Species-specific Biomarkers Research Articles

Revealing microbial community characteristics in healthy human, cat and canine salivas and looking for species-specific microbes.

As two kinds of increasingly popular pets, the saliva of cat or canine is most likely to be left at the crime scene compared with the common types of body fluids in forensics. Accurately identifying the species of saliva samples found at the crime scene involving pets will help the investigators find available testing materials, reduce the consumption of reagents and save the investigative time of the case. Therefore, it is necessary to explore the characteristics and differences of saliva microbiomes of cat, canine and human. In this study, 16S rRNA gene amplicon sequencing technology was used to reveal microbial communities of saliva samples of healthy human, cat, and canine. Alpha diversity analyses indicated that canine saliva demonstrated the highest microbial diversity, followed by cat saliva, whereas human saliva microbial diversity was the lowest. The saliva samples of the three species all had their own unique microbial community compositions, and the dominant phyla of canine and cat salivas were Proteobacteria and Bacteroidete, while the dominant phyla of human saliva were Firmicutes and Proteobacteria. There was no significant statistical difference in the salivary microbiota obtained by the two collection methods (cotton swab and liquid saliva). The gender of cats and canines might have no effect on the salivary microbiota, but the different breeds had an impact on their saliva microbiomes. Principal coordinates analysis, non-metric multidimensional scaling analysis and random forest analysis all indicated significant differences in microbial community structures among the three species, allowing inference on the species sources of saliva samples by microbiome method. Differential microbial biomarkers for the salivas of three species were screened out using a variety of bioinformatics analyses, and the results demonstrated that Prevotella melaninogenica, Veillonella parvula, and Haemophilus parainfluenzae could be used as species-specific microbial biomarkers of human saliva. The detections of human species-specific microbes provide a potential method for determining human saliva.

Highly Sensitive Immunochromatographic Detection of Porcine Myoglobin as Biomarker for Meat Authentication Using Prussian Blue Nanozyme.

This study was aimed at the sensitive immunodetection of porcine myoglobin (MG) as a species-specific biomarker in meat products. The enhanced lateral flow immunoassay (LFIA) was created in the sandwich format using monoclonal antibodies (Mab) with specificity to porcine MG and labeled by Prussian blue nanoparticles (PBNPs) as peroxidase-mimicking nanozymes. Signal amplification was provided by the colored product of oxidation catalyzed by the PBNPs. Several Mab-PBNP conjugates with different antibody loads were synthesized; the one that provided the best analytical characteristics of the LFIA was selected. Advanced optimization of the test system was carried out. As a result, the visual limit of detection (LOD) of MG was 1.5 ng/mL. Involvement of the catalytic nanozyme properties allowed the LOD to be decreased by ~9 times in comparison to the LFIA based on gold nanomarkers, and by ~27 times compared to the LFIA based on PBNP coloration. The assay time was 30 min, including catalytic enhancement. A simple technique of meat sample pre-treatment aimed at effective MG extraction and matrix disposal was proposed. The specificity of the LFIA towards the pork meat was demonstrated. The applicability of the created test system was shown by testing extracts obtained from finished meat products.

Grouping groupers in the Mediterranean: Ecological baselines revealed by ancient proteins.

Marine historical ecology provides a means to establish baselines to inform current fisheries management. Groupers (Epinephelidae) are key species for fisheries in the Mediterranean, which have been heavily overfished. Species abundance and distribution prior to the 20th century in the Mediterranean remains poorly known. To reconstruct the past biogeography of Mediterranean groupers, we investigated whether Zooarchaeology by Mass Spectrometry (ZooMS) can be used for identifying intra-genus grouper bones to species level. We discovered 22 novel, species-specific ZooMS biomarkers for groupers. Applying these biomarkers to Kinet Höyük, a Mediterranean archaeological site, demonstrated 4000 years of regional Epinephelus aeneus dominance and resiliency through millennia of fishing pressures, habitat degradation and climatic changes. Combining ZooMS identifications with catch size reconstructions revealed the Epinephelus aeneus capacity for growing 30 cm larger than hitherto documented, revising the maximum Total Length from 120 to 150 cm. Our results provide ecological baselines for a key Mediterranean fishery which could be leveraged to define and assess conservation targets.

Identification and application of species-specific peptide biomarkers from soft-shelled turtles (Pelodiscus sinensis) using post-translational modification detection-based peptidomics analysis.

The soft-shelled turtle is a commercially aquatic species in Asian countries, which serves as an important source of collagen with high nutritional and medicinal value, so it is of great significance to distinguish soft-shelled turtle derived collagen from others or adulterations. In this work, peptidomics analysis based on post-translational modification (PTM) assay was used to discover specific peptide biomarkers of soft-shelled turtle gelatin (STG). In total eight specific sequences and 74 peptides with different PTM types were screened out, and seven peptides with good signal responses and STG specificity were selected and validated as STG-specific peptide biomarkers. These peptide biomarkers could be used for distinguishing STG from other animal gelatins, and applied for ensuring the quality of collagens or gelatins from soft-shelled turtle with authenticity and traceability.

Rapid Identification of Aphid Species by Headspace GC-MS and Discriminant Analysis.

Aphids are a ubiquitous group of pests in agriculture that cause serious losses. For sustainable aphid identification, it is necessary to develop a precise and fast aphid identification tool. A new simple chemotaxonomy approach to rapidly identify aphids was implemented. The method was calibrated in comparison to the established phylogenetic analysis. For chemotaxonomic analysis, aphids were crushed, their headspace compounds were collected through closed-loop stripping (CLS) and analysed using gas chromatography-mass spectrometry (GC-MS). GC-MS data were then subjected to a discriminant analysis using CAP12.exe software, which identified key biomarkers that distinguish aphid species. A dichotomous key taking into account the presence and absence of a set of species-specific biomarkers was derived from the discriminant analysis which enabled rapid and reliable identification of aphid species. As the method overcomes the limits of morphological identification, it works with aphids at all life stages and in both genders. Thus, our method enables entomologists to assign aphids to growth stages and identify the life history of the investigated aphids, i.e., the food plant(s) they fed on. Our experiments clearly showed that the method could be used as a software to automatically identify aphids.

Shotgun Proteomics Analysis, Functional Networks, and Peptide Biomarkers for Seafood-Originating Biogenic-Amine-Producing Bacteria.

Biogenic amine-producing bacteria are responsible for the production of basic nitrogenous compounds (histamine, cadaverine, tyramine, and putrescine) following the spoilage of food due to microorganisms. In this study, we adopted a shotgun proteomics strategy to characterize 15 foodborne strains of biogenic-amine-producing bacteria. A total of 10,673 peptide spectrum matches belonging to 4081 peptides and corresponding to 1811 proteins were identified. Relevant functional pathways were determined, and strains were differentiated into hierarchical clusters. An expected protein-protein interaction network was created (260 nodes/1973 interactions). Most of the determined proteins were associated with networks/pathways of energy, putrescine metabolism, and host-virus interaction. Additionally, 556 peptides were identified as virulence factors. Moreover, 77 species-specific peptide biomarkers corresponding to 64 different proteins were proposed to identify 10 bacterial species. This represents a major proteomic dataset of biogenic-amine-producing strains. These results may also be suitable for new treatments for food intoxication and for tracking microbial sources in foodstuffs.

A novel strategy to identify the species-specific peptide biomarkers in Pheretima aspergillum (E. Perrier) based on enzymatic digestion followed by LC-MS/MS methods

Guang Dilong [P. aspergillum (E. Perrier)], is an animal-derived traditional Chinese medicine made from the dried body of Pheretima aspergillum (E. Perrier) (TCM). Due to its widely application and high medical values, preparations of P. aspergillum (E. Perrier) may be adulterated by four other species, including three crucial Pheretima species [P. vulgaris (Chen), P. pectinifera (Mkhaeken), and P. guillemi (Michaelsen)] and one considerable adulteration [Metaphire magna (Chen)]. This study developed a novel and effective strategy for analyzing and authenticating Guang Dilong based on enzymatic digestion of protein. The nanoLC-MS/MS technique used to evaluate complete peptidomics profiles of trypsin-digested samples, resulting in the identification of species-specific peptide biomarkers in P. aspergillum (E. Perrier). The significance of different samples and peptides in the target species set was then investigated using mathematical set theory. Consequently, seven peptides were chosen as prospective biomarkers. Finally, five specific peptide biomarkers for differentiating Guang Dilong with other species were confirmed and validated using UFLC-MS/MS and MRM mode. The suggested technique may also be beneficial in evaluating the quality of other animal-derived goods for safety issues in order to avoid misidentification.

