Odorant-Binding Protein 3-Oriented Rational Design and Discovery of Novel Jasmonate Derivatives as Potential Aphid-Repellent Agents.

Odorant-binding protein (OBP) is a potential target for developing insect behavior control agents due to its properties in transporting semiochemicals. In this study, 12 novel jasmonic acid (JA) derivatives were rationally designed and synthesized based on the binding features between Acyrthosiphon pisum OBP3 (ApisOBP3) and compound D1 [(E)-3,7-dimethylocta-2,6-dien-1-yl 2-(3-oxo-2-pentylcyclopentyl)acetate] with a binding affinity (Kd) of 26.79 µM. Most novel JA derivatives displayed better binding affinities than D1 (Kd = 1-26 µM). Among them, compound 6b [(E)-3,7-dimethylocta-2,6-dien-1-yl-2-((Z)-3-((acryloyloxy)imino)-2-pentylcyclopentyl)acetate] is the most promising compound with an excellent Kd of 1.33 µM and a significant repellent activity with repellent rates of 50-60% against A. pisum and Myzus persicae. Both hydrophobic and electrostatic interactions were found to contribute significantly to the binding of 6b to ApisOBP3. This study provides significant guidance for the rational design and efficient identification of novel aphid repellents based on aphid OBPs.

Identification and Behavioral Assays of Alarm Pheromone in the Vetch Aphid Megoura viciae.

Aphids are destructive pests, and alarm pheromones play a key role in their chemical ecology. Here, we conducted a detailed analysis of terpenoids in the vetch aphid, Megoura viciae, and its host plant Pisum sativum using gas chromatography/mass spectrometry. Four major components, (-)-ß-pinene (49.74%), (E)-ß-farnesene (32.64%), (-)-α-pinene (9.42%) and ( +)-limonene (5.24%), along with trace amounts of ( +)-sabinene, camphene and α-terpineol) (3.14%) were found in the aphid. In contrast, few terpenoids were found in the host plant, consisting mainly of squalene (66.13%) and its analog 2,3-epoxysqualene (31.59%). Quantitative analysis of the four major terpenes in different developmental stages of the aphid showed that amounts of the monoterpenes increased with increasing stage, while the sesquiterpene amount peaked in the 3rd instar. (-)-ß-Pinene was the most abundant terpene at all developmental stages. Behavioral assays using a three-compartment olfactometer revealed that the repellency of single compounds varied in a concentration-dependent manner, but two mixtures [(-)-α-pinene: (-)-ß-pinene: (E)-ß-farnesene: ( +)-limonene = 1:44.4:6.5:2.2 or 1:18.4:1.3:0.8], were repellent at all concentrations tested. Our results suggest that (-)-α-pinene and (-)-ß-pinene are the major active components of the alarm pheromone of M. viciae, but that mixtures play a key role in the alarm response. Our study contributes to the understanding of the chemical ecology of aphids and may help design new control strategies against this aphid pest.

Chemosensory proteins participate in insecticide susceptibility in Rhopalosiphum padi, a serious pest on wheat crops.

Rhopalosiphum padi is a worldwide agricultural pest. Chemosensory proteins (CSPs) are considered to be a type of transporters which can bind chemicals from external environments. Previous research showed that the expression of some insect CSPs were significantly increased after exposure to insecticides, and CSPs were involved in insecticide resistance or susceptibility. However, the role of CSPs in the susceptibility and response of R. padi to insecticides is still unknown. In this study, we identified eight CSP (RpCSP) from R. padi by genome-wide investigation. Seven RpCSP genes had two exons, while RpCSP7 had three exons. qPCR analyses showed that the mRNA levels of the eight RpCSP genes were significantly affected by imidacloprid and beta-cypermethrin in different post-treatment periods. Molecular docking predicted that there were hydrogen bonding sites which played key roles in binding of RpCSP4, RpCSP5, RpCSP6, RpCSP7 and RpCSP10 with imidacloprid and beta-cypermethrin. Knockdown of RpCSP4, RpCSP5, RpCSP6 and RpCSP10 by RNA interference significantly increased the aphid mortality under two sublethal concentrations of imidacloprid. Mortalities under two sublethal concentrations of beta-cypermethrin conditions were significantly higher after injection of R. padi with dsCSP4 and dsCSP6. The results indicate that some RpCSP genes are involved in the insecticide susceptibility of R. padi.

