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Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs 179PGGRFRELYYWDTY192 and 479QWDFPNAWPP489, a glycine-rich region 549GGGGEY554, and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 °C and pH 5.0, with Km and Vmax values of ~ 71 mM and ~ 126 µmol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health.

The impact of different diets on chemical defense has been extensively studied in animals that sequester defensive chemicals from food. However, there are fewer studies of diet-mediated variation in autogenously produced defenses. Ladybird beetles, which use autogenously synthesized defensive alkaloids, are used as models in a wide diversity of studies of chemical defense, specifically in studies of intraspecific variation in color pattern and chemical defense. Many aphidophagous ladybirds consume a wide diversity of aphid prey, which vary in quality and thus could affect the synthesis of chemical defense. We measured alkaloid recovery rate after reflex bleeding by the ladybird Adalia bipunctata on two different aphid diets, the high quality Acyrthosiphon pisum and the lower quality Aphis fabae. Alkaloids reaccumulated in ladybirds more slowly when they were fed A. fabae than when they were fed A. pisum and females generally had more alkaloid than males, but reaccumulated alkaloid more slowly. Recovery times were more than 12 days. There appeared to be a weak positive relationship between alkaloid level and time since reflex bleeding for eggs of A. pisum- but not A. fabae-fed females. Our findings on diet and alkaloid synthesis in ladybirds suggest that chemical defense levels are very dynamic, indicating that studies conducted at a single point in time, such as those focused on ladybird color pattern, fail to consider a wide diversity of temporal variation that occurs in the field. This is likely true for many autogenously produced chemical defense systems in a diversity of other organisms.

Chickpea chlorotic stunt virus (CpCSV, genus Polerovirus, family Solemoviridae), first reported in Ethiopia in 2006, causes an economically important yellowing and stunting disease in legume crops such as chickpea, faba bean, field pea, and lentil in most production areas of North Africa and Central and West Asia. Disease epidemics have been reported in Ethiopia, Syria, and Tunisia. The virus is transmitted persistently by aphids of the species Aphis craccivora and Acyrthosiphon pisum and naturally infects several legume and non-legume hosts. CpCSV exists as at least two geographic strain groups that differ in their genome sequence and serological and biological properties. In addition, a genetically divergent isolate proposed to be a member of a distinct polerovirus species has been reported from pea and faba bean in China. The ssRNA genome of the Ethiopian isolate has 5900 nucleotides, is encapsidated in isometric particles of ~ 28 nm diameter, and is suggested to have evolved by recombination of cucurbit aphid-borne yellows virus- and soybean dwarf virus-like parents. Moreover, a number of newly reported poleroviruses are suggested to have evolved by recombination between CpCSV and other parental poleroviruses. Identification of sources of resistance and further knowledge on disease epidemiology, including specific strains, vectors, and alternate hosts in different growing areas, are required for devising effective disease management strategies. Modern biotechnology tools such as next-generation sequencing, molecular markers, and agroinoculation-based resistance screening techniques can expedite future research and management efforts. This review addresses various aspects of CpCSV, including its properties, ecology, the disease it causes, management options, and future research perspectives.

Background: Pea aphids (Acyrthosiphon pisum) possess a weak immune system, but they can firmly mount immunological responses. However, the influence of different plants on their defense against different microbes remains largely unknown. In addition, no previous research has integrated the growth, reproduction, and defense responses of pea aphids feeding on different plants. Objective: This study aims to investigate the growth, reproduction, and defense responses of pea aphids feeding on different plants. Methods: Pea aphids were cultivated on both Medicago sativa and Vicia faba. Growth and reproduction were evaluated. Additionally, we monitored the survival and microbial loads of pea aphids after bacterial and fungal infections. Results : Pea aphids reared on M. sativa had lower growth, lower intrinsic rate of increase, and lower finite rate of increase when compared to aphids feeding on V. faba. The net reproduction was lower in aphids feeding on M. sativa, although the difference was not significant. The mean time of generation and pre-reproductive periods was longer for aphids reared on M. sativa than for aphids reared on V. faba. In the infection experiments, we found that aphid survival was not affected by the host plant. However, aphids reared on M. sativa generally harbored fewer microbial loads than those reared on V. faba. Conclusions: The growth and reproduction of pea aphids are affected by the host plant. Aphids feeding on different plants had different tolerances to microbial infections. Our study sheds light on improving biological control program for aphids.

Carotenoids are involved in many essential physiological functions and are produced from geranylgeranyl pyrophosphate through synthase, desaturase, and cyclase activities. In the pea aphid (Acyrthosiphon pisum), the duplication of carotenoid biosynthetic genes, including carotenoid synthases/cyclases (ApCscA-C) and desaturases (ApCdeA-D), through horizontal gene transfer from fungi has been detected, and ApCdeB has known dehydrogenation functions. However, whether other genes contribute to aphid carotenoid biosynthesis, and its specific regulatory pathway, remains unclear. In the current study, functional analyses of seven genes were performed using heterologous complementation and RNA interference assays. The bifunctional enzymes ApCscA-C were responsible for the synthase of phytoene, and ApCscC may also have a cyclase activity. ApCdeA, ApCdeC, and ApCdeD had diverse dehydrogenation functions. ApCdeA catalyzed the enzymatic conversion of phytoene to neurosporene (three-step product), ApCdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene (two-step product), and ApCdeD catalyzed the enzymatic conversion of phytoene to lycopene (four-step product). Silencing of ApCscs reduced the expression levels of ApCdes, and silencing these carotenoid biosynthetic genes reduced the α-, β-, and γ-carotene levels, as well as the total carotenoid level. The results suggest that these genes were activated and led to carotenoid biosynthesis in the pea aphid.

Trehalose serves multifarious roles in growth and development of insects. In this study, we demonstrated that the high trehalose diet increased the glucose content, and high glucose diet increased the glucose content but decreased the trehalose content of Acyrthosiphon pisum. RNA interference (RNAi) of trehalose-6-phosphate synthase gene (ApTPS) decreased while RNAi of trehalase gene (ApTRE) increased the trehalose and glucose contents. In the electrical penetration graph experiment, RNAi of ApTPS increased the percentage of E2 waveform and decreased the percentage of F and G waveforms. The high trehalose and glucose diets increased the percentage of E2 waveform of A. pisum red biotype. The correlation between feeding behavior and sugar contents indicated that the percentage of E1 and E2 waveforms were increased but np, C, F and G waveforms were decreased in low trehalose and glucose contents. The percentage of np, E1 and E2 waveforms were reduced but C, F and G waveforms were elevated in high trehalose and glucose contents. The results suggest that the A. pisum with high trehalose and glucose contents spent less feeding time during non-probing phase and phloem feeding phase, but had an increased feeding time during probing phase, stylet work phase and xylem feeding phase.

Bacterial symbionts in aphids are known to benefit the insect host and associated with aphid’s ecological adaptation. The pea aphid (Acyrthosiphon pisum), an important legume pest worldwide, carries at least eight endosymbionts, providing a model system to study insect–bacteria interactions. However, species diversity and geographic variations of endosymbionts are unknown in Chinese populations; therefore, we characterized symbiont communities and diversity of 27 pea aphid samples from 13 geographic populations of China. Via amplicon high-throughput sequencing and diagnostic PCR, we found that bacterial communities of Chinese populations were dominated by Proteobacteria and Firmicutes. Among eight known endosymbionts, five (Buchnera, Serratia, Hamiltonella, Regiella, and Rickettsia) were detected by both methods, with a specific geographical distribution. The obligate symbiont, Buchnera, was present in all aphid samples, while the four facultative symbionts showed a significant geographic variation. Each population was randomly infected with distinct endosymbionts, ranging from three to five species. Serratia and Rickettsia showed relatively higher abundance in central regions of China, Regiella was predominant in eastern and western China, whereas Hamiltonella showed an extremely low abundance and was absent in four populations. Samples grouped by altitudes showed a significant diversity difference, whereas there was no significant difference between red and green body colors. Bacterial community structures of the Chinese pea aphid populations were mainly influenced by environmental factors, other than body colors. These data can guide the development of potential biocontrol techniques against this aphid.

