Convergent evolution of hemoglobin switching in jawed and jawless vertebrates.

BACKGROUND: During development, humans and other jawed vertebrates (Gnathostomata) express distinct hemoglobin genes, resulting in different hemoglobin tetramers. Embryonic and fetal hemoglobin have higher oxygen affinities than the adult hemoglobin, sustaining the oxygen demand of the developing organism. Little is known about the expression of hemoglobins during development of jawless vertebrates (Agnatha). RESULTS: We identified three hemoglobin switches in the life cycle of the sea lamprey. Three hemoglobin genes are specifically expressed in the embryo, four genes in the filter feeding larva (ammocoete), and nine genes correspond to the adult hemoglobin chains. During the development from the parasitic to the reproductive adult, the composition of hemoglobin changes again, with a massive increase of chain aHb1. A single hemoglobin chain is expressed constitutively in all stages. We further showed the differential expression of other globin genes: Myoglobin 1 is most highly expressed in the reproductive adult, myoglobin 2 expression peaks in the larva. Globin X1 is restricted to the embryo; globin X2 was only found in the reproductive adult. Cytoglobin is expressed at low levels throughout the life cycle. CONCLUSION: Because the hemoglobins of jawed and jawless vertebrates evolved independently from a common globin ancestor, hemoglobin switching must also have evolved convergently in these taxa. Notably, the ontogeny of sea lamprey hemoglobins essentially recapitulates their phylogeny, with the embryonic hemoglobins emerging first, followed by the evolution of larval and adult hemoglobins.

Lampreys as Diverse Model Organisms in the Genomics Era.

Lampreys, one of the two surviving groups of ancient vertebrates, have become important models for study in diverse fields of biology. Lampreys (of which there are approximately 40 species) are being studied, for example, (a) to control pest sea lamprey in the North American Great Lakes and to restore declining populations of native species elsewhere; (b) in biomedical research, focusing particularly on the regenerative capability of lampreys; and (c) by developmental biologists studying the evolution of key vertebrate characters. Although a lack of genetic resources has hindered research on the mechanisms regulating many aspects of lamprey life history and development, formerly intractable questions are now amenable to investigation following the recent publication of the sea lamprey genome. Here, we provide an overview of the ways in which genomic tools are currently being deployed to tackle diverse research questions and suggest several areas that may benefit from the availability of the sea lamprey genome.

Recovery of a wild fish population from whole-lake additions of a synthetic estrogen.

Despite widespread recognition that municipal wastewaters contain natural and synthetic estrogens, which interfere with development and reproduction of fishes in freshwaters worldwide, there are limited data on the extent to which natural populations of fish can recover from exposure to these compounds. We conducted whole-lake additions of an active component of the birth control pill (17α-ethynylestradiol; EE2) that resulted in the collapse of the fathead minnow (Pimephales promelas) population. Here we quantify physiological, population, and genetic characteristics of this population over the 7 years after EE2 additions stopped to determine if complete recovery was possible. By 3 years post-treatment, whole-body vitellogenin concentrations in male fathead minnow had returned to baseline, and testicular abnormalities were absent. In the spring of the fourth year, adult size-frequency distribution and abundance had returned to pretreatment levels. Microsatellite analyses clearly showed that postrecovery fish were descendants of the original EE2-treated population. Results from this whole-lake experiment demonstrate that fish can recover from EE2 exposure at the biochemical through population levels, although the timelines to do so are long for multigenerational exposures. These results suggest that wastewater treatment facilities that reduce discharges of estrogens and their mimics can improve the health of resident fish populations in their receiving environments.

Gene expression during ovarian differentiation in parasitic and non-parasitic lampreys: implications for fecundity and life history types.

