Evaluation and optimization of the protocols for measuring cytochrome P450 activity in aphids.

Insect cytochrome P450 plays major roles in detoxification of phytotoxin and insecticides. However, determination of P450 activity in aphids has variable success and there is no reliable method yet. In this study, we found that homogenizing the green peach aphid, Myzus persicae, in the 96-well microplate resulted in significantly higher P450 activities than those in Eppendorf tube. Homogenizing aphids in Eppendorf tube released uncharacterized compounds that inhibited aphids and pig liver P450 activities, whereas aphids homogenized in the microplate may not be completely ground and thus released fewer such inhibitors. Then, the microplate homogenization method was optimized as follows: one or two aphids were placed in one well of the 96 well-microplate and ground in phosphate buffer using pipette tips for 20 cycles, followed by addition of 7-ethoxycoumarin, and then incubated for 1 h at room temperature, after which glycine buffer-ethanol mixture was added to stop the reaction. This method is also suitable for the pea aphid, Acyrthosiphon pisum, and the bird cherry-oat aphid, Rhopalosiphum padi. These results highlight the importance of considering inhibitory effects of endogenous compounds in insects on their P450 activities and provide one possible method to reduce these inhibitory effects.

N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide promotes bone formation via the canonical Wnt/ß-catenin signaling pathway.

Osteoporosis is characterized by low bone mineral density and microarchitectural deterioration of bone tissue. N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (MBOC) is one of the macamides isolated from Maca (Lepidium meyenii Walp.), a cruciferous plant from the Andes of Peru. In this study, C3H/10T1/2 mesenchymal stem cells were treated with MBOC in osteogenic induction medium. An ovariectomized (OVX) mouse model was used to investigate the effect of 1-month MBOC treatment on the prevention of postmenopausal osteoporosis. Remarkably, trabecular thickness, trabecular number, and bone volume/tissue volume of the distal femoral metaphysis were significantly increased in OVX + MBOC mice compared with OVX mice, as revealed by microcomputed tomography analysis. Trabecular separation was decreased in OVX + MBOC mice compared with OVX mice. Consistently, MBOC increased the levels of osteocalcin and runt-related transcription factor 2 in OVX mice, as well as the expression of runt-related transcription factor 2, osterix, and alkaline phosphatase in C3H/10T1/2 cells. Mechanistically, MBOC activates the canonical Wnt/ß-catenin signaling pathway via inhibiting phosphorylation of GSK-3ß at Tyr216 and maintaining ß-catenin expression. Collectively, the current study demonstrates the robustness of MBOC in the induction of mesenchymal stem cells osteogenic differentiation and consequent bone formation, suggesting that MBOC may be a potentially effective drug to treat postmenopausal osteoporosis.

Involvement of Laccase2 in Cuticle Sclerotization of the Whitefly, Bemisia tabaci Middle East-Asia Minor 1.

Cuticle sclerotization is critical for insect survival. Laccase2 (Lac2) is a phenol oxidase that plays a key role in cuticle formation and pigmentation in a variety of insects. However, the function of Lac2 in whitefly, Bemisia tabaci, remains unclear. In this study, we identified a BtLac2 gene in B. tabaci MEAM1 and found that BtLac2 was expressed in all stages. It was highly expressed in the egg stage, followed by nymph and adult. Moreover, the expression of BtLac2 was higher in the cuticle than in other tissues. Knockdown of BtLac2 in nymphs produced thinner and fragile cuticles, which significantly increased the mortality rate, extended the development duration of nymphs, and decreased the emergence rate of adults. This result demonstrates that BtLac2 plays an important role in the cuticle hardening of B. tabaci and suggests a potential management strategy using RNAi to knock down BtLac2 expression.

Preference and performance of the green peach aphid, Myzus persicae on three Brassicaceae vegetable plants and its association with amino acids and glucosinolates.