Determination of porcine derived components in gelatin and gelatin-containing foods by high performance liquid chromatography-tandem mass spectrometry

Detection and quantitation of porcine derived components in bovine and donkey gelatins and gelatin-containing foods is required for religious practices and consumer economic interests. In this study, shot-gun proteomics approaches were implemented to identify species-specific peptide biomarkers of three main gelatin origins, including porcine, bovine and donkey. Tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography-tandem high-resolution mass spectrometry (UPLC-HRMS), and 11 peptide biomarkers were selected for porcine, bovine and donkey. The gelatin pretreatment methods in three different food matrixes were optimized, and a label-free quantitative method using the MRM method of the most sensitive biomarker peptides was established. Among them, two porcine gelatin quantitative peptides were confirmed to have good linearity. The method has been successfully applied to the adulteration detection of porcine-derived components in gelatin and the quantification of porcine gelatin in candy and dairy products, with LODs of 0.07, 0.08 and 0.12 mg/kg, respectively. • A strategy for identification of peptide biomarkers of common gelatin species was established and improved with untargeted and targeted proteomics approaches. • Eleven species-specific peptide biomarkers were selected for porcine, bovine and donkey gelatin. • The pretreatment method of gelatin in different food matrixes has been optimized, especially for gelatin in dairy products. • The label-free MRM method is used for quantitative determination of adulterated porcine gelatin in mixed gelatin and gelatin-containing food, with good linearity, specificity, sensitivity and repeatability.

Metabolite profiling reveals insights into the species-dependent cold stress response of the green seaweed holobiont Ulva (Chlorophyta)

The green seaweed Ulva (Chlorophyta) and its associated epibacterial microbiome form a functional holobiont that adapts to stress as a whole. As the macroalga provides carbon sources for bacteria and relies on algal growth and morphogenesis-promoting factors (AGMPF) released by bacteria, those cross-kingdom interactions are especially fascinating. We hypothesized that the Ulva holobionts from the warm-temperate Mediterranean-Atlantic and cold Antarctic habitats respond to cold stress differently depending on the production of highly polar low molecular weight compounds (LMWCs) with stress-regulating activity. We compared the microbiome of and metabolic changes in U. compressa (cultivar U. mutabilis ), initially collected in Ria Formosa (Portugal), with that of U. bulbosa (strain AWI #1002) collected in Antarctica by performing a warm-cold temperature shift experiment. Microbiome analysis indicated significant differences between the two species and that the number of operational taxonomic units (OTUs) was lower in cultivated U. bulbosa than in freshly isolated Ulva ; despite this, AGMPF-producing bacteria were detected in both holobionts. Significant differences in metabolite profiles were observed between both species using hydrophilic interaction liquid chromatography coupled with electrospray ionization mass spectrometry (HILIC-ESI-MS). Biomarkers such as dimethylsulfoniopropionate (DMSP) and taurine were identified following a temperature shift from 18 °C to 5 °C in the warm-temperate U. mutabilis and the cold-adapted U. bulbosa , respectively. Our findings show that metabolic changes in the holobiont in response to cold are species-dependent. To evaluate the contribution of the metabolic changes of bacteria and algae to the stress response, the reductionistic model system of the tripartite community formed by U. mutabilis and its two essential bacteria, Roseovarius sp. strain MS2 and Maribacter sp. strain MS6, which release all essential AGMPFs, was investigated. We examined the production of polar LMWCs in the presence and absence of bacteria following a shift to cold temperatures. Among the metabolites studied, ectoine ((4 S )-2-methyl-3,4,5,6-tetrahydropyrimidine-4-carboxylic acid) was only detected in the presence of bacteria, highlighting the role of bacteria in releasing compounds that mitigate environmental stresses through cold stress adaptation factors (CSAF). Our findings suggest that microbiome engineering will allow us to improve macroalgae adaptability to stressful situations, which can be further applied to the sustainable management of (land-based) aquaculture systems. • Algal growth and morphogenesis promoting bacteria were found in cultures of Ulva independently of their origin and habitat. • Low molecular weight compounds (LMWC) of the holobiont were examined upon cold stress. • Cold stress triggers diverse potential cryoprotectants in warm- and cold-adapted Ulva. • Taurine and DMSP may be considered as species-specific stress biomarkers. • Axenic Ulva cultures elucidate the bacterial contribution to the production of LMWC.

