The clonal composition of biramous and uniramous arthropod limbs

We present the first comparative cell lineage analysis of uniramous and biramous limbs of an arthropod, the crustacean Orchestia cavimana. Via single cell labelling of the cells that are involved in limb development, we are able to present the first complete clonal composition of an arthropod limb. We show that the two main branches of crustacean limbs, exopod and endopod, are formed by a secondary subdivision of the growth zone of the main limb axis. Additional limb outgrowths such as exites result from the establishment of new axes. In contrast to general belief, uniramous limbs in Orchestia are not formed by the loss of the exopod but by suppression of the split into exopod and endopod. Our results offer a developmental approach to discriminate between the different kinds of branches of arthropod appendages. This leads to the conclusion that a 'true' biramous limb comprising an endopod and an exopod might have occurred much later in euarthropod evolution than has previously been thought, probably either in the lineage of the Mandibulata or that of the Tetraconata.     

Do males facilitate the spread of novel phenotypes within populations of the androdioecious nematode Caenorhabditis elegans?

In the androdioecious nematode Caenorhabditis elegans, self-fertilization is the predominant mode of reproduction. Nevertheless, males do occur, and it is still unclear if these represent a selective advantage or merely an evolutionary relict. In this study, we first tested the hypothesis that the production of males might benefit invaders to resident populations. We added single, GFP-marked worms to established laboratory populations and followed GFP frequencies over time. Mated hermaphrodites and also males were more successful in invading resident populations if compared to single, unmated hermaphrodites. The observed higher frequencies should increase the likelihood that any of the associated invading alleles persist. Second, we tested the hypothesis that males and, thus, higher outcrossing rates, are specifically favored under changing environmental conditions. After an outbred population was subjected to changing stress or to control laboratory conditions, we measured the male maintenance of the resulting populations. Interestingly all populations, experimental and control alike, showed high male maintenance, suggesting that persistence of males is also favored under standard laboratory conditions.     

Hymenopteran parasitoids of the ant-eating spider Zodarion styliferum (Simon) (Araneae,Zodariidae)

Calymmochilus dispar Bouček & Andriescu (Hymenoptera, Eupelmidae) and Gelis apterus (Pontoppidan) (Hymenoptera, Ichneumonidae) are newly recorded as parasitoids of the ant-eating spider Zodarion styliferum (Simon) (Araneae, Zodariidae). The larvae of both parasitoid species fed on juvenile spiders. The final instar larva and pupa of Calymmochilus dispar and the male of Gelis apterus are described for the first time. Both species represent new distribution records for Portugal. The biology and host associations of the parasitoids are discussed.     

Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288

Background

Cytoplasmic male sterility (CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. CMS may be caused by mutations, rearrangement or recombination in the mitochondrial genome. Understanding the mitochondrial genome is often the first and key step in unraveling the molecular and genetic basis of CMS in plants. Comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line in B. juneca (Brassica juncea) may help show the origin of the CMS-associated gene orf288.

Results

Through next-generation sequencing, the B. juncea hau CMS mitochondrial genome was assembled into a single, circular-mapping molecule that is 247,903 bp in size and 45.08% in GC content. In addition to the CMS associated gene orf288, the genome contains 35 protein-encoding genes, 3 rRNAs, 25 tRNA genes and 29 ORFs of unknown function. The mitochondrial genome sizes of the maintainer line and another normal type line “J163-4” are both 219,863 bp and with GC content at 45.23%. The maintainer line has 36 genes with protein products, 3 rRNAs, 22 tRNA genes and 31 unidentified ORFs. Comparative analysis the mitochondrial genomes of the hau CMS line and its maintainer line allowed us to develop specific markers to separate the two lines at the seedling stage. We also confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its maintainer lines in B. juncea. The number of repeats larger than 100 bp in the hau CMS line (16 repeats) are nearly twice of those found in the maintainer line (9 repeats). Phylogenetic analysis of the CMS-associated gene orf288 and four other homologous sequences in Brassicaceae show that orf288 was clearly different from orf263 in Brassica tournefortii despite of strong similarity.

Conclusion

The hau CMS mitochondrial genome was highly rearranged when compared with its iso-nuclear maintainer line mitochondrial genome. This study may be useful for studying the mechanism of natural CMS in B. juncea, performing comparative analysis on sequenced mitochondrial genomes in Brassicas, and uncovering the origin of the hau CMS mitotype and structural and evolutionary differences between different mitotypes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-322) contains supplementary material, which is available to authorized users.     