The green peach aphid, Myzus persicae (Sulzer) is a generalist pest of various host plants, whose feeding preference and growth performance mainly depends on the quantity and quality of nutrients and defensive metabolites in host plants. Here, we studied the preference and performance of M. persicae on three major Brassicaceae vegetables in China and measured nutrient (amino acids) and defensive metabolites (glucosinolates) in these plants. We found that M. persicae preferred and performed better on Chinese cabbage than cabbage and radish, which may be due to the relatively higher concentration of amino acids and lower levels of indole glucosinolates in their leaves. The glucosinolates level in cabbage leaves was ten times higher than the other two plants, while the amino acid concentration in radish was only half of the cabbage or Chinese cabbage. The higher concentration of indole glucosinolates in cabbage and lower levels of amino acids in radish may account for the poorer preference and growth of M. persicae on these two plants. These results suggest that both amino acids and glucosinolates in plants may play important roles in the preference and performance of M. persicae, which provide new knowledge for the cultivation and breeding of Brassicaceae vegetables.

Facultative Endosymbiont Serratia symbiotica Inhibits the Apterization of Pea Aphid To Enhance Its Spread.

Aphids display wing polyphenism, and the mother can produce a wingless morph for reproduction and a winged morph for dispersal. It is believed that the wingless morph is an adaptive status under favorable conditions and is determined prenatally. In this study, we have found that winged nymphs of the pea aphid, Acyrthosiphon pisum, can change from winged to wingless during normal development. Our results showed that winged nymphs could become the wingless morph by apterization in response to changes from stressful to favorable conditions. The acquired wingless aphids had higher fecundity than the winged morph. However, this process of regression from winged to wingless morph was inhibited by Serratia symbiotica. The existence of the symbiont did not affect the body mass and fecundity of adult aphids, but it increased the body weight of nymphs and temporally increased the quantity of a primary symbiont, Buchnera aphidicola. Our results showed that despite temporal improvement of living conditions causing the induction of apterization of winged nymphs, the inhibition effect of S. symbiotica on this process was activated simultaneously. This finding, for the first time, reveals that the wingless morph can be changed postnatally, which explains a novel regulating mechanism of wing polyphenism driven by external abiotic stimuli and internal biotic regulation together in aphids. IMPORTANCE Wing polyphenism is an important adaptative response to environmental changes for aphids. Endosymbionts are widespread in aphids and also confer the ability to withstand unfavorable conditions. However, little is known about whether endosymbionts are involved in the wing polyphenism. In this study, we report a new finding that winged nymphs of the pea aphid could turn into adults without wings or wing-related structures through apterization when winged nymphs escaped from stressful to favorable environments. Further analysis revealed that the facultative symbiont S. symbiotica could prevent the temporal determination of the host in wing suppression by inhibiting apterization, to enhance its spread. Our findings provide a novel angle to understanding the wing polyphenism regulation of aphids.

Performance and Transcriptional Response of the Green Peach Aphid Myzus persicae to the Restriction of Dietary Amino Acids.

Free amino acids in the phloem sap are the dominant nitrogen source for aphids, but their availability is usually poor. Although some studies have explored the effect of dietary amino acid restriction on aphid performance, little is known about the molecular basis of these effects. Here, we examined the performance and transcriptome of the green peach aphid, Myzus persicae, fed a standard diet (Control diet) or a diet containing 50% of the total amino acids of the Control diet (Half diet). Aphid weight and fecundity were significantly reduced in the Half diet group. Transcriptomic analysis showed that a total of 1460 genes were differentially expressed between the groups were fed on the two diets, which many of them were associated with nutrient and energy metabolism. When feeding on the Half diet, aphids upregulated genes associated with the amino acid biosynthetic pathway (predominantly amino acid biosynthesis genes and some amino acid transporter genes) as well as the cysteine and serine protease genes. Furthermore, these aphids displayed increased expression of genes associated with glycolysis, which could generate intermediates for de novo amino acid biosynthesis. Consistent with this, elevated glucose levels were observed in aphids in the Half diet group. Additionally, the expression levels of several genes associated with hormonal signaling pathway were altered. Several genes related to juvenile hormone and insulin-like peptide (ILP) signaling were downregulated, including Krüppel homolog 1 (Kr-h1) and insulin-like peptide 5 (Ilp5), respectively. In contrast, several genes related to ecdysone signaling were upregulated including broad-complex core protein (Br-c) and shade (Shd). Despite their poor performances, M. persicae adapted to dietary restriction of amino acids, through upregulation of genes involved in amino acid biosynthesis, glycolysis, and protein degradation, as well as by altering the expression level of genes involved in hormone signaling pathways.

