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.     

Discharge by aphids of soluble secretions into dietary sources

Substances secreted by aphids into diets through a rubber-wax membrane can be detected by their absorption of low wavelength ultraviolet light. Their detection can be simplified by presenting water as diet. Of the substances secreted into water by non-crowded aphids within 24 h, ca 35–55% of the absorbance at 200 nm can be accounted for by reactions with protein reagents and/or electrophoresis. Macrosiphum euphorbiae secreted over 30 ng of salivary protein in bovine serum albumin equivalents per mg fresh body weight. SDS-PAGE revealed that the proteins initially secreted had subunits mostly of >100,000 Daltons. When dead or dying aphids were present on the membrane, however, e.g. due to crowding or prolonged confinement in the feeding chamber, additional subunits of <30,000 Daltons also appeared in the water. When feeding continued for more than 24 h, proteins, separable by electrophoresis began to disappear, possibly as a result of leakage of lyzing enzymes into the water from aphids that had died with their mouth parts still inserted through the membrane.The initial secretions possessed oxidase activity. Invertase was not detected but the secretion caused non-enzymatic reduction of a copper reagent used for detection of reducing sugars. Marked contrasts were noted between the electrophoretic protein patterns of the saliva of different species of aphids. The saliva of Macrosiphum euphorbiae and Myzus persicae contained over twice as much catechol oxidase activity per unit of salivary protein as that of Nasonovia ribisnigri and Aphis fabae.The significance of these findings in relation to use of artificial diets for colony maintenance and feeding experiments and for studies of aphid-plant interactions is discussed.     

Physical and chemical interactions between aphids and plants

Aphids feed from sieve tubes deep inside the host plant. Therefore, aphids must be able to recognize their host plant(s) and to direct their stylets which must be long and thin enough to reach and puncture the sieve tubes at a particular site. Sieve tubes in angiosperms are longitudinal arrays of sieve element/companion cell modules which are highly sensitive to disturbance of any kind. The sieve tubes dispose of elaborate sealing mechanisms such as protein plugging and callose sealing which are triggered by a rise in calcium in the sieve tubes. Aphids seem to have developed a range of physical and chemical measures to limit the amount of calcium influx in response to stylet puncturing. Loss of sieve-element turgor pressure induced by stylet insertion is minimized by the minute stylet volume. Turgor-dependent Ca(2+) influx, possibly mediated by mechano sensitive Ca(2+) channels, must therefore be limited. The components of the sheath and watery saliva play a pivotal role in establishing the physical and chemical constraints on the rise of calcium. Most likely, sheath saliva prevents the influx of calcium from the apoplast by sealing the stylet puncture site while watery saliva may prevent plugging and sealing of sieve plates by potential interaction with SE sap ingredients.     

The structural sheath protein of aphids is required for phloem feeding

Aphids produce two types of saliva that mediate their interactions with plants. Watery saliva is secreted during cell penetration and ingestion, whereas gel saliva is secreted during stylet movement through the apoplast where it forms a sheath around the stylet to facilitate penetration and seal puncture sites on cell membranes. In order to study the function of the sheath when aphids interact with plants, we used RNA interference (RNAi) to silence the aphid structural sheath protein (SHP) in the pea aphid Acyrthosiphon pisum. The injection of 50 ng of double stranded RNA completely disrupted sheath formation, as confirmed by scanning electron microscopy. Aphid behavior was monitored using the electrical penetration graph technique, revealing that disrupted sheath formation prevented efficient long-term feeding from sieve tubes, with a silencing effect on reproduction but not survival. We propose that sealing the stylet penetration site in the sieve tube plasma membrane is part of a two-step mechanism to suppress sieve-tube occlusion by preventing calcium influx into the sieve tube lumen. The SHP is present in several aphid species and silencing has a similar impact to aphid-resistant plants, suggesting that SHP is an excellent target for RNAi-mediated pest control.     

