Apolygus Lucorum Research Articles

Nutritional Characteristics of Normal, Apolygus lucorum-Damaged, and Mechanically Damaged Tender Shoots of Tea (Camellia sinensis)

Apolygus lucorum is currently a major pest of tea plantations, affecting the normal growth of tea shoots and reducing yield and quality. This study measured the effect of damage by A. lucorum alongside mechanical damage using UV spectrophotometry and high performance liquid chromatography. We found that the nutritional characteristics of tea varied with the level of bug damage, which influenced levels of caffeine, theanine, and tea polyphenols. As damage increased, caffeine content gradually increased. In contrast, theanine levels in highly damaged leaves were reduced compared to other levels of damage. The polyphenol level, like caffeine, increased with increasing damage. The levels of five other substances–Epigallocatechin, Catechin, Epigallocatechin gallate, Epicatechin, and Epicatechin gallate were all higher in tea with higher damage. Damage from A. lucorum changed the chemical profile of the tea, caused tea water turbidity, taste bitterness and decreased freshness, leading to the tea quality decline.

Activities of Digestive Enzymes in the Omnivorous Pest Apolygus lucorum (Hemiptera: Miridae).

The mirid bug Apolygus lucorum Meyer-Dür, 1843, an omnivorous species that feeds on plants and animals, has become a major pest in China as production of Bt-cotton has grown to such a large scale. Its omnivory is likely to be critical for its success, but the digestive mechanism(s) underlying processing and adsorption of such diverse foods is relatively unknown. Here, we examined the activities of digestive enzymes of A. lucorum in the salivary gland complex and midgut and the effect of sex, age, and food source on these activities. Amylase and protease were present in the salivary gland complex and the midgut, but were higher in the salivary gland complex. Trypsin-like enzyme was also present in both organs, but chymotrypsin-like enzyme was present only in the midgut. Sex, age, and food source affected the activities of these digestive enzymes. In general, the activities of these enzymes peaked at 10 d after emergence, and amylase and protease activities were higher in female adults than in males. Of the food sources tested, green bean pods (Gb) induced the highest amylase activity, whereas Helicoverpa armigera Hübner, 1809 eggs (He) and a mixture of Gb and He induced higher activities of the trypsin-like and chymotrypsin-like enzymes. The results from food switching experiments confirmed that amylase activity could be induced by plant sources, and animal sources induced protease activity. Thus, the types and activities of digestive enzymes in A. lucorum provide the physiological basis of the pest’s omnivory.

Levels of Salivary Enzymes of Apolygus Lucorum (Hemiptera: Miridae), From 1st Instar Nymph to Adult, and Their Potential Relation to Bug Feeding.

In recent years, Apolygus lucorum has caused increasing damage to cotton and fruit trees in China. The salivary enzymes secreted by A. lucorum when sucking on host plants induce a series of biochemical reactions in plants, and the pre-oral digestion benefits the bug feeding. In this study, the food intake of A. lucorum from 1st instar nymphs to adults was measured, and the corresponding salivary activity of pectinase, amylase, cellulase, protease, polyphenol oxidase and peroxidase was determined. Daily food intake varied with developmental stage, peaking in 3rd and 4th instar nymphs. Pectinase, amylase, cellulase and protease were detected in both nymphal and adult saliva of A. lucorum, while neither polyphenol oxidase nor peroxidase was detected. Protease activity varied with food intake peaking at the 3rd-4th instar, and then slightly decreasing at the 5th instar. Levels of pectinase, amylase and cellulase increased significantly with the daily feeding level until the 3rd instar, corresponding with increasing damage to host plants. The activity of both cellulase and protease had a significant linear relationship with the average daily food intake. The increasing activity of enzymes in saliva explain stage-specific impacts of A. lucorum on the host plants, and suggest that optimal management of A. lucorum would be confined to its control threshold prior to the peak of daily feeding in the 3rd instar.

Identification and expression analysis of an olfactory receptor gene family in green plant bug Apolygus lucorum (Meyer-Dür).