Ecological theory predicts that host-plant traits affect herbivore population growth rates, which in turn modulates predator-prey interactions. However, while vector-borne plant pathogens often alter traits of both host plants and vectors, a few studies have assessed how pathogens may act as interaction modifiers within tri-trophic food webs. By applying a food web motif framework, we assessed how a vector-borne plant pathogen (Pea-enation mosaic virus, PEMV) modified both bottom-up (plant-herbivore) and top-down (predator-prey) interactions. Specifically, we assessed trophic interactions with PEMV-infectious Acyrthosiphon pisum (pea aphid) vectors compared to non-infectious aphids in a factorial experiment that manipulated predator and plant communities. We show that PEMV altered bi-trophic relationships, whereby on certain plant species, PEMV reduced vector performance but also increased their susceptibility to predators. However, on other plant species, PEMV weakened top-down control or increased vector performance. Our results suggest that vector-borne plant pathogens are important interaction modifiers for plant-herbivore-predator dynamics: host-plant response to viruses can decrease herbivore abundance by reducing herbivore performance, but also increase herbivore abundance by weakening top-down control. Broadly speaking, trophic interactions that regulate herbivore outbreaks appear to be modified for herbivores actively transmitting viruses to host plants. Consequently, management and monitoring of outbreaking herbivores should consider the infection status of focal populations.

Herbivores assess predation risk in their environment by identifying visual, chemical, and tactile predator cues. Detection of predator cues can induce risk-avoidance behaviors in herbivores that affect feeding, dispersal, and host selection in ways that minimize mortality and reproductive costs. For herbivores that transmit plant pathogens, including many aphids, changes in herbivore behavior in response to predator cues may also affect pathogen spread. However, few studies have assessed how aphid behavioral responses to different types of predator cues affect pathogen transmission. Here, we conducted greenhouse experiments to assess whether responses of pea aphids (Acyrthosiphon pisum) to predation risk and alarm pheromone (E-β-Farnesene), an aphid alarm signal released in response to predation risk, affected transmission of Pea enation mosaic virus (PEMV). We exposed A. pisum individuals to risk cues, and quantified viral titer in aphids and pea (Pisum sativum) host plants across several time periods. We also assessed how A. pisum responses to risk cues affected aphid nutrition, reproduction, and host selection. We show that exposure to predator cues and alarm pheromone significantly reduced PEMV acquisition and inoculation. Although vectors avoided hosts with predator cues, predator cues did not alter vector reproduction or reduce nutrient acquisition. Overall, these results suggest that non-consumptive effects of predators may indirectly decrease the spread of plant pathogens by altering vector behavior in ways that reduce vector competence and pathogen transmission efficiency.

Bu çalışmada ticari entomopatojenler olan Nibortem (Verticillium lecani), Met52 (Metarhizium anisopliae), Nostalgist (Beauveria bassiana), Priority (Paecilomyces fumosoreus) ve bitkisel bir insektisit olan Nimbecidine (Azadirachtin)’in Aphis fabae (Scopoli) ve Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) üzerindeki etkileri araştırılmıştır. Uygulanan biyolojik insektisitlerin A. fabae üzerindeki düzeltilmiş ölüm oranları 5. günde sırasıyla %78.41, %87.50, %65.91, %63.64 ve %86.36 olarak saptanmıştır. Bu değerler A. pisum için sırasıyla %63.33, %86.67, %64.44, %62.22 ve %86.67 olarak belirlenmiştir. Aynı günde (5. gün) A. fabae canlı birey sayıları sırasıyla 1.9, 1.1, 3.0, 3.2 ve 1.2 adet olurken, A. pisum için bu değerler sırasıyla 3.3, 1.2, 3.2, 3.4 ve 1.2 adet olarak saptanmıştır. Elde edilen verilere bağlı olarak her iki yaprak biti türünde de M. anisopliae ve Azadirachtin'in daha etkili olduğu saptanmıştır. Uygulanan bu iki biyolojik preparatın, başarıları arazi çalışmaları ile kanıtlandıktan sonra bu zararlılarla mücadelede kullanılabileceği kanısına varılmıştır.

The foxglove aphid (or glasshouse potato aphid, Aulacorthum solani Kaltenbach) transmits serious potato viruses (potato virus Y, and potato leaf roll virus) which cause heavy yield losses. Our aim of this study was to preliminary analysis of draft genome sequence to uncover virulence genes in the aphid. The genome assembly size (316.39 Mb) was very close to its genome size (318.19 Mb) estimated by flow cytometry. The genome completeness (81.8%) was confirmed by the Benchmarking Universal Single Copy Orthologs (BUSCO) analysis indicating 14.90% transposable elements (TEs) in the genome. Of total 22,021 predicted genes, 16,610 were annotated with putative functions of other aphids mainly Myzus persicae, Acyrthosiphon pisum and Diuraphis noxia. We identified virulence genes such as defensive and detoxification genes, salivary genes and chemoreceptors, insecticide resistance genes, virus transmission genes, transcription factors and mitochondrial genes. Importantly, analysis of detoxification genes particularly 53 cytochrome P450s (CYPs) indicated involvement of 23 CYPs families in aphid genome. Further, GO and KEGG pathways analyses showed gene enrichment predominantly with molecular function and signal transduction, respectively. Phylogeny analysis revealed genetic divergence among 12 aphid species and Au. solani is closely related with M. persicae. Further, non-synonymous (Ka)/synonymous (Ks) substitutions (Ka/Ks) indicated positive selection for 6 (Ka/Ks > 1) and 122 (Ka/Ks = 0.5–1) single copy orthologous gene pairs between Au. solani and with the pea aphid. Thus, our preliminary draft genome analysis provides new insights of Au. solani to understand molecular basis of aphid biology, host-aphid interactions and adaptation mechanism.

Background Aphids can be positioned as robust pest insects in farming and as ones of the model organisms for arthropods in molecular biology. Carotenoids are pigments that protect organisms from photooxidative damage caused by excessive light. Aphids were shown to possess genes of fungal origin for carotenoid biosynthesis, whereas a little knowledge was available about the functions of the genes and the biosynthetic pathway. Even carotenoid species contained in aphids were not enough understood. Main purpose of this study is to clarify these insufficient findings. Results The whole carotenoid biosynthetic pathway of the pea aphid (Acyrthosiphon pisum) was elucidated at the gene level, through comprehensive functional analysis of its carotenogenic genes, using Escherichia coli that synthesized carotenoid substrates, along with structural and quantitative analysis of carotenoids from various aphid species. Four genes were needed to synthesize all carotenoids accumulated in aphids from geranylgeranyl diphosphate. The tor gene mediated desaturation reaction from phytoene to 3,4-didehydrolycopene. It was revealed that a gene designated ApCrtYB3, which was considered to have functionally evolved in aphids, can convert lycopene into uncommon carotenoids with the γ-ring such as (6′S)-β,γ-carotene and γ,γ-carotene. We further demonstrated that the atypical carotenoids work as ecological indicators for estimating the food chain from aphids to predatory arthropods, and showed that aphids contributed with significant levels to the food chain from insect herbivores to several predatory arthropods, i.e., the red dragonfly (Sympetrum frequens; adults), seven-spotted ladybird (Coccinella septempunctata), and two spiders, Oxyopes sertatus and Nephila clavata. Gut microflora of the dragonfly (mature adults) was also found to include endosymbiotic bacteria such as Serratia symbiotica specific to the black bean aphid (Aphis fabae). Conclusions We revealed the whole carotenoid biosynthetic pathway of aphids, including functional identification of the corresponding genes. Subsequently, we showed that arthropodal food chain can be estimated using the uncommon carotenoids of aphids as ecological indicators. This result indicated that aphids made significant contributions to the food chain of several predatory arthropods including the red-dragonfly adults. Aphids are likely to be positioned as an important “phytochemicals” source for some predatory insects and arachnids, which are often active under bright sunlight.

Gram-negative binding proteins (GNBPs) are important in the innate immune system of insects in recognition of fungi pathogen, such as Beauveria bassiana. However, this information in aphids is not clear, which might be exploited to develop a novel aphid control strategy based on integrating RNAi and B. bassiana. Here, we firstly identified two GNBPs, ApGNBP1 and ApGNBP2, using the model aphid Acyrthosiphon pisum, and observed that two ApGNBPs were highly expressed in hemolymph and fat body as well as upon the injection of β-1,3-glucan and heat-killed B. bassiana. Intriguingly, RNAi-based silencing of ApGNBP1 but not ApGNBP2 decreased the activity of immune-related phenoloxidase. This led to the increased virulence of B. bassiana in A. pisum upon silencing of ApGNBP1, and the synergetic effects were also observed in other two aphids: Myzus persicae and Aphis citricidus. Importantly, no negative effects were detected in aphid predator Propylaea japonica under the co-application of the RNAi in targeting ApGNBP1 and B. bassiana. Taking together, we conclude that GNBP1 could be an optimal target in aphid control by combining RNAi, entomopathogenic fungi and ladybeetle predator.

