Insight into watery saliva proteomes of the grain aphid,Sitobion avenae

The grain aphid, Sitobion avenae, is an economically important cereal pest worldwide. Aphid saliva plays an essential role in the interaction between aphids and their host plants. However, limited information is available regarding the proteins found in the saliva of S. avenae. Here, the watery saliva proteins from S. avenae were collected in an artificial diet and identified using a liquid chromatography–mass spectrometry/mass spectrometry analysis. A total of 114 proteins were identified in S. avenae saliva, including several enzymes, binding proteins, and putative effectors, as well as other proteins with unknown functions. In comparison with salivary proteins from nine other aphid species, the most striking feature of the salivary protein from S. avenae was the different patterns of protein functions. Several orthologous proteins secreted by other aphid species such as glucose dehydrogenase, elongation factors, and effector C002 were also detected in S. avenae saliva and speculated to play a significant role in aphid–plant interactions. These results provide further insight into the molecular basis between aphids and cereal plant interactions.     

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE‐LC‐MS/MS and LC‐MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin‐converting enzyme (an M2 metalloprotease), an M1 zinc‐dependant metalloprotease, a glucose‐methanol‐choline (GMC)‐oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium‐binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium‐mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.     

Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113.     

Chromosome‐level genome assembly for the horned‐gall aphid provides insights into interactions between gall‐making insect and its host plant

The aphid Schlechtendalia chinensis is an economically important insect that can induce horned galls, which are valuable for the medicinal and chemical industries. Up to now, more than twenty aphid genomes have been reported. Most of the sequenced genomes are derived from free‐living aphids. Here, we generated a high‐quality genome assembly from a galling aphid. The final genome assembly is 271.52 Mb, representing one of the smallest sequenced genomes of aphids. The genome assembly is based on contig and scaffold N50 values of the genome sequence are 3.77 Mb and 20.41 Mb, respectively. Nine‐seven percent of the assembled sequences was anchored onto 13 chromosomes. Based on BUSCO analysis, the assembly involved 96.9% of conserved arthropod and 98.5% of the conserved Hemiptera single‐copy orthologous genes. A total of 14,089 protein‐coding genes were predicted. Phylogenetic analysis revealed that S. chinensis diverged from the common ancestor of Eriosoma lanigerum approximately 57 million years ago (MYA). In addition, 35 genes encoding salivary gland proteins showed differentially when S. chinensis forms a gall, suggesting they have potential roles in gall formation and plant defense suppression. Taken together, this high‐quality S. chinensis genome assembly and annotation provide a solid genetic foundation for future research to reveal the mechanism of gall formation and to explore the interaction between aphids and their host plants.     

豌豆蚜唾液蛋白家族的克隆及在不同发育阶段的表达

豌豆蚜Acyrthosiphon pisum是一种重要的刺吸式害虫, 其分泌的唾液对取食寄主植物和传播植物病毒有重要的作用。为了探讨蚜虫唾液蛋白的功能, 本研究克隆了在豌豆蚜唾液腺高表达的一个未知功能的蛋白家族, 该家族包括13个基因, 编码14种蛋白, 其中4个基因在唾液腺高表达。这个家族是蚜虫特有的蛋白家族, 富含半胱氨酸, 有14个半胱氨酸高度保守, 其中6个半胱氨酸形成3个保守的CXXC结构域。通过与基因组比对, 发现这个家族的基因没有内含子, 分布在基因组的9个scaffold上。用半定量逆转录PCR检测了每个成员在豌豆蚜不同发育阶段的表达, 结果显示这个家族没有发育阶段特异性。推测这个家族的表达可能具有组织特异性, 有氧化还原酶或DNA甲基化酶的功能。     

蚜虫唾液蛋白研究进展

蚜虫属于半翅目蚜科,多为重要的农业害虫,通过刺吸式口器吸食植物汁液,传播病毒,其爆发常常造成重大经济损失。在漫长的协同进化历程中,植物建立了高效的防御系统以应对蚜虫威胁。为了克服植物的防御反应,蚜虫也发展了相应的反制手段,其中蚜虫在取食过程中分泌的唾液蛋白能调控植物防御反应,降解植物次生物质,从而在蚜虫与植物互作中发挥着至关重要的作用。本文综述了蚜虫唾液蛋白的组分鉴定方法和相关蛋白的功能,并对唾液蛋白在蚜虫防治的应用和今后的研究方向进行了展望。常见的蚜虫唾液蛋白组分的鉴定和分析方法包括唾液蛋白的酶活性分析、唾液蛋白组学分析、唾液腺转录组学和蛋白组学分析等。但这些方法各有利弊,仅采取一种分析方法不能客观全面地反映蚜虫唾液蛋白分泌谱,多种技术手段联合分析方可提供更为逼真详实的信息。蚜虫唾液蛋白种类繁多,可分为解毒酶、保护酶、水解酶、结合功能蛋白以及分类未知的效应蛋白等。蚜虫唾液蛋白功能多样,能参与唾液鞘的形成,诱导植物防御反应,促进蚜虫取食,提高蚜虫繁殖力等。通过RNAi干扰唾液蛋白编码基因会显著改变蚜虫取食行为,并降低蚜虫存活率、产蚜量和适合度。因此,唾液蛋白是防控蚜虫的理想靶标。目前,采用寄主诱导的基因沉默(host-induced gene silencing, HIGS)技术已培育了数种靶向唾液蛋白基因的高效抗蚜作物品系,展示出了良好的应用前景。从目前研究来看,各种蚜虫唾液蛋白谱急需采用多组学手段联合分析的方法来进行完整解析。各种唾液蛋白的具体功能方面的研究还严重缺乏,需从蚜虫、植物、两者之间的互作等多维度探究唾液蛋白的作用及相关的分子机制,为发展基于蚜虫唾液蛋白调控的蚜虫防治新策略打下基础。     

