Project description:In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium bicarinatum and to unveil the potential of its products, high-throughput expressed sequence tags were generated using Illumina paired-end sequencing technology. A total of 212,371,758 pairs of quality-filtered, 100-base-pair Illumina reads were obtained. The de novo assemblies yielded 36,042 contigs for which 27,873 have at least one predicted ORF among which 59.77% produce significant hits in the available databases. The investigation of the reads mapping toxin class revealed a high diversification with the major part consistent with the classical hymenopteran venom protein signature represented by venom allergen (33.3%) followed by a diverse toxin-expression profile including several distinct isoforms of phospholipase A1 and A2, venom serine protease, hyaluronidase, protease inhibitor and secapin. Moreover, our results revealed for the first time the presence of toxin-like peptides that have been previously identified from unrelated venomous animals such as waprin-like (snakes) and agatoxins (spiders and conus). 300 ant specimens from the species Tetramorium bicarinatum were dissected in order to extract the RNA from their venom gland, The whole ant body was used as a reference,
Project description:During the nest-founding phase of the bumble bee colony cycle, queens undergo striking changes in maternal care behavior. Early in the founding phase, prior to the emergence of workers in the nest, queens are reproductive and also provision and feed their offspring. However, later in the founding phase, queens cease feeding offspring and become specialized on reproduction. This transition is synchronized with the emergence of workers in the colony, who assume the task of feeding their siblings. Using a social manipulation experiment, we tested the hypothesis that workers socially regulate the transition from feeding brood to specialization on reproduction in nest-founding bumble bee queens. Consistent with this hypothesis, we found that early-stage queens with workers prematurely added to their nests reduce their brood-feeding behavior and increase egg-laying, and likewise, late-stage queens increase their brood-feeding behavior and decrease egg-laying when workers are removed from their nests. Further, brood-feeding and egg-laying behavior were negatively correlated in these queens. We used an Agilent brain EST-based microarray to explore a second hypothesis, that workers alter brain gene expression in nest-founding queens. We found evidence that brain gene expression in nest-founding queens is altered by the presence of workers, with the effect much stronger in late-stage founding queens. Additionally, expression levels of some genes were correlated with quantitative differences in brood-feeding and egg-laying behavior. This study provides new insights into how the transition from feeding brood to specialization on reproduction in bumble bee queens is regulated during the nest initiation phase of the colony cycle.
Project description:Primary intestinal epithelial cells were isolated from the proximal part of the small intestine from either E16.5 foetal tissue or adult tissue and embedded in matrigel for culturing in in advanced F12/DMEM supplemented with EGF, R-spondin and Noggin. RNA was extracted from cultures established from independent animals and subjected to expression profiling.
Project description:We investigated environmental determinants of caste differences in paper wasps, specifically the effects of differential nutrition. We found that nutritional restriction only partially biased wasp gene expression patterns toward being worker caste-like, which highlights the complex and multifactorial nature of environmental effects on the gene expression patterns underlying plastic phenotypes PRJNA242774; We sequenced mRNA from 16 individual 5th instar larval Polistes metricus heads from 4 groups: worker-reared (n=4), foundress-reared (n=4), restricted nutrition (n=4), and ad libitum (n=4).
Project description:Sibling care is a hallmark of the social insects, but its evolution remains challenging to explain. The hypothesis that sibling care evolved from ancestral maternal care in the primitively eusocial insects has been elaborated to involve heterochronic changes in gene expression. This elaboration leads to the prediction that workers in these species will show patterns of gene expression more similar to foundress queens, who express maternal care behavior, than to established queens engaged solely in reproductive behavior. We tested this idea in the bumblebee Bombus terrestris using a microarray platform with ca. 4,500 genes. Unlike in the wasp Polistes metricus, in which support for the above prediction has been obtained, we found that patterns of brain gene expression in foundress and queen bumblebees were more similar to each other than to workers. However, comparisons of lists of differentially expressed genes derived from this study and gene lists from microarray studies in Polistes and the honeybee Apis mellifera suggest that there is a shared set of genes involved in the regulation of related social behaviors across independent eusocial lineages. Together, these results suggest that the multiple independent evolutions of eusociality in the insects involved a combination of shared and different mechanisms.