Viruses are widespread in alfalfa (Medicago sativa L.), representing a key limitation to the production of this important forage plant. Understanding the diversity of plant viruses in alfalfa and their potential vectors will play an important role in management to minimize the emergence, transmission, and impact of viruses. Next-generation sequencing (NGS) targeting the transcriptome was applied to monitor the virus communities in alfalfa and its two main pests, thrips (Odontothrips loti Haliday and Frankliniella intonsa Trybom) and aphids (Acyrthosiphon pisum Mordvilko and Therioaphis trifolii Monell). A comparison of transcriptome datasets with reference databases revealed the presence of eight candidate viruses. Five out of the eight viruses, alfalfa mosaic virus (AMV), Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), Medicago sativa amalgavirus 1 (MsAV1), and bean yellow mosaic virus (BYMV), were confirmed by RT-PCR. We identified and determined the presence of four RNA viruses from alfalfa samples, two viruses (AMV and MsAPV1) from thrips samples, and one virus (BYMV) from T. trifolii. All sequences isolated from the insect samples were more than 95% identical to the sequences from the alfalfa samples or to sequences from the National Center for Biotechnology Information (NCBI) reference database. The RNA-seq results of this study suggest that AMV and MsAPV1 are the predominant RNA plant viruses infecting alfalfa and that they are carried by the major pests. This lays the foundation for future research on the vectors and transmission of these viruses. In addition, the sequence data have enabled the assembly of the first complete genome sequence of MsDPV1 from alfalfa.

Microbial symbionts often alter the phenotype of their host. Benefits and costs to hosts depend on many factors, including host genotype, symbiont species and genotype, and environmental conditions. Here, we present a study demonstrating genotype-by-genotype (G×G) interactions between multiple species of endosymbionts harboured by an insect, and the first to quantify the relative importance of G×G interactions compared with species interactions in such systems. In the most extensive study to date, we microinjected all possible combinations of five Hamiltonella defensa and five Fukatsuia symbiotica (X-type; PAXS) isolates into the pea aphid, Acyrthosiphon pisum. We applied several ecological challenges: a parasitoid wasp, a fungal pathogen, heat shock, and performance on different host plants. Surprisingly, genetic identity and genotype × genotype interactions explained far more of the phenotypic variation (on average 22% and 31% respectively) than species identity or species interactions (on average 12% and 0.4%, respectively). We determined the costs and benefits associated with co-infection, and how these compared to corresponding single infections. All phenotypes were highly reliant on individual isolates or interactions between isolates of the co-infecting partners. Our findings highlight the importance of exploring the eco-evolutionary consequences of these highly specific interactions in communities of co-inherited species.

The aim of this study was to evaluate the antioxidant effect of selenium in Pisum sativum L. plants pre-treated with sodium selenite or sodium selenate at a concentration of 10 and 20 μM, and then colonized by pea aphid Acyrthosiphon pisum (Harris). It has been hypothesized that selenium at low concentrations alleviates oxidative stress caused by aphid feeding on pea leaves. The study focused on the generation of reactive oxygen species (superoxide anion, hydrogen peroxide and hydroxyl radical), the activities of the antioxidant enzymes (superoxide dismutase and ascorbate peroxidase) scavenging the reactive oxygen species levels, as well as on total antioxidant activity in pea leaves. Selenium in pea leaves exposed to aphid feeding affected changes in the levels of reactive oxygen species, the activity of studied antioxidant enzymes, and the total antioxidant capacity. Effects depended on the form and concentration of selenium, as well as on the time after the colonization of pea plants by aphids. Obtained results showed beneficial effects of selenium in alleviating oxidative stress in pea leaves caused by aphid feeding.

Aphids (Hemiptera: Aphididae) have a repertoire of defensive behaviors against insect predators and parasitoids that includes kicking, twitching, walking away and dropping off the plant. These defensive responses, which are most effective when aphids receive early warning of an approaching predator, are often initiated before the predator reaches the aphid. Although described before, the process of predator detection has practically never been experimentally investigated in aphids and it is currently unknown whether they can distinguish approaching predators from harmless insects, and which senses they use for pre-encounter predator detection. I therefore addressed these two questions by comparing seven behavioral responses of pea aphids (Acyrthosiphon pisum) to approaching predators (ladybugs) and non-predators (variegated caper bugs), to the plant-borne vibrations they generate and to visual cues. Aphids kicked, raised their bodies, withdrew their stylets and dropped more often in response to approaching ladybugs. Visual cues elicited only antennal movements. Body raising and antennal movements were more frequent in response to ladybug vibrations compared to caper bug vibrations. I found no evidence for predator-odor detection. Stylet withdrawal (preparation for escape) and dropping occurred in response to climbing insects but not to vibrations. I conclude that pea aphids use multimodal cues to detect approaching coccinellid predators. While visual cues alert the aphid without triggering defensive responses, vibrational cues are used to distinguish predators from non-predators. Future research on vibration patterns that aphids interpret as signs of danger may lead to the development of novel management techniques for one of the world’s worst crop pests.

A field trial was conducted at the Institute of Forage Crops (Pleven, Bulgaria) from 2015 to 2017. It studied the effects of natural products on Acyrthosiphon pisum density, as well as changes in the chemical composition, content of plant fibre components and enzyme degradability in forage pea. Treatments with the natural insecticides Madex and Agricolle, applied alone or in combination with the organic fertilizers Lithovit and Nagro were performed twice - at the beginning of the flowering stage and one week later. The fertilizers used in the trial are environmentally safe and approved for use in organic production. The synthetic products Kristalon, a foliar fertilizer, and Proteus 110 OD, an insecticide, were used for comparison. The application of natural products, either alone and in combination, resulted in a reduction in pea aphid density. Applying Agricolle with Nagro, followed by Lithovit with Agricolle, led to the highest aphid number decrease (70.0 and 51.1%, respectively). An optimal combination of decrease in the content of plant cell wall fibre components, cellulose and lignification degree with a significant increase in forage in vitro enzyme digestibility was established after applying Agricolle with Lithovit and Agricole with Nagro. Digestibility reached 71.8 and 69.8%, respectively, an increase of 8.2-5.2%, while ADF, cellulose and lignification degree decreased from 7.1 and 7.7%, 8.0 and 23.4%, amd 10.5 and 6.8% after applying Agricolle with Lithovit and Agricole with Nagro, respectively. In comparison, the synthetic products Kristalon and Kristalon with Proteus increased forage quality, but to a relatively lesser extent. A stronger linear relationship was found between aphid density and dry matter digestibility, compared to the content of neutral detergent fibres. Pea forage with low content of plant cell wall fibre components, cellulose and lignification degree, high protein content, and digestibility after treatment with the natural product Agricolle, and its combinations with Lithovit and Nagro, make it a very good complement to other forages in dairy cow rations.

Biofortification of plants with selenium (Se) is one of the current trends in agriculture. This treatment enriches plants with this essential element for humans and animals, and also increases plant resistance to stress factors. The current knowledge of the interactions between Se and plants is well documented but between the plants biofortificated with Se and phytophages is insufficient and this problem should be further investigated. The research has focused on the effect of the pre-treatment of pea with Se on the pea aphid feeding behaviour and demographic parameters of the aphid population, and also has shown the activity of antioxidant (superoxide dismutase and catalase), detoxifying (S-glutathione transferase and β-glucosidase) and redox (peroxidase and polyphenol oxidase) enzymes in tissues of Acyrthosiphon pisum. The analysis of feeding models recorded using the Electronic Penetration Graph technique has shown that the pre-treatment of peas with Se affects the activity of aphids during the feeding. The use of 10 and 20 µM sodium selenite and sodium selenate caused changes in A. pisum probing and feeding behavior. What is more, Se reduced the activity of aphids associated with the phloem phase and caused an extension of non-probing time and non-phloem penetration. The effect of Se on the demographic population parameters of the aphid was also observed in the significant reduction of female fertility and the reproductive potential of the insects. Selenium enrichment of plants colonized by aphids increased the activity of antioxidant, detoxifying and redox enzymes in the tissues of A. pisum. It was also found that both Se taken up by plants, as well as, the feeding of aphids resulted in greater synthesis of flavonols and carotenoids in pea leaves.