Genomics of Serrasalmidae teleosts through the lens of microbiome fingerprinting.

Associations between host genotype and host-associated microbiomes have been shown in a variety of animal clades, but studies on teleosts mostly show weak associations. Our study aimed to explore these relationships in four sympatric Serrasalmidae (i.e., piranha) teleosts from an Amazonian lake, using data sets from the hosts genomes (single nucleotide polymorphisms from genotyping by sequencing), skin and gut microbiomes (16S rRNA gene metataxonomics) and diets (COI metabarcoding) from the same fish individuals. First, we investigated whether there were significant covariations of microbiome and fish genotypes at the inter- and intraspecific levels. We also assessed the extent of covariation between Serrasalmidae diet and microbiome, to isolate genotypic from dietary effects on community structure. We observed a significant covariation of skin microbiomes and host genotypes at interspecific (R2 = 24.4%) and intraspecific (R2 = 6.2%) levels, whereas gut microbiomes correlated poorly with host genotypes. Serrasalmidae diet composition was significantly correlated to fish genotype only at the interspecific level (R2 = 5.4%), but did not covary with gut microbiome composition (Mantel R= -.04). Second, we investigated whether the study of interspecific differentiation could benefit from considering host-associated microbial communities in addition to host genotypes. By using a nonmetric multidimensional scaling (NMDS) ordination-based approach, we observed that ordinations from skin- and gut species-specific bacterial biomarkers identified through a random forest algorithm could significantly increase the average interspecific differentiation detected through host genotype data alone. Although future studies encompassing additional species and environments are needed, our results suggest Serrasalmidae microbiomes could constitute an insightful trait to be considered when studying the interspecific differences between members of this clade.

Anopheles salivary antigens as serological biomarkers of vector exposure and malaria transmission: A systematic review with multilevel modelling.

Entomological surveillance for malaria is inherently resource-intensive and produces crude population-level measures of vector exposure which are insensitive in low-transmission settings. Antibodies against Anopheles salivary proteins measured at the individual level may serve as proxy biomarkers for vector exposure and malaria transmission, but their relationship is yet to be quantified. A systematic review of studies measuring antibodies against Anopheles salivary antigens (PROSPERO: CRD42020185449). Multilevel modelling (to account for multiple study-specific observations [level 1], nested within study [level 2], and study nested within country [level 3]) estimated associations between seroprevalence with Anopheles human biting rate (HBR) and malaria transmission measures. From 3981 studies identified in literature searches, 42 studies across 16 countries were included contributing 393 study-specific observations of anti-Anopheles salivary antibodies determined in 42,764 samples. A positive association between HBR (log transformed) and seroprevalence was found; overall a twofold (100% relative) increase in HBR was associated with a 23% increase in odds of seropositivity (OR: 1.23, 95% CI: 1.10-1.37; p<0.001). The association between HBR and Anopheles salivary antibodies was strongest with concordant, rather than discordant, Anopheles species. Seroprevalence was also significantly positively associated with established epidemiological measures of malaria transmission: entomological inoculation rate, Plasmodium spp. prevalence, and malarial endemicity class. Anopheles salivary antibody biomarkers can serve as a proxy measure for HBR and malaria transmission, and could monitor malaria receptivity of a population to sustain malaria transmission. Validation of Anopheles species-specific biomarkers is important given the global heterogeneity in the distribution of Anopheles species. Salivary biomarkers have the potential to transform surveillance by replacing impractical, inaccurate entomological investigations, especially in areas progressing towards malaria elimination. Australian National Health and Medical Research Council, Wellcome Trust.

