Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.     

Microbiome of the larvae of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) from maize plants

Fall armyworm (FAW), Spodoptera frugiperda, is an invasive insect pest with a diverse host range threatening food security in the African continent by causing severe economic damage to staple maize crop. FAW like all other insects are associated with various microbiota that influence their several characteristics and activities. The microbiota of insects plays important roles in their growth, development and environmental adaptation to their host plants or animals and not much is known about microbiota of FAW in Nigeria. In this study, the microbial community of FAW larvae from maize plants in Nigeria was investigated using Illumina PE250 NovaSeq of 16S rRNA V3-V4 gene region. A total of 1,160,133 sequence reads that ranged from 83,794 to 145,475 were obtained. They span through 2 kingdoms: bacteria (99.59%) and archaea (0.37%), 37 phyla, 59 classes, 78 orders, 145 families and 285 genera. The dominant taxonomic group of bacteria across the larval samples were Proteobacteria (58.32%), Firmicutes (35.87%) and Bacteroidetes (4.02%). There were no significant differences in bacteria species diversity and richness among the individual fall armyworm larva and samples from different geographical regions. The relative abundance of Bacteria phyla and classes also showed no significant differences among the larval samples. Significant differences were documented in bacteria orders Acidobacteriales and Aeromonadales, family Acidobacteriaceae_ (Subgroup_1) and genera Novosphingobium and Pedobacter. The results indicate that the gut of FAW larvae harbours a diverse set of bacteria and archaea biota. Understanding their ecological and functional relevance would provide novel insight on potential approaches for their exploitation in the integrated management of FAW in the tropics.     

Influence of fall armyworm previous experience with soybean genotypes on larval feeding behavior

Previous experience on host plants can modify insect feeding behavior. Because insect habituation and induction of preference to host plants are variable across species of plants and insects, it is necessary to investigate each insect-plant interaction to determine whether this phenomenon occurs or not in the system. In this study we investigated the potential occurrence of habituation and induction of preference in fall armyworm (FAW) Spodoptera frugiperda to soybean genotypes. Neonate FAW larvae reared on artificial diet were divided into four treatment groups and fed for one generation with either the resistant soybeans PI 227687 or IAC 100 or the susceptible soybeans BRS Valiosa RR or IGRA RA 626 RR. Biological parameters of FAW were recorded. Eggs obtained from FAW of each genotype group were separated, and the newly hatched larvae were fed on the same genotypes experienced by their parents for additional 8 days. FAW larval preference and leaf area consumed were evaluated in choice feeding assays with the four soybean genotypes within a 24-h period. Genotypes PI 227687 and IAC 100 negatively affected FAW development, demonstrating they are FAW-resistant. FAW larvae exposed to both resistant genotypes consumed more foliage of genotype IGRA RA 626 RR in the choice assays, whereas larvae reared on both susceptible genotypes did not show any preference. From our preliminary study, FAW does not show habituation and induction of preference toward the experienced soybean genotypes. The importance of our findings to host plant resistance and insect-plant biology fields is discussed.     

In Vitro Identification of Histatin 5 Salivary Complexes

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein–protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein–protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance.     

A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae

Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.     

Protein networks reveal organ-specific defense strategies in maize in response to an aboveground herbivore