Aphids secrete watery saliva into plant tissues from the onset of stylet penetration

Aphid feeding requires the secretion of two types of saliva: gelling saliva (from the principal gland) that forms an intercellular sheath for the penetrating stylet, and watery saliva [from accessory salivary glands (ASGs)] that facilitates intracellular penetration and phloem feeding. Plant viruses can be used as salivary markers to investigate key steps in aphid feeding, and penetration can be monitored electrically using the electrical penetration graph (EPG) approach. We conducted a series of EPG‐controlled transmission experiments using Cucurbit aphid‐borne yellows virus [CABYV; Polerovirus spec. (Luteoviridae)], which is retained in the ASGs, as a marker for watery saliva secretions. The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), was used as a vector and melon seedlings, Cucumis melo L. (Cucurbitaceae), as host plants. Viruliferous aphids were interrupted at various stages during stylet penetration, i.e., during intercellular penetration prior to intracellular puncture and following a potential drop within the first probe. Viruliferous aphids and leaf disc samples obtained from the stylet penetration site were used to detect CABYV by quantitative real‐time RT‐PCR. Approximately half of the inoculated leaf discs were found to be infected with CABYV after very brief (12.9 ± 1.9 s) intercellular stylet probes and before intracellular stylet puncture. The number of virus particles ejected during such probes was similar to the number ejected by aphids during longer probes including a single intracellular puncture. Our results therefore suggest that watery saliva is secreted by aphids from the onset of stylet penetration.     

Mapping Relative Differences in Human Salivary Gland Secretions by Dried Saliva Spot Sampling and nanoLC–MS/MS

Dried saliva spot sampling is a minimally invasive technique for the spatial mapping of salivary protein distribution in the oral cavity. In conjunction with untargeted nano‐flow liquid chromatography tandem mass spectrometry (nanoLC–MS/MS) analysis, DSS is used to compare the proteomes secreted by unstimulated parotid and submandibular/sublingual salivary glands. Two hundred and twenty proteins show a statistically significant association with parotid gland secretion, while 30 proteins are at least tenfold more abundant in the submandibular/sublingual glands. Protein identifications and label‐free quantifications are highly reproducible across the paired glands on three consecutive days, enabling to establish the core proteome of glandular secretions categorized into eight salivary protein groups according to their biological functions. The data suggest that the relative contributions of the salivary glands fine‐tune the biological activity of human saliva via medium‐abundant proteins. A number of biomarker candidates for Sjögren's syndrome are observed among the gland‐specifically expressed proteins, which indicates that glandular origin is an important factor to consider in salivary biomarker discovery.     

Evolutionary relationships of Pemphigus and allied genera (Hemiptera: Aphididae: Eriosomatinae) and their primary endosymbiont,Buchnera aphidicola

Aphids harbor primary endosymbionts, Buchnera aphidicola, in specialized cells within their body cavities. Aphids and Buchnera have strict mutualistic relationships in nutrition exchange. This ancient association has received much attention from researchers who are interested in endosymbiotic evolution. Previous studies have found parallel phylogenetic relationships between non‐galling aphids and Buchnera at lower taxonomic levels (genus, species). To understand whether relatively isolated habitats such as galls have effect on the parallel relationships between aphids and Buchnera, the present paper investigated the phylogenetic relationships of gall aphids from Pemphigus and allied genera, which induce pseudo‐galls or galls on Populus spp. (poplar) and Buchnera. The molecular phylogenies inferred from three aphid genes (COI, COII and EF‐1α) and two Buchnera genes (gnd, 16S rRNA gene) indicated significant congruence between aphids and Buchnera at generic as well as interspecific levels. Interestingly, both aphid and Buchnera phylogenies supported three main clades corresponding to the galling locations of aphids, namely leaf, the joint of leaf blade and petiole, and branch of the host plant. The results suggest phylogenetic conservatism of gall characters, which indicates gall characters are more strongly affected by aphid phylogeny, rather than host plants.     

