Assessment of Rhodopirellula rubra as a supplementary and nutritional food source to the microcrustacean Daphnia magna.

The daily use of the planctomycete Rhodopirellula rubra as an alternative or supplementary food source for Daphnia magna and its feasibility in the nutrition of transgenerational populations were studied. The life history parameters, fatty acids (saturated, mono- and polyunsaturated; SFAs, MUFAs and PUFAs), glycogen and protein contents of organisms during feeding assays and of the first generation were analysed. An increase in the yields of D. magna with the increase of the cell concentration of R. rubra was evident, but overall, bacteria supplied as the only food source was nutritionally insufficient as observed for all the parameters analysed. However, when R. rubra was added as supplement to the microalgae Raphidocelis subcapitata a significant improvement in the life history parameters was observed namely in the reproductive output and the somatic growth rate. The identified SFAs, MUFAs and PUFAs were the fatty acids more abundant in daphniids, and the feed regimens influenced daphniids fatty acid profiles. Additionally, the mixed diet resulted in a larger number and size of offspring in the different F1 broods as also observed with the results of F0 generation. The pink colouration present in D. magna body and eggs confirmed that bacteria were absorbed, the pigment(s) retained and passed on to the next generation. Our results showed that R. rubra can play an essential role in D. magna diet as a nutritional supplement showing potential biotechnological applications.

De novo transcriptomic analysis of the venomous glands from the scorpion Heterometrus spinifer revealed unique and extremely high diversity of the venom peptides.

Scorpion, as an ancient species, has been widely used on dozens of human diseases in traditional Chinese Medicine. Although the scorpion venom from the Buthidae family with the potent toxicity attracts more interests, toxins from the non-Buthidae family draw great attention as well because of its abundance and complexity even without harm to mammals. Moreover, several toxic components of scorpion venom have been identified as valuable scaffolds for the drug design and development. Using the Next Generation Sequencing (NGS) technique, here we reported the transcriptome of the venomous glands of Heterometrus spinifer, a non-Buthidae scorpion that only a few toxic and complete components have been identified known-to-date. The total mRNA extracted from the venomous glands of H. spinifer was subjected to illumina sequencing with a strategy of de novo assembly, and a total of 54 189 transcripts were unigenes from a total of 88 311 600 determined reads. We annotated 18 567 (34.26%) unigenes from NR database, 12 258 (22.62%) from SWISSPROT database, 11 161 (20.60%) from GO database, 10 159 (18.75%) from COG database and 5059 (9.34%) from KEGG database, respectively. 2843 unigenes were further selected against the toxin-related sub-database of SWISSPROT. After removing the redundancy, 13 common toxin-related subfamilies with 62 unigenes were manually confirmed, including 8 K-toxins, 1 calcin, 3 Imperatoxin I-like, 2 La1-like, 1 scorpin-like, 3 antimicrobial peptides, two types of protease inhibitors such as 8 Kunitz-type protease inhibitors and 3 Ascaris-type protease inhibitors, and 33 proteases including 16 serine proteinases, 7 phospholipases, 5 metalloproteases, 3 hyaluronidases and 2 phosphatases. Our report is the first transcriptomic analyses of venomous glands from the scorpion H. spinifer, serving as a public information platform for the development of novel bio-therapeutics.

Transcriptome Analysis to Understand the Toxicity of Latrodectus tredecimguttatus Eggs.

Latrodectus tredecimguttatus is a kind of highly venomous black widow spider, with toxicity coming from not only venomous glands but also other parts of its body as well as newborn spiderlings and eggs. Up to date, although L. tredecimguttatus eggs have been demonstrated to be rich in proteinaceous toxins, there is no systematic investigation on such active components at transcriptome level. In this study, we performed a high-throughput transcriptome sequencing of L. tredecimguttatus eggs with Illumina sequencing technology. As a result, 53,284 protein-coding unigenes were identified, of which 14,185 unigenes produced significant hits in the available databases, including 280 unigenes encoding proteins or peptides homologous to known proteinaceous toxins. GO term and KEGG pathway enrichment analyses of the 280 unigenes showed that 375 GO terms and 18 KEGG pathways were significantly enriched. Functional analysis indicated that these unigene-coded toxins have the bioactivities to degrade tissue proteins, inhibit ion channels, block neuromuscular transmission, provoke anaphylaxis, induce apoptosis and hyperalgesia, etc. No known typical proteinaceous toxins in L. tredecimguttatus venomous glands, such as latrotoxins, were identified, suggesting that the eggs have a different toxicity mechanism from that of the venom. Our present transcriptome analysis not only helps to reveal the gene expression profile and toxicity mechanism of the L. tredecimguttatus eggs, but also provides references for the further related researches.

Cloning and Expression of Ecdysone Receptor and Retinoid X Receptor from Procambarus clarkii: Induction by Eyestalk Ablation.

