Chemical communication of queen supergene status in an ant

Traits of interest to evolutionary biologists often have complex genetic architectures, the nature of which can confound traditional experimental study at single levels of analysis. In the fire ant Solenopsis invicta, the presence of a Mendelian ‘supergene’ is both necessary and sufficient to induce a shift in a fundamental property of social organization, from single‐queen (monogyne) to multiple‐queen (polygyne) colonies. This selfish genetic element, termed the Social b (Sb) supergene, contains > 600 genes that collectively promote its fitness by inducing the characteristic polygyne syndrome, in part by causing polygyne workers to accept only queens bearing the Sb element (a behaviour termed ‘worker Sb discrimination’). Here, we employ a newly developed behavioural assay to reveal that polygyne workers, many of which bear the Sb element, employ chemical cues on the cuticle of queens to achieve worker Sb discrimination, but we found no evidence for such pheromonally mediated worker Sb discrimination in monogyne workers, which universally lack the Sb element. This polygyne worker Sb discrimination was then verified through a ‘green beard’ effect previously described in this system. We thus have demonstrated that the Sb element is required both for production of relevant chemical cues of queens and for expression of the behaviours of workers that collectively result in worker Sb discrimination. This information fills a critical gap in the map between genotype and complex phenotype in S. invicta by restricting the search for candidate genes and molecules involved in producing this complex social trait to factors associated with the Sb element itself.     

Are behavioural responses to predation cues linked across life cycle stages?

1. Prey organisms can perceive cues to predation hazard and adopt low‐risk behaviours to increase survival. Animals with complex life cycles, such as insects, can exhibit such anti‐predatory behaviours in multiple life stages. 2. Cues to predation risk may induce ovipositing females to choose habitats with low predation risk. Cues to predation risk may also induce larvae to adopt facultative behaviours that reduce risk of predation. 3. One hypothesis postulates that anti‐predation behaviours across adult and larval stages may be negatively associated because selection for effective anti‐predator behaviour in one stage leads to reduced selection for avoidance of predators in other stages. An alternative hypothesis suggests that selection by predation favours multi‐component defences, with both avoidance of oviposition and facultative adoption of low‐risk behaviours by larvae. 4. Laboratory and field experiments were used to determine whether defensive responses of adult and larval mosquitoes are positively or negatively associated. The study tested effects of waterborne cues from predatory Toxorhynchites theobaldi on oviposition choices and larval behaviours of three of its common prey: Culex mollis, Limatus durhamii and Aedes albopictus. 5. Culex mollis shows strong anti‐predator responses in both life stages, consistent with the hypothesis of a multi‐component behavioural defence. The other two species showed no detectable responses to waterborne predator cues in either adult or larval stages. Larvae of these unresponsive species were significantly more vulnerable to this predator than was C. mollis. 6. For these mosquitoes, species appear either to have been selected for multi‐component defences against predation or to act in ways that could be called predator‐naïve.     

Novel high‐throughput screens of Anopheles gambiae odorant receptors reveal candidate behaviour‐modifying chemicals for mosquitoes

Despite many decades of multilateral global efforts, a significant portion of the world population continues to be plagued with one or more mosquito‐vectored diseases. These include malaria and filariasis, as well as numerous arboviral‐associated illnesses, such as dengue and yellow fevers. The dynamics of disease transmission by mosquitoes is complex, and involves both vector competence and vectorial capacity. One area of intensive effort is the study of chemosensory‐driven behaviours in the malaria vector mosquito Anopheles gambiae Giles, the modulation of which is likely to provide opportunities for disease reduction. In this context, recent studies characterize a large divergent family of An. gambiae odorant receptors (AgORs) that play critical roles in olfactory signal transduction. This work facilitates high‐throughput, cell‐based calcium mobilization screens of AgOR‐expressing human embryonic kidney cells identifying a large number of conventional AgOR ligands, as well as the first nonconventional Orco (olfactory receptor co‐receptor) family agonist. As such, ligand‐mediated modulation serves as a proof‐of‐concept demonstration that AgORs represent viable targets for high‐throughput screening and for the eventual development of behaviour‐modifying olfactory compounds. Such attractants or repellents could foster malaria reduction programmes.     

