Selection for evasive mimicry imposed by an arthropod predator.

It has long been hypothesized that a species that is relatively easy to catch by predators may face selection to resemble a species that is harder to catch. Several experiments using avian predators have since supported this 'evasive mimicry' hypothesis. However, the sudden movement of artificial evasive prey in each of the above experiments may have startled the predators, generating an avoidance response unrelated to difficulty of capture. Additionally in the above experiments the catchability of prey was all or nothing, while in nature predators may occasionally catch evasive prey or fail to catch slower species, which might inhibit learning. Here, using mantids as predators, we conducted an experimental test of the evasive mimicry hypothesis that circumvents these limitations, using live painted calyptrate flies with modified evasive capabilities as prey. We found that mantids readily learned to avoid pursuing the more evasive prey types. Warning signals based on evasiveness and their associated mimicry may be widespread phenomena in nature. These findings not only further support its plausibility but demonstrate that even arthropod predators can select for it.

Evolutionary transitions from camouflage to aposematism: Hidden signals play a pivotal role.

The initial evolution of warning signals in unprofitable prey, termed aposematism, is often seen as a paradox because any new conspicuous mutant would be easier to detect than its cryptic conspecifics and not readily recognized by naïve predators as defended. One possibility is that permanent aposematism first evolved through species using hidden warning signals, which are only exposed to would-be predators on encounter. Here, we present a large-scale analysis of evolutionary transitions in amphibian antipredation coloration and demonstrate that the evolutionary transition from camouflage to aposematism is rarely direct but tends to involve an intermediary stage, namely cryptic species that facultatively reveal conspicuous coloration. Accounting for this intermediate step can resolve the paradox and thereby advance our understanding of the evolution of aposematism.

A synthesis of deimatic behaviour.

Deimatic behaviours, also referred to as startle behaviours, are used against predators and rivals. Although many are spectacular, their proximate and ultimate causes remain unclear. In this review we aim to synthesise what is known about deimatic behaviour and identify knowledge gaps. We propose a working hypothesis for deimatic behaviour, and discuss the available evidence for the evolution, ontogeny, causation, and survival value of deimatic behaviour using Tinbergen's Four Questions as a framework. Our overarching aim is to direct future research by suggesting ways to address the most pressing questions in this field.

The anti-predation benefit of flash displays is related to the distance at which the prey initiates its escape.

Flash behaviour is widespread in the animal kingdom and describes the exposure of a hidden conspicuous signal as an animal flees from predators. Recent studies have demonstrated that the signal can enhance survivorship by leading pursuing predators into assuming the flasher is also conspicuous at rest. Naturally, this illusion will work best if potential predators are ignorant of the flasher's resting appearance, which could be achieved if the prey flees while the predator is relatively far away. To test this hypothesis, we compared the survival of flashing and non-flashing computer-generated prey with different flight initiation distances (FIDs) using humans as model predators. This experiment found that flash displays confer a survivorship advantage only to those prey with a long FID. A complementary phylogenetic analysis of Australian bird species supports these results: after controlling for body size, species with putative flashing signals had longer FIDs than those without. Species with putative flashing signals also tended to be larger, as demonstrated in other taxa. The anti-predation benefit of flash displays is therefore related to the nature of escape behaviour. Since birds with hidden signals tend to flee at a distance, the flash display here is unlikely to function by startling would-be predators.

Consistent Associations between Body Size and Hidden Contrasting Color Signals across a Range of Insect Taxa.