Olfactory receptors are believed to play a central role in insects host-seeking, mating, and ovipositing. On the basis of male and female antennal transcriptome of adult Apolygus lucorum, a total of 110 candidate A. lucorum odorant receptors (AlucOR) were identified in this study including five previously annotated AlucORs. All the sequences were validated by cloning and sequencing. Tissue expression profiles analysis by RT-PCR indicated most AlucORs were antennal highly expressed genes. The qPCR measurements further revealed 40 AlucORs were significantly higher in the antennae. One AlucOR was primarily expressed in the female antennae, while nine AlucORs exhibited male-biased expression patterns. Additionally, both the RPKM value and RT-qPCR analysis showed AlucOR83 and AlucOR21 were much higher abundant in male antennae than in female antennae, suggesting their different roles in chemoreception of gender. Phylogenetic analysis of ORs from several Hemipteran species demonstrated that most AlucORs had orthologous genes, and five AlucOR-specific clades were defined. In addition, a sub-clade of potential male-based sex pheromone receptors were also identified in the phylogenetic tree of AlucORs. Our results will facilitate the functional studies of AlucORs, and thereby provide a foundation for novel pest management approaches based on these genes.

Effect of Water on Survival and Development of Diapausing Eggs of Apolygus lucorum (Hemiptera: Miridae)

The green mirid bug Apolygus lucorum is a regional pest of multiple crops in northern China, and the survival and development of diapausing eggs during winter plays an important role in the population dynamics of this species. The effect of water on the survival and development of A. lucorum eggs was investigated using laboratory-induced diapause. Diapausing eggs were exposed to various humidity regimes under three conditions: (1) termination of diapause with exposure to warm long-day (WLD) conditions (i.e., 26 ± 1°C and 75 ± 5% relative humidity (RH) under a photoperiod of 16 hours light and 8 hours dark), (2) termination of diapause by chilling at 4°C, or (3) during the post-diapause stage, i.e., from transfer to WLD conditions after chilling, until the hatching of nymphs. The results indicate that water availability is crucial for the post-diapause resumption of development of A. lucorum. However, exposure to excessive moisture was detrimental, as indicated by a decrease in diapause termination rate and a prolonged pre-hatching period of diapausing eggs, compared to limited moisture conditions. This implies that both too dry and too humid environmental conditions would suppress survival and postpone hatching of overwintered A. lucorum eggs, and might explain why this pest has not caused severe damage in either southern or western China where the respective climates are very humid or dry.

Central Projection of Antennal Sensory Neurons in the Central Nervous System of the Mirid Bug Apolygus lucorum (Meyer-Dür).

The mirid bug Apolygus lucorum (Meyer-Dür), a polyphagous pest, is dependent on olfactory cues to locate various host plant species and mates. In this study, we traced the projection pathway of the antennal sensory neurons and visualized their projection patterns in the central nervous system of A. lucorum through confocal microscopy and digital reconstructions. We also examined the glomerular organization of the primary olfactory center of the brain, the antennal lobe, and created a three-dimensional model of the glomeruli. We found that the axons of the sensory neurons project into the brain via the ipsilateral antennal nerve, and descend further into the gnathal ganglion, prothoracic ganglion, mesothoracic ganglion, and metathoracic ganglion, and reach as far as to the abdominal ganglion. Such a projection pattern indicates that antennal sensory neurons of A. lucorum may be potentially directly connected to motor neurons. The antennal lobe, however, is the major target area of antennal sensory neurons. The antennal lobe is composed of a large number of glomeruli, i.e. 70–80 glomeruli in one AL of A. lucorum. The results of this study which provide information about the basic anatomical arrangement of the brain olfactory center of A. lucorum, are important for further investigations of chemosensory encoding mechanisms of the mirid bug.

Predation by generalist arthropod predators on Apolygus lucorum (Hemiptera: Miridae): molecular gut-content analysis and field-cage assessment.

The mirid bug Apolygus lucorum (Meyer-Dür) is a principal pest of cotton that also causes great damage to many other crops in China. A study was conducted to assess the mortality of A. lucorum from generalist arthropod predators using both molecular methods and a field-cage trial. The species-specific primer pair for the detection of A. lucorum tissues in predators was designed according to the sequences of the cytochrome oxidase subunit I (COI) gene. A total of 2096 generalist predators that consisted of ladybeetles, lacewings and spiders were collected, and A. lucorum remains were detected using the designed primers. Only 1.6% of these predators contained A. lucorum DNA, with the highest positive proportion (6.1%) for Harmonia axyridis larvae. In the field-cage experiment, the daily predation rates of second-instar A. lucorum nymphs by H. axyridis adults and larvae were 4.7 and 5.2% respectively. The overall low positive proportion of generalist predators with A. lucorum DNA detected using the molecular method, combined with the low predation rate in the field-cage experiment, indicated that the primary generalist predators likely had a limited role in the suppression of A. lucorum in the field. © 2016 Society of Chemical Industry.

