实时荧光定量PCR及RT-PCR与常规PCR及RT-PCR检测对虾病毒效果的比较

常规PCR及RT-PCR已用于对虾DNA及RNA病毒检测,但存在费时、灵敏度较低、不能定量等问题。建立了TaqMan实时荧光定量PCR及RT-PCR方法,分别用于检测白斑综合症病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)及桃拉综合征病毒(TSV)、黄头病毒(YHV)4种对虾病毒。与常规PCR及RT-PCR比较,所建立的TaqMan实时荧光定量PCR及RT-PCR检测上述4种对虾病毒不仅有很高的特异性,检测灵敏度也提高了10～100倍,同时还具有快速、简便、不污染环境、重复性好、实时定量等优点,可明显提高对虾病毒检验检疫工作质量及效率。     

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase

Summary Nitrate reductase (NR) assays revealed a bi-specific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)⁺ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%–77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch. These sequence data appeared in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X54097     

PACAP-38 is a chemorepellent and an agonist for the lysozyme receptor in Tetrahymena thermophila

Pituitary adenylate cyclase activating peptide (PACAP-38) is a peptide hormone which functions in many mammalian systems, including the nervous and digestive systems. Using in vivo behavioral studies, we have found that this hormone functions as a chemorepellent in Tetrahymena thermophila with an EC₅₀ of 10 nM. Cells previously adapted to PACAP-38 were found to be adapted to lysozyme, and vice versa. Furthermore, the in vivo behavioral activity of PACAP-38 was blocked by addition of the anti-lysozyme receptor antibody, 5545. Chemorepellent activity of PACAP-38 was also inhibited by the addition of neomycin sulfate (inhibition constant K _i=0.080 μmol · l⁻¹), a competitive inhibitor of lysozyme binding to its receptor. PACAP-38 is a more potent and specific agonist for the lysozyme receptor than either intact lysozyme or CB2, a 24-amino acid fragment of lysozyme. Accepted: 11 October 1999     

Disruption of sensitization to methylphenidate by a single administration of MK-801

Blockade of sensitization to methylphenidate by a single injection of MK-801 was investigated using a computerized activity monitoring system. Male Sprague-Dawley rats were housed in test cages and motor activity was recorded continuously for 16 days. After 2 days of baseline recording and a saline injection on day 3, the rats were randomly divided into four experimental groups. All received 2.5 mg/kg of methylphenidate (s.c.) once a day from days 4 to 9, then after five days of no treatment, they were re-challenged with 2.5 mg/kg of methylphenidate on day 15. One group received only methylphenidate, while the other three groups also received a single i.p. injection of MK-801 (0.30 mg/kg) either 24 h (day 3) or 1 h prior to the first of the six methylphenidate injections (day 4), or 1 h prior to the second methylphenidate injection (day 5). A single injection of MK-801 on day 4 (1 h prior to methylphenidate) blocked the development of sensitization to methylphenidate, since a sensitized response could not be elicited six days after cessation of repeated methylphenidate administration (day 15). However, sensitization to methylphenidate still occurred in the groups receiving MK-801 (0.30 mg/kg) on day 5, indicating that the mechanism by which a single injection of MK-801 disrupts sensitization to methylphenidate is sensitive to timing and is not a direct long-term effect. In conclusion, a single injection of MK-801 persistently blocks the development of sensitization to methylphenidate only if it is given with methylphenidate on the first day of the repetitive treatment phase.     

Paternal genetic effects on offspring fitness are context dependent within the extrapair mating system of a socially monogamous passerine

Avian extrapair mating systems provide an interesting model to assess the role of genetic benefits in the evolution of female multiple mating behavior, as potentially confounding nongenetic benefits of extrapair mate choice are seen to be of minor importance. Genetic benefit models of extrapair mating behavior predict that females engage in extrapair copulations with males of higher genetic quality compared to their social mates, thereby improving offspring reproductive value. The most straightforward test of such good genes models of extrapair mating implies pairwise comparisons of maternal half-siblings raised in the same environment, which permits direct assessment of paternal genetic effects on offspring traits. But genetic benefits of mate choice may be difficult to detect. Furthermore, the extent of genetic benefits (in terms of increased offspring viability or fecundity) may depend on the environmental context such that the proposed differences between extrapair offspring (EPO) and within-pair offspring (WPO) only appear under comparatively poor environmental conditions. We tested the hypothesis that genetic benefits of female extrapair mate choice are context dependent by analyzing offspring fitness-related traits in the coal tit (Parus ater) in relation to seasonal variation in environmental conditions. Paternal genetic effects on offspring fitness were context dependent, as shown by a significant interaction effect of differential paternal genetic contribution and offspring hatching date. EPO showed a higher local recruitment probability than their maternal half-siblings if born comparatively late in the season (i.e., when overall performance had significantly declined), while WPO performed better early in the season. The same general pattern of context dependence was evident when using the number of grandchildren born to a cuckolding female via her female WPO or EPO progeny as the respective fitness measure. However, we were unable to demonstrate that cuckolding females obtained a general genetic fitness benefit from extrapair fertilizations in terms of offspring viability or fecundity. Thus, another type of benefit could be responsible for maintaining female extrapair mating preferences in the study population. Our results suggest that more than a single selective pressure may have shaped the evolution of female extrapair mating behavior in socially monogamous passerines.     