Lampreys diverged from the jawed vertebrate lineage approximately 500million years ago. Lampreys undergo sex differentiation much later than most other vertebrates, and ovarian differentiation occurs several years before testicular differentiation. The genetic basis of lamprey sex differentiation is of particular interest both because of the phylogenetic importance of lampreys and because of their unusual pattern of sex differentiation. As well, differences between parasitic and non-parasitic lampreys may first become evident at ovarian differentiation. However, nothing is known about the genetic basis of ovarian differentiation in lampreys. This study examined potential differences in gene expression before, during, and after ovarian differentiation in parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzonfossor. Eight target genes (17ß-hydroxysteroid dehydrogenase, germ cell-less, estrogen receptor ß, insulin-like growth factor 1 receptor, daz-associated protein 1, cytochrome c oxidase subunit III, Wilms' tumour suppressor protein 1, and dehydrocholesterol reductase 7) were examined. Northern brook lamprey displayed higher expression of cytochrome c oxidase subunit III, whereas chestnut lamprey displayed higher expression of insulin-like growth factor 1 receptor; these genes may be involved in apoptosis and oocyte growth, respectively. Presumptive male larvae had higher expression of Wilms' tumour suppressor protein 1, which may be involved in the undifferentiated gonad and/or later testicular development. Differentiated females had higher expression of 17ß hydroxysteroid dehydrogenase and daz-associated protein 1, which may be involved in female development. This study is the first to identify genes that may be involved in ovarian differentiation and fecundity in lampreys.

Population genomics of Pacific lamprey: adaptive variation in a highly dispersive species.

Unlike most anadromous fishes that have evolved strict homing behaviour, Pacific lamprey (Entosphenus tridentatus) seem to lack philopatry as evidenced by minimal population structure across the species range. Yet unexplained findings of within-region population genetic heterogeneity coupled with the morphological and behavioural diversity described for the species suggest that adaptive genetic variation underlying fitness traits may be responsible. We employed restriction site-associated DNA sequencing to genotype 4439 quality filtered single nucleotide polymorphism (SNP) loci for 518 individuals collected across a broad geographical area including British Columbia, Washington, Oregon and California. A subset of putatively neutral markers (N = 4068) identified a significant amount of variation among three broad populations: northern British Columbia, Columbia River/southern coast and 'dwarf' adults (F(CT) = 0.02, P ≪ 0.001). Additionally, 162 SNPs were identified as adaptive through outlier tests, and inclusion of these markers revealed a signal of adaptive variation related to geography and life history. The majority of the 162 adaptive SNPs were not independent and formed four groups of linked loci. Analyses with matsam software found that 42 of these outlier SNPs were significantly associated with geography, run timing and dwarf life history, and 27 of these 42 SNPs aligned with known genes or highly conserved genomic regions using the genome browser available for sea lamprey. This study provides both neutral and adaptive context for observed genetic divergence among collections and thus reconciles previous findings of population genetic heterogeneity within a species that displays extensive gene flow.

Neither philopatric nor panmictic: microsatellite and mtDNA evidence suggests lack of natal homing but limits to dispersal in Pacific lamprey.

Most species with lengthy migrations display some degree of natal homing; some (e.g. migratory birds and anadromous salmonids) show spectacular feats of homing. However, studies of the sea lamprey (Petromyzon marinus) indicate that this anadromous species locates spawning habitat based on pheromonal cues from larvae rather than through philopatry. Previous genetic studies in the anadromous Pacific lamprey (Entosphenus tridentatus) have both supported and rejected the hypothesis of natal homing. To resolve this, we used nine microsatellite loci to examine the population structure in 965 Pacific lamprey from 20 locations from central British Columbia to southern California and supplemented this analysis with mitochondrial DNA restriction fragment length polymorphism analysis on a subset of 530 lamprey. Microsatellite analysis revealed (i) relatively low but often statistically significant genetic differentiation among locations (97% pairwise F(ST) values were <0.04 but 73.7% were significant); and (ii) weak but significant isolation by distance (r(2) = 0.0565, P = 0.0450) but no geographic clustering of samples. The few moderate F(ST) values involved comparisons with sites that were geographically distant or far upstream. The mtDNA analysis--although providing less resolution among sites (only 4.7%F(ST) values were significant)--was broadly consistent with the microsatellite results: (i) the southernmost site and some sites tributary to the Salish Sea were genetically distinct; and (ii) southern sites showed higher haplotype and private haplotype richness. These results are inconsistent with philopatry, suggesting that anadromous lampreys are unusual among species with long migrations, but suggest that limited dispersal at sea precludes panmixia in this species.