A Female-Biased Odorant Receptor from Apolygus lucorum (Meyer-Dür) Tuned to Some Plant Odors.

Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) is a serious pest of cotton, jujube, grape and many other crops around the world. Understanding how olfactory information directs this insect to its host plants may provide environment-friendly approaches to the control of its population in agriculture. In our study, we cloned an odorant receptor gene, AlucOR46, that was specifically expressed in antennae and female-biased. Functional expression of AlucOR46 in Xenopus oocytes showed that it is tuned to six plant volatiles (S)-(−)-Limonene, (R)-(+)-Limonene, (E)-2-Hexenal, (E)-3-Hexenol, 1-Heptanol and (1R)-(−)-Myrtenol. Electroantennogram (EAG) recordings revealed that all six compounds could elicit electrophysiological responses from the antennae of A. lucorum, higher in females. Our results are in agreement with previous reports showing that (E)-2-Hexenal could attract female A. lucorum in behavior experiments. These results suggest that AlucOR46 might play an important role in locating the host plants of A. lucorum and therefore represents a suitable target for green pest control.

Molecular identification of closely related mirine plant bugs, Apolygus spinolae and A. lucorum (Heteroptera: Miridae) by PCR-RFLP markers

The plant bugs Apolygus spinolae (Meyer-Dür) and Apolygus lucorum (Meyer-Dür) (Heteroptera: Miridae) are well-known as economically important pests of various agricultural crops in quite a few parts of the Palearctic Region. These two species are difficult to distinguish from one another due to their similar appearances. Earlier studies revealed that the two species can be easily separated by species-specific sex pheromone composition. Segments of two mitochondrial genes, large ribosomal RNA subunit (16S) and cytochrome c oxidase subunit I (COI), were compared between A. spinolae and A. lucorum female adults that had been identified by analyzing the pheromone composition. Based on the DNA sequence differences between the two species, molecular diagnostic markers using PCR and restriction fragment length polymorphism (RFLP) were developed. This rapid and accurate method was successfully applied to verify the species identification of male adults caught by pheromone traps and of nymphs collected from various host plants. These molecular markers and diagnostic methods could provide a useful tool for the identification of these sibling species and further contribute to the development of rational pest management strategies.

Effects of intercropping tea with aromatic plants on population dynamics of arthropods in Chinese tea plantations

Tea plants (Camellia sinensis (L.) O. Kuntze) are often colonized by economically important insect pests such as tea green leafhoppers (Empoasca onukii Matsuda) and green plant bugs (Apolygus lucorum Meyer-Dur) in northern China. Aromatic plant volatiles can exert repellent effects on insect herbivores. To evaluate the impact of intercropping on the populations of two tea pests and natural enemies in tea plantations, field experiments were conducted in which four aromatic plants (Cassia tora, Medicago sativa, Leonurus artemisia, and Mentha haplocalyx) were intercropped with tea plants and compared with tea monoculture in 2014 and 2015. The counts of arthropods conducted on tea plants showed that intercropping C. tora and L. artemisia with tea plants significantly reduced the E. onukii population levels in the tea plantation. The abundance of A. lucorum was variable during the study and not significantly lower in various intercropping plots than monocropping plots. Y-tube olfactometer assays showed that volatile organic compounds derived from C. tora and M. sativa significantly affected the behavior of E. onukii and that M. haplocalyx odors can be used to mask host plant odors and are repellent to A. lucorum. Among natural enemies, coccinellids occurred often on tea plants during growing seasons. Intercropping tea plants with C. tora also promoted a marked increase in the natural enemies of tea pests, including coccinellids, lacewings, spiders, and parasitoids. Our findings suggest that the sustainable pest management achieved by intercropping offers an alternative strategy for the control of important tea pests.