Chemotyping of three Morchella species reveals species- and age-related aroma volatile biomarkers

The desirable aroma of morels is ascribed to volatiles from various metabolic pathways. Mushrooms' volatile composition varies with genetic background, age, and cultivation conditions. We aimed to differentiate mushrooms based on their aroma profiles, and identify biomarkers for chemotyping. The aroma volatile composition of Morchella rufobrunnea, Morchella importuna, and Morchella dunalii was described using headspace GC/MS. Eighty six aroma compounds were identified, comprised of alcohols, aldehydes, hydrocarbons, esters, furans, ketones, phenolic compounds, pyrazines, and terpenes. 1-Octanol, heptanal, 3-ethyl-3-methylheptane, 4,6-dimethylundecane, and 1,3-bis(1,1-dimethylethyl) benzene were identified as key species-specific biomarkers; and octanal, heptanal, 3-octen-2-one, and benzeneacetaldehyde as key age-related (young vs. mature mushroom) biomarkers. 4,6-Dimethylundecane and 1,3-bis(1,1-dimethylethyl) benzene were in accordance with the saprophytic origin of M. rufobrunnea and M. importuna. Thus, specific Morchella volatiles can serve as biomarkers in chemotyping Morchella species, indicating their age, and hinting at their saprophytic nature.

NMR-based metabolomics for frauds detection and quality control of oregano samples

In this work 1H NMR metabolomics has been employed for quality control of oregano samples. NMR data and morphological analysis (MA) were combined by PCA, obtaining a model able to individuate non-marketable samples, and to distinguish between the two marketable oregano species (Origanum vulgare and O. onites) on the basis of their metabolomic profile. Through this approach distinctive biomarkers of the two species were found, namely apigenin and p-cymene for O. onites, and salvianolic acid B for O. vulgare. Furthermore, the percentage of the samples’ impurity (evaluated by MA) and the metabolomic profiles were correlated by OPLS models, which showed that, in addition to the species-specific biomarkers, thymol and rosmarinic acid (common to both marketable species) strongly correlate to oregano degree of purity. Cistus was one of the most frequent contaminants, thus, a further OPLS model, able to detect the degree of cistus contamination in oregano samples, was also built.

Establishment and Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Detection of Shewanella Genus.

Shewanella species are widely distributed in the aquatic environment and aquatic organisms. They are opportunistic human pathogens with increasing clinical infections reported in recent years. However, there is a lack of a rapid and accurate method to identify Shewanella species. We evaluated here matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of Shewanella. A peptide mass reference spectra (PMRS) database was constructed for the type strains of 36 Shewanella species. The main spectrum projection (MSP) cluster dendrogram showed that the type strains of Shewanella species can be effectively distinguished according to the different MS fingerprinting. The PMRS database was validated using 125 Shewanella test strains isolated from various sources and periods; 92.8% (n = 116) of the strains were correctly identified at the species level, compared with the results of multilocus sequence analysis (MLSA), which was previously shown to be a method for identifying Shewanella at the species level. The misidentified strains (n = 9) by MALDI-TOF MS involved five species of two groups, i.e., Shewanella algae–Shewanella chilikensis–Shewanella indica and Shewanella seohaensis–Shewanella xiamenensis. We then identified and defined species-specific biomarker peaks of the 36 species using the type strains and validated these selected biomarkers using 125 test strains. Our study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid identification of Shewanella strains at the species level.

Phenotypic and genotypic characterization of Enterococcus spp. from yolk sac infections in broiler chicks with a focus on virulence factors

Bacterial infections of yolk sacs contribute to increased mortality of chicks, chronic infections during their rearing, or increased selection in the flock, which in turn leads to high economic losses in poultry production worldwide. The aim of this study was a phenotypic and genotypic characterization of enterococci isolated from yolk sac infections (YSI) of broiler chickens from Poland and the Netherlands. Biochemical, matrix-assisted laser desorption/ionization (MALDI)–time-of-flight (TOF) MS, and rpoA gene sequencing identification was performed. Moreover, phenotypic and genotypic characterization of virulence factors and analysis of the clonal relationship of isolates by MALDI-TOF MS and enterobacterial repetitive intergenic consensus—polymerase chain reaction (ERIC-PCR) were performed. The biochemical test identified 70 isolates as Enterococcus faecalis and 6 as Enterococcus mundtii. The results of MALDI-TOF MS were 100% concordant with those obtained by rpoA gene sequencing, and all 76 isolates were identified as E. faecalis. Differences were noted in the β-glucuronidase, β-glucosidase, α-galactosidase, phosphatase, melibiose, lactose, and raffinose tests that is going about the results of biochemical identification. None of the isolates were beta-hemolytic on blood agar in aerobic conditions, but all but one were gelatinase positive. Among biofilm-forming isolates (30/76; 39.5%), as many as 66.7% (20/30) were Polish E. faecalis strains. Most of the isolates carried virulence genes, that is gelE, ace, asa1, efaAfs, fsrA, fsrB, fsrC, cob, cpd, and ccf, but none had the hyl gene. Some isolates harbored cyl operon genes. One Polish strain (ST16) had all of the tested cyl genes and the esp gene, considered clinically important, and showed the highest biofilm-forming ability. Nearly 50% of the isolates showed close genetic relatedness in ERIC typing. In contrast with MALDI-TOF MS cluster analysis, ERIC-PCR results did not show a relationship with the origin of the strains. Using MALDI-TOF MS, 7 peaks were found in Polish and Dutch isolates, which may type them as species-specific biomarkers in E. faecalis from YSI.