Identification of a Marine Cyanophage in a Protist Single‐cell Metagenome Assembly

Analysis of microbial biodiversity is hampered by a lack of reference genomes from most bacteria, viruses, and algae. This necessitates either the cultivation of a restricted number of species for standard sequencing projects or the analysis of highly complex environmental DNA metagenome data. Single‐cell genomics (SCG) offers a solution to this problem by constraining the studied DNA sample to an individual cell and its associated symbionts, prey, and pathogens. We used SCG to study marine heterotrophic amoebae related to Paulinella ovalis (A. Wulff) P.W. Johnson, P.E. Hargraves & J.M. Sieburth (Rhizaria). The genus Paulinella is best known for its photosynthetic members such as P. chromatophora Lauterborn that is the only case of plastid primary endosymbiosis known outside of algae and plants. Here, we studied the phagotrophic sister taxa of P. chromatophora that are related to P. ovalis and found one SCG assembly to contain α‐cyanobacterial DNA. These cyanobacterial contigs are presumably derived from prey. We also uncovered an associated cyanophage lineage (provisionally named phage PoL_MC2). Phylogenomic analysis of the fragmented genome assembly suggested a minimum genome size of 200 Kbp for phage PoL_MC2 that encodes 179 proteins and is most closely related to Synechococcus phage S‐SM2. For this phage, gene network analysis demonstrates a highly modular genome structure typical of other cyanophages. Our work demonstrates that SCG is a powerful approach for discovering algal and protist biodiversity and for elucidating biotic interactions in natural samples.     

Variability in the Assessment of Snake Predation Risk by Liolaemus Lizards

The ability to assess and respond to predation risk is a strong selective force. Detection of predators is carried out by one or more sensory modalities, but the use of chemoreception has significant advantages. This study examines the chemosensorial assessment of snake predation risk and corresponding behaviours in different species and populations of Liolaemus lizards naturally exposed to different levels of snake predation pressure. The species studied were sympatric (Liolaemus lemniscatus), parapatric (L. nigroviridis) and allopatric (L. fitzgeraldi) to the saurophagous snake, Philodryas chamissonis. Additionally, two populations of L. lemniscatus from areas differing in snake densities were compared. Chemo‐assessment of predation risk was determined by comparing the number of tongue‐flicks (TF) and the behavioural responses of lizards submitted to three treatments (with semiochemicals of snake, conspecifics, and without semiochemicals – control). The results suggest that Liolaemus lizards can chemo‐assess snake predation risk, but this was modulated by the predation pressure experienced by lizards in their natural habitats. When exposed to snake semiochemicals, the sympatric prey showed less chemical exploratory behaviour (i.e. lower number of TF), a higher frequency of antipredator behaviours that would reduce its detection by a predator, and did not show the behaviour triggered by conspecific semiochemicals. The parapatric prey showed similar number of TF across different treatments, suggesting an absence of recognition of snake semiochemicals; however, it did show antipredatory behaviours when confronted with snake semiochemicals. The allopatric prey showed similar behaviour in all treatments. Both populations of the sympatric species, L. lemniscatus, showed a similar ability to detect predation risk when confronted with snake semiochemicals (i.e. similar number of TF), but antipredatory behaviours were diminished, and marking behaviours were present in the population subject to lower predation pressure. Relaxed predation pressure from a predator that releases and detects semiochemicals had similar consequences at species and population levels.     

An ADP‐ribosyltransferase Alt of bacteriophage T4 negatively regulates the Escherichia coli MazF toxin of a toxin–antitoxin module

Prokaryotic toxin–antitoxin (TA) systems are linked to many roles in cell physiology, such as plasmid maintenance, stress response, persistence and protection from phage infection, and the activities of toxins are tightly regulated. Here, we describe a novel regulatory mechanism for a toxin of Escherichia coli TA systems. The MazF toxin of MazE‐MazF, which is one of the best characterized type II TA systems, was modified immediately after infection with bacteriophage T4. Mass spectrometry demonstrated that the molecular weight of this modification was 542 Da, corresponding to a mono‐ADP‐ribosylation. This modification disappeared in cells infected with T4 phage lacking Alt, which is one of three ADP‐ribosyltransferases encoded by T4 phage and is injected together with phage DNA upon infection. In vivo and in vitro analyses confirmed that T4 Alt ADP‐ribosylated MazF at an arginine residue at position 4. Finally, the ADP‐ribosylation of MazF by Alt resulted in the reduction of MazF RNA cleavage activity in vitro, suggesting that it may function to inactivate MazF during T4 infection. This is the first example of the chemical modification of an E. coli toxin in TA systems to regulate activity.     

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Stability analysis of an artificial biomolecular oscillator with non-cooperative regulatory interactions

Oscillators are essential to fuel autonomous behaviours in molecular systems. Artificial oscillators built with programmable biological molecules such as DNA and RNA are generally easy to build and tune, and can serve as timers for biological computation and regulation. We describe a new artificial nucleic acid biochemical reaction network, and we demonstrate its capacity to exhibit oscillatory solutions. This network can be built in vitro using nucleic acids and three bacteriophage enzymes, and has the potential to be implemented in cells. Numerical simulations suggest that oscillations occur in a realistic range of reaction rates and concentrations.     

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication,SirA, with domain I of DnaA

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA‐interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N‐terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α‐helical with predominantly apolar side‐chains packing in a hydrophobic interface. Site‐directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA‐interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.     