Effects of zinc acquired through the plant-aphid-ladybug food chain on the growth, development and fertility of Harmonia axyridis.

Heavy metal pollution is an increasingly serious problem in agricultural ecosystems. Zinc accumulation in the food chain may harm the physiological functions of organisms, including herbivorous and predatory insects. Its effects on development and reproduction in Harmonia axyridis are largely unknown. In this study, five Zn solutions (25, 50, 100, and 150 mg/kg) plus control (0 mg/kg) were used to treat broad beans and to water the resulting seedlings. Aphids fed on these seedlings were eaten by H. axyridis ladybugs. Zn accumulation was found at all three trophic levels. Compared with the control group, ladybugs in the 25, 50, and 100 mg/kg groups had significantly reduced weight gain from the 4th instar to adulthood. Pupae and larvae (instars 1-4) in the 150 mg/kg group had the lowest survival of any group; pupal mortality in the 100 mg/kg group was significantly higher than that in the control group. Under Zn stress, female adults had inhibited expression of Vg1, Vg2 and VgR, reducing egg production and hatchability. Zn thus negatively affected their fertility. These results provide a theoretical basis for future exploration of soil heavy metal pollution impacts in ecosystems.

Total Syntheses and Cytotoxic Evaluations of Cryptolactones A1, A2, B1, B2, and Their Derivatives.

The cryptolactones A1, A2, B1, and B2 isolated from a Cryptomyzus sp. aphid were synthesized via the Mukaiyama aldol reaction and olefin metathesis. Their antipodes and derivatives were also synthesized by the same strategy to investigate structure-activity relationships. These compounds exhibited cytotoxic activity against human promyelocytic leukemia HL-60 cells with IC50 values of 2.1-42 µM.

Possible Insecticidal Mechanism of Cry41-Related Toxin against Myzus persicae by Enhancing Cathepsin B Activity.

Cry toxins produced by Bacillus thuringiensis are well known for their high insecticidal activities against Lepidoptera, Diptera, and Coleoptera; however, their activities against Aphididae are very low. Recently, it has been reported that a Cry41-related toxin exhibited moderate activity against the aphid Myzus persicae, and thus, it is highly desirable to uncover its unique mechanism. In this paper, we report that Cathepsin B, calcium-transporting ATPase, and symbiotic bacterial-associated protein ATP-dependent-6-phosphofructokinase were pulled down from the homogenate of M. persicae as unique proteins that possibly bound to Cry41-related toxin. Cathepsin B has been reported to cleave and inactivate antiapoptotic proteins and plays a role in caspase-initiated apoptotic cascades. In this study, Cathepsin B was expressed in Escherichia coli and purified, and in vitro interaction between recombinant Cathepsin B and Cry41-related toxin was demonstrated. Interestingly, we found that addition of Cry41-related toxin obviously enhanced Cathepsin B activity. We propose a model for the mechanism of Cry41-related toxin as follows: Cry41-related toxin enters the aphid cells and enhances Cathepsin B activity, resulting in acceleration of apoptosis of aphid cells.

Contrasting Effects of Grass - Endophyte Chemotypes on a Tri-Trophic Cascade.

Systemic grass-endophytes of the genus Epichloë symbiotically infect the above-ground plant parts of many grass species, where they produce alkaloids in a grass- and endophyte-specific manner that are toxic or deterrent to herbivores. An increasing number of studies show cascading negative effects of endophyte-derived alkaloids that extend to higher trophic levels, harming beneficial insects, including those that control aphid populations. Lacewings are one of the major biological aphid controls, and are especially resistant to insecticides and pollutants, but their susceptibility to endophyte infection in the food chain has never been studied. Our study found variability in aphid population growth depending on the endophyte-grass chemotype, where aphid population growth was lowest on chemotypes known for producing high amounts of loline alkaloids. We also showed that larval and pupal development and mortality of the Common Green Lacewing (Chrysoperla carnea) was, in a non-choice experiment, not affected by endophyte infection in the food chain. This is a first indication that lacewings might be resistant to endophyte-derived alkaloids and could be robust biocontrol agents when applied together with endophyte-infected grass, possibly replacing chemical pesticides.

Sigillins from Springtails Are Potent Natural Insecticides.