Many of the proteins and defense pathways in maize that are activated in an organ-specific manner in leaves and roots during aboveground caterpillar attack have not yet been identified. In this study, we examined systemic and organ-specific defenses in the insect-resistant maize genotype, Mp708, when infested aboveground with fall armyworm (FAW, Spodoptera frugiperda). We used proteomic and network biology analyses and then integrated these data with known FAW resistance QTL to create a protein abundance QTL (pQTL) subnetwork. Using 10-plex tandem mass spectrometry tags (TMT) proteomics technique, we identified a total of 4675 proteins in leaves and roots of control and FAW-infested plants. Among the identified proteins, 794 had significant differences in abundance in response to FAW herbivory. Proteins that were upregulated in leaves during FAW infestation included jasmonic acid biosynthetic enzymes, cysteine proteases, protease inhibitors, REDOX-related proteins, and peroxidases. In roots, highly abundant proteins were involved in ET biosynthesis, DNA expression regulation, and pyruvate biosynthesis. We found many proteins that possibly contribute different defense functions to FAW resistance in Mp708. One potential resistance mechanism identified was that trade-offs between growth and defense responses were reduced in Mp708. Some of the proteins involved in this trade-off that were found within the pQTL subnetwork were the Kinesin-like protein (GRMZM2G046186_P01) and Pi starvation-induced protein (GRMZM2G118037_P01). We proposed other mechanisms contributing to resistance that suggest that jasmonic acid and ethylene control the local accumulation of insecticidal cysteine protease (MIR1-CP) in leaves, while ethylene controlled the systemic accumulation of MIR1-CP in roots. Finally, we hypothesized that receptor kinases such as receptor protein kinase 1 (GRMZM2G055678) could be involved in the activation of root-specific defense responses during aboveground insect infestation.     

植食性昆虫唾液效应子和激发子的研究进展

植食性昆虫与寄主植物通过协同进化形成了复杂的防御和反防御机制。本文系统综述了昆虫唾液效应子和激发子在植物与昆虫互作中的作用及机理。昆虫取食中释放的唾液激发子被植物识别而激活植物早期免疫反应,昆虫也能从口腔分泌效应子到植物体内抑制免疫;抗性植物则利用抗性(R)蛋白识别昆虫无毒效应子,启动效应子诱导的免疫反应,而昆虫又进化出多种方式来躲避植物R蛋白的识别。总之,在这场军备竞赛中,昆虫的唾液成分决定着昆虫能否取食成功。取食过程中,咀嚼式口器害虫分泌大量酶类到植物体内,而刺吸式害虫则分泌胶状和水样唾液到植物中,它们都利用激发子和效应子去调控植物的免疫防御反应。分析现已报道的昆虫效应子发现其作用机制各有不同,具体表现为影响植物早期防御信号,调控植物激素通路及其他通路,或靶向小分子RNA通路。本文还综述了昆虫激发子的最新进展,揭示激发子可以通过诱导释放植物次生代谢物以及调控激素水平、Ca²⁺内流和活性氧爆发增强植物抗性。最后对昆虫效应子的分泌特性、寄主特异性和多功能性作了分析,并对无毒效应子及其对应的植物R基因,以及激发子的模式识别受体的研究进行了展望。     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Chemosensory proteins, major salivary factors in caterpillar mandibular glands

Research in the field of insect-host plant interactions has indicated that constituents of insect saliva play an important role in digestion and affect host chemical defense responses. However, most efforts have focused on studying the composition and function of regurgitant or saliva produced in the labial glands. Acknowledging the need for understanding the role of the mandibular glands in herbivory, we sought to make a qualitative and semi-quantitative comparison of soluble luminal protein fractions between mandibular and labial glands of Vanessa gonerilla butterfly larvae. Amylase and lysozyme were inspected as possible major enzymatic activities in the mandibular glands aiding in pre-digestion and antimicrobial defense. Although detected, neither of these enzymatic activities was prominent in the luminal protein preparation of a particular type of gland. Proteins isolated from the glands were identified by mass spectrometry and by searching an EST-library database generated for four other nymphalid butterfly species, in addition to the public NCBI database. The identified proteins were also quantified from the data using "Quanty", an in-house program. The proteomic analysis detected chemosensory proteins as the most abundant luminal proteins in the mandibular glands. In comparison to these proteins, the relative amounts of amylase and lysozyme were much lower in both gland types. Therefore, we speculate that the primary role of the mandibular glands in Lepidopteran larvae is chemoreception which may include the detection of microorganisms on plant surfaces, host plant recognition and communication with conspecifics.     

