Efficacy of metabarcoding for identification of fish eggs evaluated with mock communities.

There is urgent need for effective and efficient monitoring of marine fish populations. Monitoring eggs and larval fish may be more informative than that traditional fish surveys since ichthyoplankton surveys reveal the reproductive activities of fish populations, which directly impact their population trajectories. Ichthyoplankton surveys have turned to molecular methods (DNA barcoding & metabarcoding) for identification of eggs and larval fish due to challenges of morphological identification. In this study, we examine the effectiveness of using metabarcoding methods on mock communities of known fish egg DNA. We constructed six mock communities with known ratios of species. In addition, we analyzed two samples from a large field collection of fish eggs and compared metabarcoding results with traditional DNA barcoding results. We examine the ability of our metabarcoding methods to detect species and relative proportion of species identified in each mock community. We found that our metabarcoding methods were able to detect species at very low input proportions; however, levels of successful detection depended on the markers used in amplification, suggesting that the use of multiple markers is desirable. Variability in our quantitative results may result from amplification bias as well as interspecific variation in mitochondrial DNA copy number. Our results demonstrate that there remain significant challenges to using metabarcoding for estimating proportional species composition; however, the results provide important insights into understanding how to interpret metabarcoding data. This study will aid in the continuing development of efficient molecular methods of biological monitoring for fisheries management

Adaptation to a latitudinal thermal gradient within a widespread copepod species:the contributions of genetic divergence and phenotypic plasticity

Understanding how populations adapt to heterogeneous thermal regimes is essential for comprehending how latitudinal gradients in species diversification are formed, and how taxa will respond to ongoing climate change. Adaptation can occur by innate genetic factors, by phenotypic plasticity, or by a combination of both mechanisms. Yet, the relative contribution of such mechanisms to large-scale latitudinal gradients of thermal tolerance across conspecific populations remains unclear. We examine thermal performance in 11 populations of the intertidal copepod Tigriopus californicus, ranging from Baja California Sur (Mexico) to British Columbia (Canada). Common garden experiments show that survivorship to acute heat-stress differs between populations (by up to 3.8°C in LD50 values), reflecting a strong genetic thermal adaptation. Using a split-brood experiment with two rearing temperatures, we also show that developmental phenotypic plasticity is beneficial to thermal tolerance (by up to 1.3°C), and that this effect differs across populations. Although genetic divergence in heat tolerance strongly correlates with latitude and temperature, differences in the plastic response do not. In the context of climate warming, our results confirm the general prediction that low-latitude populations are most susceptible to local extinction because genetic adaptation has placed physiological limits closer to current environmental maxima, but our results also contradict the prediction that phenotypic plasticity is constrained at lower latitudes

Identification of Ambient Molecular Clouds Associated with Galactic Supernova Remnant IC443

The Galactic supernova remnant (SNR) IC443 is one of the most studied core-collapse SNRs for its interaction with molecular clouds. However, the ambient molecular clouds with which IC443 is interacting have not been thoroughly studied and remain poorly understood. Using Five College Radio Astronomy Observatory 14m telescope, we obtained fully sampled maps of ~ 1{\deg} \times 1{\deg} region toward IC443 in the 12CO J=1-0 and HCO+ J=1-0 lines. In addition to the previously known molecular clouds in the velocity range v_lsr = -6 to -1 km/s (-3 km/s clouds), our observations reveal two new ambient molecular cloud components: small (~ 1') bright clouds in v_lsr = -8 to -3 km/s (SCs), and diffuse clouds in v_lsr = +3 to +10 km/s (+5 km/s clouds). Our data also reveal the detailed kinematics of the shocked molecular gas in IC443, however the focus of this paper is the physical relationship between the shocked clumps and the ambient cloud components. We find strong evidence that the SCs are associated with the shocked clumps. This is supported by the positional coincidence of the SCs with shocked clumps and other tracers of shocks. Furthermore, the kinematic features of some shocked clumps suggest that these are the ablated material from the SCs upon the impact of the SNR shock. The SCs are interpreted as dense cores of parental molecular clouds that survived the destruction by the pre-supernova evolution of the progenitor star or its nearby stars. We propose that the expanding SNR shock is now impacting some of the remaining cores and the gas is being ablated and accelerated producing the shocked molecular gas. The morphology of the +5 km/s clouds suggests an association with IC443. On the other hand, the -3 km/s clouds show no evidence for interaction.Comment: Accepted for publication in ApJ. 15 pages (with emulateapj.cls), 17 figures, and 2 table