Sigillins are highly chlorinated natural products from the springtail Ceratophysella sigillata (Collembola) that are used to deter arthropod predators. We report here the isolation and structure elucidation of sigillin F, a hydrogenated benzopyranone compound bearing two trichloromethyl groups, and the synthesis of trideoxysigillin (8), a non-natural compound representing the basic scaffold of the sigillins. Sigillins A and F showed insecticidal activity toward various insects, similar to the commercial insecticide imidacloprid, whereas 8 was inactive. The highest mortality was observed for the aphids Megoura viciae and Myzus persicae, but other insect species were also susceptible. Sigillins act as noncompetitive antagonists of the GABA receptor. This mode of action is identical to that of known insecticides with high chlorine content such as dieldrin or endosulfan. The high content of sigillins in C. sigillata, more than 4 mM in concentration, indicates self-resistance. Strikingly, the Collembola and humans have both arrived at the same target with related types of compounds to combat insects.

Growth and Fatty Acid Metabolism of Aphis gossypii Parasitized by the Parasitic Wasp Lysiphlebia japonica.

Parasitism usually causes considerable changes in lipids and fatty acids by redirecting the development of the host. In this study, changes in weight and in free fatty acid content of cotton aphids were recorded after aphids had been parasitized. Results showed that the weight of parasitized Aphis gossypii was increased compared to nonparasitized aphids, and significantly increased weights were detected at 1, 2, and 3 instars after parasitization by Lysiphlebia japonica. Free fatty acid test kits and GC-MS showed that the fatty acid content increased in the early stage of parasitization but decreased after 3 days of parasitization. Seven genes related to the fatty acid synthesis pathway were significantly upregulated in the parasitized aphids, where they were 1.96-10.97 times greater. Our data described the change that occurs in the fatty acid content of parasitized A. gossypii.

Liquid Marbles in Nature: Craft of Aphids for Survival.

Some aphids that live in the leaf galls of the host plant are known to fabricate liquid marbles consisting of honeydew and wax particles as an inner liquid and a stabilizer, respectively. In this study, the liquid marbles fabricated by the galling aphids, Eriosoma moriokense, were extensively characterized with respect to size and size distribution, shape, nanomorphology, liquid/solid weight ratio, and chemical compositions. The stereo microscopy studies confirmed that the liquid marbles have a near-spherical morphology and that the number-average diameter was 368 ± 152 µm, which is 1 order of magnitude smaller than the capillary length of the honeydew. The field emission scanning electron microscopy studies indicated that micrometer-sized wax particles with fiber- and dumpling-like shapes coated the honeydew droplets, which rendered the liquid marbles hydrophobic and nonwetting. Furthermore, the highly magnified scanning electron microscopy images confirmed that the wax particles were formed with assemblage of submicrometer-sized daughter fibers. The contact angle measurements indicated that the wax was intrinsically hydrophobic and that the liquid marbles were stabilized by the wax particles in the Cassie-Baxter model. The weight ratio of the honeydew and the wax particles was determined to be 96/4, and the honeydew consisted of 19 wt % nonvolatile components and 81 wt % water. The 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy, and mass spectroscopy studies confirmed that the wax mainly consisted of triglycerides and that the honeydew mainly consisted of saccharides (glucose and fructose) and ribitol. The atomic force microscopy studies confirmed that honeydew is sticky in nature.

Integrative transcriptomic and genomic analysis of odorant binding proteins and chemosensory proteins in aphids.

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play essential roles in insect chemosensory recognition. Here, we identified nine OBPs and nine CSPs from the Myzus persicae transcriptome and genome. Genomic structure analysis showed that the number and length of the introns are much higher, and this appears to be a unique feature of aphid OBP genes. Three M. persicae OBP genes (OBP3/7/8) as well as CSP1/4/6, CSP2/9 and CSP5/8 are tandem arrayed in the genome. Phylogenetic analyses of five different aphid species suggest that aphid OBPs and CSPs are conserved in single copy across all aphids (with occasional losses), indicating that each OBP and CSP class evolved from a single gene in the common ancestor of aphids without subsequent duplication. Motif pattern analysis revealed that aphid OBP and CSP motifs are highly conserved, and this could suggest the conserved functions of aphid OBPs and CSPs. Three OBPs (MperOBP6/7/10) are expressed antennae specifically, and five OBPs (MperOBP2/4/5/8/9) are expressed antennae enriched, consistent with their putative olfactory roles. M. persicae CSPs showed much broader expression profiles in nonsensory organs than OBPs. None of the nine MperCSPs were found to be antennae specific, but five of them (MperCSP1/2/4/5/6) showed higher expression levels in the legs than in other tissues. MperCSP10 mainly expressed in the antennae and legs. The broad and diverse expression patterns of M. persicae CSPs suggest their multifunctions in olfactory perception, development and other processes.

