Factorial Microarray Analysis of Zebra Mussel Adhesion Process under the Impact of Multiple Environmental Factors
ABSTRACT: Factorial Microarray Analysis of Zebra Mussel (Dreissena polymorpha) Adhesion Process under the Impact of Multiple Environmental Factors The expression profiles of the zebra mussel byssus unique genes in our cDNA microarray can be influenced by multiple factors. Three environmental factors plus adhesion status were considered as four main factors in this study. Two different levels in each factor were created, therefore, a 2x2x2x2 factorial experimental design was made by sixteen treatment groups with four biological replicates in each group. There were 32 dual-channel microarray slides included in this study and the effects of main factors on gene expression profiles as well as the two-way, three-way, and four-way interactions of the main factors were analyzed. Factors: temperature, water agitation, oxygen level, and adhesion status
Project description:To identify the expression profiles of zebra mussel byssus unique genes with the regeneration of the byssal threads, a time-course study was performed. The RNA samples from the feet of fully attached zebra mussels (maintained attachment for more than 4 weeks) were extracted as common reference. The detached mussels were allowed to re-attach on underwater surface. Then the RNA samples from feet of the mussels at different time points post detachment were taken, namely, 12 hours, 1 day, 2 days, 3 days, 4 days, 7 days, and 21 days post detachment. The comparisons of the genes expression profiles were made between different time points and reference, as well as the any two connected time points (expect for 7 days and 21 days). The dual-channel microarray hybridizations were performed as following: 12h→R, 12h←R, 1D→R, 1D←R, 2D→R, 2D←R, 3D→R, 3D←R, 4D→R 4D←R, 7D→R, 7D←R, 21D→R, 21D←R, 12h→1D, 12h←1D, 1D→2D, 1D←2D, 2D→3D, 2D←3D, 3D→4D, 3D←4D, 4D→7D, 4D←7D, 7D→21D, 7D←21D. The direction of the arrows stand for the way of labeling: Alexa555→Alexa647. Six biological replicates of 1D, 2D, 3D, 4D, and 7D were used; four biological repllicates of 12h and 21D were taken; fourteen biological replicates of reference were applied. The whole experiment includes 26 microarray slide hybridizations.
Project description:Factorial Microarray Analysis of Zebra Mussel (Dreissena polymorpha) Adhesion Process under the Impact of Multiple Environmental Factors The expression profiles of the zebra mussel byssus unique genes in our cDNA microarray can be influenced by multiple factors. Three environmental factors plus adhesion status were considered as four main factors in this study. Overall design: Two different levels in each factor were created, therefore, a 2x2x2x2 factorial experimental design was made by sixteen treatment groups with four biological replicates in each group. There were 32 dual-channel microarray slides included in this study and the effects of main factors on gene expression profiles as well as the two-way, three-way, and four-way interactions of the main factors were analyzed. Factors: temperature, water agitation, oxygen level, and adhesion status
Project description:BACKGROUND:The zebra mussel (Dreissena polymorpha) has been well known for its expertise in attaching to substances under the water. Studies in past decades on this underwater adhesion focused on the adhesive protein isolated from the byssogenesis apparatus of the zebra mussel. However, the mechanism of the initiation, maintenance, and determination of the attachment process remains largely unknown. RESULTS:In this study, we used a zebra mussel cDNA microarray previously developed in our lab and a factorial analysis to identify the genes that were involved in response to the changes of four factors: temperature (Factor A), current velocity (Factor B), dissolved oxygen (Factor C), and byssogenesis status (Factor D). Twenty probes in the microarray were found to be modified by one of the factors. The transcription products of four selected genes, DPFP-BG20_A01, EGP-BG97/192_B06, EGP-BG13_G05, and NH-BG17_C09 were unique to the zebra mussel foot based on the results of quantitative reverse transcription PCR (qRT-PCR). The expression profiles of these four genes under the attachment and non-attachment were also confirmed by qRT-PCR and the result is accordant to that from microarray assay. The in situ hybridization with the RNA probes of two identified genes DPFP-BG20_A01 and EGP-BG97/192_B06 indicated that both of them were expressed by a type of exocrine gland cell located in the middle part of the zebra mussel foot. CONCLUSIONS:The results of this study suggested that the changes of D. polymorpha byssogenesis status and the environmental factors can dramatically affect the expression profiles of the genes unique to the foot. It turns out that the factorial design and analysis of the microarray experiment is a reliable method to identify the influence of multiple factors on the expression profiles of the probesets in the microarray; therein it provides a powerful tool to reveal the mechanism of zebra mussel underwater attachment.