Parasitism of Overwintering Eggs of Apolygus lucorum by Telenomus sp. in Jujube Orchard

Parasitism of Overwintering Eggs of Apolygus lucorum by Telenomus sp. in Jujube Orchard

The Joint Toxicity of Different Temperature Coefficient Insecticides onApolygus lucorum(Hemiptera: Miridae)

The effect of temperature on the cotoxicity coefficient (CTC) value was used to evaluate mixture efficacy of different temperature coefficient chemicals from 15 to 35°C by exposing third-instar Apolygus lucorum (Meyer-Dür) to dip-treated asparagus bean pods. The results indicated the joint toxicity of same temperature coefficient insecticide (TCI) types were unaffected by temperature. This means that even when temperatures change, the mixture ratios of the highest CTC values remained the same, and the effect of temperature on the joint toxicity of same TCI types was only on the CTC values. However, the effect of temperature was variable when considering the joint toxicity of different TCI types. The effect of temperature on the joint toxicity of both strong positive and strong negative TCI types was clear, and the highest CTC values of mixture ratios changed with temperature regularly. When comparing the influence of temperature between strong/slight positive/negative insecticides, the results indicated a greater influence of the strong TCI. Paradoxically, the highest CTC value of the imidacloprid and methomyl mixture did not change with temperature changes consistently, even with the variance of imidacloprid ratios, a strong TCI. These results will guide pest managers in choosing the most effective insecticide mixtures for A. lucorum control under given environmental conditions.

Survey of organophosphate resistance and an Ala216Ser substitution of acetylcholinesterase-1 gene associated with chlorpyrifos resistance in Apolygus lucorum (Meyer-Dür) collected from the transgenic Bt cotton fields in China

The mirid bug is frequently controlled by the application of organophosphorus insecticides in the transgenic Bt cotton field of China. A topical bioassay method was performed to evaluate the toxicities of chlorpyrifos and malathion towards field-collected Chinese populations of Apolygus lucorum from transgenic Bt cotton fields. For chlorpyrifos, the resistance ratios ranged from 0.8 to 9.4-fold compared to a susceptible strain. For malathion, the resistance levels relative to the susceptible strain ranged from 1.2 to 14.4-fold. Compared to a susceptible strain, the Cangzhou population from Hebei province showed the highest resistance ratios towards these insecticides. A comparison of the detoxifying and target enzyme activities between the Cangzhou population and a susceptible strain revealed that altered acetylcholinesterase possibly account for the chlorpyrifos and malathion resistance in the Cangzhou population. Two acetylcholinesterase (AChE-encoding) genes (designated Alace1 and Alace2) from the green mirid bug (A. lucorum) were identified. The Alace1 and Alace2 genes encoded 597 and 645 amino acids, respectively. Both AChE proteins had conserved motifs including a catalytic triad, a choline-binding site, and an acyl pocket. Quantitative real-time PCR analysis showed that Alace1 had a much higher transcriptional level than Alace2, for the expression profiles of both spatial and time distributions. One amino acid substitution, Ala216Ser in Alace1, was found in the Cangzhou population. These results suggest that the mutation Ala216Ser should be most likely involved in organophosphorus resistance in A. lucorum.

Identification and functional characterization of four transient receptor potential ankyrin 1 variants in Apolygus lucorum (Meyer-Dür).

As signal integrators that respond to various physical and chemical stimuli, transient receptor potential (TRP) channels fulfil critical functional roles in the sensory systems of both vertebrate and invertebrate organisms. Here, four variants of TRP ankyrin 1 (TRPA1) were identified and cloned from the green plant bug, Apolygus lucorum. Spatiotemporal expression profiling across development and in different adult tissues revealed that the highest relative-transcript levels occurred in first-instar nymphs and antennae, respectively. In Xenopus laevis-based functional assays, Apo.lucorum TRPA1-A (AlucTRPA1-A), AlucTRPA1-B and AlucTRPA1-C were activated by increasing the temperature from 20 to 40°C with no significant desensitization observed after repeated temperature stimuli. The activation temperature of AlucTRPA1-A and AlucTRPA1-B was < 25°C, whereas the activation temperature of AlucTRPA1-C was between 25 and 30°C. Amongst the variants, only AlucTRPA1-A and AlucTRPA1-C were directly activated by high concentrations of allyl isothiocyanate, cinnamaldehyde and citronellal. Taken together, these results suggest that AlucTRPA1 variants may function in vivo as both thermal and chemical sensors, with the four variants potentially mediating different physiological functions. This study not only enriches our understanding of TRPA1 function in Hemiptera (Miridae), but also offers a foundation for developing new pest control strategies.