Project description:To identify peanut Aspergillus-interactive and Aspergillus-resistance genes, we carried out a large scale peanut Expressed Sequence Tag (EST) project followed by a peanut microarray study. For expression profiling, resistant and susceptible peanut cultivars were infected with a mixture of Aspergillus flavus and parasiticus spores. Microarray analysis identified 65 and 1 genes in resistant (C20) and susceptible (TF) cultivars, respectively, that were up-regulated in response to Aspergillus infection. In addition we identified 40 putative Aspergillus-resistance genes that were constitutively up-expressed in the resistant cultivar in comparison to the susceptible cultivar. Some of these genes were homologous to peanut, corn, and soybean genes previously shown to confer resistance to fungal infection. These results provide a comprehensive genome-scale platform for future studies focused on developing Aspergillus-resistant peanut cultivars through conventional breeding, marker-assisted breeding, or biotechnological methods by gene manipulation. Four samples were analyzed with four hybs. Two samples were obtained from resistant (C20) and and susceptible (TF) cultivars. Two factors were varied in the experimental design: (i) peanut cultivars (resistant (GT-C20) and susceptible (TF)) and (ii) Aspergillus exposure. A combination of these factors produced four hybridizations as follows: (1) C20Y vs. TFY (GT-C20 infected vs. TF infected) (2) C20Y vs. C20N (GT-C20 infected vs. not infected) (3) TFY vs. TFN (TF infected vs. not infected) (4) C20N vs. TFN (GT-C20 not infected vs. TF not infected)
Project description:Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an “orthogonal UB transfer (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2 that are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.
Project description:The molecular mechanisms underlying aflatoxin production have been well-studied in strains of the fungus Aspergillus flavus (A. flavus) under artificial conditions. However, aflatoxin biosynthesis has rarely been studied in natural isolates of A. flavus strains. In the present study, tandem mass tag (TMT) labeling and high-performance liquid chromatography (HPLC) coupled with tandem-mass spectrometry analysiswere used for proteomic quantification in natural isolates of high- and low-aflatoxin-yield A. flavus strains.
Project description:Animal venoms are a rich source of novel biomolecules with tremendous potential in medicine and agriculture. Ants represent one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom compositions from Myrmica rubra and Myrmica ruginodis, two members of the Myrmicinae subfamily of ants. We applied a proteo-transcriptomics based venomics workflow. Our analysis revealed that venoms of both species are composed of several protein classes, such as venom serine protease, cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins (CAP), Kunitz-type serine protease inhibitors or venom acid phosphatase. Several protein classes identified are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel toxins of the epidermal growth factor (EGF) family in its venom proteome and an array of additional EGF-like toxins in venom gland transcriptomes of both species. They display similarity to known toxins from the related myrmecine Manica rubida and the Australian red bulldog ant Myrmecia gullosa of the Myrmeciinae subfamily and may serve as nociceptive weapons in defensive scenarios. Our work suggests that the venoms of M. rubra and M. ruginodis contain many high molecular proteins and enzymes with putatively cell damaging functions. Nevertheless, the presence of EGF-like toxins underpins that myrmicine ants also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of these EGF-like toxins, their presence in Myrmecinae and Myrmeciinae suggests that they play an important role for the venom systems of Formicoidea. Our work adds to the emerging picture of ant venoms as a source for novel biomolecules. This underlines the importance to incorporate such taxa in future venom bioprospecting programs.
Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first large-scale study of protein turnover rates in which we distinguish between N-terminal proteoforms pointing to translation initiation events. Using pulsed SILAC combined with N-terminal COFRADIC we monitored the stability of 1,941human N-terminal proteoforms, including 147 proteoform pairs with heterogeneous N-termini originating from the same gene that result from alternative translation initiation and incomplete processing of the initiator methionine. N-terminally truncated proteoforms were on average less abundant than canonical proteoforms, many had different stabilities and exhibited both faster and slower turnover rates compared to their canonical counterparts. These differences in stability did not depend on the length of truncation but on individual protein characteristics. In silico simulation of N-terminal proteoforms in macromolecular complexes revealed possible consequences for complex integrity such as replacement of unstable canonical subunits. The extent of intrinsic disorder in N-terminal protein structures correlated with turnover times, indicating that a change in the structural flexibility of protein N-termini in truncated proteoforms might impact proteoform stability. Interestingly, removal of the initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms while susceptibility for N-terminal acetylation, another common co-translational modification, did not seem to impact on turnover rates. Taken together, our findings reveal differences in protein stability between N-terminal proteoforms and point to a role for alternative translation initiation and co-translational initiator methionine removal in the overall regulation of proteome homeostasis.