Ecdysone receptor and retinoid X receptor are key regulators in molting. Here, full length ecdysone receptor (PcEcR) and retinoid X receptor (PcRXR) cDNAs from Procambarus clarkii were cloned. Full length cDNA of PcEcR has 2500 bp, encoding 576 amino acid proteins, and full length cDNA of PcRXR has 2593 bp, in which a 15 bp and a 204 bp insert/deletion splice variant regions in DNA binding domain and hinge domain were identified. The two splice variant regions in PcRXR result four isoforms: PcRXR1-4, encoding 525, 520, 457 and 452 amino acids respectively. PcEcR was highly expressed in the hepatopancreas and eyestalk and PcRXR was highly expressed in the eyestalk among eight examined tissues. Both PcEcR and PcRXR had induced expression after eyestalk ablation (ESA) in the three examined tissues. In muscle, PcEcR and PcRXR were upregulated after ESA, PcEcR reached the highest level on day 3 after ESA and increased 33.5-fold relative to day 0, and PcRXR reached highest the level on day 1 after ESA and increased 2.7-fold relative to day 0. In the hepatopancreas, PcEcR and PcRXR dEcReased continuously after ESA, and the expression levels of PcEcR and PcRXR were only 0.7% and 1.7% on day 7 after ESA relative to day 0, respectively. In the ovaries, PcEcR was upregulated after ESA, reached the highest level on day 3 after ESA, increased 3.0-fold relative to day 0, and the expression level of PcRXR changed insignificantly after ESA (p > 0.05). The different responses of PcEcR and PcRXR after ESA indicates that different tissues play different roles (and coordinates their functions) in molting.

Recent Advances in Research on Widow Spider Venoms and Toxins.

Widow spiders have received much attention due to the frequently reported human and animal injures caused by them. Elucidation of the molecular composition and action mechanism of the venoms and toxins has vast implications in the treatment of latrodectism and in the neurobiology and pharmaceutical research. In recent years, the studies of the widow spider venoms and the venom toxins, particularly the α-latrotoxin, have achieved many new advances; however, the mechanism of action of the venom toxins has not been completely clear. The widow spider is different from many other venomous animals in that it has toxic components not only in the venom glands but also in other parts of the adult spider body, newborn spiderlings, and even the eggs. More recently, the molecular basis for the toxicity outside the venom glands has been systematically investigated, with four proteinaceous toxic components being purified and preliminarily characterized, which has expanded our understanding of the widow spider toxins. This review presents a glance at the recent advances in the study on the venoms and toxins from the Latrodectus species.

Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism.

Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

BACKGROUND: Animal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution. RESULTS: We estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression. CONCLUSIONS: Quantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.

Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders.

The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders.

Isoforms of gelsolin from lobster striated muscles differ in calcium-dependence.

Two distinct isoforms of the Ca-dependent actin filament severing protein gelsolin were identified in cross-striated muscles of the American lobster. The variants (termed LG1 and LG2) differ by an extension of 18 AA at the C-terminus of LG1, and by two substitutions at AA735 and AA736, the two C-terminal amino acids of LG2. Functional comparison of the isolated and purified proteins revealed gelsolin-typical properties for both with differences in Ca(2+)-sensitivity, LG2 being activated at significant lower Ca-concentration than LG1: Half maximal activation for both filament severing and G-actin binding was ∼4×10(-7)M Ca(2+) for LG2 vs. ∼2×10(-6)M Ca(2+) for LG1. This indicates a differential activation for the two isoproteins in vivo where they are present in almost equal amounts in the muscle cell. Structure prediction modeling on the basis of the known structure of mammalian gelsolin shows that LG2 lacks the C-terminal alpha-helix which is involved in contact formation between domains G6 and G2. In both mammalian gelsolin and LG1, this "latch bridge" is assumed to play a critical role in Ca(2+)-activation by keeping gelsolin in a closed, inactive conformation at low [Ca(2+)]. In LG2, the reduced contact between G6 and G2 may be responsible for its activation at low Ca(2+)-concentration.

Electrochemical impedimetric biosensor based on a nanostructured polycarbonate substrate.

This study integrates the techniques of nanoelectroforming, hot-embossing, and electrochemical deposition to develop a disposable, low-cost, and high sensitivity nanostructure biosensor. A modified anodic aluminum oxide barrier-layer surface was used as the template for thin nickel film deposition. After etching the anodic aluminum oxide template off, a three-dimensional mold of the concave nanostructure array was created. The fabricated three-dimensional nickel mold was further used for replica molding of a nanostructure polycarbonate substrate by hot-embossing. A thin gold film was then sputtered onto the polycarbonate substrate to form the electrode, followed by deposition of an orderly and uniform gold nanoparticle layer on the three-dimensional gold electrode using electrochemical deposition. Finally, silver nanoparticles were deposited on the uniformly deposited gold nanoparticles to enhance the conductivity of the sensor. Electrochemical impedance spectroscopy analysis was then used to detect the concentration of the target element. The sensitivity of the proposed scheme on the detection of the dust mite antigen, Der p2, reached 0.1 pg/mL.

A novel nanostructured biosensor for the detection of the dust mite antigen Der p2.

The group 2 allergen, Der p2, has been reported to activate innate toll-like receptors (TLRs) on respiratory epithelial cells and thus aggravate respiratory diseases. In this study, a highly sensitive nanostructured biosensor based on a 3D sensing element with uniformly deposited gold nanoparticles is proposed for the detection of the dust mite antigen Der p2. The barrier layer comprises an anodic aluminum oxide (AAO) film which is used as the template in this highly sensitive nanostructured biosensor. Simple electrochemical deposition without reducing agent and stabilizer was enough to uniformly synthesize gold nanoparticles on the surface of the barrier layer. The size and the distribution density of the nanoparticles can be well controlled by the applied potential during electrochemical deposition. Following this procedure, the dust mite monoclonal antibodies (IgG) were then immobilized through the 11-MUA (11-mercaptoundecanoic acid), (1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide)/(N-hydroxysuccinimide) self-assembled monolayer approach for the dust mite antigen Der p2 detection. The detection limit of the proposed 3D gold nanoparticle-based nanostructured biosensor was examined using electrochemical impedance spectroscopy analysis and found to be 1 pg/mL. The dynamic range was found to be 5 µg/mL. The proposed nanostructured biosensor would be useful for fast detection of rare molecules in a solution.