Apolygus lucorum genome provides insights into omnivorousness and mesophyll feeding

Apolygus lucorum (Miridae) is an omnivorous pest that occurs worldwide and is notorious for the serious damage it causes to various crops and substantial economic losses. Although some studies have examined the biological characteristics of the mirid bug, no reference genome is available in Miridae, limiting in‐depth studies of this pest. Here, we present a chromosome‐scale reference genome of A. lucorum, the first sequenced Miridae species. The assembled genome size was 1.02 Gb with a contig N50 of 785 kb. With Hi‐C scaffolding, 1,016 Mb contig sequences were clustered, ordered and assembled into 17 large scaffolds with scaffold N50 length 68 Mb, each corresponding to a natural chromosome. Numerous transposable elements occur in this genome and contribute to the large genome size. Expansions of genes associated with omnivorousness and mesophyll feeding such as those related to digestion, chemosensory perception, and detoxification were observed in A. lucorum, suggesting that gene expansion contributed to its strong environmental adaptability and severe harm to crops. We clarified that a salivary enzyme polygalacturonase is unique in mirid bugs and has significantly expanded in A. lucorum, which may contribute to leaf damage from this pest. The reference genome of A. lucorum not only facilitates biological studies of Hemiptera as well as an understanding of the damage mechanism of mesophyll feeding, but also provides a basis on which to develop efficient control technologies for mirid bugs.     

Host plant use of a polyphagous mirid,Apolygus lucorum: Molecular evidence from migratory individuals

While the host plant use of insect herbivores is important for understanding their interactions and coevolution, field evidence of these preferences is limited for generalist species. Molecular diet analysis provides an effective option for gaining such information, but data from field‐sampled individuals are often greatly affected by the local composition of their host plants. The polyphagous mirid bug Apolygus lucorum (Meyer‐Dür) seasonally migrates across the Bohai Sea, and molecular analysis of migrant bugs collected on crop‐free islands can be used to estimate the host plant use of A. lucorum across the large area (northern China) from where these individuals come. In this study, the host plant use of A. lucorum adults was determined by identifying plant DNA using a three‐locus DNA barcode (rbcL, trnH‐psbA, and ITS) in the gut of migrant individuals collected on Beihuang Island. We successfully identified the host plant families of A. lucorum adults, and the results indicated that captured bugs fed on at least 17 plant families. In addition, gut analyses revealed that 35.9% of A. lucorum individuals fed on multiple host plants but that most individuals (64.1%) fed on only one plant species. Cotton, Gossypium hirsutum L., DNA was found in 35.8% of the A. lucorum bugs examined, which was much higher than the percentage of bugs in which other host plants were found. Our work provides a new understanding of multiple host plant use by A. lucorum under natural conditions, and these findings are available for developing effective management strategies against this polyphagous pest species.     

Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference

Salivary enzymes of many piercing–sucking insects lead to host plant injury. The salivary enzymes, polygalacturonase (PGs), act in insect feeding. PG family genes have been cloned from the mirid bug Apolygus lucorum, a pest of cotton and other host crops in China. We investigated the function of two PG genes that are highly expressed in A. lucorum nymphs (PG3‐4) and adults (PG3‐5), using siRNA injection‐based RNA interference (RNAi). Accumulation of mRNA encoding both genes and their cognate proteins was significantly reduced (>60%) in experimental compared control green fluorescent protein (GFP) siRNA‐treated mirids at 48 h post injection. Injury levels of cotton buds were also significantly reduced after injecting saliva isolated from PG3‐4 and PG3‐5 siRNA‐treated A. lucorum. These results demonstrate that these two PG act in A. lucorum elicitation of plant injury.     

Response of mirid predators to synthetic herbivore‐induced plant volatiles

Zoophytophagous plant bugs feed on plant tissue as a source of water and nutrients, besides feeding on prey. By phytophagy, mirid predators activate plant defense responses through different pathways, resulting, among others, in the release of herbivore‐induced plant volatiles (HIPVs). These compounds could repel herbivores and attract parasitoids and predators, and synthetic versions could potentially be used in biological control. Nevertheless, little is known about the influence of synthetic volatiles on mirid attraction. Using Y‐tube olfactometer trials, we evaluated the responses of Nesidiocoris tenuis (Reuter), Macrolophus pygmaeus (Rambur), and Dicyphus bolivari Lindberg (Hemiptera: Miridae), important natural enemies used to control various greenhouse pests, to 10 synthetic versions of HIPVs released from tomato (Solanum lycopersicum L., Solanaceae) plants induced by N. tenuis and M. pygmaeus. Nesidiocoris tenuis responded to five of the 10 HIPVs, whereas M. pygmaeus and D. bolivari responded to four of the 10 HIPVs. Two green leaf volatiles, (Z)‐3‐hexenyl propanoate and (Z)‐3‐hexenyl acetate, and the ester methyl salicylate (MeSA) were attractive to all three mirid predator species. Our results demonstrate that the volatiles released by tomato plants activated by N. tenuis and M. pygmaeus phytophagy are attractive to their conspecifics and also to D. bolivari. Further studies should evaluate the potential of these compounds to attract predatory mirids in the field.     

From lynx spiders to cotton: Behaviourally mediated predator effects over four trophic levels

Food web studies often examine density and behaviourally mediated effects of predators on herbivores, but are less likely to assess the plant targeted by the herbivore. We conducted a study that incorporated four trophic levels examining the effect of two generalist predators (damsel bugs, Nabis kinbergii Reuter; and lynx spiders, Oxyopes molarius L. Koch) on damage to cotton bolls caused by green mirids (Creontiades dilutus (Stål)). First we tested whether lynx spiders and damsel bugs could control mirid numbers and cotton boll damage in field cages. We found that in cages containing mirids and only lynx spiders, lynx spiders reduced both mirid numbers and boll damage. However, in cages which contained mirids and both predators (lynx spiders and damsel bugs) only mirid numbers were reduced. To explain the negative effect of damsel bugs on boll damage, we examined the interactions between lynx spiders, damsel bugs and mirids. We found that lynx spiders were better mirid predators than damsel bugs, and that lynx spiders attacked damsel bugs, but not vice versa. Behaviourally, mirids responded to increasing predator pressure regardless of whether the predators were lynx spiders or damsel bugs. However, damsel bugs seemed to alter the behaviour of lynx spiders because in their presence, a higher proportion of lynx spiders moved to the top of the plant, towards the damsel bugs but away from the bolls found lower on the plant. These results suggest that the most likely explanation for the increase in boll damage in the presence of damsel bugs was that lynx spiders moved to the top of the plant in the presence of damsel bugs, which then exposed the bolls lower down on the plant to mirid attack. This work emphasizes the importance of behaviourally mediated effects in food webs extending over four trophic levels.     

绿盲蝽和中黑盲蝽对不同抗性和虫害处理棉花的选择趋性

研究绿盲蝽Apolygus lucorum和中黑盲蝽Adelphocoris suturalis对不同抗性和虫害处理棉花的选择趋性,为棉盲蝽的综合治理提供理论依据。本研究以抗性棉花品种BR-S-10（抗性品种）和感性棉花品种科林08-15（感性品种）,以及绿盲蝽和中黑盲蝽分别危害处理后的该两种棉花植株为试验材料,以纯净空气为空白对照,共成对设置15个气味源组合,采用“Y”型昆虫嗅觉仪室内研究了绿盲蝽和中黑盲蝽对不同抗性和虫害处理棉花的选择趋性。结果表明,绿盲蝽显著选择感性品种、中黑盲蝽危害感性品种和绿盲蝽危害感性品种。中黑盲蝽显著选择绿盲蝽危害感性品种、中黑盲蝽危害感性品种和感性品种。总的来说,两种棉盲蝽趋向于选择敏感棉花品种,抗性棉花品种对供试昆虫有显著趋避性;绿盲蝽显著趋向选择中黑盲蝽危害感性品种和绿盲蝽危害感性品种,中黑盲蝽显著趋向选择绿盲蝽危害感性品种和中黑盲蝽危害感性品种。     

Males of the predatory mirid bug Macrolophus caliginosus exploit plant volatiles induced by conspecifics as a sexual synomone

The olfactory responses of male and female Macrolophus caliginosus Wagner (Heteroptera: Miridae) adults towards volatiles from green bean plants previously exposed to feeding by conspecifics and to direct odours from conspecifics were tested in a Y-tube olfactometer. Female M. caliginosus did not respond to volatiles from plants exposed to mirid feeding or to odours emitted directly by adult mirids. In contrast, male mirid bugs were attracted both to volatiles from plants previously exposed to feeding by conspecific females and to odours emitted by conspecifics only with a marginally significant preference for the former. The gas chromatography-mass spectrometry analysis showed that mirid feeding induced the release of 11 additional compounds as compared to the volatiles emitted from clean plants. Three of these substances (5-ethyl-2(5H)-furanone, Z-3-hexenyl tiglate, and E,E-α-farnesene) were released only after feeding by females. Furthermore, 21 compounds were identified in volatiles emitted directly by mirids, 12 of which were unique to the mirids (i.e., not present in clean plants or plants previously exposed to mirid feeding). The results suggest that female-specific herbivore-induced plant volatiles play a role as mate-finding cues by the male mirids. The ecological implications of the findings are discussed, and the term ‘sexual synomone’ is introduced.     

Host location in Oomyzus gallerucae (Hymenoptera: Eulophidae), an egg parasitoid of the elm leaf beetle Xanthogaleruca luteola (Coleoptera: Chrysomelidae)

Eggs of the elm leaf beetle Xanthogaleruca luteola are often heavily attacked by the chalcidoid wasp Oomyzus gallerucae. We studied the chemical signals mediating interactions between the egg parasitoid, its host, and the plant Ulmus campestris. Olfactometer bioassays with O. gallerucae showed that volatiles of the host-plant complex attract the parasitoid. In order to determine the source of attractive volatiles within this host-plant-complex, we tested separately the effect of odours of eggs, gravid elm leaf beetle females, faeces of the beetles and elm twigs (with undamaged leaves and leaves damaged either mechanically or by feeding of the beetles). Odours of faeces of the elm leaf beetle were attractive, whereas neither volatiles from eggs nor from gravid females acted as attractants. Volatiles from undamaged or damaged plants did not elicit a positive reaction in O. gallerucae, whereas volatiles from feeding-damaged plants onto which host eggs had been deposited were attractive. This latter result suggests that it is not feeding but deposition of host eggs onto elm leaves that induces the production of plant volatiles attractive to the egg parasitoid. Investigations of the search patterns of O. gallerucae within the habitat by laboratory bioassays revealed that the egg parasitoid encounters host eggs by chance. Contact kairomones from faeces were demonstrated to be important in microhabitat acceptance, while contact kairomones isolated from the host eggs are relevant for host recognition. Received: 12 February 1997 / Accepted: 29 April 1997     

不同棉花品种(系)抗盲蝽田间鉴定与评价

采用目测法调查棉田盲蝽混合种群发生数量,并结合盲蝽对棉花植株的危害程度,对162个供试棉花品种(系)进行了抗盲蝽鉴定及评价。共计筛选获得包括亚洲棉、灵-29、07生试6号等16个对盲蝽具有抗性潜力的棉花品种(系),以及辽阳多毛棉、冀丰989、08生试6号、大铃668、石抗338、HB5 6个对盲蝽敏感的棉花品种(系)。初步建立了棉花品种(系)抗盲蝽田间鉴定与评价技术规程,为棉花抗盲蝽育种提供了种质资源及技术参考。     

A PCR-based analysis of plant DNA reveals the feeding preferences of <Emphasis Type="Italic">Apolygus lucorum</Emphasis> (Heteroptera: Miridae)

The mirid bug Apolygus lucorum (Meyer-Dür) (Heteroptera: Miridae) is a severe pest of cotton and other crops in China. The feeding preferences of this pest are unclear due to its frequent movement among different host plants and the inconspicuous signs of its feeding. Here, we present results of a field trial that used direct observation of bug densities and a PCR-based molecular detection assay to detect plant DNA in bugs to explore relationships between A. lucorum population abundance and its feeding preference between two host plants, Humulus scandens (Loureiro) Merrill and Medicago sativa L. The field-plot samples showed that A. lucorum adults generally prefer flowering host plants. Its density was significantly higher on flowering H. scandens than on seedlings of M. sativa, and a similarly higher bug density was observed on flowering M. sativa than on seedlings of H. scandens. In the laboratory, we designed two pairs of species-specific primers targeting the trnL-F region for H. scandens and M. sativa, respectively. The detectability of plant DNA generally decreased with time post-feeding, and the half-life of plant DNA detection (DS₅₀) in the gut was estimated as 6.26 h for H. scandens and 3.79 h for M. sativa with significant differences between each other. In mirid bugs exposed to seedlings of H. scandens and flowering M. sativa, the detection rate of M. sativa DNA was significantly higher than that of H. scandens. Meanwhile, in mirid bugs exposed to seedlings of M. sativa and flowering H. scandens, a significantly higher detection rate of H. scandens DNA was found. We developed a useful tool to detect the remaining plant food species specifically from the gut of A. lucorum in the current study. We provided direct evidence of its feeding preference between H. scandens and M. sativa at different growth stages, which strongly supported a positive correlation between population abundance and feeding preference of A. lucorum on different plants under field conditions. The findings provide new insights into the understanding of A. lucorum’s feeding preference, and are helpful for developing the strategies to control this pest.     