While there have been a number of recent advances in our understanding of the evolution of animal color patterns, much of this work has focused on color patterns that are constantly displayed. However, some animals hide functional color signals and display them only transiently through behavioral displays. These displays are widely employed as a secondary defense following detection when fleeing (flash display) or when stationary (deimatic display). Yet if displays of hidden colors are so effective in deterring predation, why have not all species evolved them? An earlier study suggested that the hidden antipredatory color signals in insects are more likely to have evolved in species with large size because either (or both) (i) large cryptic prey are more frequently detected and pursued or (ii) hidden color signals in large prey are more effective in deterring predation than in small prey. These arguments should apply universally to any prey that use hidden signals, so the association between large size and hidden contrasting color signals should be evident across diverse groups of prey. In this study, we tested this prediction in five different groups of insects. Using phylogenetically controlled analysis to elucidate the relationship between body size and color contrast between forewings and hind wings, we found evidence for the predicted size-color contrast associations in four different groups of insects, namely, Orthoptera, Phasmatidae, Mantidae, and Saturniidae, but not in Sphingidae. Collectively, our study indicates that body size plays an important role in explaining variation in the evolution of hidden contrasting color signals in insects.

Mitochondrial drug targets in apicomplexan parasites.

In evolutionary terms, mitochondria in apicomplexan parasites appear to be "relicts-in-the-making": they possess the smallest mitochondrial genomes known, encoding only three proteins, and in one genus, Cryptosporidium, the genome is eliminated altogether. Several features of mitochondrial physiology provide validated or potential targets for antiparasitic drugs. Atovaquone, a broad spectrum antiparasitic drug, selectively inhibits mitochondrial electron transport at the cytochrome bc(1) complex and collapses mitochondrial membrane potential. Recent investigations using model systems provide important insights into the mechanism of action for this drug, which may prove valuable for development of other selective inhibitors of mitochondrial electron transport. Although mitochondria do not appear to be a source of ATP during the erythrocytic stages in Plasmodium species, they do serve other critical functions, including the assembly of iron-sulfur clusters and various other biosynthetic processes depending on the species. To serve these metabolic functions, parasites need to maintain the apparatus for mitochondrial genome replication, repair, recombination, transcription, and translation, components of which are encoded in the nucleus and imported into the mitochondrion. Several unusual aspects of the components of this apparatus are coming to light through genome sequence analyses, and could provide potential targets for antiparasitic drug discovery and development.

Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast.

PCR product-based gene disruption has greatly accelerated molecular analysis of Saccharomyces cerevisiae. This approach involves amplification of a marker gene (e.g., URA3) including its flanking regulatory (promoter and polyadenylation) regions using primers that include at their 5' ends about 50 bases of homology to the targeted gene. Unfortunately, this approach has proved less useful in organisms with higher rates of non-homologous recombination; e.g., in the yeast Candida glabrata, desired recombinants represent < or =2% of transformants. We modified the PCR-based approach by eliminating marker-flanking regions and precisely targeting recombination such that marker expression depends on the regulatory sequences of the disrupted gene. Application of this promoter-dependent disruption of genes (PRODIGE) method to three C. glabrata genes (SLT2, LEM3, and PDR1) yielded desired recombinants at frequencies of 20, 31, and 11%, the latter representing a weakly expressed gene. For Candida albicans LEM3 and RHO1, specificity was 79-95% for one or both alleles, >sixfold higher than the published results with conventional PCR-based gene disruption. All 5 C. glabrata and C. albicans mutants had predicted phenotypes of calcofluor hypersensitivity (slt2Delta and RHO1/rho1Delta), cycloheximide hypersensitivity (pdr1Delta), or miltefosine resistance (lem3Delta and lem3Delta/lem3Delta). PRODIGE application to the S. cerevisiae PDR5 gene in strains with and without the Pdr1-Pdr3 transcriptional activators of this gene confirmed that transformant yield and growth rate depend on promoter strength. Using this PDR5 promoter-URA3 recombinant, we further demonstrate a simple extension of the method that yields regulatory mutants via 5-fluoroorotic acid selection. PRODIGE warrants testing in other yeast, molds, and beyond.

Histone H3 phosphorylation can promote TBP recruitment through distinct promoter-specific mechanisms.

Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Previously, we showed that histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase regulates INO1 gene activation in part via Gcn5/SAGA complex-mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. Here we examine the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA-bound activators, and in its role in modifying histones and recruiting TATA-binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10's role in recruiting SAGA may substitute for recruitment by DNA-bound activator. Our results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter-specific requirements.

Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing.

Low levels of histone covalent modifications are associated with gene silencing at telomeres and other regions in the yeast S. cerevisiae. Although the histone deacetylase Sir2 maintains low acetylation, mechanisms responsible for low H2B ubiquitylation and low H3 methylation are unknown. Here, we show that the ubiquitin protease Ubp10 targets H2B for deubiquitylation, helping to localize Sir2 to the telomere. Ubp10 exhibits reciprocal Sir2-dependent preferential localization proximal to telomeres, where Ubp10 serves to maintain low H2B Lys123 ubiquitylation in this region and, through previously characterized crosstalk, maintains low H3 Lys4 and Lys79 methylation in a slightly broader region. Ubp10 is also localized to the rDNA locus, a second silenced domain, where it similarly maintains low histone methylation. We compare Ubp10 to Ubp8, the SAGA-associated H2B deubiquitylase involved in gene activation, and show that telomeric and gene-silencing functions are specific to Ubp10. Our results suggest that these H2B-deubiquitylating enzymes have distinct genomic functions.

H2B ubiquitylation and de-ubiquitylation in gene activation.

Previous models for the role of histone modifications suggest that adding and removing modifications, such as acetylation/deacetylation in gene regulation, are functionally antagonistic. We have investigated a transcriptional role of H2B C-terminal ubiquitylation and de-ubiquitylation in Saccharomyces cerevisiae. H2B ubiquitylation is required for optimal transcription of SUC2 and GAL1 genes. The ubiquitin hydrolase Ubp8 is a stable component of SAGA but not ADA complexes, and is not required for overall integrity of SAGA. Biochemical and genetic evidence indicates that Ubp8 targets H2B for deubiquitylation. The dynamic balance of H2B ubiquitylation/deubiquitylation is important for GAL1 transcription since either substitution of the ubiquitylation site in H2B (Lys123), or loss of Ubp8, lowers GAL1 expression. Further, this balance of ubiquitylation appears to set the balance of histone H3 methylation at Lys4 relative to Lys36. Thus, unlike acetylation/deacetylation whose functions are mutually opposing, both ubiquitylation and de-ubiquitylation are required for gene activation. These results suggest that ubiquitylation of histones has a unique role among histone modifications, possibly to orchestrate an ordered pathway of chromatin alterations.

Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B.

Covalent modifications of the histone N tails play important roles in eukaryotic gene expression. Histone acetylation, in particular, is required for the activation of a subset of eukaryotic genes through the targeted recruitment of histone acetyltransferases. We have reported that a histone C tail modification, ubiquitylation of H2B, is required for optimal expression of several inducible yeast genes, consistent with a role in transcriptional activation. H2B was shown to be ubiquitylated and then deubiquitylated at the GAL1 core promoter following galactose induction. We now show that the Rad6 protein, which catalyzes monoubiquitylation of H2B, is transiently associated with the GAL1 promoter upon gene activation, and that the period of its association temporally overlaps with the period of H2B ubiquitylation. Rad6 promoter association depends on the Gal4 activator and the Rad6-associated E3 ligase, Bre1, but is independent of the histone acetyltransferase, Gcn5. The SAGA complex, which contains a ubiquitin protease that targets H2B for deubiquitylation, is recruited to the GAL1 promoter in the absence of H2B ubiquitylation. The data suggest that Rad6 and SAGA function independently during galactose induction, and that the staged recruitment of these two factors to the GAL1 promoter regulates the ubiquitylation and deubiquitylation of H2B. We additionally show that both Rad6 and ubiquitylated H2B are absent from two regions of transcriptionally silent chromatin but present at genes that are actively transcribed. Thus, like histone H3 lysine 4 and lysine 79 methylation, two modifications that it regulates, Rad6-directed H2B ubiquitylation defines regions of active chromatin.

Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8.

