been discriminated by a quantitative real-time PCR protocol. Taken together, these markers constitute useful tools for marker-assisted selection breeding programs to improve genetic resistance to TYLCV, and also to initiate map-based cloning approaches to isolate the resistance genes.     

大鳞副泥鳅和泥鳅GNB2L1基因的克隆、表达及系统进化分析

由G蛋白β2亚基类似物1基因(GNB2L1)编码的蛋白激酶C受体(RACK1)是一个高度保守的锚定蛋白,属于WD40结构域蛋白家族成员,在细胞信号转导等生命过程中发挥着重要作用。本文采用RACE技术和基因克隆技术分别对大鳞副泥鳅(Paramisgurnus dabryanus)和泥鳅(Misgurnus anguillicaudatus)精巢组织的GNB2L1基因c DNA序列进行了克隆。序列分析表明,大鳞副泥鳅GNB2L1基因c DNA序列全长1 115 bp,开放阅读框(ORF)长965 bp,编码317个氨基酸;泥鳅GNB2L1基因c DNA序列的开放阅读框长965 bp,编码317个氨基酸;两种泥鳅GNB2L1基因编码的蛋白与其他鱼类的RACK1蛋白的同源性为94%~97%,且不同进化地位物种的GNB2L1基因均由8个外显子和7个内含子组成。以GNB2L1基因为标记基因,构建的鱼类系统发育树显示,大鳞副泥鳅和泥鳅在进化上的亲缘关系最近。RT-PCR结果显示,GNB2L1基因在大鳞副泥鳅成体各组织中均有表达,且在脑组织的表达量高于其他组织。以上结果表明,GNB2L1基因为一个进化保守基因,可能在大鳞副泥鳅的细胞活动中发挥着重要作用。     

Changes in circadian parameters of humbug damselfish,Dascyllus aruanus according to lunar phase shifts in Micronesia

The objectives of this study were to test the nighttime effects of the lunar phase on circadian rhythm in the humbug damselfish, Dascyllus aruanus. We measured moonlight intensities at eight different phases across the lunar cycle. At each lunar phase, the circadian rhythm was evaluated by measuring the clock genes cryptochrome 1 and period 2. In addition, we measured arylalkylamine N-acetyltransferase 2 (AANAT2), melatonin and melatonin receptor 1 (MT-R1). The moonlight intensity was highest at full moon and lowest during the waning crescent. Clock gene expression was highest during the full moon compared to the other phases. By contrast, the plasma concentrations of AANAT2 and melatonin and the MT-R1 mRNA expression were highest during the full moon phase. Our results suggest that moonlight affects circadian rhythm patterns in the humbug damselfish. There is a need to investigate potential other physiological effects of lunar phase shifts.     

Age dependent expression of melatonin membrane receptor (MT1, MT2) and its role in regulation of nitrosative stress in tropical rodent Funambulus pennanti

Age-dependent declining level of melatonin induces free radical load and thereby deteriorates immune function. However, reports are lacking about age-dependent melatonin membrane receptor (MT1 & MT2) expression, their role in regulation of reactive nitrogen species (RNS) and eventually how they affect immunity of a tropical rodent F. pennanti. We checked MT1R, MT2R and iNOS expression in lymphoid organs of young middle and old aged squirrels. Nitrite and nitrate ion concentration (NOx) in lymphoid organs, testes and plasma, lymphocyte proliferation and IL-2 level was recorded. Age-dependent decrease in MT1 and MT2 receptor expression, lymphocyte proliferation, IL-2 level and increased RNS in lymphoid organs, testes and plasma was observed with decreased circulatory melatonin. Androgen and AR expression was increased in middle-aged while declined in old-aged squirrels. Present study suggests that age associated immunosenescence is consequence of increased RNS which might have important relationship with melatonin membrane receptors in F. pennanti.