Trehalose-6-phosphate synthase (TPS) and trehalase (TRE) directly regulate trehalose metabolism and indirectly regulate chitin metabolism in insects. Real-time quantitative PCR (RT-qPCR) and RNA interference (RNAi) were used to detect the expressions and functions of the ApTPS and ApTRE genes. Abnormal phenotypes were found after RNAi of ApTRE in the Acyrthosiphon pisum. The molting deformities were observed in two color morphs, while wing deformities were only observed in the red morphs. The RNAi of ApTPS significantly down-regulated the expression of chitin metabolism-related genes, UDP-N-acetyglucosamine pyrophosphorylase (ApUAP), chitin synthase 2 (Apchs-2), Chitinase 2, 5 (ApCht2, 5), endo-beta-N-acetylglucosaminidase (ApENGase) and chitin deacetylase (ApCDA) genes at 24 h and 48 h; The RNAi of ApTRE significantly down-regulated the expression of ApUAP, ApCht1, 2, 8 and ApCDA at 24 h and 48 h, and up-regulated the expression of glucose-6-phosphate isomerase (ApGPI) and Knickkopf protein (ApKNK) genes at 48 h. The RNAi of ApTRE and ApTPS not only altered the expression of chitin metabolism-related genes but also decreased the content of chitin. These results demonstrated that ApTPS and ApTRE can regulate the chitin metabolism, deepen our understanding of the biological functions, and provide a foundation for better understanding the molecular mechanism of insect metamorphosis.

Nesidiocoris tenuis (Reuter) is an efficient predatory biological control agent used throughout the Mediterranean Basin in tomato crops but regarded as a pest in northern European countries. From the family Miridae, it is an economically important insect yet very little is known in terms of genetic information and no genomic or transcriptomic studies have been published. Here, we use a linked‐read sequencing strategy on a single female N. tenuis. From this, we assembled the 355 Mbp genome and delivered an ab initio, homology‐based and evidence‐based annotation. Along the way, the bacterial “contamination” was removed from the assembly. In addition, bacterial lateral gene transfer (LGT) candidates were detected in the N. tenuis genome. The complete gene set is composed of 24 688 genes; the associated proteins were compared to other hemipterans (Cimex lectularis, Halyomorpha halys and Acyrthosiphon pisum). We visualized the genome using various cytogenetic techniques, such as karyotyping, CGH and GISH, indicating a karyotype of 2n = 32. Additional analyses include the localization of 18S rDNA and unique satellite probes as well as pooled sequencing to assess nucleotide diversity and neutrality of the commercial population. This is one of the first mirid genomes to be released and the first of a mirid biological control agent., The annotated genome of predatory mirid Nesidiocoris tenuis presented here was generated from a single female insect using a linked‐read sequencing and assembly strategy.Potential contaminants and/or endosymbionts were identified during assembly decontamination along with putative lateral gene transfer candidates.Additional cytogenetic analysis includes karyotyping and investigations into the ancestral insect telomeric motif, sex chromosomes, rDNA and a very abundant DNA repeat scattered throughout the genome.

Aphids are a diverse family of crop pests. Aphids formed a complex relationship with intracellular bacteria. Depending on the region of study, the species composition of both aphids and their facultative endosymbionts varies. The aim of the work was to determine the occurrence and genetic diversity of Wolbachia, Spiroplasma and Rickettsia symbionts in aphids collected in 2018-2019 in Moscow. For these purposes, 578 aphids from 32 collection sites were tested by PCR using specific primers. At least 21 species of aphids from 14 genera and four families were identified by barcoding method, of which 11 species were infected with endosymbionts. Rickettsia was found in six species, Wolbachia in two species, Spiroplasma in one species. The presence of Rickettsia in Impatientinum asiaticum, Myzus cerasi, Hyalopterus pruni, Eucallipterus tiliae, Chaitophorus tremulae and Wolbachia in Aphis pomi and C. tremulae has been described for the first time. A double infection with Rickettsia and Spiroplasma was detected in a half of pea aphid (Acyrthosiphon pisum) individuals. For the first time was found that six species of aphids are infected with Rickettsia that are genetically different from previously known. It was first discovered that A. pomi is infected with two Wolbachia strains, one of which belongs to supergroup B and is genetically close to Wolbachia from C. tremulae. The second Wolbachia strain from A. pomi belongs to the supergroup M, recently described in aphid species. Spiroplasma, which we observed in A. pisum, is genetically close to male killing Spiroplasma from aphids, ladybirds and moths. Both maternal inheritance and horizontal transmission are the pathways for the distribution of facultative endosymbiotic bacteria in aphids.Тли – разнообразное семейство вредителей сельскохозяйственных культур. Тли сформировали сложную взаимосвязь с внутриклеточными бактериями, известными как эндосимбионты, которые оказывают как положительное, так и отрицательное влияние на хозяина, что может иметь практическое значение. В раз- ных регионах мира состав факультативных симбионтов в популяциях тлей варьирует. Задачей работы было установить распространение и генетическое разнообразие симбионтов Wolbachia, Spiroplasma и Rickettsia в тлях, собранных в 2018–2019 гг. в Москве и Подмосковье. Для этого 578 тлей из 32 мест сбора тестировали методом ПЦР, используя специфические праймеры для мтДНК тлей, Wolbachia, Spiroplasma и Rickettsia. Мето- дом молекулярно-генетического анализа определено не менее 21 вида тлей из 14 родов и четырех семейств. Одиннадцать видов оказались инфицированы эндосимбионтами, а именно: у шести видов обнаружены Rickettsia, у двух видов – Wolbachia, у одного – Spiroplasma. Впервые выявлено заражение бактерией Rickettsia у Impatientinum asiaticum, Myzus cerasi, Hyalopterus pruni, Eucallipterus tiliae, Chaitophorus tremulae и бактерией Wolbachia у Aphis pomi и C. tremulae. У половины особей гороховой тли Acyrthosiphon pisum установлено двой- ное заражение Rickettsia и Spiroplasma. Впервые выявлены риккетсии у шести видов тлей, которые генетиче- ски отличаются от известных ранее. Впервые обнаружено заражение яблонной тли A. pomi двумя штаммами Wolbachia, причем один из штаммов относится к супергруппе В и генетически близок с Wolbachia из осино- вой тли C. tremulae, а второй штамм относится к супергруппе М, недавно описанной у видов тлей. Spiroplasma, найденная нами у A. pisum, генетически близка Spiroplasma, вызывающей андроцид у тлей, божьих коровок и молей, и кластеризуется с S. ixodetis. Разнообразие ДНК симбионтов убедительно свидетельствует о том, что как материнское наследование, так и горизонтальный перенос являются путями распространения факульта- тивных бактерий у тлей.

Large-scale chromosome rearrangements are arguably the most dramatic type of mutations, often leading to rapid evolution and speciation. However, chromosome dynamics have only been studied at the sequence level in a small number of model systems. In insects, Diptera (flies and mosquitoes) and Lepidoptera (butterflies and moths) have high levels of chromosome conservation. Whether this truly reflects the diversity of insect genome evolution is questionable given that many species exhibit rapid karyotype evolution. Here, we investigate chromosome evolution in aphids – an important group of hemipteran plant pests – using newly generated chromosome-scale genome assemblies of the green peach aphid (Myzus persicae) and the pea aphid (Acyrthosiphon pisum), and a previously published chromosome-scale assembly of the corn-leaf aphid (Rhopalosiphum maidis). We find that aphid autosomes have undergone dramatic reorganisation over the last 30 million years, to the extent that chromosome homology cannot be determined between aphids from the tribes Macrosiphini (M. persicae and A. pisum) and Aphidini (R. maidis). In contrast, gene content of the aphid sex (X) chromosome remained unchanged despite rapid sequence evolution, low gene expression and high transposable element load. To test whether rapid evolution of genome structure is a hallmark of Hemiptera, we compared our aphid assemblies to chromosome-level assemblies of two blood-feeding Hemiptera (Rhodnius prolixus and Triatoma rubrofasciata). Despite being more diverged, the blood-feeding hemipterans have conserved synteny and we detect only two chromosome fusion or fission events. The exceptional rate of structural evolution of aphid autosomes renders them an important emerging model system for studying the role of large-scale genome rearrangements in evolution.