Pervasive male-biased expression throughout the germline-specific regions of the sea lamprey genome supports key roles in sex differentiation and spermatogenesis.

Sea lamprey undergo programmed genome rearrangement (PGR) in which ∼20% of the genome is jettisoned from somatic cells during embryogenesis. Although the role of PGR in embryonic development has been studied, the role of the germline-specific region (GSR) in gonad development is unknown. We analysed RNA-sequence data from 28 sea lamprey gonads sampled across life-history stages, generated a genome-guided de novo superTranscriptome with annotations, and identified germline-specific genes (GSGs). Overall, we identified 638 GSGs that are enriched for reproductive processes and exhibit 36x greater odds of being expressed in testes than ovaries. Next, while 55% of the GSGs have putative somatic paralogs, the somatic paralogs are not differentially expressed between sexes. Further, putative orthologs of some the male-biased GSGs have known functions in sex determination or differentiation in other vertebrates. We conclude that the GSR of sea lamprey plays an important role in testicular differentiation and potentially sex determination.

Transcriptomic analysis of gonadal development in parasitic and non-parasitic lampreys (Ichthyomyzon spp.), with a comparison of genomic resources in these non-model species.

Lampreys are jawless fishes that diverged ∼550 million years ago from other vertebrates. Sequencing of the somatic and the germline genomes of the sea lamprey (Petromyzon marinus) in 2013 and 2018, respectively, has helped to improve our understanding of the genes and gene networks that control many aspects of lamprey development. However, little is known about the genetic basis of gonadal differentiation in lampreys, partly due to the prolonged period during which their gonads remain sexually indeterminate. We performed RNA-sequencing on gonadal samples from four chestnut lamprey (Ichthyomyzon castaneus) and six northern brook lamprey (I. fossor) to identify differentially expressed genes (DEG's) and pathways associated with transcriptomic differences in: (1) larvae during early gonadal differentiation versus definitive females (i.e., with oocytes in the slow cytoplasmic growth phase); and (2) females versus definitive males undergoing spermatogonial proliferation. We compared the mapping percentages of these transcriptomes to the two available sea lamprey reference genomes and three annotation files (Ensembl and UCSC for the somatic genome and SIMRbase for the germline genome). We found that mapping the RNA-seq reads to the germline genome gave superior results and, using Trinotate, we provided new putative annotations for 8161 genes in the somatic assembly and 880 genes for the germline assembly. We identified >2000 DEG's between stages and sexes, as well as biological pathways associated with each. Interestingly, some of the upregulated genes (e.g., DEG's associated with spermiation) suggest that changes in gene expression can precede morphological changes by several months. In contrast, only 81 DEG's were evident between the chestnut lamprey (that remains sexually immature during an extended post-metamorphic parasitic feeding phase) and the nonparasitic northern brook lamprey (that undergoes sexual maturation near the end of metamorphosis), but few replicates were available for comparable stages and sexes. This work lays the foundation for identifying and confirming the orthology and the function of genes involved in gonadal development in these and other lamprey species across more developmental stages.

Development and testing of a simple field-based intermittent-flow respirometry system for riverine fishes.