Usefulness of matrix-assisted laser desorption ionization/time of flight mass spectrometry for the identification of Streptococcus mutans.

This study evaluated the reliability of MALDI-TOF MS coupled with statistical tools for the identification of Streptococcus mutans in comparison with PCR-based techniques. Bacterial isolates were identified and serotyped by conventional PCR, using S. mutans species and serotype-specific primers. For bacterial identification, mass spectra data from S. mutans and other streptococci were compared with Biotyper V 3.1 database and the mass peak lists were examined by cluster and principal component (PCA) analysis. Identification of potential biomarkers was performed using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and BLAST tool of the NCBI database. PCR identified 100% of the isolates as S. mutans. S. mutans strains were typed as serotypes c (85.6%), e (8.6%), k (4.8%), and f (0.9%). Although only the 70% of the strains tested were identified at species level by the Biotyper database, PCA and cluster analysis of mass peaks allowed the identification of 100% S. mutans isolates and its differentiation from the other oral and non-oral streptococci. One mass peak at m/z value of 9572.73 was identified as species-specific biomarker for S. mutans. No biomarkers were identified for S. mutans serotypes. KEY POINTS: • MALDI-TOF MS coupled with statistical tools for the identification of S. mutans. • Detection of species identifying biomarkers by MALDI-TOF MS. • PCR identification and serotyping of S. mutans from saliva samples.

Serum Health Biomarkers in African and Asian Elephants: Value Ranges and Clinical Values Indicative of the Immune Response.

Simple SummaryBiomarkers are biological molecules found in the blood or other fluids or tissues that can indicate normal or abnormal processes or disease. Developing tools to measure biomarkers that indicate immune function and establishing concentrations observed within a species is an important first step in their use for managing health and understanding disease processes. Here we report assays, observed value ranges, and concentrations during illness or injury for seven immune biomarkers measured in the serum of African and Asian elephants under human care. Concentrations were variable in both clinical and non-clinical samples, but all seven biomarkers were elevated in at least one case and most increased in response to routine vaccination in a single Asian elephant. These tools provide an exciting avenue for monitoring health status and helping diagnose and treat health problems in wildlife species, like elephants.Serum biomarkers indicative of inflammation and disease can provide useful information regarding host immune processes, responses to treatment and prognosis. The aims of this study were to assess the use of commercially available anti-equine reagents for the quantification of cytokines (tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukins (IL) 2, 6, and 10) in African (Loxodonta africana, n = 125) and Asian (Elephas maximus, n = 104) elephants, and alongside previously validated anti-human reagents for acute-phase proteins (serum amyloid A and haptoglobin), calculate species-specific biomarker value ranges. In addition, we used opportunistically collected samples to investigate the concentrations of each biomarker during identified clinical cases of illness or injury, as a first step to understanding what biomarkers may be useful to managing elephant health. Immune biomarkers were each elevated above the calculated species-specific value ranges in at least one clinical case, but due to variability in both clinical and non-clinical samples, only serum amyloid A was significantly higher in clinical compared to non-clinical paired samples, with tendencies for higher TNF-α and IL-10. We also detected increased secretion of serum amyloid A and all five cytokines following routine vaccination of a single Asian elephant, indicating that these biomarkers can be beneficial for studying normal immune processes as well as pathology. This study indicates that assays developed with commercial reagents can be used to quantify health biomarkers in wildlife species and identifies several that warrant further investigation to elucidate immune responses to various pathologies.

Core Mycobiome and Their Ecological Relevance in the Gut of Five Ips Bark Beetles (Coleoptera: Curculionidae: Scolytinae).