Large‐scale gene expression reveals different adaptations of Hyalopterus persikonus to winter and summer host plants

Host alternation, an obligatory seasonal shifting between host plants of distant genetic relationship, has had significant consequences for the diversification and success of the superfamily of aphids. However, the underlying molecular mechanism remains unclear. In this study, the molecular mechanism of host alternation was explored through a large‐scale gene expression analysis of the mealy aphid Hyalopterus persikonus on winter and summer host plants. More than four times as many unigenes of the mealy aphid were significantly upregulated on summer host Phragmites australis than on winter host Rosaceae plants. In order to identify gene candidates related to host alternation, the differentially expressed unigenes of H. persikonus were compared to salivary gland expressed genes and secretome of Acyrthosiphon pisum. Genes involved in ribosome and oxidative phosphorylation and with molecular functions of heme–copper terminal oxidase activity, hydrolase activity and ribosome binding were potentially upregulated in salivary glands of H. persikonus on the summer host. Putative secretory proteins, such as detoxification enzymes (carboxylesterases and cytochrome P450s), antioxidant enzymes (peroxidase and superoxide dismutase), glutathione peroxidase, glucose dehydrogenase, angiotensin‐converting enzyme, cadherin, and calreticulin, were highly expressed in H. persikonus on the summer host, while a SCP GAPR‐1‐like family protein and a salivary sheath protein were highly expressed in the aphids on winter hosts. These results shed light on phenotypic plasticity in host utilization and seasonal adaptation of aphids.     

Phylogeny of Rhus gall aphids (Hemiptera : Pemphigidae) based on combined molecular analysis of nuclear EF1α and mitochondrial COII genes

Rhus gall aphids (Fordinae : Melaphidini) have a disjunct distribution in East Asia and North America and have specific host plant relationships. Some of them are of economic importance and all species form sealed galls which show great variation in shape, size, structure, and galling‐site. We present a phylogeny incorporating ten species and four subspecies of Rhus gall aphids based on 1694 base pairs of nuclear elongation factor‐1α (EF1α) and mitochondrial cytochrome oxidase subunit II (COII) DNA sequence data. The results suggest that Melaphidini is monophyletic and at the genus level, Schlechtendalia, Nurudea, and Floraphis were each monophyletic. Kaburagia and Meitanaphis were not monophyletic and therefore inconsistent with the current classification. The North American sumac gall aphid, Melaphis rhois, was most closely related to the East Asian Floraphis species, although this was poorly supported. The conservation of gall morphology with respect to aphid phylogeny rather than their host plants suggests that gall morphology is largely determined by the aphids. While there is no evidence of strict co‐speciation between the aphids and their primary host plants, switching between recently diverged host plants may be involved in the speciation process in Melaphidini.     

Insight into Salivary Gland Proteomes of Two Polyphagous Stink Bugs: Nezara viridula L. and Halyomorpha halys Stål

The invasive brown marmorated stink bug (BMSB), Halyomorpha halys Stål, and the southern green stink bugs (SGSBs), Nezara viridula L., are widely distributed in Europe, even if the date of introduction and the diet differ. Saliva of Hemipteran pests plays essential roles in the interaction between insects and their host plants. The salivary proteomes of several aphid species have been studied and found to differ according to the species, while no comparative investigation between phytophagous stink bugs has been performed yet. Here, the salivary proteins from two bugs, BMSB and SGSB, are analyzed using LC‐MS/MS. Data are available via ProteomeXchange with identifiers PXD011920 and PXD011976. A total of 238 and 305 proteins are identified in salivary glands of BMSB and SGSB, respectively. In comparison with salivary proteome from other Hemiptera, the most striking feature of the salivary gland proteomes of SGSB and BMSB is the similar pattern of protein functions between both species. Some of the proteins are speculated to play a significant role in plant–insect interactions. The results herein provide a framework for future research to elucidate the molecular basis of differential impact of piercing–sucking insects on host plants.     

Proteomic analysis of secreted saliva from Russian Wheat Aphid (Diuraphis noxia Kurd.) biotypes that differ in virulence to wheat

Diuraphis noxia, Russian Wheat Aphid (RWA), biotypes are classified by their differential virulence to wheat varieties containing resistance genes. RWA salivary proteins, unlike those of most aphid species, cause foliar damage and physiological alterations in plants. A comparative proteomic analysis of secreted saliva from four differentially virulent RWA biotypes identified thirty-four individual proteins. The five major proteins were glucose dehydrogenase, lipophorin, chitinase, CiV16.8g1-like, and lava lamp. Fourteen proteins quantitatively varied among biotypes; trehalase, β-N-acetylglucosaminidase (chitinase), two separate glucose dehydrogenases, calreticulin, aminopeptidase, acetylglucosaminyltransferase, hydroxymethylglutaryl-CoA lyase, acyltransferase, ficolin-3, lava lamp, retinaldehyde-binding protein, and two proteins of unknown function. Fifty-four percent of spectral counts were associated with glucose dehydrogenase, which is thought to detoxify plant defensive compounds. One-dimensional electrophoresis detected nine protein bands from 9 to 60 kDa that quantitatively differed. Two-dimensional electrophoresis identified six major gel zones with quantitative and qualitative variance in proteins. Our findings reveal that the salivary proteome of RWA, a phytotoxic aphid, differs considerably from those reported for nonphytotoxic aphids. The potential roles of proteins used in the general plant feeding processes of aphids and those that are potential phytotoxins related to aphid virulence are discussed.