A proteomic approach to the identification of salivary proteins from the argasid ticks Ornithodoros moubata and Ornithodoros erraticus

The saliva of ticks contains anti-haemostatic, anti-inflammatory and immunomodulatory molecules that allow these parasites to obtain a blood meal from the host and help tick-borne pathogens to infect the vertebrate host more efficiently. This makes the salivary molecules attractive targets to control ticks and tick-borne pathogens. Although Ornithodoros moubata and O. erraticus are important argasid ticks that transmit severe diseases, to date only a few of their salivary proteins have been identified. Here we report our initial studies using proteomic approaches to characterize the protein profiles of salivary gland extracts (SGE) from these two argasids. The present work describes the proteome of the SGEs of both tick species, their antigenic spots, and the identification of several of their proteins. The whole number of identifications was low despite the good general quality of the peptide mass maps obtained. In the O. moubata SGE, 18 isoforms of a protein similar to O. savignyi TSGP1 were identified. In the O. erraticus SGE we identified 6 novel proteins similar to unknown secreted protein DS-1 precursor, NADPH dehydrogenase subunit 5, proteasome alpha subunit, ATP synthase F0 subunit 6, lipocalin and alpha tubulin. Finally, the current drawbacks of proteomics when applied to the identification of acarine peptides and proteins are discussed.     

Proteomic analysis of Lyme disease: global protein comparison of three strains of Borrelia burgdorferi

The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric analysis. Protein identification was facilitated by the availability of the complete B31 genome sequence. A total of 665 Borrelia proteins were identified representing approximately 38% coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.     

Proteome Analysis of Watery Saliva Secreted by Green Rice Leafhopper,Nephotettix cincticeps

The green rice leafhopper, Nephotettix cincticeps, is a vascular bundle feeder that discharges watery and gelling saliva during the feeding process. To understand the potential functions of saliva for successful and safe feeding on host plants, we analyzed the complexity of proteinaceous components in the watery saliva of N. cincticeps. Salivary proteins were collected from a sucrose diet that adult leafhoppers had fed on through a membrane of stretched parafilm. Protein concentrates were separated using SDS-PAGE under reducing and non-reducing conditions. Six proteins were identified by a gas-phase protein sequencer and two proteins were identified using LC-MS/MS analysis with reference to expressed sequence tag (EST) databases of this species. Full -length cDNAs encoding these major proteins were obtained by rapid amplification of cDNA ends-PCR (RACE-PCR) and degenerate PCR. Furthermore, gel-free proteome analysis that was performed to cover the broad range of salivary proteins with reference to the latest RNA-sequencing data from the salivary gland of N. cincticeps, yielded 63 additional protein species. Out of 71 novel proteins identified from the watery saliva, about 60 % of those were enzymes or other functional proteins, including GH5 cellulase, transferrin, carbonic anhydrases, aminopeptidase, regucalcin, and apolipoprotein. The remaining proteins appeared to be unique and species- specific. This is the first study to identify and characterize the proteins in watery saliva of Auchenorrhyncha species, especially sheath-producing, vascular bundle-feeders.     

草地贪夜蛾对5种寄主植物和6种杂草的取食选择性和适应性

为明确草地贪夜蛾Spodoptera frugiperda J. E. Smith对不同寄主植物和主要杂草的取食选择及适应性,以玉米Zea mays、甘蔗Saccharum officinarum、花生Arachis hypogaea、大豆Glycine max、香蕉Musa nana、稗草Echinochloa crusgalli、马唐Digitaria sanguinalis、牛筋草Eleusine indica、莎草Cyperus rotundus、马齿苋Portulaca oleracea、鹅肠草Malachium aquaticum作为寄主,采用叶碟法测定草地贪夜蛾各龄幼虫对5种寄主植物和6种杂草的取食偏好,并测定了不同虫态的发育历期、单头蛹重、化蛹率以及单雌产卵量等。结果表明,草地贪夜蛾在5种寄主植物和6种杂草上均可完成世代发育。寄主作物中,香蕉处理的草地贪夜蛾成虫前期最长,为29.66 d;甘蔗处理下草地贪夜蛾蛹重和化蛹率分别为250.44 mg和71.67%,显著低于玉米和花生处理的蛹重和化蛹率;草地贪夜蛾取食花生后单雌产卵量最高,为768.93粒,与玉米上产卵量差异不显著,香蕉上最低,为498.76粒;1~2龄幼虫对玉米和香蕉的取食选择率有显著差异,3~6龄幼虫对玉米取食选择率显著高于其他寄主植物。6种杂草中取食莎草的成虫前期最长30.21 d,且单雌产卵量最低526.33粒;取食鹅肠草蛹重最低188.00 mg;取食马齿苋化蛹率最低为72.37%;草地贪夜蛾取食马唐后其蛹重、化蛹率、单雌产卵量最高,与取食其它5种杂草有显著性差异;2~4龄幼虫更偏向取食马唐,与其它5种差异显著。研究结果表明：寄主植物种类对草地贪夜蛾的生长发育有显著影响,其中草地贪夜蛾对玉米和马唐具有较高的取食偏好。     