Structural insights into the conformational diversity of ClpP from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

ClpP is a cylindrical protease that is tightly regulated by Clp-ATPases. The activation mechanism of ClpP using acyldepsipeptide antibiotics as mimics of natural activators showed enlargement of the axial entrance pore for easier processing of incoming substrates. However, the elimination of degradation products from inside the ClpP chamber remains unclear since there is no exit pore for releasing these products in all determined ClpP structures. Here we report a new crystal structure of ClpP from Bacillus subtilis, which shows a significantly compressed shape along the axial direction. A portion of the handle regions comprising the heptameric ring-ring contacts shows structural transition from an ordered to a disordered state, which triggers the large conformational change from an extended to an overall compressed structure. Along with this structural change, 14 side pores are generated for product release and the catalytic triad adopts an inactive orientation. We have also determined B. subtilis ClpP inhibited by diisopropylfluoro-phosphate and analyzed the active site in detail. Structural information pertaining to several different conformational steps such as those related to extended, ADEP-activated, DFP-inhibited and compressed forms of ClpP from B. subtilis is available. Structural comparisons suggest that functionally important regions in the ClpP-family such as N-terminal segments for the axial pore, catalytic triads, and handle domains for the product releasing pore exhibit intrinsically dynamic and unique structural features. This study provides valuable insights for understanding the enigmatic cylindrical degradation machinery of ClpP as well as other related proteases such as HslV and the 20S proteasome.     

Reproductive compensation favours male-killing Wolbachia in a live-bearing host

Wolbachia are maternally inherited, cellular endosymbionts that can enhance their fitness by biasing host sex ratio in favour of females. Male killing (MK) is an extreme form of sex-ratio manipulation that is selectively advantageous if the self-sacrifice of Wolbachia in males increases transmission through females. In live-bearing hosts, females typically produce more embryos than can be carried to term, and reproductive compensation through maternal resource reallocation from dead males to female embryos could increase the number of daughters born to infected females. Here, we report a new strain of MK Wolbachia (wCsc2) in the pseudoscorpion, Cordylochernes scorpioides, and present the first empirical evidence that reproductive compensation favours the killing of males in a viviparous host. Females infected with the wCsc2 strain produced 26 per cent more and significantly larger daughters than tetracycline-cured females. In contrast to the previously described wCsc1 MK Wolbachia strain in C. scorpioides, wCsc2 infection was not accompanied by an increase in the rate of spontaneous brood abortion. Characterization of the wCsc1 and wCsc2 strains by multi-locus sequence typing and by Wolbachia surface protein (wsp) gene sequencing indicates that the marked divergence between these two MK strains in their impact on host reproductive success, and hence in their potential to spread, has occurred in association with homologous recombination in the wsp gene.     

A marine snail neurotoxin shares with scorpion toxins a convergent mechanism of blockade on the pore of voltage-gated K channels.

kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. kappa-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in kappa-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, kappa-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, zdelta, is approximately 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) M-1 s-1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced kappa-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the alpha-KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.     

Genotoxicity assessment of a pharmaceutical effluent using four bioassays

Pharmaceutical industries are among the major contributors to industrial waste. Their effluents when wrongly handled and disposed of endanger both human and environmental health. In this study, we investigated the potential genotoxicity of a pharmaceutical effluent, by using the Allium cepa, mouse- sperm morphology, bone marrow chromosome aberration (CA) and micronucleus (MN) assays. Some of the physico-chemical properties of the effluent were also determined. The A. cepa and the animal assays were respectively carried out at concentrations of 0.5, 1, 2.5, 5 and 10%; and 1, 5, 10, 25 and 50% of the effluent. There was a statistically different (p < 0.05), concentration-dependent inhibition of onion root growth and mitotic index, and induction of chromosomal aberrations in the onion and mouse CA test. Assessment of sperm shape showed that the fraction of the sperm that was abnormal in shape was significantly (p < 0.05) greater than the negative control value. MN analysis showed a dose-dependent induction of micronucleated polychromatic erythrocytes across the treatment groups. These observations were provoked by the toxic and genotoxic constituents present in test samples. The tested pharmaceutical effluent is a potentially genotoxic agent and germ cell mutagen, and may induce adverse health effects in exposed individuals.     

Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.     

Different functions for homologous serotonergic interneurons and serotonin in species-specific rhythmic behaviours

Closely related species can exhibit different behaviours despite homologous neural substrates. The nudibranch molluscs Tritonia diomedea and Melibe leonina swim differently, yet their nervous systems contain homologous serotonergic neurons. In Tritonia, the dorsal swim interneurons (DSIs) are members of the swim central pattern generator (CPG) and their neurotransmitter serotonin is both necessary and sufficient to elicit a swim motor pattern. Here it is shown that the DSI homologues in Melibe, the cerebral serotonergic posterior-A neurons (CeSP-As), are extrinsic to the swim CPG, and that neither the CeSP-As nor their neurotransmitter serotonin is necessary for swim motor pattern initiation, which occurred when the CeSP-As were inactive. Furthermore, the serotonin antagonist methysergide blocked the effects of both the serotonin and CeSP-As but did not prevent the production of a swim motor pattern. However, the CeSP-As and serotonin could influence the Melibe swim circuit; depolarization of a cerebral serotonergic posterior-A was sufficient to initiate a swim motor pattern and hyperpolarization of a CeSP-A temporarily halted an ongoing swim motor pattern. Serotonin itself was sufficient to initiate a swim motor pattern or make an ongoing swim motor pattern more regular. Thus, evolution of species-specific behaviour involved alterations in the functions of identified homologous neurons and their neurotransmitter.     