By understanding range-wide intraspecific variation in metabolic rate we can better understand how organisms have adapted to their environment. However, methods to quantify metabolic rate of fishes from remote areas or those that cannot be brought back to the laboratory because of imperilment status are lacking. Consequently, practical and reliable field-based methods are needed. To address this need, we developed a simple yet robust intermittent-flow respirometry system, adapted from a design commonly used in the laboratory that is readily suited for field use. Standard metabolic rate (SMR), maximum metabolic rate (MMR) and aerobic scope (AS) estimates were obtained from juvenile lake trout (Salvelinus namaycush) and brook trout (Salvelinus fontinalis) using both field- and laboratory-based systems. Whole-fish SMR, MMR and AS estimates from the field and laboratory methods did not differ from one another (ANCOVA and LMM: all P > 0.05) for either species and were comparable to estimates previously reported. Our field setup is a simpler system than the conventional laboratory-based system that requires less power and equipment to operate, yet still offers users the ability to: (1) acclimate fish to the respirometry chamber; (2) measure oxygen consumption during a shorter period (1 h), which yield metabolic rate estimates comparable to systems that take measurements over longer periods; and (3) take repeated oxygen consumption measurements with manual user-defined flush and measurement phase routines. Developing practical and reliable field respirometry methods, as demonstrated here, is important if we wish to improve our ability to predict how imperiled species will respond to changes in their environment. Such knowledge is critical for informing conservation strategies.

Use of genotyping by sequencing data to develop a high-throughput and multifunctional SNP panel for conservation applications in Pacific lamprey.

Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species.

RNA interference technology to control pest sea lampreys--a proof-of-concept.

The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

Erosion of interspecific reproductive barriers resulting from hatchery supplementation of rainbow trout sympatric with cutthroat trout.

The frequency of hybridization between cutthroat (Onchorhynchus clarki clarki) and rainbow (O. mykiss irideus) trout from coastal habitats in British Columbia, Canada, was examined in seven populations where the two species are sympatric with no history of rainbow trout stocking and compared with areas where native rainbow trout populations have been supplemented with hatchery fish (three populations). Four nuclear markers were used to identify each species and interspecific hybrids and one mitochondrial marker showed the direction of gene exchange between species. The frequency of hybrids was significantly higher (Fisher exact test, P < 0.001) in river systems where hatchery rainbow trout have been introduced (50.6% hybrids) than in populations where the two species naturally co-occur without supplementation (9.9% hybrids).

Molecular resolution of the family Dreissenidae (Mollusca: Bivalvia) with emphasis on Ponto-Caspian species, including first report of Mytilopsis leucophaeata in the Black Sea basin.

Considerable uncertainty exists in determination of the phylogeny among extant members of the Dreissenidae, especially those inhabiting the Ponto-Caspian basin, as multiple systematic revisions based on morphological characteristics have failed to resolve relationships within this group of bivalves. In this study we use DNA sequence analyses of two mitochondrial gene fragments, 16S rRNA and cytochrome c oxidase subunit I (COI), to determine phylogenetic relationships among Dreissena rostriformis, D. bugensis, D. polymorpha, D. stankovici, Congeria kusceri, and Mytilopsis leucophaeata. Dreissena stankovici was determined to represent a sister taxa to D. polymorpha and both are more closely related to other extant Dreissena species than Congeria or Mytilopsis. Sequence divergence between D. rostriformis and D. bugensis was relatively low (0.3-0.4%), suggesting that these two taxa constitute a single species. However, environmental differences suggest two races of D. rostriformis, a brackish water race (rostriformis) and a freshwater race (bugensis). Spread of bugensis-type individuals into habitats in the Caspian Sea that are occupied by rostriformis-type individuals may create novel hybridization opportunities. Species-specific molecular markers also were developed in this study since significant intraspecific variation in morphological features complicates dreissenid identification. Using two gene fragments (nuclear 28S and 16S), we identified restriction fragment length polymorphisms (RFLPs) that distinguish among D. rostriformis/bugensis, D. polymorpha, and D. stankovici and revealed the presence of a cryptic invader to the Black Sea basin, Mytilopsis leucophaeata. This is the first report of this North American native in southern Europe.