Project description:We used a cDNA microarray previously defined for the marine sentinel organism Mytilus galloprovincialis (MytArray1.0) to evaluate the effects of nanomolar doses of combined metal salts (50, 100 and 200 nM mixtures of Cd, Cu and Hg) after 48 hours of mussel exposure. Pointing to the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we found significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel pulp by atomic absorption spectrometry. Following gill RNA purification and DNA microarray analysis, individual gene expression profiles revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses with roughly similar amounts of up- and down-regulated signals. The functional annotation of transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. Three-condition experiment (50, 100 and 200 nM doses), individual treated mussel vs. pooled control mussels (N=5), 3 biological replicates with dye-swap competitive hybridization (array A and B, 2 technical replicates/array).
Project description:The European population of Greater Scaup Aythya marila has experienced an alarming, ~60% decline in numbers over the last two decades. The brackish lagoons of the Odra River Estuary (ORE) in the south-western Baltic Sea, represent an important area for the species during the non-breeding season in Europe. The lagoons regularly support over 20 000 Scaup, with peaks exceeding 100 000 (38%-70% of the population wintering in NW Europe and the highest number recorded in April 2011-105 700). In the ORE, Scaup feed almost exclusively on the non-native Zebra Mussel Dreissena polymorpha. This mussel was present in the ORE already in the 19th century and continues to be superabundant. Using the results of 22 Scaup censuses (November to April 2002/2003 to 2013/2014) from the whole ORE (523 km2 of water), we show that Scaup flocks follow areas with the greatest area of occurrence and biomass of the Zebra Mussel, while areas with low mussel densities are ignored. The numbers of Scaup in the ORE are primarily related to the area of Zebra Mussel occurrence on the lagoon's bottom (km2) in a non-linear fashion. Zebra Mussels were absolutely prevalent (97% of biomass) in the digestive tracts of birds unintentionally by-caught in fishing nets (n = 32). We estimate that Scaup alone consume an average of 5 400 tons of Zebra Mussels annually, which represents 5.6% of the total resources of the mussel in the ORE. Our results provide a clear picture of the strong dependence of the declining, migratory duck species on the non-native mussel, its primary food in the ORE. Our findings are particularly important as they can form the basis for the conservation action plan aimed at saving the north-western European populations of Scaup.
Project description:Invasion facilitation, whereby one species has a positive effect on the establishment of another species, could help explain the rapid colonisation shown by some freshwater invasive species, but the underlying mechanisms remain unclear. We employed two-choice test arenas to test whether the presence of zebra mussel (Dreissena polymorpha) could facilitate the establishment of the killer shrimp (Dikerogammarus villosus). Killer shrimp preferred to settle on mats of zebra mussel, but this was unrelated to mat size, and was not different from attraction shown to artificial grass, suggesting that zebra mussel primarily provides substrate and refuge to the killer shrimp. Killer shrimp were strongly attracted to water scented by zebra mussel, but not to water scented by fish. Chemical attraction to the zebra mussel's scent did not differ between sympatric and allopatric populations of killer shrimp, suggesting that chemical attraction is not an acquired or learned trait. Our study shows, for the first time, chemical attraction between two highly invasive freshwater species, thereby providing a plausible mechanism for invasion facilitation. This has implications for managing the spread of killer shrimp, and perhaps other freshwater invasive species, because chemical attraction could significantly increase establishment success in mutualistic systems. Failure to consider invasion facilitation may underestimate the risk of establishment, and likely also the impact of some aquatic invaders.
