We will discover, design, and validate two RT-PCR markers that are expected to be differentially expressed acrosslife stages (maturity) and sexes (male/female) in PL using a combination of bioinformatics, common garden (i.e.,hatchery) experiments, and laboratory analysis. To provide a gene expression profile against which differential geneexpression will be analyzed, we will also develop an RT-PCR marker for a common housekeeping gene, such asbeta2-microglobulin (b2m)5.Housekeeping genes are genes that are expressed by all tissues because they are involved in the maintenance of basic cellular function under normal conditions.  Our marker discovery effort will only target DEGs with demonstrably high likelihood (i.e., via bioinformatic analysis) of diagnosing species, life stage, and sex in PL.  We will validate taxon-specificity of the markers using mRNA sequences of PL and nontarget species (e.g., Lampetra) using the nucleotide sequence archive of the National Center for Biotechnology Information (NCBI: https://www.ncbi.nlm.nih.gov/nucleotide/). All lamprey species with target gene sequences registered at NCBI will be set as nontarget species to ensure specificity.  To further validate specificity, we will also utilize the Sea Lamprey (Petromyzon marinus) and newly assembled Western Brook Lamprey (L. richardsoni) genomes.  Primer pairs will be designed to have at least two nucleotide substitutions within five bases from the 3′- end against the other nontarget species in at least one of the forward and reverse primers, and the opposite primer was set on the exon–exon junction of the target gene to suppress the undesirable amplification of genomic DNA (gDNA) which includes intron sequences. The primer sets will be tested by in silico PCR using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/ prime r-blast/) and the results will be supported by the species and gene specificity of each primer set. As a final check of specificity, we will also analyze RNA extracted from tissues of Western Brook Lamprey.   

We will use a common garden experiment to analyze the effect of life stage (maturity) and sex (male/female) on gene expression.  Approximately equal densities (mass of PL per volume of water) of live larval, pre-spawn female, pre-spawn male, spawned female, and spawned male PL will be placed separately into experimental tanks. A tank containing no fish will be used as a negative control; a tank containing Lampetra spp. will also be used to check species specificity (i.e., for cross species amplification in Lampetra); and a tank containing an intermediate life stage (eyed juvenile) of PL will be used to understand if there is a spectrum of gene expression across life stages.  We will aerate and maintain tanks at 15°C while maintaining a 12-hr/12-hr light/dark cycle. We will treat all equipment, such as air stones, air tubing, and tanks with 10% bleach solution for at least 5 minutes and thoroughly rinse equipment with running tap water. Water will be collected from tanks at a set duration after the introduction of lamprey. Specifically, 3.0 L of water will be collected into a 10-L plastic container by scooping water with a disposable polyethylene cup. The water sample will be stirred well and dispensed in 1-L amounts into disposable polyamide packs to create duplicate subsamples. The water samples will be immediately filtered using a Sterivex filter cartridge with a nominal pore size of 0.45 μm. After filtration, each cartridge will be injected with RNALater, sealed, frozen, and mailed to the genetics laboratory. Methods of eRNA extraction and RT-PCR processes are available upon request.  In addition to eRNA samples, one to two individuals from each tank (depending on the life stage) will be sampled for gonadal tissue (i.e., the target organ) and for a non-target tissue (i.e., negative control) to confirm the source of mRNA in the eRNA samples. 

We have two research questions: (1) Are the recently discovered DEGs between life stage and sex in Icthyomyzonspp. orthologous (i.e., do they share ancestry and function) in PL? What differences in gene expression betweenmaturity and sex are detectable in samples of PL eRNA? Given the2000orthologous DEGs were discovered in twospecies of lamprey separated by ~8million years of evolution, and the zebrafish eRNA from six different tissues weredetectable in sampled water, we expect some DEGs to be orthologous in PL and that we might detect thecorresponding eRNA in water.There are three project milestones: (1) discover diagnostic RNA sequences in Ichthyomyzon that are orthologous inEntosphenus; (2) design RT-PCR primers for the two most diagnostic DEGs (i.e., one for life stage, one for sex); and(3) validate markers using hatchery experiments