Microbial associates are widespread in insects, some conferring a protection to their hosts against natural enemies like parasitoids. These protective symbionts may affect the infection success of the parasitoid by modifying behavioral defenses of their hosts, the development success of the parasitoid by conferring a resistance against it or by altering life-history traits of the emerging parasitoids. Here, we assessed the effects of different protective bacterial symbionts on the entire sequence of the host-parasitoid interaction (i.e., from parasitoid attack to offspring emergence) between the pea aphid, Acyrthosiphon pisum, and its main parasitoid, Aphidius ervi and their impacts on the life-history traits of the emerging parasitoids. To test whether symbiont-mediated phenotypes were general or specific to particular aphid–symbiont associations, we considered several aphid lineages, each harboring a different strain of either Hamiltonella defensa or Regiella insecticola, two protective symbionts commonly found in aphids. We found that symbiont species and strains had a weak effect on the ability of aphids to defend themselves against the parasitic wasps during the attack and a strong effect on aphid resistance against parasitoid development. While parasitism resistance was mainly determined by symbionts, their effects on host defensive behaviors varied largely from one aphid–symbiont association to another. Also, the symbiotic status of the aphid individuals had no impact on the attack rate of the parasitic wasps, the parasitoid emergence rate from parasitized aphids nor the life-history traits of the emerging parasitoids. Overall, no correlations between symbiont effects on the different stages of the host–parasitoid interaction was observed, suggesting no trade-offs or positive associations between symbiont-mediated phenotypes. Our study highlights the need to consider various sequences of the host-parasitoid interaction to better assess the outcomes of protective symbioses and understand the ecological and evolutionary dynamics of insect–symbiont associations.

This paper presents analysis of the effects of the alfalfa crop age, sowing method, phenological development phases, and green mass mowing on the composition and density of insects and spiders on the aboveground organs of alfalfa grown in crop rotation without irrigation, pest control measures, and fertilizers. The main inhabitants of alfalfa crops comprised over 50 species, with prevalence of the chewing herbivores: alfalfa weevil Hypera postica, alfalfa seed chalcid Bruchophagus roddi, nodule weevils Sitona lineatus and S. inops; the sucking herbivores: flower thrips Odontothrips sp., gorse shield bug Piezodorus lituratus, lucerne bug Adelphocoris lineolatus, plant bug Lygus pratensis, cowpea aphid Aphis craccivora, and pea aphid Acyrthosiphon pisum; the predators: spiders, predatory bug Orius niger, predatory thrips Aeolothrips sp., and variegated ladybug Hippodamia variegata. The seasonal dynamics of the composition and density of dominant groups was more pronounced in the first-year crops than in 2–4 year old ones. In field crop rotations without the use of insecticides, plots with periodic green mass mowing during the budding and flowering phases of alfalfa seed crops should alternate with unmown plots, so that plots with blossoming plants should be present in the field throughout the summer. This measure would promote the development and maintenance of highly diverse insect complexes, attract predators and pollinators, provide additional feeding for parasitic Hymeno­ptera, and reduce the number and harmfulness of herbivores as the result of greater trophic competition between them as well as of the impact of entomophages.

The maintenance of sexuality is a puzzling phenomenon in evolutionary biology. Many universal hypotheses have been proposed to explain the prevalence of sex despite its costs, but it has been hypothesized that sex could be also retained by lineage‐specific mechanisms that would confer some short‐term advantage. Aphids are good models to study the maintenance of sex because they exhibit coexistence of both sexual and asexual populations within the same species and because they invade a large variety of ecosystems. Sex in aphids is thought to be maintained because only sexually produced eggs can persist in cold climates, but whether sex is obligate or facultative depending on climatic conditions remains to be elucidated. In this study, we have inferred the reproductive mode of introduced populations of the pea aphid Acyrthosiphon pisum in Chile along a climatic gradient using phenotypic assays and genetic‐based criteria to test the ecological short‐term advantage of sex in cold environments. Our results showed a latitudinal trend in the reproductive mode of Chilean pea aphid population from obligate parthenogenesis in the north to an intermediate life cycle producing both parthenogenetic and sexual progeny in the southernmost locality, where harsh winters are usual. These findings are congruent with the hypothesis of the ecological short‐term advantage of sex in aphids., Invasive population of Acyrthosiphon pisum in Chile is structured along a latitudinal transect. The southernmost one, where winters are harsh, is the only one that exhibits genetic and phenotypic features of sexual reproduction.

Faba bean is an important position between leguminous food crops in Egypt. A piercing-sucking pest associated with broad bean plants causes serious damage, directly by sucking plant juices or indirectly as the aphid insects are vectors to plant diseases. A field experiment was conducted at Wadi Elmalak, Ismailia Governorate, Egypt in winter growing seasons 2018/2019 and 2019/2020. To evaluate the population fluctuation of some piercing-sucking pests and the common natural enemies and relation to weather factors on broad bean plants. Results indicated that the important piercing-sucking pests, pea aphid, Acyrthosiphon pisum (Harris) were the most dominant, followed by Aphis craccivora Koch, while green peach aphid, Myzus persicae (Sulzer), Thrips tabaci Lind. and Jassid, Empoasca discipiens Poali were found in low densities throughout the two seasons. The eleven spotted lady beetle, Coccinella undecimpunctata L., green lacewing, Chrysoperla carnea (Stephens), and seven-spotted lady beetle, Coccinella septempunctata L. are the most important predators which associated with the sucking insect pests on faba bean. The population fluctuation of piercing-sucking pests and associated predators were higher in the first season than in the second season. The pea aphid, A. pisum started with attacks broad bean in the second week of November and reached it has two peaks early January and in mid-May in the two successive years. The relationship between both of the population of piercing-sucking pests and associated predators and weather factors were studied. The loss in seed yield in the two successive seasons was evaluated all together being (330.0 kg. /fed). The avoidable losses in seeds yield were (19.18% per fed.) during two seasons

To provide fundamental information for the biological control of aphids in vegetable greenhouses, we compared the host ranges of four aphid parasitoid species, Aphidius colemani Viereck, Aphidius gifuensis Ashmead, Diaeretiella rapae (M’Intosh), and Ephedrus nacheri Quilis (Hymenoptera: Braconidae: Aphidiinae). The acceptability as host of 11 vegetable-pest aphids, Acyrthosiphon pisum (Harris), Aphis craccivora Koch, Aphis gossypii Glover, Aulacorthum solani (Kaltenbach), Brevicoryne brassicae (Linnaeus), Chaetosiphon fragaefolii (Cockerell), Lipaphis erysimi (Kaltenbach), Macrosiphoniella sanborni (Gillette), Macrosiphum euphorbiae (Thomas), Myzus persicae (Sulzer), and Uroleucon formosanum (Takahashi), in addition to five aphid species, Melanaphis sacchari (Zehntner), Rhopalosiphum maidis (Fitch), Rhopalosiphum padi (Linnaeus), Schizaphis graminum (Rondani), and Sitobion akebiae (Shinji) (Hemiptera: Aphididae) that serve as alternative hosts in banker–plant systems for the four aphid parasitoid species, were investigated. A newly emerged pair of parasitoid adults were provided to 100 aphids of each species on caged host plants in a 25 °C chamber for 24 h. The numbers of mummified aphids and emerged adults were counted in 10 trials for each aphid species. Aphidius colemani, A. gifuensis, D. rapae and E. nacheri parasitized four, two, three, and eight pest species, respectively, and four, three, three, and five alternative host species, respectively. Ephedrus nacheri had the broadest host range among the four species, and all the four species parasitized M. persicae, R. maidis, and S. graminum. This information will be useful for selecting candidate of biological control agents for aphids and for constructing banker–plant systems.

Wing dimorphism is a phenomenon of phenotypic plasticity in aphid dispersal. However, the signal transduction for perceiving environmental cues (e.g., crowding) and the regulation mechanism remain elusive. Here, we found that aci-miR-9b was the only down-regulated microRNA (miRNA) in both crowding-induced wing dimorphism and during wing development in the brown citrus aphid Aphis citricidus . We determined a targeted regulatory relationship between aci-miR-9b and an ABC transporter ( AcABCG4 ). Inhibition of aci-miR-9b increased the proportion of winged offspring under normal conditions. Overexpression of aci-miR-9b resulted in decline of the proportion of winged offspring under crowding conditions. In addition, overexpression of aci-miR-9b also resulted in malformed wings during wing development. This role of aci-miR-9b mediating wing dimorphism and development was also confirmed in the pea aphid Acyrthosiphon pisum . The downstream action of aci-miR-9b- AcABCG4 was based on the interaction with the insulin and insulin-like signaling pathway. A model for aphid wing dimorphism and development was demonstrated as the following: maternal aphids experience crowding, which results in the decrease of aci-miR-9b. This is followed by the increase of ABCG4 , which then activates the insulin and insulin-like signaling pathway, thereby causing a high proportion of winged offspring. Later, the same cascade, “ miR-9b-ABCG4-insulin signaling ,” is again involved in wing development. Taken together, our results reveal that a signal transduction cascade mediates both wing dimorphism and development in aphids via miRNA. These findings would be useful in developing potential strategies for blocking the aphid dispersal and reducing viral transmission.

1. Generalist predators are important contributors to reliable conservation biological control. Indirect interactions between prey species that share a common generalist predator can influence both community dynamics and the efficacy of biological control.2. Laboratory cage experiments investigated the impact of the combined consumptive and non-consumptive effects of predation by adult Hippodamia convergens as a shared predator on the population growth and relative abundance of Acyrthosiphon pisum and Aphis gossypii as prey species. Predation pressure and prey density were varied.3. At low predation pressure the indirect interaction between aphid species was asymmetrical with a proportionally greater negative impact of predation on A. gossypii than on A. pisum. At intermediate predation pressure, the indirect interaction became symmetrical. At high predation pressure and higher levels of prey density, it was asymmetrical with greater negative impact on A. pisum, often driven to local extinction while A. gossypii populations persisted.4. A linear mixed-effects model including early population growth of both aphid species and predation pressure explained 96% and 92% of the variation in the population growth of A. pisum and A. gossypii, respectively, over an 8-day period. The overall effect of shared predation on the indirect interaction between the two aphid species is best described as apparent commensalism, where A. pisum benefited from early population growth of A. gossypii, while A. gossypii was unaffected by early population growth of A. pisum. Considering these indirect interactions is important for conservation biological control efforts to be successful.

The biocontrol values of natural enemies are strongly correlated to their ability to regulate the density of their host/prey. For parasitoids, apart from parasitism and host feeding, unsuccessful host stinging (i.e., stings that were aborted, abandoned, or discontinued without oviposition or host feeding) can also negatively affect their hosts and host populations. Although several studies have reported unsuccessful host stinging and its impacts on hosts, the effects of this type of attack on host life table parameters are still unclear. In the present study, we used the parasitoid Aphelinus asychis Walker (Hymenoptera: Aphelinidae) and its host Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) to investigate the influence of unsuccessful host stinging on host populations under laboratory conditions at. Biological parameters of A. pisum were analyzed using an age stage, two-sex life table. The results of this study showed that unsuccessful host stinging was prevalent under laboratory conditions, and the frequency of this type of attack on third- and fourth-instar hosts was higher than the frequencies of parasitism and host feeding. Unsuccessful host stinging adversely impacted aphid populations, by decreasing aphid survival and reproduction, and impacts were greatest in hosts attacked at the first and fourth instars. These results indicate that unsuccessful host stinging enhances the biological control impact of A. asychis attacking A. pisum, and its effect on host populations should also be considered when selecting and mass rearing of parasitoids for biological control.

Invasive species are a major driver of global biodiversity loss. However, we often lack a mechanistic understanding why some non-native species become invasive. Missing coevolutionary history between invasive predators and native prey can lead to a lack of predator avoidance by native prey and consequently higher consumption rates by invasive predators. We compared predation rates of four native European lady beetle species and the invasive lady beetle species Harmonia axyridis Pallas (Coleoptera: Coccinellidae), using the native pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) for prey. Here, we also quantified initial dropping of A. pisum from host plants. In an additional plant-choice experiment, we tested for aphid avoidance of plants bearing lady beetle cues. Differences in predation were determined by predator body size. Initial dropping of aphids did not differ between the invasive lady beetle species H. axyridis and the remaining native lady beetle species. However, A. pisum showed no avoidance behavior towards H. axyridis cues, but to cues of the most voracious native species. Thus, relatively large body size and missing chemical cue avoidance by aphids can benefit the invasive H. axyridis.

In the present study, the natural enemies of aphids (Hemiptera: Aphididae) and their host plants including herbaceous plants, shrubs and trees were analysed to reveal their biodiversity and disclose tritrophic associations in different habitats of the South Marmara region of northwest Turkey. As a result of field surveys, 58 natural enemy species associated with 43 aphids on 58 different host plants were identified in the region between March of 2017 and November of 2018. In 173 tritrophic natural enemy-aphid-host plant interactions including association records new for Europe and Turkey, there were 21 representatives of the family Coccinellidae (Coleoptera), 14 of the family Syrphidae (Diptera) and 15 of the subfamily Aphidiinae (Hymenoptera), as well as eight other generalist natural enemies. In these interactions, a total of 37 aphid-natural enemy associations–including 19 associations of Acyrthosiphon pisum (Harris) with natural enemies, 16 associations of Therioaphis trifolii (Monell) with natural enemies and two associations of Aphis craccivora Koch with natural enemies–were detected on Medicago sativa L. during the sampling period. Similarly, 12 associations of Myzus cerasi (Fabricius) with natural enemies were revealed on Prunus avium (L.), along with five associations of Brevicoryne brassicae (Linnaeus) with natural enemies (including mostly parasitoid individuals) on Brassica oleracea L. Also in the study, reduviids of the species Zelus renardii (Kolenati) are reported for the first time as new potential aphid biocontrol agents in Turkey. The results of field surveys show that the natural enemies of aphids have high biodiversity, which should be considered in the management of biological pest control.

Symbiotic viruses exist in many insects; however, their functions in host insects are not well understood. In this study, we explored the role of acyrthosiphon pisum virus (APV) in the interaction of its host aphid Acyrthosiphon pisum with plants. APV is primarily located in aphid salivary glands and gut and propagated in the insect. APV is horizontally transmitted to host plants during aphid feeding, but the virus does not replicate in the host plant. When the pea host race of aphids colonized two low-fitness plants, Medicago truncatula and Vicia villosa, the virus titers in both the aphids and plants significantly increased. Furthermore, APV infection strongly promoted the survival rate of the pea host race on V. villosa. Transcriptomic analysis showed that only 0.85% of aphid genes responded to APV infection when aphids fed on V. villosa, with a fold change in transcript levels of no more than fourfold. The improved survival due to APV infection was apparently related to the inhibitory effect of the virus on levels of phytohormone jasmonic acid (JA) and JA-isoleucine. Our data suggest a benefit of the symbiotic virus to its aphid host and demonstrate a novel case of symbiotic virus-mediated three-species interaction.

The pea aphid complex [Acyrthosiphon pisum (Harris 1776)] (Hemiptera, Aphididae), listed among the 14 aphid species of the greatest economic importance, is a globally distributed key pest of pulse crops. Since the pea aphid is a well-studied model organism, an enormous amount of literature has been published about this species. However, comprehensive morphological analyses using scanning electron microscopy (SEM), focused on all body parts of adult viviparous and sexual generations of A. pisum, are lacking. Thus, we present the first detailed morphological description of all body parts of adult wingless and winged viviparous females (parthenogenetic generation) as well as oviparous females, winged and wingless males (sexual generation), representing the most common A. pisum pea biotype. In particular, the sensilla of the body surface, antennae, mouthparts, legs and male genitalia are mapped by the high-resolution scanning electron microscope. The morphological diversity of sensilla is discussed in the context of their function and the biology of aphids.

Background RNA interference (RNAi) has potential as a pest insect control technique. One possible RNAi target is the cuticle protein, which is important in insect molting and development. As an example, here we evaluate the possibility of designing double-stranded RNA (RNA) that is effective for silencing the cuticle protein 19 gene (CP19) in aphids but is harmless to non-target predator insects. Results The sequences of CP19s were similar (86.6-94.4%) among the tested aphid species (Aphis citricidus, Acyrthosiphon pisum, and Myzus persicae) but different in the predator Propylaea japonica. Ingestion of species-specific dsRNAs of CP19 by the three aphids produced 39.3-64.2% gene silencing and 45.8-55.8% mortality. Ingestion of non-species-specific dsRNA (dsAcCP19) by Ac. pisum and M. persicae gave gene silencing levels ranging from 40.4% to 50.3% and 43.3-50.8% mortality. The dsApCP19 did not affect PjCP19 expression or developmental duration in P. japonica. Conclusion The results demonstrate that CP19 is a promising RNAi target for aphid control via one dsRNA design. The targeting of genes that are conserved in insect pests but not present in beneficial insects is a useful RNAi-based pest control strategy. © 2019 Society of Chemical Industry.

Soybean dwarf virus (SbDV, genus Luteovirus) is a single-stranded positive-sense RNA virus able to infect several legume species. SbDV was first reported in Japan where it was associated with significant yield losses in soybean (Tamada, 1969). Since then the virus has been detected worldwide. In Europe, the virus has only been reported from Germany (Abraham et al. 2007; Gaafar et al. 2020). In July 2018, several white clover plants (Trifolium repens L.) with leaf discoloration were observed in different locations in Oulu region in northern Finland. Individual plants were collected and analysed for the presence of viruses using small-RNA (sRNA) sequencing (Kreuze et. al. 2009) and reverse transcription-PCR (RT-PCR). Total RNA was extracted using EZNA micro RNA kit (Omega Bio-Tek, GA, USA). For sRNA analysis, sequencing libraries were constructed using the TruSeq small RNA library prep kit (Illumina, CA, USA) and sequenced on Illumina MiSeq platform. On average, 1.3 million single-end reads were obtained per sample, of which 27% were 18-25 nt long and used for the subsequent analysis. Contig assembly and virus identification with VirusDetect software (Zheng et al. 2017) detected SbDV in five out of six white clover samples analysed. Depending on the sample, 26-39 contigs (with lengths up to 301-469 nt) aligned to complete genome of a SbDV isolate previously described from white clover in USA (accession no. JN674402). The cumulative alignment coverage ranged from 35.5 % to 65.3 % with nucleotide identities between 94.4 % and 97.3 %. Additionally, two samples seemed to contain an unidentified closterovirus and one contained White clover cryptic virus 2. No additional viruses were detected from two of the samples.To confirm the presence of SbDV, the samples were tested by RT-PCR using primers MDF, MYF and MUR in multiplex (Schneider et al. 2011) together with SuperScript III One-Step RT-PCR System with the Platinum Taq DNA polymerase kit (Thermo Fisher Scientific, USA), essentially as instructed by the manufacturer. RT-PCR product of approximately 400 bp was produced from each of the five samples previously tested SbDV positive by sRNA analysis. No products were produced from the sample that was SbDV negative in sRNA analysis. Direct sequencing of two of the PCR products produced 347 and 361 bp sequences (GenBank: MZ355392 and MW929169) that were 95.7 % and 95.2 % identical, respectively, to a SbDV isolate (accession no. AB038148) that causes yellowing on soybean and is transmitted by Acyrthosiphon pisum (Terauchi et al. 2003). To our knowledge this is the first report of SbDV in Finland. SbDV is transmitted only by aphids (neither mechanical nor seed transmission occurs). In siRNA analysis all the isolates from Finland formed contigs that aligned almost perfectly (100 % coverage with ≥ 99 % nucleotide identity) to the coat protein (accession no. EF466131) of an SbDV isolate transmittable from white clover to faba bean by A. pisum (Abraham et al. 2007), an aphid common in Finland. Although significant yield losses by SbDV have only been reported on soybean (Tamada, 1969), the virus also causes symptoms in other legume crops, such as growth reduction on pea (Tian et al. 2017) and faba bean (Abraham et al. 2007), both of which are cultivated in Finland. References: Abraham et al. 2007. Plant Dis. 91: 1059. Gaafar et al. 2020. Front microbiol. 11: 583242. Kreuze et al. 2009. Virology 388:1. Schneider et al. 2011. Virology 412: 46. Tamada. 1969. Ann Phytopathol Soc Jpn. 35: 282. Terauchi et al. 2003. Phytopathology 93: 1560. Tian et al. 2017. Viruses 9: 155. Zheng et al. 2017. Virology 500: 130.

Simple Summary Programmed cell death is a universal mechanism in animal development and tissue maintenance, which facilitates the elimination of surplus or poorly functioning cells. Many conserved regulators of programmed cell death have been identified in model organisms including the fruit fly Drosophila melanogaster. In the latter, the four members of the RHG gene family function as critical inducers of programmed cell death. Despite this important role, RHG genes had thus far only been found in a surprisingly small number of insect groups, i.e., other flies and butterflies. This study reports the much deeper conservation of RHG genes in winged insects, ranging from cockroaches to beetles. In addition to opening new opportunities to study programmed cell death in a wide range of insects, the bioinformatic search strategy developed for this work will be of general use for studying gene families with challenging sequence evolution dynamics. Abstract Together with sickle (skl), the Drosophila paralogs reaper (rpr), head involution defective (hid), and grim (RHG) control a critical switch in the induction of programmed cell death. RHG homologs have been identified in other dipteran and lepidopteran species but not beyond. Revisiting this issue with a “taxon hopping” BLAST search strategy in current genome and transcriptome resources, I detected high confidence RHG homologs in Coleoptera, Hymenoptera, Hemiptera, and Dictyoptera. Analyses of gene structure and protein sequence conservation revealed aconserved splicing pattern and highly conserved amino acid residues at both the N- and C-terminal ends that identify hid as the most ancestrally organized RHG gene family member in Drosophila. hid-like RHG homologs were also detected in mosquitoes, redefining their michelob_x (mx) genes as an expansion of derived RHG homologs. Only singleton homologs were detected in the large majority of other insect clades. Lepidopteran RHG homologs, however, stand out by producing an evolutionarily-derived splice isoform, identified in previous work, in addition to the newly detected hid-like isoform. Exceptional sequence diversification of select RHG homologs at the family- and genus-level explain their previous elusiveness in important insect genome model species like the red flour beetle Tribolium castaneum and the pea aphid Acyrthosiphon pisum. Combined, these findings expand the minimal age of the RHG gene family by about 100 million years and open new avenues for molecular cell death studies in insects.

The pea aphid Acyrthosiphon pisum has a worldwide distribution and causes serious losses for agricultural production. Drunken horse grass Achnatherum inebrians is a widely distributed perennial poisonous grass on the grasslands of Northern and Northwestern China. The present study focused on contact toxicity activity of aqueous extracts of endophyte-infected (E+) and endophyte-free (E−) A. inebrians in different growth periods of pea aphids, and the growth and development of two color morphs of F1 generation nymphs. Both of the color morphs had development durations in E+ treatments that tended to be longer at 1st, 2nd, 3rd, and 4th instars than E− and control (CK). The E+ treated aphids also showed decreased weights at maturity with over all lower mean relative growth rates (MRGR). Aphid survival of E+ treated aphids was lower than that of E− and CK at all growth periods. Seeding stage E+ extracts showed a greater propensity for negatively affecting aphids than did E+ extract at maturity and the yellowing stage. These results show that extracts from endophyte-containing plants may contain compounds that may be used to control insects.

NADPH-cytochrome P450 reductase (CPR) is involved in the metabolism of endogenous and exogenous substances, and detoxification of insecticides. RNA interference (RNAi) of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides. However, the CPR of Acyrthosiphon pisum has not been characterized, and its function is still not understood. In this study, we investigated the biochemical functions of A. pisum CPR (ApCPR). ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage, and in the gut, head, and abdominal cuticle. After optimizing the dose and silencing duration of RNAi for downregulating ApCPR, we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents, and of cuticular waxy coatings. Deficiency in cuticular hydrocarbons decreased the survival rate of A. pisum under desiccation stress, and increased its susceptibility to contact insecticides. Moreover, desiccation stress induced a significant increase in ApCPR mRNA levels. We further confirmed that ApCPR participates in cuticular hydrocarbon production. These results indicate that ApCPR modulates cuticular hydrocarbon production, desiccation tolerance, and insecticide susceptibility in A. pisum, and presents a novel target for pest control. This article is protected by copyright. All rights reserved.

Aphids are paradigmatic photoperiodic animals often used to study the role of the circadian clock in the seasonal response. Previously, we described some elements of the circadian clock core (genes period and timeless) and output (melatonin, AANATs and PTTH) that could have a role in the regulation of the aphid seasonal response. More recently we identified two opsins (C-ops and SWO4) as candidate input photoperiodic receptors. In the present report, we focus on the study of cryptochromes (cry) as photoreceptors of the circadian clock and discuss their involvement in the seasonal response. We analyze the expression of cry1 and cry2 genes in a circadian and seasonal context, and map their expression sites in the brain. We observe a robust rhythmic expression of cry2 peaking at dusk in phase with core clock genes period and timeless, while cry1 shows a weaker rhythm. Changes in cry1 and cry2 expression correlate with activation of the seasonal response, suggesting a possible link. Finally, we map the expression of cry1 and cry2 genes to clock neurons in the pars lateralis, a region essential for the photoperiodic response. Our results support a role for cryptochromes as elements of the aphid circadian clock and suggest a role in photoreception for cry1 and in clock repression for cry2. This article is protected by copyright. All rights reserved.