植物和刺吸式口器昆虫的诱导防御与反防御研究进展

刺吸式口器昆虫在长期的进化过程中形成特殊的口针结构,用于专门吸食植物韧皮部筛管细胞的汁液成分.以蚜虫为例,它们在取食过程中分泌的胶状唾液和水状唾液将有效的降低植物防御反应,其中水状唾液包含的大量酶类不仅可以帮助蚜虫穿刺植物韧皮部,刺探到筛管细胞,同时也是植物感受蚜虫为害的激发因子,诱导出植物防御反应和相关抗性基因的表达...     

Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps

Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from “fed diet” and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84 kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5′- and 3′-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061 bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca²⁺ -binding proteins. As predicted, NcSP84 exhibited Ca²⁺-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca²⁺ tended to decline discretely, depending on the concentration of CaCl₂ with which it was mixed for 1 h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca²⁺ ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.     

Characterization of EPG waveforms for the tea green leafhopper, Empoasca vitis Göthe (Hemiptera: Cicadellidae), on tea plants and their correlation with stylet activities

The stylet probing activities of the tea green leafhopper Empoasca vitis Gothe (Hemiptera: Cicadellidae) were studied using the DC electrical penetration graph (EPG) technique. Seven different EPG waveforms (i.e., Np, E1, E2, E3, E4, E5 and E6) were distinguished and characterized on susceptible tea leaves. In addition, four of them (i.e., Np, E1, E2, E3), together accounting for 97.08% of the total recording time, were behaviorally correlated with probing and non-probing activities using artificial diet observation with high-magnification video recording. At the start of stylet probing, waveform E1 always occurred at a variable voltage. E1, with all three of its waveform sub-types (E1-A to E1-C), was correlated with production of the salivary sheath trunk, stylet laceration, and channel cutting in viscous artificial diet. Afterwards, two types of high-amplitude waveforms, E2 and E3, followed. E2 had a highly regular, quasi-square wave, repetitive appearance, and lasted the longest duration of all E. vitis probing waveforms. E3 usually appeared after E2, and also exhibited a quasi-square wave feature similar to E2, but had much higher amplitude. Both waveforms E2 and E3 were correlated with active ingestion in liquid artificial diet. In addition, secretion of watery, enzymatic saliva was likely during E2. The active stylet movements and channel-cutting observed during the probing process indicate that E. vitis is a cell rupture feeder, not a salivary sheath feeder, as aphids and other leafhoppers. Thus, hopperburn damage to the tea plant is probably due to the cell rupture feeding strategy, similar to other hopperburning Empoasca species.     

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.     

The influence of some honeybee products as a diet substitute on the different stages of Coccinella undecimpunctata L. in Egypt

Several natural diets of honeybee products were tested for rearing Coccinella undecimpunctata L. under laboratory conditions. Two groups of tested adult and larval insect have been fed on the tested diets, the first group were fed on the tested honey bee products diets without adding aphids, while the second group fed on the same honeybee products with aphids added. The oviposition period in the first group was significantly shorter than control. The tested diets supplied with royal jelly + aphid and/or pollen grains + aphid caused the longest oviposition period. Diets of aphid alone and bee honey with aphid recorded the shortest oviposition period. Fecundity of ladybird was the highest in the tested diets with aphids than diets without aphid and/or aphid diet alone. The durations of larval and pupal stages were significantly different when compared with the diets with or without aphid and/or aphid diet alone. Further results indicated that the diets of royal jelly caused the shortest duration in larval and pupal stages.     

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.     

Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.     

Nutrients Differentially Regulate Nucleobindin-2/Nesfatin-1 In Vitro in Cultured Stomach Ghrelinoma (MGN3-1) Cells and In Vivo in Male Mice

