Towards a comprehensive barcode library for arctic life - Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada

Background

In order to understand the role of herbivores in trophic webs, it is essential to know what they feed on. Diet analysis is, however, a challenge in many small herbivores with a secretive life style. In this paper, we compare novel (high-throughput pyrosequencing) DNA barcoding technology for plant mixture with traditional microhistological method. We analysed stomach contents of two ecologically important subarctic vole species, Microtus oeconomus and Myodes rufocanus, with the two methods. DNA barcoding was conducted using the P6-loop of the chloroplast trnL (UAA) intron.

Results

Although the identified plant taxa in the diets matched relatively well between the two methods, DNA barcoding gave by far taxonomically more detailed results. Quantitative comparison of results was difficult, mainly due to low taxonomic resolution of the microhistological method, which also in part explained discrepancies between the methods. Other discrepancies were likely due to biases mostly in the microhistological analysis.

Conclusion

We conclude that DNA barcoding opens up for new possibilities in the study of plant-herbivore interactions, giving a detailed and relatively unbiased picture of food utilization of herbivores.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

RU-486 inhibits rat gonadal steroidogenesis

RU-486 is a synthetic steroid analogue that can inhibit adrenal steroid synthesis in the rat and rhesus monkey. We measured the activities of five testicular and two ovarian microsomal steroidogenic enzymes to assess the potential effect of RU-486 on rat gonadal steroidogenesis. Hypophysectomized, gonadotropin-replaced rats received RU-486 or a vehicle solution twice daily for seven days. The animals were sacrificed and their gonads were resected, weighed, and microsomal enzyme activities were measured according to RU-486 treatment. Testicular 17-hydroxylase and aromatase activity decreased in RU-486 treated animals whereas 17,20-desmolase, 3 beta-hydroxysteroid dehydrogenase and 17-ketosteroid reductase activities were unaffected. Ovarian 17-hydroxylase but not 3 beta-hydroxysteroid dehydrogenase activity was decreased in the animals receiving the drug. We conclude that RU-486 inhibits both testicular and ovarian steroidogenesis in the rat.     

A novel approach to the generation of high affinity class II-binding peptides

We have found that if core regions crucial for class II binding are incorporated in multiple copies in the same peptide molecule ("reiterative motifs"), marked enhancement of the binding capacity occurs. Isotype specificity (IAd vs IEd binding capacities) is retained in all three antigenic determinants so far analyzed (lambda rep 12-26, OVA 323-339, and hen egg lysozyme 105-120). The mechanism involved in such an effect is not clear, but experiments involving introduction of a peptide spacer between two repeated core regions do not support the notion that the effect is mediated by cross-linking of more than one MHC molecule, favoring the possibility that conformational effects or distinct subsites of interaction on the MHC molecule may be involved. Based on reiterative structures, a peptide molecule composed of only two different amino acids (Ala and His) has been produced that still retains a very high binding affinity. An 125I-radiolabeled form of this peptide has been used to demonstrate that the high binding detected is mediated by the same binding site involved in the interaction of IAd and OVA 323-339. Inhibition of Ag presentation studies further supports the immunologic relevance of the phenomena observed. Finally, we observed naturally occurring clustered binding sites in proximity of immunodominant protein regions, raising the possibility that the phenomenon might have a physiologic counterpart.     

Sensitive and high resolution in situ hybridization to human chromosomes using biotin labelled probes: assignment of the human thymocyte CD1 antigen genes to chromosome 1.

A method for in situ hybridization originally developed for mapping genes in the nematode, Caenorhabditis elegans has been adapted for high resolution cytological mapping of genes in the human. The probe DNAs are labelled by incorporation of biotin dUTP and the site of hybridization detected by immunofluorescence. For the accurate assignment of the hybridization signal to chromosome bands, visualized by staining with Hoechst 33258, a heterologous ribosomal DNA probe is also included in the hybridization reaction. These rDNA signals are used as fiducial markers when aligning the two fluorescent images. We demonstrate the method by assignment of the human thymocyte CD1 antigen genes to human chromosome 1q22-23.     

A new, improved technique for automated sequencing of non-polar peptides

A method for the automated sequential degradation of non-polar peptides is reported. Both the polarity and film-forming properties of these peptides are increased by the attachment of 2-amino-1,5 napthalene disulfonic acid to the C-terminal residue via a water-soluble carbodiimide. The sequences of three individual peptides modified by the procedure are reported with high yields while these same peptides could not be successfully sequenced if no modification was made.     