唾液成分在刺吸式昆虫与植物关系中的作用

近年来,人们对刺吸式昆虫唾液成分的研究,揭示出其在刺吸式昆虫与植物关系中的重要作用。对多数刺吸式昆虫而言,他们取食时会分泌胶状和水状两种唾液,其中胶状唾液会在取食早期分泌形成唾液鞘来围绕并保护口针,通过直接和间接的作用来帮助取食;而水状唾液中则包含了果胶酶、纤维素酶、多酚氧化酶、过氧化物酶、碱性磷酸酯酶、蔗糖酶等组分,来帮助刺吸式昆虫对植物穿刺、消化食物、解毒次生物质并破坏植物的防御反应。有趣的是,唾液成分同时还可以诱导植物的防御反应,包括诱导植物的伤信号引起直接防御反应和诱导植物产生挥发物吸引植食者的天敌引起间接防御反应。并且,许多刺吸式昆虫取 食能够特异性地引发植物的病理反应,有研究推测刺吸式昆虫唾液中多聚半乳糖醛酸酶、碱性磷酸酯酶、蔗糖酶、多酚氧化酶等成分可能是某些植物特定病理反应的激发子,但是目前还没有定论,同时许多刺吸式昆虫唾液中的氨基酸和蛋白酶还是引起植物虫瘿的原因之一。 迄今的研究表明,刺吸式昆虫会根据不同的寄主植物和不同的生理需要,通过唾液组分的改变,来达到取食和发育的目的。对刺吸式昆虫唾液成分和作用机理的研究,可以为揭示刺吸式昆虫致害机理特别是传毒机理、指导害虫有效治理、阐明其与植物的协同进化等提供一定的思路。     

Unraveling persistent host cell infection with Coxiella burnetii by quantitative proteomics

The interaction between the immune system and invading bacteria is sufficient to eradicate microorganisms for the majority of bacterial infections, but suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. To identify the cellular pathways affected by bacterial persistence, we applied the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique for a comparative study of protein expression in the C. burnetii strains Nine Mile (NM) and its respective strain (NMper) isolated from 18 months persistently infected cell cultures. In total, three different proteome comparisons were performed with the total bacterial proteome, potentially secreted bacterial proteins, and the eukaryotic infected proteome being assessed. Our results revealed that among the 547 identified bacterial proteins, 53 had significantly altered levels of expression and indicated potential metabolic differences between the two strains. Regarding differences in the secreted proteins between both strains and different modulation of the host cell, machineries reflect at least large rearrangements of both bacterial and eukaryotic proteomes during the persistent model of infection when compared to the acute one, which emphasizes that C. burnetii orchestrates a vast number of different bacterial and eukaryotic host cell processes to persist within its host.     

Insight into the salivary transcriptome and proteome of Dipetalogaster maxima

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.     

Analysis of the human saliva proteome

Interest in the characterization of the salivary proteome has increased in the last few years. This review discusses the different techniques and methodologies applied to the separation and identification of salivary proteins. Nowadays, proteomic techniques are the state of the art for the analysis of biologic materials and saliva is no exception. 2D electrophoresis and tryptic digest analysis by mass spectrometry are the typical methodology, but new approaches using 2D liquid chromatography/mass spectrometry methods have already been introduced for saliva analysis. Due to their important physiologic role in the oral cavity, low-molecular-weight proteins and peptides are also included in this article and the methodologies discussed.