Nutrition versus defense: Why Myzus persicae (green peach aphid) prefers and performs better on young leaves of cabbage.

Plant leaves of different ages differ in nutrients and toxic metabolites and thus exhibit various resistance levels against insect herbivores. However, little is known about the influence of leaf ontogeny on plant resistance to phloem-feeding insects. In this study, we found that the green peach aphid, Myzus persicae, preferred to settle on young cabbage leaves compared with mature or old leaves, although young leaves contained the highest concentration of glucosinolates. Furthermore, aphids feeding on young leaves had higher levels of glucosinolates in their body, but aphids performed better on young leaves in terms of body weight and population growth. Phloem sap of young leaves had higher amino acid:sugar molar ratio than mature leaves, and aphids feeding on young leaves showed two times longer phloem feeding time and five times more honeydew excretion than on other leaves. These results indicate that aphids acquired the highest amount of nutrients and defensive metabolites when feeding on young cabbage leaves that are strong natural plant sinks. Accordingly, we propose that aphids generally prefer to obtain more nutrition rather than avoiding host plant defense, and total amount of nutrition that aphids could obtain is significantly influenced by leaf ontogeny or source-sink status of feeding sites.

Leaf Chemical Compositions of Different Eggplant Varieties Affect Performance of Bemisia tabaci (Hemiptera: Aleyrodidae) Nymphs and Adults.

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) MEAM1 is a serious pest of many crops worldwide, and its control mostly depends on insecticides. One of the most preferred host plants of B. tabaci is eggplant, Solanum melongena, although preferences among different cultivars of the whiteflies vary. We hypothesized that certain nutritional and defensive chemicals of plant leaves, such as nitrogen, glucose, fructose, sucrose, amino acids, total phenolic components, and moisture content may affect whitefly's feeding and ovipositional preference, fecundity and longevity, nymph development, and survival among different eggplant varieties. To seek the most susceptible eggplant variety for use as an attractive trap crop for whitefly adults, we determined the variation of leaf chemical compositions among six eggplant varieties (H149, JSZ, JGL, TLB, DYZ, and QXN) and evaluated the effects of their leaf chemicals to the performance of nymphs and adults of B. tabaci. In choice feeding and oviposition tests, the varieties 'H149' and 'JSZ' had the most eggs. The whiteflies had significantly higher fecundity, longevity, lowest nymph development period, and higher survivals on JSZ than on other varieties. The least preferred TLB variety possessed the lowest adults and eggs, the lowest fecundity and longevity, and nymph development period. JGL, DYZ, and QXN were considered as a moderately preferred variety. Leaf chemistry revealed that highly susceptible variety possessed higher concentration of nitrogen, glucose, amino acids, and lower moisture content. The resistant variety possessed higher amount of total phenolic component. Both nutritional and defensive chemicals combined associated with nymph and adult performance of whitefly.

The green peach aphid Myzus persicae perform better on pre-infested Chinese cabbage Brassica pekinensis by enhancing host plant nutritional quality.