Bioaccumulation of cadmium, lead, and zinc in agriculture-based insect food chains.

Globally, the metal concentration in soil is increasing due to different anthropogenic and geogenic factors. These metals are taken up by plants and further transferred in the food chain through different routes. The present study was designed to assess the transfer and bioaccumulation of the heavy metals, cadmium (Cd), lead (Pb), and zinc (Zn), in food chains from soil to berseem plants (Triofolium alexandrinum), to insect herbivores (the grasshopper Ailopus thalassinus and the aphid Sitobion avenae) and to an insect carnivore (the ladybird beetle Coccinella septempunctata). The soil of studied berseem fields were slightly alkaline, silty loam in texture and moderate in organic matter. In soil, the concentration of Zn and Pb were under permissible level while Cd was above the permissible level. The accumulation of metals in T. alexandrinum were found in the order Zn > Cd > Pb. Grasshoppers showed higher accumulation of Pb than of Cd and Zn. In the soil-berseem-aphid-beetle food chain, metal enrichment was recorded. However, aphids did not show bioaccumulation for Cd. Metals accumulation in beetles showed that translocation of Zn, Cd, and Pb was taking place in the third trophic level. Our study highlights the mobility of metals in insect food chains and showed that insect feeding style greatly influenced the bioaccumulation. However, different metals showed variable bioaccumulation rates depending on their toxicity and retention.

Laser surgery reveals the biomechanical and chemical signaling functions of aphid siphunculi (cornicles).

Aphids are an attractive food source to many predators and parasitoids because of their small size, soft bodies and slow movement. To combat predation, aphids evolved both behavioral and chemical defensive mechanisms that are operated via siphunculi (cornicles), differently developed structures that more or less extend from their abdomen. Although both direct and indirect linkages between siphunculi and their defensive mechanisms have been explored, their ultimate effects on aphid fitness are still broadly debated. To explicitly test the influence of siphunculi on brown citrus aphid, Aphis (Toxoptera) citricida (Kirkaldy), fitness, we razor-cut and laser-sealed the siphunculi. Siphunculi removal resulted in two distinct behavior modifications, (false aggregation and increased drop-off rates) that led to decreased survival and the loss of the ability to right themselves from an inverted position. These results together indicate that siphunculi play an important role in survival, and removal of these organs will have negative effect on aphid fitness. Furthermore, results suggested that released alarm pheromone may play an important role in communication among aphid clone-mate, and omitting it results in miscommunication and competition among clonemates. These findings will help in better understanding the aphid biology.

A role of uroleuconaphins, polyketide red pigments in aphid, as a chemopreventor in the host defense system against infection with entomopathogenic fungi.

Four red polyketide pigments, uroleuconaphins A1 (1) and B1 (2) and their glucosides 3 and 4, were isolated from the red goldenrod aphid Uroleucon nigrotuberculatum. Although these red pigments exist only as glucosides 3 and 4 in the intact insect body, 3 and 4 convert instantly to aglycones 1 and 2 at death. Pigments 1 and 2 inhibited the growth of Lecanicillium sp. (Ascomycota: Cordycipitaceae) and 1, 2, and 3 were active against Conidiobolus obscurus (Entomophthoromycota; Entomophthorales); these fungal species are pathogenic. We therefore regard aphid pigments 1-4 as chemopreventive agents that aid in the resistance of infection by entomopathogenic fungi at the level of the individual aphid and/or at the species level.

Genomic Insight into Symbiosis-Induced Insect Color Change by a Facultative Bacterial Endosymbiont, "Candidatus Rickettsiella viridis".