盲蝽类害虫种群密度与危害的调查方法

盲蝽是棉花、果树等作物上的一类重要害虫。盲蝽的卵产在植物组织中,若虫活动性和隐蔽性强,成虫善于飞行扩散。针对盲蝽各个虫态的习性,发展了田间种群发生与危害调查技术,为其种群监测提供了科学支撑。     

Olfactory response of a predatory mirid to herbivore induced plant volatiles: multiple herbivory vs. single herbivory

Plants infested with a single herbivore species can attract natural enemies through the emission of herbivore‐induced plant volatiles (HIPVs). However, under natural conditions plants are often attacked by more than one herbivore species. We investigated the olfactory response of a generalist predators Macrolophus caliginosus to pepper infested with two‐spotted spider mites, Tetranychus urticae, or green peach aphid, Myzus persicae, vs. plants infested with both herbivore species in a Y‐tube olfactometer set up. In addition, the constituents of volatile blends from plants exposed to multiple or single herbivory were identified by gas chromatography‐mass spectrometry (GC‐MS). The mirid bugs showed a stronger response to volatiles emitted from plants simultaneously infested with spider mites and aphids than to those emitted from plants infested by just one herbivore, irrespective of the species. Combined with results from previous studies under similar conditions we infer that this was a reaction to herbivore induced plant volatiles. The GC‐MS analysis showed that single herbivory induced the release of 22 additional compounds as compared with the volatiles emitted from clean plants. Quantitative analyses revealed that the amount of volatile blends emitted from pepper infested by both herbivores was significantly higher than that from pepper infested by a single herbivore. Moreover, two unique substances were tentatively identified (with a probability of 94% and 91%, respectively) in volatiles emitted by multiple herbivory damaged plants: α‐zingiberene and dodecyl acetate.     

Sex Pheromones and Reproductive Isolation in Five Mirid Species

Mate location in many mirid bugs (Heteroptera: Miridae) is mediated by female-released sex pheromones. To elucidate the potential role of the pheromones in prezygotic reproductive isolation between sympatric species, we investigated differences in the pheromone systems of five mirid species, Apolygus lucorum, Apolygus spinolae, Orthops campestris, Stenotus rubrovittatus and Taylorilygus apicalis. GC/MS analyses of metathoracic scent gland extracts of virgin females showed that all five species produced mixtures of hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal, but in quite different ratios. (E)-2-hexenyl butyrate was the major component of A. spinolae, while hexyl butyrate was the most abundant component in the pheromone blends of the other four species. In addition to the three compounds, a fourth component, (E)-2-octenyl butyrate, was present in the gland extracts of A. lucorum and T. apicalis females. Field tests suggest that the ternary blends of hexyl butyrate, (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal as found in the extracts of the females of each species do not inhibit attraction of conspecific males but ensure species-specificity of attraction between A. lucorum, O. campestris and T. apicalis. Furthermore, (E)-2-octenyl butyrate was essential for attraction of A. lucorum and T. apicalis males, but strongly inhibited attraction of male A. spinolae, O. campestris and S. rubrovittatus. The combined results from this study and previous studies suggest that the minor component and pheromone dose in addition to the relative ratio of the major components play an important role in reproductive isolation between mirid species.     

Complex Odor from Plants under Attack: Herbivore's Enemies React to the Whole, Not Its Parts

Background

Insect herbivory induces plant odors that attract herbivores'' natural enemies. Assuming this attraction emerges from individual compounds, genetic control over odor emission of crops may provide a rationale for manipulating the distribution of predators used for pest control. However, studies on odor perception in vertebrates and invertebrates suggest that olfactory information processing of mixtures results in odor percepts that are a synthetic whole and not a set of components that could function as recognizable individual attractants. Here, we ask if predators respond to herbivore-induced attractants in odor mixtures or to odor mixture as a whole.

Methodology/Principal Findings

We studied a system consisting of Lima bean, the herbivorous mite Tetranychus urticae and the predatory mite Phytoseiulus persimilis. We found that four herbivore-induced bean volatiles are not attractive in pure form while a fifth, methyl salicylate (MeSA), is. Several reduced mixtures deficient in one component compared to the full spider-mite induced blend were not attractive despite the presence of MeSA indicating that the predators cannot detect this component in these odor mixtures. A mixture of all five HIPV is most attractive, when offered together with the non-induced odor of Lima bean. Odors that elicit no response in their pure form were essential components of the attractive mixture.