Project description:Aggregations of the Ponto-Caspian invasive zebra mussel (Dreissena polymorpha) constitute a suitable habitat for macroinvertebrates, considerably increasing their abundance and providing effective antipredator protection. Thus, the overall effect of a mussel bed on particular predator species may vary from positive to negative, depending on both prey density increase and predator ability to prey in a structurally complex habitat. Alien Ponto-Caspian goby fish are likely to be facilitated when introduced into new areas by zebra mussels, provided that they are capable of utilizing mussel beds as habitat and feeding grounds. We ran laboratory experiments to find which prey (chironomid larvae) densities (from ca. 500 to 2,000 individuals m-2) in a mussel bed make it a more beneficial feeding ground for the racer goby Babka gymnotrachelus (RG) and western tubenose goby Proterorhinus semilunaris (WTG) compared to sandy and stone substrata (containing the basic prey density of 500 ind. m-2). Moreover, we checked how food availability affects habitat selection by fish. Mussel beds became more suitable for fish than alternative mineral substrata when food abundance was at least two times higher (1,000 vs. 500 ind. m-2), regardless of fish size and species. WTG was associated with mussel beds regardless of its size and prey density, whereas RG switched to this habitat when it became a better feeding ground than alternative substrata. Larger RG exhibited a stronger affinity for mussels than small individuals. WTG fed more efficiently from a mussel bed at high food abundances than RG. A literature review has shown that increasing chironomid density, which in our study was sufficient to make a mussel habitat an attractive feeding ground for the gobies, is commonly observed in mussel beds in the field. Therefore, we conclude that zebra mussels may positively affect the alien goby species and are likely to facilitate their establishment in novel areas, contributing to an invasional meltdown in the Ponto-Caspian invasive community.
Project description:The zebra mussel is present in Spain since early 2000,s, when it was discovered in the lower part of the Ebro river. To study the gene expression pattern of different populations of zebra mussel a long the Ebro River we use a custom microarray developed in our laboratory, using 4057 publicly available DNA sequences from Dreissena polymorpha and other related genera. Also it was used an external sampling site located in Sitjar Dam, about 200km form the Ebro river. Transcriptome profiles were analysed using the gills of individuals collected in the same period (20-23 March) to diminish seasonal effects. A total of 755 transcripts changed significantly their mRNA levels among the sites of the study (ANOVA p<0.01, fc ±1.5). Genes encoding for xenobiotic, energetic and calcium metabolism and cell proliferation were those showing the highest differences among populations. Geographical origin appeared as the major driver of the differences among the studied populations, as the transcriptomic profiles from four populations collected within a radius of few km around the Flix factory clustered together and separated from those from other distant populations both upstream the Ebro River or in the Sitjar dam. Differences on gene expression pattern were measure in gills of Dreissena polymorpha in different populations in five populations along the Ebro river and one population in Sitjar dam. The collection of samples were done during Srping (March). For the microarray it was used 2 replicates of each sites of the study.
Project description:Songbirds provide rich natural models for studying the relationships of brain anatomy, behavioral function, environmental signals and gene expression. Under the Songbird Neurogenomics Initiative, investigators from more than a dozen laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by the 18,848 non-redundant cDNAs spotted on the microarray. Social and environmental manipulations had highly variable impact, ranging from nil in some cases to robust in others. Several specific genes were identified as points of interest in the evolution of mechanisms by which environmental signals influence behavior. The data were also analyzed using Weighted Gene Co-expression Network Analysis (WGCNA) followed by Gene Ontology (GO) analysis. This revealed modules of co-regulated genes that are also enriched for specific functional annotations such as “ribosome” (expressed more highly in juvenile brain) and “dopamine metabolic process” (expressed more highly in striatal song control nucleus Area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience. RNA samples were collected from six different songbird species (phylogeny in SI Appendix, Figure S1) and representing 80 different “treatments”, i.e., combinations of species, brain region, sex, age, and behavioral state. The tissue samples were organized around 15 standalone experiments (Table 1 and SI Appendix, Table S1), contributed by investigators in a dozen different laboratories but analyzed under uniform conditions in a single laboratory as originally planned under the Songbird Neurogenomics Initiative. Each sample was hybridized to a zebra finch cDNA array along with a universal reference (a pool of zebra finch telencephalic RNA). e10: NCM and L2a from adult female zebra finches, 30 or 90 minutes after onset of novel song playback, or silence control, 6 biological replicates per group. All samples were hybridized against the SoNG universal RNA reference pool.