Variation in Thermal Tolerance and Its Relationship to Mitochondrial Function Across Populations of Tigriopus californicus

Variation in thermal tolerance plays a key role in determining the biogeographic distribution of organisms. Consequently, identifying the mechanistic basis for thermal tolerance is necessary for understanding not only current species range limits but also the capacity for range limits to shift in response to climate change. Although variation in mitochondrial function likely contributes to variation in thermal tolerance, the extent to which mitochondrial function underlies local thermal adaptation is not fully understood. In the current study, we examine variation in thermal tolerance and mitochondrial function among three populations of the intertidal copepod Tigriopus californicus found across a latitudinal thermal gradient along the coast of California, USA. We tested (1) acute thermal tolerance using survivorship and knockdown assays, (2) chronic thermal tolerance using survivorship of nauplii and developmental rate, and (3) mitochondrial performance at a range of temperatures using ATP synthesis fueled by complexes I, II, and I&II, as well as respiration of permeabilized fibers. We find evidence for latitudinal thermal adaptation: the southernmost San Diego population outperforms the northernmost Santa Cruz in measures of survivorship, knockdown temperature, and ATP synthesis rates during acute thermal exposures. However, under a chronic thermal regime, survivorship and developmental rate are more similar in the southernmost and northernmost population than in the mid-range population (Abalone Cove). Though this pattern is unexpected, it aligns well with population-specific rates of ATP synthesis at these chronic temperatures. Combined with the tight correlation of ATP synthesis decline and knockdown temperature, these data suggest a role for mitochondria in setting thermal range limits and indicate that divergence in mitochondrial function is likely a component of adaptation across latitudinal thermal gradients

Interpopulation hybridization results in widespread viability selection across the genome in Tigriopus californicus

<p>Abstract</p> <p>Background</p> <p>Genetic interactions within hybrids influence their overall fitness. Understanding the details of these interactions can improve our understanding of speciation. One experimental approach is to investigate deviations from Mendelian expectations (segregation distortion) in the inheritance of mapped genetic markers. In this study, we used the copepod <it>Tigriopus californicus</it>, a species which exhibits high genetic divergence between populations and a general pattern of reduced fitness in F2 interpopulation hybrids. Previous studies have implicated both nuclear-cytoplasmic and nuclear-nuclear interactions in causing this fitness reduction. We identified and mapped population-diagnostic single nucleotide polymorphisms (SNPs) and used these to examine segregation distortion across the genome within F2 hybrids.</p> <p>Results</p> <p>We generated a linkage map which included 45 newly elucidated SNPs and 8 population-diagnostic microsatellites used in previous studies. The map, the first available for the Copepoda, was estimated to cover 75% of the genome and included markers on all 12 <it>T. californicus </it>chromosomes. We observed little segregation distortion in newly hatched F2 hybrid larvae (fewer than 10% of markers at p < 0.05), but strikingly higher distortion in F2 hybrid adult males (45% of markers at p < 0.05). Hence, segregation distortion was primarily caused by selection against particular genetic combinations which acted between hatching and maturity. Distorted markers were not distributed randomly across the genome but clustered on particular chromosomes. In contrast to other studies in this species we found little evidence for cytonuclear coadaptation. Instead, different linkage groups exhibited markedly different patterns of distortion, which appear to have been influenced by nuclear-nuclear epistatic interactions and may also reflect genetic load carried within the parental lines.</p> <p>Conclusion</p> <p>Adult male F2 hybrids between two populations of <it>T. californius </it>exhibit dramatic segregation distortion across the genome. Distorted loci are clustered within specific linkage groups, and the direction of distortion differs between chromosomes. This segregation distortion is due to selection acting between hatching and adulthood.</p

