Phenotypic variation for adhesive tenacity in the barnacle Balanus amphitrite

Silicone fouling-release coatings represent a non-toxic alternative to biocide-containing ship hull paints. These coatings allow fouling organisms to attach to the hull surface, but prevent firm adhesion. Adhesive tenacity to fouling-release materials varies both among and within species. We quantified broad-sense genetic and environmental sources of intraspecific variation in tenacity to two silicone substrata, for the barnacle Balanus amphitrite. For both materials tenacity varied over an order of magnitude; however, the partitioning of this variation differed between the substrata. For International Veridian, a commercially-available fouling-release coating, removal stress varied significantly among maternal families and replicate barnacle cultures. Variation among the maternal families was associated with previously observed differences among these families in the condition of the adhesive plaque. Additional experiments suggested that variation among the replicate cultures arose from heterogeneity between replicate coatings in properties that affect tenacity. We could not attribute variation in removal stress for Dow Corning Silastic T-2, a silicone rubber used for mold-making, to any of the genetic or environmental sources tested. Instead, variation may have been due to measurement error or heterogeneity within replicate coatings in properties affecting tenacity. Differences among maternal families in removal stress may stem from variation in the interaction between the adhesive and the substratum, or in the viscoelastic properties of the adhesive plaque.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

The screening of sponge extracts for antifouling activity using a bioassay with laboratory-reared cyprid larvae of the barnacle Balanus amphitrite

Antifouling coatings based on organotin compounds possess a world-wide threat to the environment and due to growing restrictions there is a need for environmentally safe antifouling systems. TNO is working on the development of novel antifouling systems based on secondary metabolites from sponges. Screening for natural antifoulants is conducted using a settlement assay with cyprid larvae of the barnacle Balanus amphitrite Darwin. Forty-four sponges (35 species) were collected from around the island of Curaçao in the Caribbean and settlement assays were performed with the ethyl-acetate extracts. Thirty-one extracts significantly inhibited cyprid settlement at 0.1 mg ml⁻¹, of which 22 significantly inhibited settlement at 0·01 mg ml⁻¹.     

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

Background  

Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.     

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

Background  

Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae.     

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase^plus software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results     

Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus drought-stressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

草豆蔻cDNA文库的构建及鉴定

以草豆蔻花序原基为材料,构建了cDNA文库。原始文库滴度为0.8×106pfu/mL,扩增后滴度为4.23×1011pfu/mL。插入片段大小在500bp～1.5kb之间,重组率为95.3%。以水稻RAP1A基因中包含MADS-box保守区段的序列为探针对该文库进行筛选,获得的阳性克隆经测序及序列比对分析,确认其中共有10个含MADS-box的阳性克隆。     

Validation of Zebrafish （Danio redo） Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR （qRT-PCR） is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The fl-actin, EFlot and Rpll3ot genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1 or, Rpll3α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

Comparison of nutritional status of field and laboratory reared Balanus amphitrite Darwin (Cirripedia: Thoracica) larvae and implication of starvation

Experiments were carried out to evaluate the influence of rearing temperature and food concentration (20 and 30 °C, 1×10⁵ and 2×10⁵ cells ml⁻¹) on the starvation threshold and nucleic acid content of the larvae of Balanus amphitrite. The larvae were also field-reared using micro-enclosures. Laboratory-reared larvae were larger in size than the field-reared larvae. An increase in size, DNA content and instar index of the starved II instar larvae was observed indicating that the absence of food may not be fatal to this early instar. The temperature at which larvae were raised and the food concentration had variable influence on the capacity to withstand starvation. Exposure to increased temperatures during starvation eliminated the effect of doubling food concentration during their feeding period prior to starvation. The larvae reared at 20 °C had comparatively lower nucleic acid content. The laboratory-reared larvae had ca. 1.7 times greater RNA:DNA ratio than larvae raised at comparable temperature in the field.     

Selection of optimal reference genes for normalization in quantitative RT-PCR

Background  

Normalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.     

Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris

Background  

Quantitative real-time polymerase chain reaction (RT-qPCR) is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc Octopus vulgaris, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization.     

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

Background  

Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR.