Cryptic Variation in Morphological Evolution: HSP90 as a Capacitor for Loss of Eyes in Cavefish

In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting

Fundamental research questions in subterranean biology

Five decades ago, a landmark paper inSciencetitledThe Cave Environmentheralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field.Peer reviewe

Inhibition of Competence Development, Horizontal Gene Transfer and Virulence in Streptococcus pneumoniae by a Modified Competence Stimulating Peptide

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae

The Streptococcus pneumoniae Competence Regulatory System Influences Respiratory Tract Colonization ▿

The Streptococcus pneumoniae ComDE two-component signaling system controls the development of genetic competence in the bacterium and affects virulence in models of pneumonia and bacteremia. We have investigated the impact of the competence pathway during colonization of the nasopharynx, the principal ecological niche of the pneumococcus. Previous work showed that deletion of the pneumococcal CiaRH signaling system inhibited colonization and increased expression of genes required for competence. We anticipated that signaling by the competence pathway might similarly reduce carriage. Consistent with this expectation, a comE deletion that blocked transformation increased colonization fitness such that the mutant outcompeted the wild type in an infant rat model of asymptomatic carriage. Deletion of comD—immediately upstream of comE and likewise required for competence—similarly increased colonization fitness if the orientation of the antibiotic resistance cassette inserted into the comD locus was such that it reduced transcription of comE. However, an alternative comD deletion mutation that caused an increase in comE transcription impaired colonization instead. Activation of the competence system through a comE(D143Y) mutation did not affect colonization, but an inability to secrete the competence-stimulating peptide due to deletion of comAB produced a density-dependent reduction in colonization fitness. These results suggest a model in which signaling by the unactivated form of ComE reduces colonization fitness compared to that of bacteria in which it is either activated or absent entirely, with the most substantial fitness gain accompanying deletion of comE. This observation demonstrates that the pneumococcus incurs a substantial fitness cost in order to retain a functional competence regulatory system

Percentage of surviving embryos at 9–12 hours post injection.

<p>The number of total and surviving embryos were calculated for control and injected embryos in the morning after three sets of injections for each TALEN pair. mRNA is the total amount of mRNA injected per embryo. Percentage survival was calculated by dividing the number of living embryos by the total number of embryos. Survival of embryos for each trial can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119370#pone.0119370.s002" target="_blank">S1 Table</a>.</p><p>Percentage of surviving embryos at 9–12 hours post injection.</p

Analysis of mutagenesis in <i>oca2</i> TALEN-injected F₀ fish.

<p>A. Genotyping gel of uninjected control and embryos injected with 400 pg and 600 pg of the <i>oca2</i> TALEN targeting exon 9. A portion of the <i>oca2</i> genomic region was amplified by PCR around exon 9 from 10 individual control embryos or individual injected embryos, and half of the PCR product was digested (D) with <i>Bsr</i>I. Wild type DNA digests completely with <i>Bsr</i>I whereas alleles with mutations induced by the TALEN pair are resistant to restriction digest, indicated by the arrow. The percentage of mutant alleles is indicated below the band. Note that some mutant alleles may not have lost the restriction site, and may still be sensitive to restriction digest. B. Genotyping gel of uninjected control and embryos injected with 800 pg of the <i>oca2</i> TALEN targeting exon 9. A portion of the <i>oca2</i> genomic region was amplified by PCR around exon 9 from individual control embryos or individual injected embryos and half of the PCR product was digested (D) with <i>Bsr</i>I. Wild type DNA digests completely with <i>Bsr</i>I, whereas alleles with mutations induced by the TALEN pair are resistant to restriction digestion, as indicated by the arrow. The percentage of mutant alleles is indicated below the band. Note that some mutant alleles may not have lost the restriction site, and may still be sensitive to restriction digest. C. Sequence of a wild type surface fish exon 9 and of five clones from the restriction enzyme resistant band from an <i>oca2</i>-injected individual. The dashed lines indicate missing nucleotides, and the lower case letters indicate mismatches. The total number of base pairs less than the wild type sequence is indicated to the right of each clone. Note that two of the five clones were identical. D. Genotyping gel of uninjected control and embryos injected with 400 pg total mRNA of the <i>oca2</i> TALENs targeting either side of exon 21. A portion of the <i>oca2</i> genomic region was amplified by PCR around exon 21 from 10 pooled control embryos or individual injected embryos. A band approximately 100 base pairs lower than the wild type band (arrow) indicates mutant alleles in injected embryos. The percentage of mutant alleles is indicated below the band. E. Sequence of a wild type surface fish around exon 21 and of five clones from 100 base pair smaller band from an <i>oca2</i>-injected individual. The dashed lines indicate missing nucleotides. The lower case letters are intron sequence, and the upper case letters are exon sequence. Sequence between the TALEN pairs is not shown and is indicated by the three dots. The total number of base pairs less than the wild type sequence is indicated to the right of each clone. Note that the starred clone contains a large deletion (321 bp) and an insertion (209 bp). F. Sequence of a wild type surface fish exon 9 and of four mutant clones from the restriction enzyme resistant band from pools of F₁s from a cross between an <i>oca2</i> exon 9 400 pg-injected F₀ individual and a wildtype surface fish. The dashed lines indicate missing nucleotides, and the red letters indicate mismatches. The total number of base pairs less than the wild type or the mutation compared to wildtype sequence is indicated to the right of each clone. Note that two of the four clones were identical.</p