Discovery of the male of Lobrathium rotundiceps (Koch), and a new species of Lobrathium from Jiangxi,East China (Coleoptera,Staphylinidae, Paederinae)

The male of Lobrathium rotundiceps (Koch, 1939) from Zhejiang and L. luoxiaoense sp. n. from Jiangxi are described and illustrated.     

Evidence for phosphorylation in the MSP cytoskeletal filaments of amoeboid spermatozoa

Nematode spermatozoa are highly specialized cells that lack flagella and, instead, extend a pseudopod to initiate motility. Crawling spermatozoa display classic features of amoeboid motility (e.g. protrusion of a pseudopod that attaches to the substrate and the assembly and disassembly of cytoskeletal filaments involved in cell traction and locomotion), however, cytoskeletal dynamics in these cells are powered exclusively by Major Sperm Protein (MSP) rather than actin and no other molecular motors have been identified. Thus, MSP-based motility is regarded as a simple locomotion machinery suitable for the study of plasma membrane protrusion and cell motility in general. This recent focus on MSP dynamics has increased the necessity of a standardized methodology to obtain C. elegans sperm extract that can be used in biochemical assays and proteomic analysis for comparative studies. In the present work we have modified a method to reproducibly obtain relative high amounts of proteins from C. elegans sperm extract. We show that these extracts share some of the properties observed in sperm extracts from the parasitic nematode Ascaris including Major Sperm Protein (MSP) precipitation and MSP fiber elongation. Using this method coupled to immunoblot detection, Mass Spectrometry identification, in silico prediction of functional domains and biochemical assays, our results indicate the presence of phosphorylation sites in MSP of Caenorhabditis elegans spermatozoa.     

Unravelling the Wolbachia evolutionary role: the reprogramming of the host genomic imprinting

Environmental factors can induce significant epigenetic changes that may also be inherited by future generations. The maternally inherited symbiont of arthropods Wolbachia pipientis is an excellent candidate as an ‘environmental’ factor promoting trans-generational epigenetic changes: by establishing intimate relationships with germ-line cells, epigenetic effects of Wolbachia symbiosis would be manifested as a ‘maternal effect’, in which infection of the mother modulates the offspring phenotype. In the leafhopper Zyginidia pullula, Wolbachia feminizes genetic males, leaving them as intersexes. With the exception of male chitinous structures that are present in the last abdominal segment, feminized males display phenotypic features that are typical of females. These include ovaries that range from a typical histological architecture to an altered structure. Methylation-sensitive random amplification of polymorphic DNA profiles show that they possess a female genomic imprint. On the other hand, some rare feminized males bear testes instead of ovaries. These specimens possess a Wolbachia density approximately four orders of magnitude lower than feminized males with ovaries and maintain a male genome—methylation pattern. Our results indicate that Wolbachia infection disrupts male imprinting, which dramatically influences the expression of genes involved in sex differentiation and development, and the alteration occurs only if Wolbachia exceeds a density threshold. Thus, a new Wolbachia''s role as an environmental evolutionary force, inducing epigenetic trans-generational changes, should now be considered.     

Ultrastructure of Gonionchus australis (Xyalidae,Nematoda)

Observations are reported on the ultrastructure of the buccal cavity, body cuticle, spermatids, spermatozoa, male genitalia, and caudal glands of Gonionchus australis. The buccal cuticle is a continuation of the pharyngeal cuticle. Anteriorly it is secreted by arcade tissue and overlaps the mouth rim; laterally it forms longitudinal tooth ridges. The non-annulated cephalic cuticle differs sharply from the remainder of the body wall cuticle. The cortical and basal zones become much thinner, while a largely structureless, lucent median zone expands to fill the bulk of the lips and lip flaps. Spermatids possess fibrous bodies, multimembrane organelles, mitochondria, and compact chromatin. The spermatozoa of G. australis resemble those of most other nematodes by the absence of the nuclear envelope and presence of fibrous bodies, mitochondria, and compact chromafin. The ejaculatory duct possesses microvilli. Two ejaculatory glands lie beside the duct. Two neurons are located within each spicule and each part of the paired gubernaculum. Caudal gland nuclei are large, with dispersed chromatin. The ducts of all three caudal glands are filled with secretory vesicles.