Population structure and behaviour of crop-raiding elephants in Kibale National Park, Uganda

We examined patterns of crop raiding by elephants across gender and age classes in relation to elephant life history and sociobiology and estimated the quantitative contribution of crops to elephant diet in Kibale National Park (KNP). Elephant dung‐boli sizes were used to estimate age and sex, while the presence of crop remains in the dung of crop‐raiding elephants was used as evidence of repeated raiding. To estimate the expected proportion of elephants raiding per age class, the age distribution of raiders was compared with the age distribution of all KNP elephants. Elephants raiding crops were predominantly males. They began raiding in expected proportions at 10–14 years while a higher than expected proportion raided crops at 20–24 years. These results suggest that crop raiding is initiated at an age when male elephants leave their families and a large proportion of elephants raid when they are approaching reproductive competition. Evidence from dung of crop raiders, suggests that repeated raiding increases with age. Crop raiders derived 38% of their daily forage from the short time spent raiding, consistent with expectations of foraging theory. Males may be more likely to learn crop raiding because they are socially more independent and experience intense mating competition than females.     

N-Terminal-oriented Proteogenomics of the Marine Bacterium Roseobacter Denitrificans Och114 using N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) Labeling and Diagonal Chromatography

Given the ease of whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now purely based on automated prediction. However, errors in gene structure are frequent, the correct determination of start codons being one of the main concerns. Here, we combine protein N termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP Ac-OSu) as a labeling reagent with the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting method to enrich labeled N-terminal peptides for mass spectrometry detection. Protein digestion was performed in parallel with three proteases to obtain a reliable automatic validation of protein N termini. The analysis of these N-terminal enriched fractions by high-resolution tandem mass spectrometry allowed the annotation refinement of 534 proteins of the model marine bacterium Roseobacter denitrificans OCh114. This study is especially efficient regarding mass spectrometry analytical time. From the 534 validated N termini, 480 confirmed existing gene annotations, 41 highlighted erroneous start codon annotations, five revealed totally new mis-annotated genes; the mass spectrometry data also suggested the existence of multiple start sites for eight different genes, a result that challenges the current view of protein translation initiation. Finally, we identified several proteins for which classical genome homology-driven annotation was inconsistent, questioning the validity of automatic annotation pipelines and emphasizing the need for complementary proteomic data. All data have been deposited to the ProteomeXchange with identifier PXD000337.Recent developments in mass spectrometry and bioinformatics have established proteomics as a common and powerful technique for identifying and quantifying proteins at a very broad scale, but also for characterizing their post-translational modifications and interaction networks (1, 2). In addition to the avalanche of proteomic data currently being reported, many genome sequences are established using next-generation sequencing, fostering proteomic investigations of new cellular models. Proteogenomics is a relatively recent field in which high-throughput proteomic data is used to verify coding regions within model genomes to refine the annotation of their sequences (2–8). Because genome annotation is now fully automated, the need for accurate annotation for model organisms with experimental data is crucial. Many projects related to genome re-annotation of microorganisms with the help of proteomics have been recently reported, such as for Mycoplasma pneumoniae (9), Rhodopseudomonas palustris (10), Shewanella oneidensis (11), Thermococcus gammatolerans (12), Deinococcus deserti (13), Salmonella thyphimurium (14), Mycobacterium tuberculosis (15, 16), Shigella flexneri (17), Ruegeria pomeroyi (18), and Candida glabrata (19), as well as for higher organisms such as Anopheles gambiae (20) and Arabidopsis thaliana (4, 5).The most frequently reported problem in automatic annotation systems is the correct identification of the translational start codon (21–23). The error rate depends on the primary annotation system, but also on the organism, as reported for Halobacterium salinarum and Natromonas pharaonis (24), Deinococcus deserti (21), and Ruegeria pomeroyi (18), where the error rate is estimated above 10%. Identification of a correct translational