Soil microbiome responses to the short‐term effects of Amazonian deforestation

Slash‐and‐burn clearing of forest typically results in increase in soil nutrient availability. However, the impact of these nutrients on the soil microbiome is not known. Using next generation sequencing of 16S rRNA gene and shotgun metagenomic DNA, we compared the structure and the potential functions of bacterial community in forest soils to deforested soils in the Amazon region and related the differences to soil chemical factors. Deforestation decreased soil organic matter content and factors linked to soil acidity and raised soil pH, base saturation and exchangeable bases. Concomitant to expected changes in soil chemical factors, we observed an increase in the alpha diversity of the bacterial microbiota and relative abundances of putative copiotrophic bacteria such as Actinomycetales and a decrease in the relative abundances of bacterial taxa such as Chlamydiae, Planctomycetes and Verrucomicrobia in the deforested soils. We did not observe an increase in genes related to microbial nutrient metabolism in deforested soils. However, we did observe changes in community functions such as increases in DNA repair, protein processing, modification, degradation and folding functions, and these functions might reflect adaptation to changes in soil characteristics due to forest clear‐cutting and burning. In addition, there were changes in the composition of the bacterial groups associated with metabolism‐related functions. Co‐occurrence microbial network analysis identified distinct phylogenetic patterns for forest and deforested soils and suggested relationships between Planctomycetes and aluminium content, and Actinobacteria and nitrogen sources in Amazon soils. The results support taxonomic and functional adaptations in the soil bacterial community following deforestation. We hypothesize that these microbial adaptations may serve as a buffer to drastic changes in soil fertility after slash‐and‐burning deforestation in the Amazon region.     

A detailed analysis of the leaf rolling mutant sll2 reveals complex nature in regulation of bulliform cell development in rice (Oryza sativa L.)

Bulliform cells are large, thin‐walled and highly vacuolated cells, and play an important role in controlling leaf rolling in response to drought and high temperature. However, the molecular mechanisms regulating bulliform cell development have not been well documented. Here, we report isolation and characterisation of a rice leaf‐rolling mutant, named shallot‐like 2 (sll2). The sll2 plants exhibit adaxially rolled leaves, starting from the sixth leaf stage, accompanied by increased photosynthesis and reduced plant height and tiller number. Histological analyses showed shrinkage of bulliform cells, resulting in inward‐curved leaves. The mutant is recessive and revertible at a rate of 9%. The leaf rolling is caused by a T‐DNA insertion. Cloning of the insertion using TAIL‐PCR revealed that the T‐DNA was inserted in the promoter region of LOC_Os07 g38664. Unexpectedly, the enhanced expression of LOC_Os07 g38664 by the 35S enhancer in the T‐DNA is not responsible for the leaf rolling phenotype. Further, the enhancer also exerted a long‐distance effect, including up‐regulation of several bulliform cell‐related genes. sll2 suppressed the outward leaf rolling of oul1 in the sll2oul1 double mutant. We conclude that leaf rolling in sll2 could be a result of the combined effect of multi‐genes, implying a complex network in regulation of bulliform cell development.     

Consequences of temperature and temperature variability on swimming activity,group structure,and predation of endangered delta smelt

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Mycobacterium tuberculosis UvrB forms dimers in solution and interacts with UvrA in the absence of ligands

During its life cycle Mycobacterium tuberculosis (MTB) must face a variety of environmental and endogenous physical and chemical stresses that could produce genotoxic damage. However, MTB possesses efficient systems to counteract the harmful effects of DNA‐damaging assaults. The nucleotide excision repair (NER) is a highly conserved multi‐enzymatic cascade that is initiated by the concerted action of three core proteins, that is UvrA, UvrB, and UvrC. Although the functional roles of these enzymes are well characterized, the intra‐pathway coordination of the NER components and the dynamics of their association is still a matter of debate. In the presented study, we analyzed the hydrodynamic properties and the oligomeric state of the MTB UvrB protein (MtUvrB) that we expressed and purified to homogeneity in a tag‐free form. Our results show that, differently to what has been previously observed for the His‐tagged version of the protein, MtUvrB forms dimers in solution, which are characterized by an elongated shape, as determined by small‐angle X‐ray scattering analysis. Moreover, to gain insights into the mycobacterial UvrA/UvrB lesion sensing/tracking complex we adopted a size‐exclusion chromatography‐based approach, revealing that the two proteins interact in the absence of ligands, leading to the assembling of A₂B₂ hetero‐tetramers in solution. Surface plasmon resonance analysis showed that the dissociation constant of the MtUvrA/MtUvrB complex falls in the low micromolar range that could represent the basis for a fine modulation of the complex architecture accompanying the multi‐step DNA repair activity of mycobacterial NER.     