Members of the genus Rickettsiella are bacterial pathogens of insects and other arthropods. Recently, a novel facultative endosymbiont, "Candidatus Rickettsiella viridis," was described in the pea aphid Acyrthosiphon pisum, whose infection causes a striking host phenotype: red and green genetic color morphs exist in aphid populations, and upon infection with the symbiont, red aphids become green due to increased production of green polycyclic quinone pigments. Here we determined the complete genome sequence of the symbiont. The 1.6-Mb circular genome, harboring some 1,400 protein-coding genes, was similar to the genome of entomopathogenic Rickettsiella grylli (1.6 Mb) but was smaller than the genomes of phylogenetically allied human pathogens Coxiella burnetii (2.0 Mb) and Legionella pneumophila (3.4 Mb). The symbiont's metabolic pathways exhibited little complementarity to those of the coexisting primary symbiont Buchnera aphidicola, reflecting the facultative nature of the symbiont. The symbiont genome harbored neither polyketide synthase genes nor the evolutionarily allied fatty acid synthase genes that are suspected to catalyze the polycyclic quinone synthesis, indicating that the green pigments are produced not by the symbiont but by the host aphid. The symbiont genome retained many type IV secretion system genes and presumable effector protein genes, whose homologues in L. pneumophila were reported to modulate a variety of the host's cellular processes for facilitating infection and virulence. These results suggest the possibility that the symbiont is involved in the green pigment production by affecting the host's metabolism using the secretion machineries for delivering the effector molecules into the host cells.IMPORTANCE Insect body color is relevant to a variety of biological aspects such as species recognition, sexual selection, mimicry, aposematism, and crypsis. Hence, the bacterial endosymbiont "Candidatus Rickettsiella viridis," which alters aphid body color from red to green, is of ecological interest, given that different predators preferentially exploit either red- or green-colored aphids. Here we determined the complete 1.6-Mb genome of the symbiont and uncovered that, although the red-green color transition was ascribed to upregulated production of green polycyclic quinone pigments, the symbiont genome harbored few genes involved in the polycyclic quinone biosynthesis. Meanwhile, the symbiont genome contained type IV secretion system genes and presumable effector protein genes, whose homologues modulate eukaryotic cellular processes for facilitating infection and virulence in the pathogen Legionella pneumophila We propose the hypothesis that the symbiont may upregulate the host's production of polycyclic quinone pigments via cooption of secretion machineries and effector molecules for pathogenicity.

Heritable variation in prey defence provides refuge for subdominant predators.

Generalist predators with broadly overlapping niches commonly coexist on seemingly identical sets of prey. Here, we provide empirical demonstration that predators can differentially exploit fine-grained niches generated by variable, heritable and selective defences within a single prey species. Some, but not all, clones of the aphid Aphis craccivora are toxic towards the dominant invasive predatory ladybeetle, Harmonia axyridis However, other less competitive ladybeetle species are not affected by the aphid's toxic trait. In laboratory and open field experiments, we show: (i) that subdominant ladybeetle species were able to exploit the toxic aphids, benefitting from the suppression of the dominant predator; and (ii) that this narrow-spectrum toxicity can function as an anti-predator defence for the aphid, but depends on enemy community context. Our results demonstrate that niche differentiation among generalist predators may hinge upon previously underappreciated heritable variation in prey defence, which, in turn, may promote diversity and stability of enemy communities invaded by a dominant predator.

Computational investigation of the molecular conformation-dependent binding mode of (E)-ß-farnesene analogs with a heterocycle to aphid odorant-binding proteins.

Odorant-binding proteins (OBPs) play an important role as ligand-transfer filters in olfactory recognition in insects. (E)-ß-farnesene (EBF) is the main component of the aphid alarm pheromone and could keep aphids away from crops to prevent damage. Computational insight into the molecular binding mode of EBF analogs containing a heterocycle based on the structure of Megoura viciae OBP 3 (MvicOBP3) was obtained by molecular docking and molecular dynamics simulations. The results showed that high affinity EBF analogs substituted with an aromatic ring present a unique binding conformation in the surface cavity of MvicOBP3. A long EBF chain was located inside the cavity and was surrounded by many hydrophobic residues, while the substituted aromatic ring was exposed to the outside due to limitations from the formation of multiple hydrogen bonds. However, the low activity EBF analogs displayed an exactly inverted binding pose, with EBF loaded on the external side of the protein cavity. The affinity of the recently synthesized EBF analogs containing a triazine ring was evaluated in silico based on the binding modes described above and in vitro through fluorescence competitive binding assay reported later. Compound N1 not only showed a similar binding conformation to that of the high affinity analogs but was also found to have a much higher docking score and binding affinity than the other analogs. In addition, the docking score results correlated well with the predicted logP values for these EBF analogs, suggesting highly hydrophobic interactions between the protein and ligand. These studies provide an in silico screening model for the binding affinity of EBF analogs in order to guide their rational design based on aphid OBPs.