Nesfatin-1 is secreted, meal-responsive anorexigenic peptide encoded in the precursor nucleobindin-2 [NUCB2]. Circulating nesfatin-1 increases post-prandially, but the dietary components that modulate NUCB2/nesfatin-1 remain unknown. We hypothesized that carbohydrate, fat and protein differentially regulate tissue specific expression of nesfatin-1. NUCB2, prohormone convertases and nesfatin-1 were detected in mouse stomach ghrelinoma [MGN3-1] cells. NUCB2 mRNA and protein were also detected in mouse liver, and small and large intestines. MGN3-1 cells were treated with glucose, fatty acids or amino acids. Male C57BL/6 mice were chronically fed high fat, high carbohydrate and high protein diets for 17 weeks. Quantitative PCR and nesfatin-1 assays were used to determine nesfatin-1 at mRNA and protein levels. Glucose stimulated NUCB2 mRNA expression in MGN3-1 cells. L-Tryptophan also increased NUCB2 mRNA expression and ghrelin mRNA expression, and nesfatin-1 secretion. Oleic acid inhibited NUCB2 mRNA expression, while ghrelin mRNA expression and secretion was enhanced. NUCB2 mRNA expression was significantly lower in the liver of mice fed a high protein diet compared to mice fed other diets. Chronic intake of high fat diet caused a significant reduction in NUCB2 mRNA in the stomach, while high protein and high fat diet caused similar suppression of NUCB2 mRNA in the large intestine. No differences in serum nesfatin-1 levels were found in mice at 7 a.m, at the commencement of the light phase. High carbohydrate diet fed mice showed significantly elevated nesfatin-1 levels at 1 p.m. Serum nesfatin-1 was significantly lower in mice fed high fat, protein or carbohydrate compared to the controls at 7 p.m, just prior to the dark phase. Mice that received a bolus of high fat had significantly elevated nesfatin-1/NUCB2 at all time points tested post-gavage, compared to control mice and mice fed other diets. Our results for the first time indicate that nesfatin-1 is modulated by nutrients.     

Ant and silkworm pupae as convenient diets for the development and reproduction of big-eyed bug Geocoris ochropterus (Hemiptera: Geocoridae)

We investigated the influence of convenient diets on big-eyed bug Geocoris ochropterus. Development and reproduction of G. ochropterus fed on convenient diets of ant pupae Oecophylla smaragdina and silkworm pupae Bombyx mori were examined using aphids Aphis gossypii as the control diet. Results showed that Geocoris ochropterus nymphs completed development to adults on all diets. Total average development period was 35.1 days fed on ant pupae, 35.9 days fed on silkworm pupae, and 36.0 days fed on aphids. Head width, body length, forewing length, and fresh body weight of adults were not affected by diets, except for females reared on ant pupae that were significantly heavier than those fed on aphids. There was no significant difference in offspring sex ratio. Total number of eggs deposited per female fed on ant pupae was significantly larger than when fed on aphids, while eggs laid by females fed on silkworm pupae were significantly longer than eggs laid by females fed on aphids. Results suggest that ant pupae and silkworm pupae could be effectively used for mass rearing of G. ochropterus.     

人工饲料pH值对桃蚜存活、繁殖及抗氧化酶活性的影响

通过测定在不同pH值饲料上桃蚜Myzus persicae（Sulzer）的存活率、繁殖率及其抗氧化酶活性的变化,评价饲料pH值对桃蚜生态和生理指标的影响,筛选扩繁桃蚜饲料最适pH值。结果显示：不同pH值人工饲料饲喂桃蚜,其存活率3d后即呈现显著差异,在试验调查时间内,饲料pH值为7.0处理的存活率、单雌产蚜量和内禀增长率均为最高;不同处理桃蚜体内的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性与对照处理相比均存在显著性差异,在24h左右达到最大值,而后有所下降;饲料pH值为7.0和7.2两处理的酶活性变化幅度较小,在48h后接近正常水平。试验结果表明,扩繁桃蚜较适合的人工饲料pH值为7.0左右。     

Phenol oxidising enzymes in the grain aphid’s saliva

Sucrose-agarose gels and sucrose liquid diets were used to study the phenol oxidising enzymes in the salivary secretions of the grain aphid, Sitobion avenae (Fabricius). Activity indicating the presence of two oxidoreductases, polyphenol oxidase (PPO) and peroxidase (Px), was found. Both enzymes were present in the aphids stylet sheath (gelling saliva) but only polyphenol oxidase activity was found in the halos around sheaths and thus in watery saliva. Electrical penetration graphs (EPG) revealed that the secretion of these enzymes into the gels, by an individual aphid, was associated with its probing activity observed during penetration of the epidermal and mesophyll tissues. The grain aphids PPO, secreted in its saliva reacted with a range of phenolic compounds. As most of these phenolics occur naturally in cereals, the grain aphid could modify its host-plants phenolic composition. The importance of the grain aphids polyphenol oxidase and peroxidase in detoxifying cereal phenolics is discussed.     