start site is essential for the genetic and biochemical analysis of a protein because errors can seriously impact subsequent biological studies. If the N terminus is not correctly identified, the protein will be considered in either a truncated or extended form, leading to errors in bioinformatic analyses (e.g. during the prediction of its molecular weight, isoelectric point, cellular localization) and major difficulties during its experimental characterization. For example, a truncated protein may be heterologously produced as an unfolded polypeptide recalcitrant to structure determination (25). Moreover, N-terminal modifications, which are poorly documented in annotation databases, may occur (26, 27).Unfortunately, the poor polypeptide sequence coverage obtained for the numerous low abundance proteins in current shotgun MS/MS proteomic studies implies that the overall detection of N-terminal peptides obtained in proteogenomic studies is relatively low. Different methods for establishing the most extensive list of protein N termini, grouped under the so-called “N-terminomics” theme, have been proposed to selectively enrich or improve the detection of these peptides (2, 28, 29). Large N-terminome studies have recently been reported based on resin-assisted enrichment of N-terminal peptides (30) or terminal amine isotopic labeling of substrates (TAILS) coupled to depletion of internal peptides with a water-soluble aldehyde-functionalized polymer (31–35). Among the numerous N-terminal-oriented methods (2), specific labeling of the N terminus of intact proteins with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinamide (TMPP-Ac-OSu)¹ has proven reliable (21, 36–39). TMPP-derivatized N-terminal peptides have interesting properties for further LC-MS/MS mass spectrometry: (1) an increase in hydrophobicity because of the trimethoxyphenyl moiety added to the peptides, increasing their retention times in reverse phase chromatography, (2) improvement of their ionization because of the introduction of a positively charged group, and (3) a much simpler fragmentation pattern in tandem mass spectrometry. Other reported approaches rely on acetylation, followed by trypsin digestion, and then biotinylation of free amino groups (40); guanidination of lysine lateral chains followed by N-biotinylation of the N termini and trypsin digestion (41); or reductive amination of all free amino groups with formaldehyde preceeding trypsin digestion (42). Recently, we applied the TMPP method to the proteome of the Deinococcus deserti bacterium isolated from upper sand layers of the Sahara desert (13). This method enabled the detection of N-terminal peptides allowing the confirmation of 278 translation initiation codons, the correction of 73 translation starts, and the identification of non-canonical translation initiation codons (21). However, most TMPP-labeled N-terminal peptides are hidden among the more abundant internal peptides generated after proteolysis of a complex proteome, precluding their detection. This results in disproportionately fewer N-terminal validations, that is, 5 and 8% of total polypeptides coded in the theoretical proteomes of Mycobacterium smegmatis (37) and Deinococcus deserti (21) with a total of 342 and 278 validations, respectively.An interesting chromatographic method to fractionate peptide mixtures for gel-free high-throughput proteome analysis has been developed over the last years and applied to various topics (43, 44). This technique, known as COmbined FRActional DIagonal Chromatography (COFRADIC), uses a double chromatographic separation with a chemical reaction in between to change the physico-chemical properties of the extraneous peptides to be resolved from the peptides of interest. Its previous applications include the separation of methionine-containing peptides (43), N-terminal peptide enrichment (45, 46), sulfur amino acid-containing peptides (47), and phosphorylated peptides (48). COFRADIC was identified as the best method for identification of N-terminal peptides of two archaea, resulting in the identification of 240 polypeptides (9% of the theoretical proteome) for Halobacterium salinarum and 220 (8%) for Natronomonas pharaonis (24).Taking advantage of both the specificity of TMPP labeling, the resolving power of COFRADIC for enrichment, and the increase in information through the use of multiple proteases, we performed the proteogenomic analysis of a marine bacterium from the Roseobacter clade, namely Roseobacter denitrificans OCh114. This novel approach allowed us to validate and correct 534 unique proteins (13% of the theoretical proteome) with TMPP-labeled N-terminal signatures obtained using high-resolution tandem mass spectrometry. We corrected 41 annotations and detected five new open reading frames in the R. denitrificans genome. We further identified eight distinct proteins showing direct evidence for multiple start sites.     