Identification of the Alarm Pheromone of Cowpea Aphid, and Comparison With Two Other Aphididae Species.

In response to a predator attack, many Aphidinae species release an alarm pheromone, which induces dispersal behavior in other individuals within the colony. The major component of this pheromone is the sesquiterpene (E)-ß-farnesene (Eßf), but variations occur between aphid species. In the present work, we collected, identified, and quantified the alarm pheromone of Aphis craccivora Koch (Hemiptera: Aphididae), before quantifying the escape behavior induced in the neighboring individuals. We compared the semiochemistry and associated behavior of alarm signaling with two other aphid species: Myzus persicae (Sulzer) (Hemiptera: Aphididae) and Aphis fabae Scopoli (Hemiptera: Aphididae). Eßf was the only volatile found for each species. M. persicae produces a higher quantity of Eßf (8.39 ± 1.19 ng per individual) than A. craccivora (6.02 ± 0.82 ng per individual) and A. fabae (2.04 ± 0.33 ng per individual). Following exposure to natural doses of synthetic Eßf (50 ng and 500 ng), A. craccivora respond more strongly than the two other Aphidinae species with 78% of the individuals initiated alarm behavior for 500 ng of Eßf.

Nano-formulation enhances insecticidal activity of natural pyrethrins against Aphis gossypii (Hemiptera: Aphididae) and retains their harmless effect to non-target predators.

The insecticidal activity of a new nano-formulated natural pyrethrin was examined on the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), and the predators Coccinella septempunctata L. (Coleoptera: Coccinellidae) and Macrolophus pygmaeus Rambur (Hemiptera: Miridae), in respect with the nano-scale potential to create more effective and environmentally responsible pesticides. Pyrethrin was nano-formulated in two water-in-oil micro-emulsions based on safe biocompatible materials, i.e., lemon oil terpenes as dispersant, polysorbates as stabilizers, and mixtures of water with glycerol as the dispersed aqueous phase. Laboratory bioassays showed a superior insecticidal effect of the pyrethrin micro-emulsions compared to two commercial suspension concentrates of natural pyrethrins against the aphid. The nano-formulated pyrethrins were harmless, in terms of caused mortality and survival time, to L3 larvae and four-instar nymphs of the predators C. septempunctata and M. pygmaeus, respectively. We expect that these results can contribute to the application of nano-technology in optimization of pesticide formulation, with further opportunities in the development of effective plant protection products compatible with integrated pest management practices.

Adaptation of Defensive Strategies by the Pea Aphid Mediates Predation Risk from the Predatory Lady Beetle.

Within a species, individual animals adopt various defensive strategies to resist natural enemies, but the defensive strategies that are adopted in response to variations in predation risk are poorly understood. Here, we assessed consecutive foraging processes on cohorts of two biotypes (green and red) of the pea aphid, Acyrthosiphon pisum, by the predatory lady beetle Propylea japonica, to investigate the adaptive mechanism underlying the defensive strategy. We observed the behavioral responses of individuals (continue feeding or escape, i.e., walk away or drop off from initial feeding site), simultaneously quantified the amount of alarm pheromone, (E)-ß-farnesene (EßF) released from cohorts using gas chromatography-mass spectrometry (GC-MS), and recorded the foraging times of predators in intervals. The results indicated that: (1) the anti-predator responses differed markedly between biotypes and among the stages of the consecutive foraging processes. (2) Few green cohorts tended to release EßF during the first foraging; those that did released only a low dose that did not increase the number of escapes. However, the amount of EßF rose rapidly following the second foraging process, which caused an intense escape response. In contrast, more red cohorts released greater amounts of EßF, which caused more individuals to escape from their innate feeding sites during the first foraging. During the second foraging, more red individuals tended to escape without releasing EßF in greater quantities. (3) The foraging time was effectively shortened in each biotype cohort that adopted diverse defensive strategies. This study of the defensive strategies of the pea aphid may contribute to understanding the intraspecific differences in aphid defense mechanisms.