Gene activation and repression regulated by acetylation and deacetylation represent a paradigm for the function of histone modifications. We provide evidence that, in contrast, histone H2B monoubiquitylation and its deubiquitylation are both involved in gene activation. Substitution of the H2B ubiquitylation site at Lys 123 (K123) lowered transcription of certain genes regulated by the acetylation complex SAGA. Gene-associated H2B ubiquitylation was transient, increasing early during activation, and then decreasing coincident with significant RNA accumulation. We show that Ubp8, a component of the SAGA acetylation complex, is required for SAGA-mediated deubiquitylation of histone H2B in vitro. Loss of Ubp8 in vivo increased both gene-associated and overall cellular levels of ubiquitylated H2B. Deletion of Ubp8 lowered transcription of SAGA-regulated genes, and the severity of this defect was exacerbated by codeletion of the Gcn5 acetyltransferase within SAGA. In addition, disruption of either ubiquitylation or Ubp8-mediated deubiquitylation of H2B resulted in altered levels of gene-associated H3 Lys 4 methylation and Lys 36 methylation, which have both been linked to transcription. These results suggest that the histone H2B ubiquitylation state is dynamic during transcription, and that the sequence of histone modifications helps to control transcription.

ROX1 and ERG regulation in Saccharomyces cerevisiae: implications for antifungal susceptibility.

Yeasts respond to treatment with azoles and other sterol biosynthesis inhibitors by upregulating the expression of the ERG genes responsible for ergosterol production. Previous studies on Saccharomyces cerevisiae implicated the ROX1 repressor in ERG regulation. We report that ROX1 deletion resulted in 2.5- to 16-fold-lower susceptibilities to azoles and terbinafine. In untreated cultures, ERG11 was maximally expressed in mid-log phase and expression decreased in late log phase, while the inverse was observed for ROX1. In azole-treated cultures, ERG11 upregulation was preceded by a decrease in ROX1 RNA. These inverse correlations suggest that transcriptional regulation of ROX1 is an important determinant of ERG expression and hence of azole and terbinafine susceptibilities.

Antifungal activity in Saccharomyces cerevisiae is modulated by calcium signalling.

The most important group of antifungals is the azoles (e.g. miconazole), which act by inhibiting lanosterol demethylase in the sterol biosynthesis pathway. Azole activity can be modulated through structural changes in lanosterol demethylase, altered expression of its gene ERG11, alterations in other sterol biosynthesis enzymes or altered expression of multidrug transporters. We present evidence that azole activity versus Saccharomyces cerevisiae is also modulated by Ca2+-regulated signalling. (i) Azole activity was reduced by the addition of Ca2+. Conversely, azole activity was enhanced by the addition of Ca2+ chelator EGTA. (ii) Three structurally distinct inhibitors (fluphenazine, calmidazolium and a W-7 analogue) of the Ca2+-binding regulatory protein calmodulin enhanced azole activity. (iii) Two structurally distinct inhibitors (cyclosporin and FK506) of the Ca2+-calmodulin-regulated phosphatase calcineurin enhanced azole activity. (iv) Strains in which the Ca2+ binding sites of calmodulin were eliminated and strains in which the calcineurin subunit genes were disrupted demonstrated enhanced azole sensitivity; conversely, a mutant with constitutively activated calcineurin phosphatase demonstrated decreased azole sensitivity. (v) CRZ1/TCN1 encodes a transcription factor regulated by calcineurin phosphatase; its disruption enhanced azole sensitivity, whereas its overexpression decreased azole sensitivity. All the above treatments had comparable effects on the activity of terbinafine, an inhibitor of squalene epoxidase within the sterol biosynthesis pathway, but had little or no effect on the activity of drugs with unrelated targets. (vi) Treatment of S. cerevisiae with azole or terbinafine resulted in transcriptional upregulation of genes FKS2 and PMR1 known to be Ca2+ regulated. A model to explain the role of Ca2+-regulated signalling in azole/terbinafine tolerance is proposed.