Conclusions/Significance

We conclude that the predatory mites perceive odors as a synthetic whole and that the hypothesis that predatory mites recognize attractive HIPV in odor mixtures is unsupported.     

Nymphal performance correlated with adult preference for flowering host plants in a polyphagous mirid bug, Apolygus lucorum (Heteroptera: Miridae)

Apolygus lucorum (Heteroptera: Miridae), a polyphagous mirid bug, often shows a strong preference for flowering host plants. It is hypothesized that host plant selection by phytophagous insects results in a variety of behavioral trade-offs. In this study, the relationship between adult preference and nymphal performance was examined on host plants with their flowers left intact and with flowers removed to investigate potential trade-offs in A. lucorum’s preference for flowering plants. Field trials in 2010 and 2011 showed that adults and nymphs were significantly more abundant on plants with flowers left intact than on those with flowers removed, while oviposition choice trials in field cages revealed that female adults preferred to lay eggs on plants with flowers. Laboratory performance trials demonstrated that adult lifespan and fecundity and nymphal development rate and survival were all significantly higher on plants with flowers compared with plants without flowers. In contrast, sex ratio was not significantly different. Simple linear regression analysis showed positive correlations between (1) adult and nymphal abundances in the field, (2) nymphal development rates and survival with adult fecundity in the laboratory, and (3) adult lifespan with fecundity in the laboratory. Since a positive preference–performance correlation was found for A. lucorum, we conclude that there are no evident behavioral trade-offs for host plant selection by this polyphagous mirid bug.     

Response of a phytoseiid predator to herbivore-Induced plant volatiles: Selection on attraction and effect on prey exploitation

Bean plants infested with herbivorous spider mites emit volatile chemicals that are attractive toP. persimilis, a predator of spider mites. In Y-tube olfactometer tests we evaluated involvement of a genetic component in predator response to herbivore-induced plant volatiles. Replicated bidirectional selection resulted in a significant increase in attraction after one generation of selection, but no decrease even after three generations of selection, indicating significant, but unbalanced, additive genetic variation in predator perception of, or response to, herbivore-induced plant volatiles. Selected lines responded differently than an unselected population to food deprivation, pointing to an interaction between their internal state and response to plant volatiles. Selected lines also differed from unselected ones in behaviors associated with local prey exploitation, such as residence time, prey consumption, and reproduction. At lower prey densities,P. persimilis from both “+” lines left spider mite-infested leaves more rapidly and consumed fewer prey eggs than an unselected population. Defining olfactory components of predator search behavior is one step in understanding the effect of plant volatiles on predator foraging efficiency. By selecting lines differing in their attraction to herbivore-induced plant volatiles we may experimentally investigate the link between this behavior, predator foraging efficiency, and local and regional predator-prey population dynamics. The impact of significant additive genetic variation in predator response to plant volatiles on evolution in a tritrophic context also remains to be uncovered.     

伤害和挥发物对棉花花外蜜的诱导效应

植物花外蜜的分泌是一种植物间接防御反应。为了明确植食性昆虫、机械伤和机械伤诱导的挥发性气体在植物花外蜜诱导分泌中的作用,分析了咀嚼式口器昆虫棉铃虫Helicoverpa armigera(Hübner)、刺吸式口器昆虫棉蚜Aphis gossypii Glover取食、剪刀机械伤、剪刀机械伤+棉铃虫反吐物、针刺机械伤以及机械伤诱导挥发物、顺式-茉莉酮对棉花Gossypium hirsutum L.叶片花外蜜分泌量的影响。结果表明,棉铃虫取食、剪刀机械伤、剪刀机械伤+棉铃虫反吐物处理均显著增加了被处理叶片花外蜜的分泌量。棉花花外蜜的诱导效应在处理叶片上表现明显,并且在较幼嫩的第3片真叶上也有系统性增长。顺式-茉莉酮和机械伤挥发物处理1 d对棉花较幼嫩的第4、5片真叶花外蜜有诱导效应。棉花叶片花外蜜的诱导主要与植物组织损伤有关;不同口器类型的昆虫对棉花叶片花外蜜的诱导量不同,咀嚼式口器的棉铃虫对棉花花外蜜的诱导强度显著高于刺吸式口器的棉蚜;顺式-茉莉酮和机械伤诱导的挥发物能作为棉花植株间交流的信息物质诱导棉花幼嫩叶片花外蜜的分泌。