A gene-based SNP resource and linkage map for the copepod Tigriopus californicus

<p>Abstract</p> <p>Background</p> <p>As yet, few genomic resources have been developed in crustaceans. This lack is particularly evident in Copepoda, given the extraordinary numerical abundance, and taxonomic and ecological diversity of this group. <it>Tigriopus californicus </it>is ideally suited to serve as a genetic model copepod and has been the subject of extensive work in environmental stress and reproductive isolation. Accordingly, we set out to develop a broadly-useful panel of genetic markers and to construct a linkage map dense enough for quantitative trait locus detection in an interval mapping framework for <it>T. californicus--</it>a first for copepods.</p> <p>Results</p> <p>One hundred and ninety Single Nucleotide Polymorphisms (SNPs) were used to genotype our mapping population of 250 F₂larvae. We were able to construct a linkage map with an average intermarker distance of 1.8 cM, and a maximum intermarker distance of 10.3 cM. All markers were assembled into linkage groups, and the 12 linkage groups corresponded to the 12 known chromosomes of <it>T. californicus</it>. We estimate a total genome size of 401.0 cM, and a total coverage of 73.7%. Seventy five percent of the mapped markers were detected in 9 additional populations of <it>T. californicus</it>. Of available model arthropod genomes, we were able to show more colocalized pairs of homologues between <it>T. californicus </it>and the honeybee <it>Apis mellifera</it>, than expected by chance, suggesting preserved macrosynteny between Hymenoptera and Copepoda.</p> <p>Conclusions</p> <p>Our study provides an abundance of linked markers spanning all chromosomes. Many of these markers are also found in multiple populations of <it>T. californicus</it>, and in two other species in the genus. The genomic resource we have developed will enable mapping throughout the geographical range of this species and in closely related species. This linkage map will facilitate genome sequencing, mapping and assembly in an ecologically and taxonomically interesting group for which genomic resources are currently under development.</p

Diversifying Selection Underlies the Origin of Allozyme Polymorphism at the Phosphoglucose Isomerase Locus in Tigriopus californicus

The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations

Genomic signatures of mitonuclear coevolution across populations of Tigriopus californicus

The copepod Tigriopus californicus shows extensive population divergence and is becoming a model for understanding allopatric differentiation and the early stages of speciation. Here, we report a high-quality reference genome for one population (similar to 190 megabases across 12 scaffolds, and similar to 15,500 protein-coding genes). Comparison with other arthropods reveals 2,526 genes presumed to be specific to T. californicus, with an apparent proliferation of genes involved in ion transport and receptor activity. Beyond the reference population, we report re-sequenced genomes of seven additional populations, spanning the continuum of reproductive isolation. Populations show extreme mitochondrial DNA divergence, with higher levels of amino acid differentiation than observed in other taxa. Across the nuclear genome, we find elevated protein evolutionary rates and positive selection in genes predicted to interact with mitochondrial DNA and the proteins and RNA it encodes in multiple pathways. Together, these results support the hypothesis that rapid mitochondrial evolution drives compensatory nuclear evolution within isolated populations, thereby providing a potentially important mechanism for causing intrinsic reproductive isolation

Exposure to fluctuating salinity enhances free amino acid accumulation in Tigriopus californicus (Copepoda)

Intracellular concentrations of free amino acids (FAA) in the intertidal copepod Tigriopus californicus increase in response to hyperosmotic stress and decrease in response to hypo-osmotic stress. The purpose of this study was to determine if exposure to repeated bouts of osmotic stress resulted in changes in FAA accumulation or the degree of FAA retention in subsequent episodes. Five groups of T. californicus were exposed for 22 days to a fluctuating salinity regime which consisted of 24 h at 100% seawater followed by 24 h at either 90, 80, 70, 60 or 50% seawater (11 cycles). After the tenth exposure to 100% seawater, individuals from each treatment group were analyzed for alanine and proline concentration. Alanine and proline accumulation generally increased in proportion to the osmotic stress up to 60–100% seawater — additional osmotic stress failed to increase total accumulation. Prior exposure to fluctuating salinity increased the extent of alanine and proline retention observed upon transfer to a hypo-osmotic medium. The treatment group which had experienced the most extreme fluctuation (50–100% seawater) retained alanine and proline levels approximately 10- and 20-fold higher, respectively, than controls. A less severe salinity fluctuation was required to elicit this response for alanine (90–100% seawater) than for proline (60–100% seawater). Previous exposure to fluctuating salinity also resulted in increased alanine and proline accumulation in subsequent episodes of hyperosmotic stress. 24 h after transfer from 50 to 100% seawater, alanine and proline levels in the conditioned copepods were approximately 3- and 7-fold higher, respectively, than in copepods which had not been cycled. This facilitation in alanine and proline accumulation occurred after 10 and 11 cycles, respectively. Of the increased accumulation in alanine and proline, 7.0% and 22.5%, respectively, could be accounted for by the higher degree of FAA retention while under hypo-osmotic conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47130/1/360_2004_Article_BF00692733.pd

The Neonatal Fc Receptor (FcRn) Enhances Human Immunodeficiency Virus Type 1 (HIV-1) Transcytosis across Epithelial Cells

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure