Nanoencapsulated deltamethrin combined with indoxacarb: An effective synergistic association against aphids.

Widespread pesticide use for decades has caused environmental damage, biodiversity loss, serious human and animal health problems, and resistance to insecticides. Innovative strategies are needed to reduce treatment doses in pest management and to overcome insecticide resistance. In the present study, combinations of indoxacarb, an oxadiazine insecticide, with sublethal concentrations of deltamethrin encapsulated in lipid nanocapsules, have been tested on the crop pest Acyrthosiphon pisum. In vivo toxicological tests on A. pisum larvae have shown a synergistic effect of nanoencapsulated deltamethrin with a low dose of indoxacarb. Furthermore, the stability of deltamethrin nanoparticles has been demonstrated in vitro under different mimicking environmental conditions. In parallel, the integrity and stability of lipid nanoparticles in the digestive system of aphid larvae over time have been observed by Förster Resonance Energy Transfer (FRET) imaging. Thus, the deltamethrin nanocapsules/indoxacarb synergistic association is promising for the development of future formulations against pest insects to reduce insecticide doses.

Nutritional quality regulates postnatal wing morph in pea aphids.

Aphids can produce winged or wingless offspring in response to environmental changes. Host nutrition is one of the extensively studied environmental factors influencing the plasticity of wing morphs of aphids. In this study, we found that the pea aphid, Acyrthosiphon pisum, produced a low proportion of winged offspring when fed on plants, but a significantly higher proportion on the artificial diet. Interestingly, when newly born nymphs were transferred back to the artificial diet after feeding on plants for six hours or longer, most nymphs became wingless. These results suggest that the wing morph state of pea aphids can change postnatally, potentially determined by the nutritional quality of their food. Furthermore, aphids feeding on the artificial diet exhibited higher levels of glucose and stronger insulin signaling activity compared with aphids on plants. Conversely, the amino acid levels were lower, and TOR signaling was weaker in aphids fed on the artificial diet. Insulin and the target of rapamycin (TOR) are the primary nutrient-sensing signaling pathways involved in controlling organism growth and have been implicated in regulating aphid wing morph plasticity. We tested whether these nutrient responsive pathways were involved in postanal wing determination of aphids. However, reducing amino acid content in the diet or inhibiting TOR with rapamycin resulted in a decrease of the winged morph, suggesting that the lower amino acid levels or TOR activity was not responsible for the higher proportion of winged morph on the artificial diet. These results suggest that nutritional quality, particularly sugars like sucrose and glucose, may regulate the postnatal wing morph of the pea aphid, likely via the insulin signaling pathway.

Enhancing protein signal detection in asexual and viviparous pea aphids: A guided protocol for tissue dissection and proteinase K treatment.

Aphids, as hemipteran insects, reproduce via parthenogenesis and viviparity, resulting in rapid and exponential offspring production. To investigate the molecular mechanisms underlying parthenogenetic viviparity in asexual aphids, precise protein detection through immunostaining is essential. Our previous research demonstrated the need for proteinase K (PK) treatment to improve tissue permeability, enabling antibodies targeting the germ-cell marker Ap-Vas1 to access gastrulating and later-stage embryos. However, optimal PK digestion protocols have not been thoroughly explored. In this study, we propose strategies to optimize PK digestion conditions for early, middle, and late-stage pea aphid embryos, which have varying tissue thicknesses. Additionally, we extend the application of PK treatment to salivary glands, a representative somatic tissue, by optimizing conditions for antibody penetration against the salivary gland marker C002. To enhance spatial precision in signal detection, we provide a detailed protocol for tissue dissection specific to pea aphids, focusing on the preservation of tissue integrity. These comprehensive guidelines, covering tissue dissection and PK titration, are expected to improve the specificity and intensity of protein signals in pea aphids and other aphid species.•Provide aphid-specific dissection methods to obtain intact embryos and salivary glands.•Present strategies for optimizing PK treatment conditions across different tissue types.

Lipopeptides from Bacillus velezensis ZLP-101 and their mode of action against bean aphids Acyrthosiphon pisum Harris.

BACKGROUND: Natural products are important sources for the discovery of new biopesticides to control the worldwide destructive pests Acyrthosiphon pisum Harris. Here, insecticidal substances were discovered and characterized from the secondary metabolites of the bio-control microorganism Bacillus velezensis strain ZLP-101, as informed by whole-genome sequencing and analysis. RESULTS: The genome was annotated, revealing the presence of four potentially novel gene clusters and eight known secondary metabolite synthetic gene clusters. Crude extracts, prepared through ammonium sulfate precipitation, were used to evaluate the effects of strain ZLP-101 on Acyrthosiphon pisum Harris aphid pests via exposure experiments. The half lethal concentration (LC50) of the crude extract from strain ZLP-101 against aphids was 411.535 mg/L. Preliminary exploration of the insecticidal mechanism revealed that the crude extract affected aphids to a greater extent through gastric poisoning than through contact. Further, the extracts affected enzymatic activities, causing holes to form in internal organs along with deformation, such that normal physiological activities could not be maintained, eventually leading to death. Isolation and purification of extracellular secondary metabolites were conducted in combination with mass spectrometry analysis to further identify the insecticidal components of the crude extracts. A total of 15 insecticidal active compounds were identified including iturins, fengycins, surfactins, and spergualins. Further insecticidal experimentation revealed that surfactin, iturin, and fengycin all exhibited certain aphidicidal activities, and the three exerted synergistic lethal effects. CONCLUSIONS: This study improved the available genomic resources for B. velezensis and serves as a foundation for comprehensive studies of the insecticidal mechanism by Bacillus velezensis ZLP-101 in addition to the active components within biological control strains.

Dynamic response of essential amino acid biosynthesis in Buchnera aphidicola to supplement sub-optimal host nutrition.

The endosymbiotic bacterium Buchnera aphidicola allows its host Acyrthosiphon pisum to utilise a nutritionally limited phloem sap diet without significant mortality by providing essential amino acids (EAAs), which it biosynthesises de novo via complex pathways consisting of multiple enzymes. Previous studies have reported how non-essential amino acids (NEAAs) provided by the host are utilised by B. aphidicola, along with how genes within the biosynthetic pathways respond to amino acid deficiency. Although the effect on B. aphidicola gene expression upon the removal of a single EAA and multiple NEAAs from the A. pisum diet has been reported, little is known about the effects of the complete simultaneous removal of multiple EAAs, especially branched-chain amino acids (BCAAs). To investigate this, A. pisum was provided with amino acid deficient diets ilv- (lacking isoleucine, leucine, valine) or thra- (lacking threonine, methionine, lysine). Due to their involvement in the production of several amino acids, the expression of genes ilvC, ilvD (both involved in isoleucine, leucine and valine biosynthesis) and thrA (involved in threonine, methionine and lysine biosynthesis) was analysed and the expression of trpC (involved in tryptophan biosynthesis) was used as a control. Survival was reduced significantly when A. pisum was reared on ilv- or thra- (P < 0.001 and P = 0.000 respectively) compared to optimal artificial diet and was significantly lower on ilv- (P < 0.001) than thra-. This is likely attributed to the EAAs absent from ilv- being required at higher concentrations for aphid growth, than those EAAs absent from thra-. Expression of ilvC and ilvD were upregulated 2.49- and 2.08-fold (respectively) and thrA expression increased 2.35- and 2.12-fold when A. pisum was reared on ilv- and thra- (respectively). The surprisingly large upregulation of thrA when reared on ilv- is likely due to threonine being an intermediate in isoleucine biosynthesis. Expression of trpC was not affected by rearing on either of the two amino acid deficient diets. To our knowledge this study has shown, for the first time, how genes within the biosynthetic pathways of an endosymbiont respond to the simultaneous complete omission of multiple EAAs as well as all three BCAAs (leucine, isoleucine, valine), from the host diet.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to the defense against bacterial infection in the pea aphid.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is activated by infections of bacteria, fungi, viruses and parasites and mediated cellular and humoral immune responses. In the pea aphid Acyrthosiphon pisum little is known about the function of JAK/STAT signaling in its immune system. In this study, we first showed that expression of genes in the JAK/STAT signaling, including the receptors Domeless1/2, Janus kinase (JAK) and transcriptional factor Stat92E, is up-regulated upon bacteria Escherichia coli and Staphylococcus aureus and fungus Beauveria bassiana infections. After knockdown of expression of these genes by means of dsRNA injection, the aphids harbored more bacteria and suffered more death after infected with E. coli and S. aureus, but showed no significant change after B. bassiana infection. Our study suggests the JAK/STAT signaling contributes to the defense against bacterial infection in the pea aphid.

Transgenerational plasticity in aphids reared in a poor-resource environment.

The changing environmental conditions can affect insect biology over multiple generations and phenotypic plasticity is important for coping with these changes. Transgenerational plasticity occurs when the environment in which the parents developed influences the plastic response of the offspring phenotype. In the present study, the plastic effects of resource limitation on important life history traits such as body size, fecundity, survival, and resistance to starvation of the pea aphid Acyrthosiphon pisum were investigated over two generations. This study focused on understanding how resource limitation can determine an adaptive expression of maternal effects and transgenerational plasticity in fitness-related traits. Aphids showed phenotypic plasticity for the life history traits investigated, as they performed better when grown in an optimal environment than in a resource-poor one. Also, aphids had a poorer performance if their mothers were raised in a resource-poor environment. The effects of transgenerational plasticity were observed only in response to resistance to starvation, through increased survival in the offspring of the mother reared in a resource-poor environment, suggesting an evolutionary bet-hedging strategy. The results of this study showed that the effects of adaptive transgenerational plasticity may be partially masked in stressful environments, where developmental problems instead predominate. More information on the transgenerational response to resource limitation across generations can contribute to a better understanding of aphid biology.

Toxicological, biochemical, and in silico investigations of three trehalase inhibitors for new ways to control aphids.

Insect trehalases have been identified as promising new targets for pest control. These key enzymes are involved in trehalose hydrolysis and plays an important role in insect growth and development. In this contribution, plant and microbial compounds, namely validamycin A, amygdalin, and phloridzin, were evaluated for their effect, through trehalase inhibition, on Acyrthosiphon pisum aphid. The latter is part of the Aphididae family, main pests as phytovirus vectors and being very harmful for crops. Validamycin A was confirmed as an excellent trehalase inhibitor with an half maximal inhibitory concentration and inhibitor constant of 2.2 × 10-7 and 5 × 10-8 M, respectively, with a mortality rate of ~80% on a A. pisum population. Unlike validamycin A, the insect lethal efficacy of amygdalin and phloridzin did not correspond to their trehalase inhibition, probably due to their hydrolysis by insect ß-glucosidases. Our docking studies showed that none of the three compounds can bind to the trehalase active site, unlike their hydrolyzed counterparts, that is, validoxylamine A, phloretin, and prunasin. Validoxylamine A would be by far the best trehalase binder, followed by phloretin and prunasin.

Self-Recognition and Allorecognition Mechanisms Exert a Significant Influence on the Sex Allocation Patterns of the Pea Aphid.

The mechanism controlling sex allocation in the pea aphid, Acyrthosiphon pisum (Harris), remains a crucial yet unresolved issue in the field of evolutionary ecology. This study aims to assess the influence of the presence of both self and non-self clones, along with juvenile hormone III (JH III) titer, on the sex allocation of aphid offspring. To this end, red and green clones were utilized as experimental subjects, and the agar method was employed. Initially, three distinct experimental treatments were established using sexuparae, and the daily offspring count and sex allocation in each treatment zone were recorded. Subsequently, an additional experimental condition involving mixed-clone treatments was introduced. This procedure entailed the transfer of a single sexupara and 20 oviparous females from either the red (1G + 20Rov) or green clone (1G + 20Gov) onto a leaf on agar medium. Simultaneously, a control setup with a new sexupara (1G) was established. Three days following sexupara production, a dose of 0, 25, or 50 ng of JH III was applied to the aphids' abdomens. Subsequently, the titers of JH III in the sexuparae across each treatment group were quantified, and the extent of sex allocation was tallied. The findings demonstrated pronounced disparities in sex allocation among the various treatments and, notably, a substantial increase in the total offspring and oviparous number in the mixed-clone treatment group. The effects of mixed-clone treatment on the sex allocation patterns of the sexupara progeny could be determined by the application of exogenous JH III, indicating that JH may mediate the effects of mixed-clone treatment on sex allocation. Consequently, it can be concluded that A. pisum sexuparae possess the capability to modulate their sex allocation in response to the nature of adjacent competitor clones, thereby demonstrating a variety of sex allocation patterns. Throughout this process, JH III plays a pivotal role.

Use of double-stranded RNA targeting ß2 divergent nicotinic acetylcholine receptor subunit to control pea aphid Acyrthosiphon pisum at larval and adult stages.

BACKGROUND: In recent years, the use of the RNA interference technology (RNAi) has emerged as one of the new strategies for species-specific control of insect pests. Its specificity depends on the distinctiveness of the target gene sequence for a given species. In this work, we assessed in the pea aphid Acyrthosiphon pisum (A. pisum) the use of a double-stranded RNA (dsRNA) that targets the ß2 divergent nicotinic acetylcholine receptor (nAChR) subunit (dsRNA-ß2), which shares low sequence identity with other subunits, to control populations of this pest at different developmental stages. Because nAChRs are targeted by neonicotinoid insecticides such as imidacloprid, we also assessed the effect of dsRNA-ß2 coupled to this insecticide on aphid survival. Finally, because the effect of a control agent on beneficial insect must be considered before any use of new pest management strategies, the acute toxicity of dsRNA-ß2 combined with imidacloprid was evaluated on honeybee Apis mellifera. RESULTS: In this work, we demonstrated that dsRNA-ß2 alone has an insecticidal effect on aphid larvae and adults. Moreover, dsRNA-ß2 and imidacloprid effects on aphid larvae and adults were additive, meaning that dsRNA-ß2 did not alter the efficacy of imidacloprid on these two developmental stages. Also, no obvious acute toxicity on Apis mellifera was reported. CONCLUSION: Using RNAi that targets ß2 divergent nAChR subunit is effective alone or combined with imidacloprid to control A. pisum at larval and adult stages. Because no obvious Apis mellifera mortality has been reported, this RNAi-based pest management strategy should be considered to control insect pest. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Markers of resistance to pea aphid, Acyrthosiphon pisum Harris in Pisum sativum L. accessions.

One of the major insect pests in Pisum sativum L. (is Acyrthosiphon pisum Harris (Hemiptera: pests in Pisum sativum L. (Hemiptera: Aphididae) is Acyrthosiphon pisum Harris (Hemiptera: Aphididae). An effective strategy for aphid control is the resistant host plant use. The current study aimed to identify resistance mechanisms and assess biochemical and morphological markers of pea aphid resistance in pea accessions. Meteorological variables affected the pea aphid density, which positively correlated with temperature, while precipitation amount and humidity negatively impacted. The aphid number was significantly and positively associated with the leaf area and the nitrogen content but negatively correlated with calcium and phosphorus levels. The pea aphid-resistant cultivars L 123-7-11, L 128-1and L 125-5 had small leaf areas, and high phosphorus and calcium content but a low nitrogen level. In the mutual influence of the plant indicators, phosphorus concentration had the highest negative impact on pea aphid density, followed by calcium. The plant marker inclusion in the pea breeding process is an efficient tool for a substantial selection program improvement for aphid resistance. Therefore, resistant host plants are essential tools promoting considerable selection program improvement for aphid resistance in the P. sativum breeding process and helping develop sustainable and environmentally friendly agriculture.

Host plant-mediated effects on Buchnera symbiont: implications for biological characteristics and nutritional metabolism of pea aphids (Acyrthosiphon pisum).

Introduction: The pea aphid, Acyrthosiphon pisum, is a typical sap-feeding insect and an important worldwide pest. There is a primary symbiont-Buchnera aphidicola, which can synthesize and provide some essential nutrients for its host. At the same time, the hosts also can actively adjust the density of bacterial symbiosis to cope with the changes in environmental and physiological factors. However, it is still unclear how symbionts mediate the interaction between herbivorous insects' nutrient metabolism and host plants. Methods: The current study has studied the effects of different host plants on the biological characteristics, Buchnera titer, and nutritional metabolism of pea aphids. This study investigated the influence of different host plants on biological characteristics, Buchnera titer, and nutritional metabolism of pea aphids. Results and discussion: The titer of Buchnera was significantly higher on T. Pretense and M. officinalis, and the relative expression levels were 1.966±0.104 and 1.621±0.167, respectively. The content of soluble sugar (53.46±1.97µg/mg), glycogen (1.12±0.07µg/mg) and total energy (1341.51±39.37µg/mg) of the pea aphid on V. faba were significantly higher and showed high fecundity (143.86±11.31) and weight (10.46±0.77µg/mg). The content of total lipids was higher on P. sativum and T. pretense, which were 2.82±0.03µg/mg and 2.92±0.07µg/mg, respectively. Correlation analysis found that the difference in Buchnera titer was positively correlated with the protein content in M. officinalis and the content of total energy in T. pratense (P < 0.05). This study confirmed that host plants not only affected the biological characteristics and nutritional metabolism of pea aphids but also regulated the symbiotic density, thus interfering with the nutritional function of Buchnera. The results can provide a theoretical basis for further studies on the influence of different host plants on the development of pea aphids and other insects.

Arbuscular mycorrhizal fungus changes alfalfa (Medicago sativa) metabolites in response to leaf spot (Phoma medicaginis) infection, with subsequent effects on pea aphid (Acyrthosiphon pisum) behavior.

Plant disease occurs simultaneously with insect attack. Arbuscular mycorrhizal fungi (AMF) modify plant biotic stress response. Arbuscular mycorrhizal fungi and pathogens may modify plant volatile organic compound (VOC) production and insect behavior. Nevertheless, such effects are rarely studied, particularly for mesocosms where component organisms interact with each other. Plant-mediated effects of leaf pathogen (Phoma medicaginis) infection on aphid (Acyrthosiphon pisum) infestation, and role of AMF (Rhizophagus intraradices) in modifying these interactions were elucidated in a glasshouse experiment. We evaluated alfalfa disease occurrence, photosynthesis, phytohormones, trypsin inhibitor (TI) and total phenol response to pathogen and aphid attack, with or without AMF, and aphid behavior towards VOCs from AMF inoculated and non-mycorrhizal alfalfa, with or without pathogen infection. AM fungus enhanced alfalfa resistance to pathogen and aphid infestation. Plant biomass, root : shoot ratio, net photosynthetic rate, transpiration rate, stomatal conductance, salicylic acid, and TI were significantly increased in AM-inoculated alfalfa. Arbuscular mycorrhizal fungi and pathogen significantly changed alfalfa VOCs. Aphids preferred VOCs of AM-inoculated and nonpathogen-infected to nonmycorrhizal and pathogen-infected alfalfa. We propose that AMF alter plant response to multiple biotic stresses in ways both beneficial and harmful to the plant host, providing a basis for strategies to manage pathogens and herbivore pests.

Aphidius ervi venom regulates Buchnera contribution to host nutritional suitability.

The association between the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), and the endophagous parasitoid wasp Aphidius ervi Haliday (Hymenoptera: Braconidae) offers a unique model system for studying the molecular mechanisms underlying the complex interactions between the parasitoid, its host and the associated primary symbiont. Here, we investigate in vivo the functional role of the most abundant component of A. ervi venom, Ae-γ-glutamyl transpeptidase (Ae-γ-GT), which is known to induce host castration. Microinjections of double-stranded RNA into A. ervi pupae stably knocked down Ae-γ-GT1 and Ae-γ-GT2 paralogue genes in newly emerged females. These females were used to score the phenotypic changes both in parasitized hosts and in the parasitoid's progeny, as affected by a venom blend lacking Ae-γ-GT. Ae-γ-GT gene silencing enhanced growth both of host and parasitoid, supported by a higher load of the primary bacterial symbiont Buchnera aphidicola. Emerging adults showed a reduced survival and fecundity, suggesting a trade-off with body size. This demonstrates in vivo the primary role of Ae-γ-GT in host ovary degeneration and suggests that this protein counterbalances the proliferation of Buchnera likely triggered by other venom components. Our study provides a new approach to unravelling the complexity of aphid parasitoid venom in vivo, and sheds light on a novel role for Ae-γ-GT in host regulation.

Factors affecting host plant selection in alfalfa aphids.

Alfalfa (Medicago sativa L.) hosts several species of aphid, Acyrthosiphon pisum (Harris), Aphis craccivora Koch and Therioaphis trifolii (Monell). The preference of the aphids of alfalfa plants for dense assemblies or individual plants, as well as for healthy or infested plants, was investigated in the field as in the laboratory. Years of field research have revealed the specific preferences of all three species of aphid. A. pisum and T. trifolii are most commonly found in alfalfa crops, while A. craccivora is mostly found on alfalfa weeds. Also, a single species of aphid alone is usually present on a plant. In order to determine the reason for this clear preference and to establish whether at the very beginning, i.e. at the stage of choosing a host, aphid species distance themselves from each other, we tested the effect of the volatiles of healthy and infested plants on their attractiveness to aphids. A. craccivora is repelled by the volatiles of dense crops and plants previously infested with one of the other two species. A. pisum and T. trifolii choose a dense assembly of plants, repelled by the volatiles of plants previously infested with A. craccivora. A. pisum displays the weakest competitive traits, and A. craccivora the strongest. This research showed that competition between aphid species does not occur only when they find themselves on the same plant at the same time, fighting for resources, but also in the choice of plant, in order to avoid later competition.

An aphid symbiont confers protection against a specialized RNA virus, another increases vulnerability to the same pathogen.

Insects often harbour heritable symbionts that provide defence against specialized natural enemies, yet little is known about symbiont protection when hosts face simultaneous threats. In pea aphids (Acyrthosiphon pisum), the facultative endosymbiont Hamiltonella defensa confers protection against the parasitoid, Aphidius ervi, and Regiella insecticola protects against aphid-specific fungal pathogens, including Pandora neoaphidis. Here, we investigated whether these two common aphid symbionts protect against a specialized virus A. pisum virus (APV), and whether their antifungal and antiparasitoid services are impacted by APV infection. We found that APV imposed large fitness costs on symbiont-free aphids and these costs were elevated in aphids also housing H. defensa. In contrast, APV titres were significantly reduced and costs to APV infection were largely eliminated in aphids with R. insecticola. To our knowledge, R. insecticola is the first aphid symbiont shown to protect against a viral pathogen, and only the second arthropod symbiont reported to do so. In contrast, APV infection did not impact the protective services of either R. insecticola or H. defensa. To better understand APV biology, we produced five genomes and examined transmission routes. We found that moderate rates of vertical transmission, combined with horizontal transfer through food plants, were the major route of APV spread, although lateral transfer by parasitoids also occurred. Transmission was unaffected by facultative symbionts. In summary, the presence and species identity of facultative symbionts resulted in highly divergent outcomes for aphids infected with APV, while not impacting defensive services that target other enemies. These findings add to the diverse phenotypes conferred by aphid symbionts, and to the growing body of work highlighting extensive variation in symbiont-mediated interactions.

Identification of azukisapogenol triterpenoid saponins from Oxytropis hirta Bunge and their aphicidal activities against pea aphid Acyrthosiphon pisum Harris.

BACKGROUND: Acyrthosiphon pisum Harris is the most destructive pest worldwide because of its ability to feed on plants directly and transmit plant viruses as a vector. This study aims to identify triterpenoid saponins from Oxytropis hirta Bunge as biopesticides to control aphids. RESULTS: Three new azukisapogenol triterpenoid saponins (1-3), a new pinoresinol lignan glycoside (8), and four known saponins (4-7) were identified from the root of O. hirta. Compounds 4-7 displayed significant aphicidal activities against A. pisum with oral toxicities (LC50  = 51.10-147.43 µg/mL, 72 h), deterrent effects (deterrence index = 1.00, 100-200 µg/mL, 24 h), and aphid reproduction inhibitory effects (inhibition rates = 75.91-86.73%, 400 µg/mL, 24 h), respectively. The carboxyl groups at C-3 GlcA and C-30 were functional groups for their aphicidal activities. The toxic symptoms caused by the optimal 5 involved insect body-color changes from light green to dark or gray-green, and then brown until death. The intestinal cavity, apical microvilli, nuclei, mitochondria, and electron dense granules in the midgut tissues of A. pisum were the target sites showing aphicidal activity. The suppression of pepsin and α-amylase, and the activation of lipase and trypsin could be the signs of organelle damage in the midgut tissues. CONCLUSION: Azukisapogenol triterpenoid saponins from O. hirta could be used as biopesticides to control aphids for their multiple efficacies, including oral toxicity, deterrent activity, and reproduction inhibitory activity. The toxic symptoms involved insect body-color changes. Midgut tissues and their related enzymes were the targets for saponins showing aphicidal activities. © 2022 Society of Chemical Industry.

Functional evaluation of the insulin/insulin-like growth factor signaling pathway in determination of wing polyphenism in pea aphid.

Wing polyphenism is a common phenomenon that plays key roles in environmental adaptation of insects. Insulin/insulin-like growth factor signaling (IIS) pathway is a highly conserved pathway in regulation of metabolism, development, and growth in metazoans. It has been reported that IIS is required for switching of wing morph in brown planthopper via regulating the development of the wing pad. However, it remains elusive whether and how IIS pathway regulates transgenerational wing dimorphism in aphid. In this study, we found that pairing and solitary treatments can induce pea aphids to produce high and low percentage winged offspring, respectively. The expression level of ILP5 (insulin-like peptide 5) in maternal head was significantly higher upon solitary treatment in comparison with pairing, while silencing of ILP5 caused no obvious change in the winged offspring ratio. RNA interference-mediated knockdown of FoxO (Forkhead transcription factor subgroup O) in stage 20 embryos significantly increased the winged offspring ratio. The results of pharmacological and quantitative polymerase chain reaction experiments showed that the embryonic insulin receptors may not be involved in wing polyphenism. Additionally, ILP4 and ILP11 exhibited higher expression levels in 1st wingless offspring than in winged offspring. We demonstrate that FoxO negatively regulates the wing morph development in embryos. ILPs may regulate aphid wing polyphenism in a developmental stage-specific manner. However, the regulation may be not mediated by the canonical IIS pathway. The findings advance our understanding of IIS pathway in insect transgenerational wing polyphenism.

Functional Analysis of Amino Acid Transporter Genes ACYPI000536 and ACYPI004320 in Acyrthosiphon pisum.

In recent years, pea aphids have become major pests of alfalfa. Our previous study found that "Gannong 5" is a highly aphid-resistant alfalfa variety and that "Lie Renhe" is a susceptible one. The average field susceptibility index of "Gannong 5" was 31.31, and the average field susceptibility index of "Lie Renhe" was 80.34. The uptake and balance of amino acids in insects are usually dependent on amino acid transporters. RT-qPCR was used to detect the relative expression levels of seven amino acid transporter differential genes in the different instar pea aphids fed on resistant and susceptible alfalfa varieties after 24 h, and two key genes were selected. When pea aphids fed on "Gannong 5", the expression of ACYPI004320 was significantly higher than that in pea aphids fed on "Lie Renhe"; however, the expression of ACYPI000536 was significantly lower than that in pea aphids fed on "Lie Renhe". Afterward, the RNA interference with pea aphid ACYPI000536 and ACYPI004320 genes was performed using a plant-mediated method, and gene function was verified via liquid chromatography-mass spectrometry and pea aphid sensitivity to aphid-resistant and susceptible alfalfa varieties. The results showed that the down-regulation of the ACYPI000536 gene expression led to an increase in the histidine and lysine contents in pea aphids, which, in turn, led to an increase in mortality when pea aphids fed on the susceptible variety "Lie Renhe". The down-regulation of the ACYPI004320 gene expression led to an increase in phenylalanine content in pea aphids, which, in turn, led to a decrease in mortality when pea aphids fed on the resistant variety "Gannong 5".

Effects of abiotic stresses on the expression of chitinase-like genes in Acyrthosiphon pisum.

Insect chitinases play a crucial part to digest chitin in the exoskeleton during the molting process. However, research on insect chitinase related to the environmental stress response is very limited. This study was the first conducted to expression analysis of chitinase- related genes in A. pisum under abiotic stresses. Here, we identified five chitinase-like proteins (ApIDGF, ApCht3, ApCht7, ApCht10 and ApENGase), and clustered them into five groups (group II, III, V, Ⅹ, and ENGase). Developmental expression analysis revealed that the five A. pisum chitinase-related genes were expressed at whole developmental stages with different relative expression patterns. When aphids were exposed to various abiotic stresses including temperature, insecticide and the stress 20-hydroxyecdysone (20E), all five chitinase genes were differentially expressed in A. pisum. The results showed that insecticide such as imidacloprid down-regulated the expression of these five Cht-related genes. Analysis of temperature stress of A. pisum chitinase suggested that ApCht7 expression was high at 10°C, which demonstrates its important role in pea aphids under low temperature. Conversely, ApCht10 was more active under high temperature stress, as it was significantly up-regulated at 30°C. Besides, 20E enhanced ApCht3 and ApCht10 expression in A. pisum, but reduced ApCht7 expression. These findings provide basic information and insights for the study of the role of these genes under abiotic stress, which advances our knowledge in the management of pea aphids under multiple stresses.