Sequence analysis and receptor specificity of the hemagglutinin of a recent influenza H2N2 virus isolated from chicken in North America

Influenza viruses bind host cells following an interaction between the viral hemagglutinin (HA) protein and host cell sialylated glycoproteins and glycolipids. Differences in binding affinities of the HAs for different types of sialic acid linkages (α2-3 vs. α2-6) contribute to determining the host range of an influenza virus. The ability of an avian influenza virus HA to bind the human form of the receptor may be one requirement for an avian virus to propagate in the human population. In this paper, we describe the characterization of the HA from an H2N2 virus isolated from a Pennsylvania chicken farm in 2004. Sequence analysis revealed that this HA is a member of the Eurasian clade, and receptor binding studies show that it maintains its specificity for the avian influenza virus α2-3 linked sialic acid receptor.     

The effects of geographic origin and antibiotic treatment on the gut symbiotic communities of Bactrocera oleae populations

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major insect pest of olive orchards (Olea europaea L.), causing extensive damages on cultivated olive crops worldwide. Due to its economic importance, it has been the target species for a variety of population control approaches including the sterile insect technique (SIT). However, the inefficiency of the current mass‐rearing techniques impedes the successful application of area‐wide integrated pest management programs with an SIT component. It has been shown that insect mass rearing and quality of sterile insects can be improved by the manipulation of the insect gut microbiota and probiotic applications. In order to exploit the gut bacteria, it is important to investigate the structure of the gut microbial community. In the current study, we characterized the gut bacterial profile of two wild olive fruit fly populations introduced in laboratory conditions using next generation sequencing of two regions of the 16S rRNA gene. We compared the microbiota profiles regarding the geographic origin of the samples. Additionally, we investigated potential changes in the gut bacteria community before and after the first exposure of the wild adult flies to artificial adult diet with and without antibiotics. Various genera – such as Erwinia, Providencia, Enterobacter, and Klebsiella – were detected for the first time in B. oleae. The most dominant species was Candidatus Erwinia dacicola Capuzzo et al. and it was not affected by the antibiotics in the artificial adult diet used in the first generation of laboratory rearing. Geographic origin affected the overall structure of the gut community of the olive fruit fly, but antibiotic treatment in the first generation did not significantly alter the gut microbiota community.     

Lianas as a food resource for herbivorous insects: a comparison with trees

Woody climbers or, ‘lianas’, are one of the features that characterise rainforests. They contribute substantially to plant diversity and leaf biomass which makes them a potentially important food source for herbivores. Here, we focus on insect herbivores, folivores in particular, to show how disparities in the quantitative and qualitative availability of leaves between lianas and trees may differentially influence insect folivory and the herbivore communities themselves. We develop a conceptual model and show that lianas in general have lower structural and chemical defences, a greater nutritional profile and a preferable phenology in comparison with trees, which, contrary to our expectations, has led to assemblages of more‐specialised insects. The impacts this has on higher trophic levels and broader ecological networks, however, are poorly known. We show through a study of four tropical floras from different biogeographic realms that lianas are likely to be a target for a wide range of insect herbivore taxa as they are a phylogenetically diverse group and increase diversity of higher taxa at local scales. This, in combination with their highly palatable leaves, may also make them a suitable temporary food source for insects during times when preferred host plants are scarce. This phenomenon has been observed in mammalian herbivores but awaits investigation in insects as does the effects this may have on survival and fitness. Apparent recent increases in liana abundances in some forests, likely due to climate change, makes understanding their role in supporting and maintaining biodiversity an increasingly important and necessary challenge. Since trees or saplings have usually been the subject of studies on insect herbivory, major knowledge gaps remain about the ways in which lianas contribute to, support and maintain the ecosystems in which they exist. We use our conceptual model to guide future research directions and express the necessity for caution when extrapolating explanations of herbivory derived from data on trees to growth forms with fundamentally different ecologies.