Targeting insect mitochondrial complex I for plant protection.

SummaryPlant engineered to express double‐stranded RNA (dsRNA) can target the herbivorous insect gene for silencing. Although mounting evidence has emerged to support feasibility of this new pest control technology, field application is slow largely due to lack of potent targets. Here, we show that suppression of the gene encoding NDUFV2, a subunit of mitochondrial complex I that catalyses NADH dehydrogenation in respiratory chain, was highly lethal to insects. Feeding cotton bollworm (Helicoverpa armigera) larvae with transgenic cotton tissues expressing NDUFV2 dsRNA led to mortality up to 80% within 5 days, and almost no larvae survived after 7 days of feeding, due to the altered mitochondrial structure and activity. Transcriptome comparisons showed a drastic repression of dopa decarboxylase genes. Reciprocal assays with Asian corn borer (Ostrinia furnacalis), another lepidopteran species, revealed the sequence‐specific effect of NDUFV2 suppression. Furthermore, the hemipteran lugus Apolygus lucorum was also liable to NDUFV2 repression. These data demonstrate that the mitochondrial complex I is a promising target with both sequence specificity and wide applicability for the development of new‐generation insect‐proof crops.

Flowers promote ovarian development and vitellogenin gene expression in Apolygus lucorum (Heteroptera: Miridae)

Apolygus lucorum (Heteroptera: Miridae), an important herbivore feeding on a broad range of cultivated and wild plants, always shows a strong preference for flowering host plants. In a prior study, we found A. lucorum fecundity to be higher on flowering plants than on plants still at the vegetative stage. This led us to hypothesize that ovarian development and vitellogenin (Vg) expression in A. lucorum, which are highly correlated with its fecundity, might respond positively to the presence of host plant flowers as food. In this study, two types of plant foods, i.e., cotton terminals and flowers, and cotton terminals only without flowers, were examined. Adult females on terminals with flowers had higher ovarian development levels than those of similar stage held on terminals without flowers. Moreover, adults on terminals with flowers had more follicles per ovary throughout adult life than those held on terminals without flowers, and the length of the most developed ovariole of the former treatment was significantly longer than that of the latter diet. Use of qRT-PCR showed that the expression level of Vg was significantly higher in adults raised on a diet with flowers compared with those raised on the diet without flowers. These results suggest that host plant flowers significantly facilitate ovarian development and Vg gene expression of A. lucorum adults.

Molecular Cloning and the Expression Profile of Vitellogenin in Relation to Tissue and Food Source inApolygus lucorum(Hemiptera: Miridae)

Vitellogenin proteins (Vgs) are precursors of the major egg yolk proteins in many oviparous vertebrates and invertebrates. Here we cloned the full-length cDNA of the Vg gene of Apolygus lucorum , an omnivorous mirid bug that feeds on various agricultural crops and small insects. The gene has 6,131 base pairs (bp) with an open reading frame (ORF) of 5,988 bp, encoding a protein with 1,996 amino acids, with molecular mass 218.3 kDa and isoelectric point 9.02. Phylogenetic analysis showed that Vg gene has a high similarity with that in Hemiptera insects and was most closely related to the rice leaf bug, Trigonotylus caelestialium . The expression of Vg gene was significantly higher in the fat body than in other tissues in females (ovary, hemolymph, gut, and malpighian tubule). Vg gene expression was extremely low in newly emerged adult females and increased with age. The effect of different food sources on the expression level of Vg in adult females was investigated after they were fed on green bean pods (Gb), H. armigera eggs (He), or a combination of green bean pods and H. armigera eggs (GbHe). The results showed that transcript level of Vg gene in females was significantly higher on the GbHe diet than on Gb or He. Our results demonstrate that A. lucorum can acquire nutrients from both plant and animal origin food sources, and the combined food sources significantly facilitated Vg synthesis.