麦长管蚜唾液中几种酶的鉴定、活力测定与功能分析

用Parafilm膜夹营养液法,以两种食料介质饲喂麦长管蚜Macrosiphum avenae 3龄若蚜并收集其唾液,对唾液中的酶类进行了鉴定、活力测定和功能分析。结果表明,在20%蔗糖介质提取液中,鉴定有果胶酶、多酚氧化酶和纤维素酶; 在水介质提取液中鉴定有纤维素酶; 两种介质提取液中都未鉴定出过氧化物酶。酶活力测定结果表明, 在20%蔗糖介质提取液中, 每30头蚜虫分泌的果胶酶、多酚氧化酶和纤维素酶的酶活力分别为2.59×10^-3 U/g、7×10^-3 U/g和7.89×10^-3 U/g; 在水介质提取液中,纤维素酶活力为3.68×10^-3 U/g。行为反应试验结果表明,果胶酶处理麦苗的挥发物组分能引起麦长管蚜寄生性天敌燕麦蚜茧蜂Aphidius avenae和捕食性天敌七星瓢虫Coccinella septempunctata 的嗅觉偏好反应,因此,果胶酶在麦长管蚜取食诱导小麦植株的间接防御反应中具有重要作用。     

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management.     

Development of probiotic diets for the olive fly: evaluation of their effects on fly longevity and fecundity

One possible control strategy against the olive fly, Bactrocera oleae, the most serious olive crop pest, is the Sterile Insect Technique (SIT) application. However, a number of problems associated with this method remain that decrease the effectiveness of SIT, including the quality of reared insects. Taking the importance of the relationship between the olive fly and bacteria into consideration, the effects of probiotic diets enriched with Pseudomonas putida on B. oleae longevity and fecundity were evaluated. First, we found that the probiotic bacterium, P. putida, is conveyed from diets to the oesophageal bulb as well as to the fly midgut after feeding on the probiotic diet. Subsequently, B. oleae adults fed on either: (a) a standard full protein and sugar diet; (b) a sugar only diet; (c) a probiotic standard full protein and sugar diet; or (d) a probiotic sugar diet. Flies fed on probiotic diets were supplied with an inoculated gel containing P. putida; non‐inoculated gel was provided to the flies fed on non‐probiotic diets. B. oleae males and females that fed on sugar diets did not survive as long as those that fed on protein diets. A comparison of the longevity of adults fed on full diet and sugar with their respective probiotic diets revealed no significant difference. Males fed on the sugar only diet survived longer than males fed on probiotic sugar diet, and females fed on the full protein and sugar diet survived much longer than females fed on the full probiotic diet. As regarding fecundity, both full diets resulted in a higher number of eggs laid per female. Females fed on the probiotic sugar diet laid a higher number of eggs than females that fed on sugar only. The inoculated gel of the probiotic sugar diet contained a significantly higher quantity of leucine, isoleucine and proline than the non‐inoculated gel of the sugar only diet. The possible role of dietary bacteria in relation to functional aspects of olive fly physiology is discussed.     

人工饲料对龟纹瓢虫捕食功能的影响

研究不同成分的人工饲料对龟纹瓢虫Propylaea japonica(Thunberg)捕食功能的影响。结果表明:不同成分的人工饲料对龟纹瓢虫捕食棉蚜的功能反应类型没有影响,与对照组瓢虫一样均符合HollingⅡ型模型;添加蔗糖的饲料2组的a’/Th(瞬间攻击率/处理时间)要高于其他人工饲料组,表明其对棉蚜的捕食效应较强;各人工饲料组喂养的龟纹瓢虫在捕食功能上要明显劣于野生型龟纹瓢虫。