Ciliary Rootlet Coiled-Coil 2 (crocc2) Is Associated with Evolutionary Divergence and Plasticity of Cichlid Jaw Shape

Cichlid fishes exhibit rapid, extensive, and replicative adaptive radiation in feeding morphology. Plasticity of the cichlid jaw has also been well documented, and this combination of iterative evolution and developmental plasticity has led to the proposition that the cichlid feeding apparatus represents a morphological “flexible stem.” Under this scenario, the fixation of environmentally sensitive genetic variation drives evolutionary divergence along a phenotypic axis established by the initial plastic response. Thus, if plasticity is predictable then so too should be the evolutionary response. We set out to explore these ideas at the molecular level by identifying genes that underlie both the evolution and plasticity of the cichlid jaw. As a first step, we fine-mapped an environment-specific quantitative trait loci for lower jaw shape in cichlids, and identified a nonsynonymous mutation in the ciliary rootlet coiled-coil 2 (crocc2), which encodes a major structural component of the primary cilium. Given that primary cilia play key roles in skeletal mechanosensing, we reasoned that this gene may confer its effects by regulating the sensitivity of bone to respond to mechanical input. Using both cichlids and zebrafish, we confirmed this prediction through a series of experiments targeting multiple levels of biological organization. Taken together, our results implicate crocc2 as a novel mediator of bone formation, plasticity, and evolution.     

Bentho-Pelagic Divergence of Cichlid Feeding Architecture Was Prodigious and Consistent during Multiple Adaptive Radiations within African Rift-Lakes

Background

How particular changes in functional morphology can repeatedly promote ecological diversification is an active area of evolutionary investigation. The African rift-lake cichlids offer a calibrated time series of the most dramatic adaptive radiations of vertebrate trophic morphology yet described, and the replicate nature of these events provides a unique opportunity to test whether common changes in functional morphology have repeatedly facilitated their ecological success.

Methodology/Principal Findings

Specimens from 87 genera of cichlid fishes endemic to Lakes Tanganyka, Malawi and Victoria were dissected in order to examine the functional morphology of cichlid feeding. We quantified shape using geometric morphometrics and compared patterns of morphological diversity using a series of analytical tests. The primary axes of divergence were conserved among all three radiations, and the most prevalent changes involved the size of the preorbital region of the skull. Even the fishes from the youngest of these lakes (Victoria), which exhibit the lowest amount of skull shape disparity, have undergone extensive preorbital evolution relative to other craniofacial traits. Such changes have large effects on feeding biomechanics, and can promote expansion into a wide array of niches along a bentho-pelagic ecomorphological axis.

Conclusions/Significance

Here we show that specific changes in trophic anatomy have evolved repeatedly in the African rift lakes, and our results suggest that simple morphological alterations that have large ecological consequences are likely to constitute critical components of adaptive radiations in functional morphology. Such shifts may precede more complex shape changes as lineages diversify into unoccupied niches. The data presented here, combined with observations of other fish lineages, suggest that the preorbital region represents an evolutionary module that can respond quickly to natural selection when fishes colonize new lakes. Characterizing the changes in cichlid trophic morphology that have contributed to their extraordinary adaptive radiations has broad evolutionary implications, and such studies are necessary for directing future investigations into the proximate mechanisms that have shaped these spectacular phenomena.     

Evolution of novelty in the cichlid dentition

The shape of teeth occupies a central position in various biological disciplines, from paleo-ecology to molecular biology to cosmetic and reconstructive dentistry. Despite a long tradition of study in mammals, important questions remain regarding the genetic and developmental basis of differences in tooth shape. Here, we use natural mutants of cichlid fish from East Africa, which exhibit tremendous dental diversity, to help fill the gaps in our understanding of vertebrate odontogenesis. We employ an expanded genetic linkage map to demonstrate that cusp number segregates as a gene of major effect, which explains approximately 40% of the phenotypic variance, on cichlid chromosome 5. Furthermore, we examine patterns of Bmp4 expression in early odontogenesis to address and refine predictions of models linking tooth shape and tooth number. Mutations in the Bmp4 cistron do not control tooth shape in this mapping cross. Our data suggest that the evolution of novelty in the cichlid dentition is galvanized by a small number of genetic changes, echoing similar conclusions from recent studies of other vertebrate adaptive morphologies.     

Impacts of Channel Reconstruction on Invertebrate Assemblages in a Restored River

Ecosystem restoration often aims to recreate the physical habitat needed to support a particular life‐stage of a focal species. For example, river channel reconstruction, a common restoration practice along the Pacific coast, is typically used to enhance spawning habitat for adult Chinook salmon, a species experiencing large population declines. These restoration efforts rarely consider, however, that altering spawning habitat could have indirect effects on other life‐stages, such as juveniles, which might occur if, e.g. reconstruction alters the benthic food web. To determine how channel reconstruction impacts benthic macroinvertebrates, juvenile Chinook's primary prey, we conducted two studies at a restoration site in the Merced River, California. We asked (1) has gravel enhancement altered invertebrate assemblages in the restored reach compared with an unrestored reach? and, if so, (2) can shifts in the invertebrate community be explained by increased substrate mobility and by reduced heterogeneity that results from restoration? We show that invertebrate abundance and biomass were lower in the restored reach and that these changes were accompanied by a shift from dominance by filter‐feeding caddisflies (Hydropsyche) in the unrestored reach to grazing mayflies (Baetis) in the restored reach. Using an in situ manipulation, we demonstrated that this trend was driven by increased substrate mobility that reduces the abundance of Hydropsyche and by decreased substrate heterogeneity that reduces the abundance of Baetis. Our studies suggest that geomorphic changes typical of reconstructed rivers can alter food webs in ways that may have important implications for supporting the focal species of restoration efforts.