A pair of receptor‐like kinases is responsible for natural variation in shoot growth response to mannitol treatment in Arabidopsis thaliana

Growth is a complex trait that adapts to the prevailing conditions by integrating many internal and external signals. Understanding the molecular origin of this variation remains a challenging issue. In this study, natural variation of shoot growth under mannitol‐induced stress was analyzed by standard quantitative trait locus mapping methods in a recombinant inbred line population derived from a cross between the Col‐0 and Cvi‐0 Arabidopsis thaliana accessions. Cloning of a major QTL specific to mannitol‐induced stress condition led to identification of EGM1 and EGM2, a pair of tandem‐duplicated genes encoding receptor‐like kinases that are potentially involved in signaling of mannitol‐associated stress responses. Using various genetic approaches, we identified two non‐synonymous mutations in the EGM2[Cvi] allele that are shared by at least ten accessions from various origins and are probably responsible for a specific tolerance to mannitol. We have shown that the enhanced shoot growth phenotype contributed by the Cvi allele is not linked to generic osmotic properties but instead to a specific chemical property of mannitol itself. This result raises the question of the function of such a gene in A. thaliana, a species that does not synthesize mannitol. Our findings suggest that the receptor‐like kinases encoded by EGM genes may be activated by mannitol produced by pathogens such as fungi, and may contribute to plant defense responses whenever mannitol is present.     

Synthesis and structure of a new trinuclear nickel(II) complex bridged by N‐[3‐(Dimethylamino)propyl]‐N′‐(2‐hydroxyphenyl)oxamido: in vitro anticancer activities,and reactivities toward DNA and protein

A new trinickel(II) complex bridged by N‐[3‐(dimethylamino)propyl]‐ N ′‐(2‐hydroxylphenyl)oxamido (H₃pdmapo), namely [Ni₃(pdmapo)₂(H₂O)₂]?4CH₃OH, was synthesized and characterized by X‐ray single‐crystal diffraction and other methods. In the molecule, two symmetric cis‐ pdmapo^3? mononickel(II) complexes as a “complex ligand” using the carbonyl oxygen atoms coordinate to the center nickel(II) ion situated on an inversion point. The Ni···Ni distance through the oxamido bridge is 5.2624(4) Å. The center nickel(II) ion and the lateral ones have octahedral and square‐planar coordination geometries, respectively. In the crystal, a three‐dimensional supramolecular network dominated by hydrogen bonds is observed. The reactivity toward DNA/protein bovine serum albumin (BSA) revealed that the complex could interact with herring sperm DNA (HS‐DNA) through the intercalation mode and quench the intrinsic fluorescence of BSA via a static mechanism. The in vitro anticancer activities suggested that the complex is active against the selected tumor cell lines.     

Specific and sensitive detection of Alcaligenes species from an agricultural environment

A quantitative real‐time PCR assay to specifically detect and quantify the genus Alcaligenes in samples from the agricultural environment, such as vegetables and farming soils, was developed. The minimum detection sensitivity was 106 fg of pure culture DNA, corresponding to DNA extracted from two cells of Alcaligenes faecalis. To evaluate the detection limit of A. faecalis, serially diluted genomic DNA from this organism was mixed with DNA extracted from soil and vegetables and then a standard curve was constructed. It was found that Alcaligenes species are present in the plant phytosphere at levels 10²–10⁴ times lower than those in soil. The approach presented here will be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.     

Host‐searching responses to herbivory‐associated chemical information and patch use depend on mating status of female solitary parasitoid wasps

1. In a tritrophic interaction system consisting of plants, herbivores, and their parasitoids, chemicals released from plants after herbivory are known to play important roles for many female parasitoids to find their hosts efficiently. On the plant side, chemical information associated with herbivory can act as an indirect defence by attracting the natural enemies of the host herbivores. 2. However, mated and virgin females of haplodiploid parasitoids might not necessarily respond to such chemical cues in the same way. Since virgin females can produce only sons, they might refrain from searching for hosts to invest eggs until copulation, in order to produce both sexes. 3. Here, we investigated differential host‐searching behaviours shown by mated and virgin females in the solitary parasitoid wasp, Cotesia vestalis, in response to herbivory‐associated chemical information from cruciferous plants infested by their host larvae, Plutella xylostella. 4. Mated females showed a significantly higher flight preference for host‐infested plants over intact plants, while no preference was observed with virgin females. Mated females also showed more intensive antennal searching and ovipositor probing behaviours to leaf squares with wounds caused by hosts than did virgin females. Furthermore, mated females stayed longer in host patches with higher parasitism rates than virgin females. 5. These results indicate that mating status of C. vestalis females clearly influences their host‐searching behaviour in response to herbivory‐associated chemical information and patch exploitation. Female parasitoids seem to forage for hosts depending on their own physiological condition in a tritrophic system.