Trichinella spiralis: comparison of stages in host intestine with those of an Arctic Trichinella sp

The intestinal phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic were compared in experimental animals. Reproductive capacity, pathogenicity, distribution, and persistence of adults in the small intestine, morphological measurements, and release of newborn larvae in vitro were examined. Numerous passages of 40 days each for T. spiralis and the Trichinella sp. isolate in mice did not affect reproductive capacity, distribution of adults in the small intestine, and size of worms. Reproductive capacity index for T. spiralis, I = 151.27 ± 27.30 was significantly higher compared to the Trichinella sp. isolate index, I = 63.46 ± 19.34. The Trichinella sp. isolate was more pathogenic to mice and wild rodents compared to T. spiralis during the intestinal phase. Both parasites were located in the anterior part of the small intestine but the position of T. spiralis adults (P = 17.08) differed from position of the Trichinella sp. isolate adults (P = 23.46) in the small intestine. Intestinal phase of T. spiralis was longer (20 days) compared to the Trichinella sp. isolate (15 days) but sex ratios (:♂) were similar for both parasites. T. spiralis females released significantly higher numbers of larvae in vitro/24 hr compared to the Trichinella sp. isolate. Release of larvae was continuous during the intestinal phase and the average fecundity for T. spiralis was 335 larvae/female and for the Trichinella sp. isolate 114 larvae/female. Adults of T. spiralis and the Trichinella sp. isolate were morphologically indistinguishable and did not differ in size. A comparative index for the intestinal phase is proposed for comparison of any Trichinella spp. isolates and standard T. spiralis.     

Nucleotide and deduced amino acid sequence of bovine adrenal medulla chromogranin B (secretogranin I)

1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain.     

A forceful upper jaw facilitates picking-based prey capture: biomechanics of feeding in a butterflyfish,Chaetodon trichrous

Biomechanical models of feeding mechanisms elucidate how animals capture food in the wild, which, in turn, expands our understanding of their fundamental trophic niche. However, little attention has been given to modeling the protrusible upper jaw apparatus that characterizes many teleost species. We expanded existing biomechanical models to include upper jaw forces using a generalist butterflyfish, Chaetodon trichrous (Chaetodontidae) that produces substantial upper jaw protrusion when feeding on midwater and benthic prey. Laboratory feeding trials for C. trichrous were recorded using high-speed digital imaging; from these sequences we quantified feeding performance parameters to use as inputs for the biomechanical model. According to the model outputs, the upper jaw makes a substantial contribution to the overall forces produced during mouth closing in C. trichrous. Thus, biomechanical models that only consider lower jaw closing forces will underestimate total bite force for this and likely other teleost species. We also quantified and subsequently modeled feeding events for C. trichrous consuming prey from the water column versus picking attached prey from the substrate to investigate whether there is a functional trade-off between prey capture modes. We found that individuals of C. trichrous alter their feeding behavior when consuming different prey types by changing the timing and magnitude of upper and lower jaw movements and that this behavioral modification will affect the forces produced by the jaws during prey capture by dynamically altering the lever mechanics of the jaws. In fact, the slower, lower magnitude movements produced during picking-based prey capture should produce a more forceful bite, which will facilitate feeding on benthic attached prey items, such as corals. Similarities between butterflyfishes and other teleost lineages that also employ picking-based prey capture suggest that a suite of key behavioral and morphological innovations enhances feeding success for benthic attached prey items.     

Elemental composition of seston and nutrient dynamics in the Sea of Marmara

The Sea of Marmara, an intercontinental basin with shallow and narrowstraits, connects the Black and Mediterranean Seas. Data obtained during1991–1996 have permitted the determination of the elementalcomposition of seston in the euphotic zone and the N:P ratio of thesubhalocline waters of the Marmara Sea. Since primary production is alwayslimited to the less saline upper layer (15–20 m), of the Marmara Sea,the subhalocline waters of Mediteranean origin are always rich in nutrients(NO₃ + NO₂ = 8–10 μm, PO₄ = 0.8–1.2 μm) but depleted in dissolvedoxygen (30–50 μm) throughout the basin, yielding an -O_{_2} : N : P ratio of 178 : 9 : 1. Pollution of the surfacewaters since the 60s has modified the subhalocline nutrient chemistryslightly. In the euphotic zone, the N : P ratio of the seston changes from5.9 to 9.5 between the less and more productive periods. Though the biologyof the Marmara has changed significantly during the previous two decades,the close relationship observed between the elemental composition of thesurface seston and the NO₃ : PO₄ ratio of thesubhalocline waters strongly suggests that during the whole year primaryproduction throughout the basin and POM export to the lower layer remainnitrogen-limited. This suggestion needs to be confirmed by bio-assays,biological studies and sediment trap data from the upper subhaloclinedepths. Nonetheless, the counterflows in the Marmara basin possessrelatively low N : P ratios in both dissolved and particulate nutrients andextend as far as the adjacent seas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Species richness of sawfly–host plant associations at higher taxonomic levels

Insect–plant interactions are important to understanding the evolutionary mechanisms responsible for most of the diverse plant‐feeding insects. We aggregated data on sawfly–host plant associations and other resource associations from existing sources to address the following questions: (i) Is there a general correlation between host diversity and sawfly species richness? (ii) Is the pattern of host plant use consistent across sawfly lineages? and (iii) Is there a phylogenetically significant shift in species richness among sawflies? Our analysis comprised 8567 sawfly species, including 2087 species with host plant and other records. In total, there were 2126 records of host usage for sawflies, the overwhelming majority of which were sawflies using angiosperms as resources. Rosales are used by most of the species in sawfly families or subfamilies. We found that there was a strong correlation between the number of host plant orders and the species richness of sawfly families and subfamilies. To examine the points at which sawflies have experienced significant shifts in species richness, we compared sister taxon species richness. Several positive and negative shifts in species richness among sawflies were related to their range of host plant usage and specialized niche, respectively. In general, we found that most of the sawfly families and subfamilies used several orders as host plants, but mainly core eudicots, although some families or subfamilies were specialized on pteridophytes or gymnosperms.