Aphids are prominent phloem-feeding insect pests. Myzus persicae and Acyrthosiphon pisum are generalist and specialist species, respectively. In this study, these two aphid species were used to investigate host acceptance and plant resistance to aphid feeding. M.persicae survived and reproduced on rape (Brassica rapa), but few individuals (9%) survived on faba bean (Vicia faba). A.pisum survived and reproduced on faba bean, but no A.pisum survived on rape. The probing and feeding behavior of M. persicae and A. pisum on rape and faba bean was examined using an electrical penetration graph (EPG) technique. The results demonstrated the feeding preferences of these two aphid species. The EPG results suggest that the resistance of faba bean to M. persicae and that of rape to A. pisum are likely residing in the phloem and mesophyll tissues, respectively. Due to the distinct probing and feeding behaviors, specialist and generalist aphids would have different impacts on the epidemiology of plant viral diseases. The findings can be applied to the management of viral diseases transmitted by specialist or generalist aphids in crop production.

Simple Summary Successful foraging behavior of parasitoids depends on specific organic information emitted by host-infested plants. For instance, the emission of volatile compounds increases in infested plants, and these are the first indicator of host presence. Parasitoids are attracted by these volatiles in a quite specific way. By combining behavioral and chemical studies, we showed bottom-up effects in a broad bean Vicia faba (Fabaceae)–pea aphid Acyrthosiphon pisum (Homoptera: Aphididae)–parasitoid Aphidius ervi (Hymenoptera: Braconidae) model system. We found that behavioral selection of parasitoid females toward plants with a high density of aphid infestation was reduced, and this can be linked to reduced emission of volatile compounds. In practice, if parasitoids are less attracted to plants with high-density aphid infestations, there may be potential negative impacts on biological control. Therefore, the common recommendation in biological control is to release parasitoids early in the season when aphid density on crop plants is still low. Abstract Herbivore-induced plant volatiles constitute the first indicators of insect host presence, and these can affect the foraging behavior of their natural enemies. The density of insect hosts may affect the nature and concentration of these plant-induced volatiles. We tested the impact of infestation density (low, intermediate, and high) of the pea aphid, Acyrthosiphon pisum (Homoptera: Aphididae), feeding on the broad bean Vicia faba, on the attractiveness of the parasitoid Aphidius ervi (Hymenoptera: Braconidae), using a Y-tube olfactometer (infested vs. non-infested plants). The emitted volatile compounds from both infested and non-infested plants were collected and identified. In addition, two series of experiments were carried out to test the impact of the presence of a conspecific female parasitoid within the aphid/plant complex on the attractiveness to other females. Parasitoids were significantly more attracted to the plants with low and intermediate aphid infestation levels. The volatile blend composition of the infested plants changed in relation to aphid density and may explain the low attraction of parasitoids toward high aphid density. The presence of conspecific females on the aphid patch had no apparent impact on the behavioral choices of other parasitoid females. Our study adds a new aspect to understanding plant–aphid–parasitoid interactions, including the possibility that aphids may manipulate chemical cues of host plants affecting the orientation of parasitoids.

A growing body of evidence points to a role for horizontal gene transfer (HGT) in the evolution of animal novelties. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (CdtB) from the Acyrthosiphon pisum Secondary Endosymbiont (APSE) bacteriophage to drosophilid and aphid genomes. Here, we report that cdtB is also found in the nuclear genome of the gall-forming ‘swede midge’ Contarinia nasturtii (Diptera: Cecidomyiidae). We subsequently searched genome sequences of all available cecidomyiid species for evidence of microbe-to-insect HGT events. We found evidence of pervasive transfer of APSE-like toxin genes to cecidomyiid nuclear genomes. Many of the toxins encoded by these horizontally transferred genes target eukaryotic cells, rather than prokaryotes. In insects, catalytic residues important for toxin function are conserved. Phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor of the toxin gene to cecidomyiid genomes, suggesting a broader pool of microbial donor lineages. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for HGT, which showed, that prokaryotic-to-insect HGT was more likely to occur between taxa in common environments. Our study highlights the horizontal transfer of genes encoding a new functional class of proteins in insects, toxins that target eukaryotic cells, which is potentially important in mediating interactions with eukaryotic pathogens and parasites.Significance StatementThe diversity of genes encoded by phages infecting bacterial symbionts of eukaryotes represents an enormous, relatively unexplored pool of new eukaryotic genes through horizontal gene transfer (HGT). In this study, we discovered pervasive HGT of toxin genes encoded by Acyrthosiphon pisum secondary endosymbiont (APSE) bacteriophages and other microbes to the nuclear genomes of gall midges (Diptera: Cecidomyiidae). We found five toxin genes were transferred horizontally from phage, bacteria, or fungi into genomes of several cecidomyiid species. These genes were aip56, cdtB, lysozyme, rhs, and sltxB. Most of the toxins encoded by these genes antagonize eukaryotic cells, and we posit that they may play a protective role in the insect immune system.

Simple Summary Flavonoids are plant phenolic compounds whose biological activities include participation in plant responses to various stresses of biological and environmental origins, including protection against insect herbivore attack. We were interested in whether the specific flavonoids detected in soybean leaves have the potential to discourage the pea aphid from infesting other leguminous plants, peas in particular. We immersed the pea leaves in ethanolic solutions of the flavonoids apigenin, daidzein, genistein and kaempferol, offered them to the pea aphids and observed their behavior when they probed plant tissues with their piercing–sucking mouthparts. We discovered that aphids readily probed the pea leaves whether they were treated with flavonoids or not. However, later on, the behavior of the aphids changed depending on the flavonoid applied. Apigenin, daidzein and kaempferol caused a decrease in the intensity of plant sap ingestion. In addition, daidzein and kaempferol made the finding of sap-transporting vessels more difficult for aphids. In contrast, genistein did not affect the pea aphids’ feeding activity. Our findings provide the plant breeders and plant protection services with information on what direction their efforts should take to protect leguminous plants against aphids in a sustainable and environmentally friendly way. Abstract Flavonoids detected in soybean Glycine max (L.) Merr. (Fabaceae) cause various alterations in the metabolism, behavior, and development of insect herbivores. The pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) poses potential threat to soybeans, but the effect of individual flavonoids on its feeding-associated behavior is relatively unknown. We monitored probing behavior (stylet penetration activities) of A. pisum on its preferred host plant, Pisum sativum L. untreated (control) and treated with 0.1% ethanolic solutions of flavonoids apigenin, daidzein, genistein, and kaempferol. We applied the electrical penetration graph (electropenetrography, EPG) technique, which visualizes the movements of aphid stylets within plant tissues. None of the applied flavonoids affected the propensity to probe the plants by A. pisum. However, apigenin enhanced the duration of probes in non-phloem tissues, which caused an increase in the frequency and duration of stylet mechanics derailment and xylem sap ingestion but limited the ingestion of phloem sap. Daidzein caused a delay in reaching phloem vessels and limited sap ingestion. Kaempferol caused a reduction in the frequency and duration of the phloem phase. Genistein did not affect aphid probing behavior. Our findings provide information for selective breeding programs of resistant plant cultivars to A. pisum.

The green peach aphid, Myzus persicae (Sulzer), is a serious agricultural pest with a worldwide distribution. Catalase (CAT), which is encoded by the catalase (Cat) gene, is an extremely important antioxidant enzyme that plays a pivotal role in protecting cells against the toxic effects of hydrogen peroxide. The Cat gene has not been characterized in M. persicae; therefore, this study describes the identification of the Cat (MpCat) gene from M. persicae. MpCat contains an open reading frame of 1515 bp and encodes a MpCAT protein consisting of 504 amino-acid residues. MpCAT possesses features typical of other insect catalases, including 7 conserved amino acids involved in binding heme and 15 involved in binding nicotinamide adenine dinucleotide phosphate. Phylogenetic analysis showed that MpCAT was closely related to orthologs from other aphid species. MpCat consisted of nine exons and eight introns, and the number and insertion sites of introns are consistent with those of Cat genes from Acyrthosiphon pisum (Harris) and Aphis gossypii Glover. The mRNA transcripts of MpCat were detected at all tested developmental stages, with the highest mRNA level in alate adults. The expression of MpCat was significantly upregulated when M. persicae was exposed to low and high temperatures, ultraviolet radiation, Beauveria bassiana, and permethrin. The transcription of MpCat and the activity of catalase were suppressed by RNA interference, and knockdown of MpCat significantly reduced the survival rate in M. persicae under heat stress. The results provide valuable information for further study on the physiological functions of MpCat.