Analysis of mutagenesis in <i>mc1r</i> TALEN F₀ fish.

<p>A. Genotyping gel of uninjected control and 800 pg-injected embryos. A portion of the <i>mc1r</i> genomic region was amplified by PCR from individual embryos, and half of the PCR product was digested (D) with <i>Bss</i>SI. Wild type DNA digests completely with <i>Bss</i>SI whereas alleles with mutations induced by the TALEN pair are resistant to restriction digest, indicated by the arrow. The percentage of mutant alleles is indicated below the band. Note that some mutant alleles may not have lost the restriction site and may still be sensitive to restriction digest. B. Sequence of a wild type surface fish and sequence of four clones from the restriction enzyme resistant band from a <i>mc1r</i>-injected individual. The dashed lines indicate missing nucleotides, the lower case letters indicate mismatches, and the sequence below the last clone is additional nucleotide sequence. The total number of base pairs more or less than the wild type sequence is indicated to the right of each clone.</p

<i>mc1r</i> and <i>oca2</i> TALEN targeting design.

<div><p>A. Diagram of the <i>mc1r</i> annotated coding sequence with the location and sequences of the Pachón, Japonés and Yerbaniz mutations highlighted. The transmembrane domains are indicated in light gray. The two base pair deletion in Pachón is indicated by dashed lines. The single nucleotide change in the Japonés and Yerbaniz populations is in red. The sequence targeted by the TALEN is indicated below. The TALEN 1 binding site is in green and the TALEN 2 binding site is in blue. The spacer region is gray. The underlined sequence is the <i>Bss</i>SI restriction enzyme recognition sequence used for genotyping. The gene structure is based on the <i>Astyanax</i> genome sequence database and Gross et al. 2009 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119370#pone.0119370.ref019" target="_blank">19</a>].</p> <p>B. Diagram of the <i>oca2</i> gene. Boxes indicate exons and lines indicate introns. The empty boxes are UTR and the closed boxes are coding sequence. The slanted lines indicate a region of the genome left out because the distance is unknown. Note that the small scale of the figure resulted in some exons not being to scale and the size of some introns being so small that they appear to be one continuous exon. The exon 24 Pachón and the exon 21 Molino deletions are indicated in red. The amount of intronic sequence deleted in these populations is currently unknown. TALENs were designed targeting exon 9 and the either end of exon 21. The TALEN 1 binding sites are in green and the TALEN 2 binding sites are in blue. The spacer regions are gray. The underlined sequence in the exon 9 TALEN is the <i>Bsr</i>I restriction enzyme recognition sequence used for genotyping. The exon sequence is capitalized and the intronic sequence in the two exon 21 TALENs is lower case. The genomic structure is based on the <i>Astyanax</i> genome sequence database and Protas et al. 2006 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119370#pone.0119370.ref017" target="_blank">17</a>]. Both gene structures were generated using <a href="http://wormweb.org/exonintron" target="_blank">http://wormweb.org/exonintron</a> and then modified.</p></div

Analysis of pigmentation in <i>oca2</i>-injected F0s.

<p>A. Pigmentation in an uninjected surface fish. B. Close up of the dorsal region of the uninjected surface fish from A. C. Pigmentation in a 400 pg <i>oca2</i> exon 9 injected F0 surface fish. D. Close up of pigmentation patch lacking melanin pigmentation from panel C.</p