Bark beetles are destructive forest pests considering their remarkable contribution to forest depletion. Their association with fungi is useful against the challenges of survival on the noxious and nutritionally limited substrate, i.e., conifer tissues. Fungal symbionts help the beetles in nutrient acquisition and detoxification of toxic tree secondary metabolites. Although gut is the prime location for food digestion and detoxification, limited information is available on gut-mycobiome of bark beetles. The present study screened the gut-mycobiont from six bark beetles (five Ips and one non-Ips) from Scolytinae subfamily using high-throughput sequencing and explored their putative role in symbiosis with the host insect. Results revealed the predominance of four fungal classes- Sordariomycetes, Saccharomycetes, Eurothiomycetes, and Dothidomycetes in all bark beetles. Apart from these, Agaricomycetes, Leothiomycetes, Incertae sedis Basidiomycota, Tremellomycetes, Lecanoromycetes, and Microbotryomycetes were also documented in different beetles. Five Ips bark beetles share a consortium of core fungal communities in their gut tissues consisting of 47 operational taxonomic units (OTUs) belonging to 19 fungal genera. The majority of these core fungal genera belong to the phylum Ascomycota. LEfSe analysis revealed a set of species-specific fungal biomarkers in bark beetles. The present study identified the gut mycobiont assemblage in bark beetles and their putative ecological relevance. An enriched understanding of bark beetle-fungal symbiosis is not only filling the existing knowledge gap in the field but may also unleash an unforeseen potential for future bark beetle management.

Testing algal-based pCO2 proxies at a modern CO2 seep (Vulcano, Italy)

Understanding long-term trends in atmospheric concentrations of carbon dioxide (pCO2) has become increasingly relevant as modern concentrations surpass recent historic trends. One method for estimating past pCO2, the stable carbon isotopic fractionation associated with photosynthesis (Ɛp) has shown promise over the past several decades, in particular using species-specific biomarker lipids such as alkenones. Recently, the Ɛp of more general biomarker lipids, organic compounds derived from a multitude of species, have been applied to generate longer-spanning, more ubiquitous records than those of alkenones but the sensitivity of this proxy to changes in pCO2 has not been constrained in modern settings. Here, we test Ɛp using a variety of general biomarkers along a transect taken from a naturally occurring marine CO2 seep in Levante Bay of the Aeolian island of Vulcano in Italy. The studied general biomarkers, loliolide, cholesterol, and phytol, all show increasing depletion in 13C over the transect from the control site towards the seep, suggesting that CO2 exerts a strong control on isotopic fractionation in natural phytoplankton communities. The strongest shift in fractionation was seen in phytol, and pCO2 estimates derived from phytol confirm the utility of this biomarker as a proxy for pCO2 reconstruction.

Analysis of cicada wing surface constituents by comprehensive multidimensional gas chromatography for species differentiation

Cicada wings exhibit several intriguing properties that arise from a combination of nanopillar structures and chemical constituents, including superhydrophobicity, as well as antimicrobial, antireflective, and self-cleaning functions. While the physical dimensions of the nanofeatures are relatively simple to characterize through microscopy, the chemicals that cover these pillars are more difficult to characterize due to the variety and complexity of the mixture. Here, we compared the extractable chemicals from the wing surfaces of two different cicada species using both gas chromatography time-of-flight mass spectrometry (GC-TOFMS) and two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) platforms. Chemical extracts from Neotibicen pruinosus and Magicicada septendecim cicada wings were separated and analyzed. The GC × GC-TOFMS platform was able to isolate and identified roughly three times the number of constituents as the GC-TOFMS platform at a signal-to-noise ratio (SNR) ≥10.0 and spectral similarity ≥800. When comparing the two cicada species wing extracts, the two-dimensional platform was able to expose differences in the chemical composition that were undetectable by the one-dimensional technique. GC × GC-TOFMS revealed nearly four times the number of unique species-specific compounds as compared to the number identified by GC-TOFMS. Further, surface chemicals were identified that are likely xenobiotics and can pinpoint location and contamination from where the cicada was collected. While the advantages of GC × GC-TOFMS over GC-TOFMS have been documented in the past, our work presents a powerful biological application of GC × GC-TOFMS with promise to reveal both organism species-specific biomarkers while providing insight into the environmental conditions of individual organisms.