The green peach aphid, Myzus persicae Sulzer, is a notorious pest on vegetables, which often aggregates in high densities on crop leaves. In this study, we investigated whether M. persicae could suppress the resistance level of Chinese cabbage Brassica pekinensis. M. persicae performed better in terms of weight gain (~33% increase) and population growth (~110% increase) when feeding on previously infested (pre-infested) Chinese cabbage compared with those on non-infested plants. However, when given a choice, 64% of the aphids preferred to settle on non-infested leaves, while 29% of aphids chose pre-infested leaves that had a 2.9 times higher concentration of glucosinolates. Aphid feeding significantly enhanced the amino acid:sugar ratio of phloem sap and the absolute amino acid concentration in plant leaves. Aphid infestation significantly increased the expression levels of salicylic acid (SA) marker genes, while it had marginal effects on the expression of jasmonate marker genes. Exogenously applied SA or methyl jasmonate had no significant effects on M. persicae performance, although these chemicals increased glucosinolates concentration in plant leaves. M. persicae infestation increase amino acid:sugar ratio and activate plant defenses, but aphid performed better on pre-infested plants, suggesting that both nutrition and toxics should be considered in insect-plant interaction.

Jasmonate- and salicylate-induced defenses in wheat affect host preference and probing behavior but not performance of the grain aphid, Sitobion avenae.

Jasmonate- and salicylate-mediated signaling pathways play significant roles in induced plant defenses, but there is no sufficient evidence for their roles in monocots against aphids. We exogenously applied methyl jasmonate (MeJA) and salicylic acid (SA) on wheat seedlings and examined biochemical responses in wheat and effects on the grain aphid, Sitobion avenae (Fab.). Application of MeJA significantly increased levels of wheat's polyphenol oxidase, peroxidase and proteinase inhibitor 1, 2 and 6 days after treatment. In two-choice tests, adult aphids preferred control wheat leaves to MeJA- or SA-treated leaves. Electrical penetration graph (EPG) recordings of aphid probing behavior revealed that on MeJA-treated plants, the duration of aphid's first probe was significantly shorter and number of probes was significantly higher than those on control plants. Also total duration of probing on MeJA-treated plants was significantly shorter than on control plants. Total duration of salivation period on SA-treated plants was significantly longer, while mean phloem ingestion period was significantly shorter than on control plants. However, no significant difference in total duration of phloem sap ingestion period was observed among treatments. The EPG data suggest that MeJA-dependent resistance factors might be due to feeding deterrents in mesophyll, whereas the SA-mediated resistance may be phloem-based. We did not observe any significant difference of MeJA and SA application on aphid development, daily fecundity, intrinsic growth rate and population growth. The results indicate that both MeJA- and SA-induced defenses in wheat deterred S. avenae colonization processes and feeding behavior, but had no significant effects on its performance.

Deciphering the mechanism of ß-aminobutyric acid-induced resistance in wheat to the grain aphid, Sitobion avenae.

The non-protein amino acid ß-aminobutyric acid (BABA) can induce plant resistance to a broad spectrum of biotic and abiotic stresses. However, BABA-induced plant resistance to insects is less well-studied, especially its underlying mechanism. In this research, we applied BABA to wheat seedlings and tested its effects on Sitobion avenae (F.). When applied as a soil drench, BABA significantly reduced weights of S. avenae, whereas foliar spray and seed treatment had no such effects. BABA-mediated suppression of S. avenae growth was dose dependent and lasted at least for 7 days. The aminobutyric acid concentration in phloem sap of BABA-treated plants was higher and increased with BABA concentrations applied. Moreover, after 10 days of treatment, the aminobutyric acid content in BABA-treated plants was still higher than that in control treatment. Sitobion avenae could not discriminate artificial diet containing BABA from standard diet, indicating that BABA itself is not a deterrent to this aphid. Also S. avenae did not show preference for control plants or BABA-treated plants. Consistent with choice test results, S. avenae had similar feeding activities on control and BABA-treated plants, suggesting that BABA did not induce antifeedants in wheat seedlings. In addition, aminobutyric acid concentration in S. avenae feeding on BABA-treated plants was significantly higher than those feeding on control plants. Sitobion avenae growth rate was reduced on the artificial diet containing BABA, indicating that BABA had direct toxic effects on this aphid species. These results suggest that BABA application reduced S. avenae performance on wheat seedlings and the mechanism is possibly due to direct toxicity of high BABA contents in plant phloem.