Molecular characterisation of the vitellogenin gene (AlVg) and its expression after Apolygus lucorum had fed on different hosts.

The polyphagous pest Apolygus lucorum is now the dominant pest of Bacillus thuringiensis (Bt) cotton in China. In this study, the transcriptional and translational profiles of AlVg influenced by different hosts were identified, and then the correlations between AlVg gene or AlVg protein expression and key population proliferation parameters of A. lucorum were further clarified. AlVg or AlVg expression can be significantly regulated by different host nutrients (P < 0.05). AlVg or AlVg expression was significantly higher in A. lucorum reared on Bt and conventional cotton than in A. lucorum reared on garland chrysanthemum and broad bean (P < 0.05), but there was no significant difference between AlVg or AlVg expression in A. lucorum reared on Bt and conventional cotton (P > 0.05). In addition, there were significant linear regression correlations between AlVg or AlVg expression and total mortality rate of nymphs, female lifespan, per female fecundity and egg hatching rates (P < 0.05). Our results confirm that AlVg or AlVg is the key parameter affecting female fertility of A. lucorum. AlVg and AlVg expression can be influenced by different host nutrients except for Bt toxin. © 2015 Society of Chemical Industry.

Does feeding behavior of a zoophytophagous mirid differ between host plant and insect prey items?

Many mirid bugs are omnivores, and several species are key pests of agricultural crops. While mirid feeding behavior on plant hosts is relatively well studied, little attention has been paid to foraging, acceptance, and consumption of insect prey items. In this study, we investigated the feeding behavior and stylet penetration of the green mirid bug Apolygus lucorum on insect prey (Helicoverpa armigera eggs), plant hosts (green bean Phaseolus vulgaris pod), and combined plant + prey diets. Direct observation and electrical penetration graph (EPG) assays were used to assess feeding on either food item. Overall feeding time was highest on combined diets (48.18 ± 4.92 % of total time) and plant foods (42.23 ± 3.36 %), compared with only 28.95 ± 2.27 % feeding time on H. armigera eggs. When exposed to combined diets, A. lucorum spent 3.8 times longer feeding on green bean pod than on H. armigera eggs. The overall duration of stylet penetration was 5866.27 ± 800.39 s on green bean pod versus 3644.81 ± 715.19 s on H. armigera eggs, representing a 37.87 % reduction in duration. Similarly, the duration of a single probing event was significantly higher on green bean pod (355.76 ± 50.13 s) versus H. armigera eggs (236.20 ± 25.17 s), with a 33.61 % reduction. This first work in which omnivory is studied by combined use of observational studies and EPG assays (1) provides further insights into A. lucorum omnivory, (2) further elucidates its trophic position in agroecosystems, and (3) guides the development of artificial diets for this pest species.

Insecticide susceptibility of the green plant bug, Apolygus lucorum Meyer-Dür (Homoptera: Miridae) and two predatory arthropods

Abstract The green plant bug (Apolygus lucorum Meyer-Dür) is a key pest of Bt cotton in China. Along with biological control, chemical control is one of the most important strategies in A. lucorum Integrated Pest Management (IPM). The goal of this study was to evaluate the toxicity of eight conventional insecticides to A. lucorum and to assess the susceptibility of two generalist predators Chrysopa sinica (Jieder) and Propylaea japonica (Thunbery) to insecticides that are commonly used in A. lucorum management. Via glass-vial and leaf-dip bioassay, toxicity tests with selected insecticides at two different life-stages of A. lucorum indicated significant differences between the LD50 or LC50 values for these compounds within different insecticidal classes. Phenylpyrazole fipronil had the highest toxicity to 4th-instar nymphs and adults of A. lucorum, whereas neonicotinoid imidacloprid had the lowest toxicity among the insecticides. Females were more tolerant to insecticides than were males, as shown by the higher LD50 values for females. Furthermore, laboratory tests showed that endosulfan had the highest selectivity to C. sinica and P. japonica: the selective toxicity ratios (STRs) were superior to other tested insecticides, particularly imidacloprid, and were 5.396 and 4.749-fold higher than baseline STRs, respectively. From this study, we conclude that fipronil can potentially be used to efficiently control A. lucorum. An alternative control agent worth consideration is endosulfan, owing to its relative safety to non-targeted natural enemies.