Role of the amygdala in disrupted integration and effective connectivity of cortico-subcortical networks in apathy.

BACKGROUND: Apathy is a quantitative reduction in motivation and goal-directed behaviors, not only observed in neuropsychiatric disorders, but also present in healthy populations. Although brain abnormalities associated with apathy in clinical disorders have been studied, the organization of brain networks in healthy individuals has yet to be identified. METHOD: We examined properties of intrinsic brain networks in healthy individuals with varied levels of apathy. By using functional magnetic resonance imaging in combination with graph theory analysis and dynamic causal modeling analysis, we tested communications among nodes and modules as well as effective connectivity among brain networks. RESULTS: We found that the average participation coefficient of the subcortical network, especially the amygdala, was lower in individuals with high than low apathy. Importantly, we observed weaker effective connectivity fromthe hippocampus and parahippocampal gyrus to the amygdala, and from the amygdala to the parahippocampal gyrus and medial frontal cortex in individuals with apathy. CONCLUSION: These findings suggest that individuals with high apathy exhibit aberrant communication within the cortical-to-subcortical network, characterized by differences in amygdala-related effective connectivity. Our work sheds light on the neural basis of apathy in subclinical populations and may have implications for understanding the development of clinical conditions that feature apathy.

Connectome-based individualized prediction of reciprocity propensity and sensitivity to framing: a resting-state functional magnetic resonance imaging study.

BACKGROUND: The social representation theory states that individual differences in reciprocity decisions are composed of a stable central core (i.e., reciprocity propensity, RP) and a contextual-dependent periphery (i.e., sensitivity to the framing effect; SFE, the effect by how the decision is presented). However, the neural underpinnings that explain RP and SFE are still unknown. METHOD: Here, we employed prediction and lesion models to decode resting-state functional connectivity (RSFC) of RP and SFE for reciprocity decisions of healthy volunteers who underwent RS functional magnetic resonance imaging and completed one-shot trust (give frame) and distrust (take frame) games as trustees. RESULTS: Regarding the central core, reciprocity rates were positively associated between the give and take frame. Neuroimaging results showed that inter-network RSFC between the default-mode network (DMN; associated with mentalizing) and cingulo-opercular network (associated with cognitive control) contributed to the prediction of reciprocity under both frames. Regarding the periphery, behavioral results demonstrated a significant framing effect-people reciprocated more in the give than in the take frame. Our neuroimaging results revealed that intra-network RSFC of DMN (associated with mentalizing) contributed dominantly to the prediction of SFE. CONCLUSION: Our findings provide evidence for distinct neural mechanisms of RP and SFE in reciprocity decisions.

Moral Conflict in Economic Decision Making: The Role of the Anterior Cingulate Cortex-Striatum Pathway.

The present study combined a novel hypothetical investment game with functional magnetic resonance imaging to examine how moral conflict biases our real decision preference when it is not obvious or explicitly presented. Investment projects were chosen based on their prior subjective morality ratings to fit into 2 categories: a high level of moral conflict (HMC) or a low level of moral conflict (LMC). Participants were instructed to invest high or low amounts of capital into different projects. Behavioral and neural responses during decision making were recorded and compared. Behaviorally, we observed a significant decision bias such that investments were lower for HMC projects than for LMC projects. At the neural level, we found that moral conflict-related activity in the anterior cingulate cortex (ACC) was higher in the HMC condition than in the LMC condition and that reward-related activity in bilateral striatum was lower. Dynamic causal modeling further suggested that the moral conflict detected in the ACC influenced final decisions by modulating the representation of subjective value through the ACC's connection to the reward system.

A Novel Peptide-Equipped Exosomes Platform for Delivery of Antisense Oligonucleotides.

Exosomes are natural delivery vehicles because of their original feature such as low immunogenicity, excellent biocompatibility, and migration capability. Engineering exosomes with appropriate ligands are effective approaches to improve the low cellular uptake efficiency of exosomes. However, current strategies face considerable challenges due to the tedious and labor-intensive operational process. Here, we designed a novel peptides-equipped exosomes platform which can be assembled under convenient and mild reaction condition. Cell-penetrating peptides (CPPs) was conjugated on HepG2 cells-derived exosomes surface which can not only enhance the penetrating capacity of exosomes but also assist exosomes in loading antisense oligonucleotides (ASOs). The cellular uptake mechanism was investigated and we compared the difference between natural exosomes and modified exosomes. The resulting nanosystem demonstrated a preferential tropism for cells that are parented to their source tumor cells and could remarkably increase the cellular delivery of G3139 with efficient downregulation of antiapoptotic Bcl-2. This work developed a rapid strategy for intracellular delivery of nucleic acids, thus providing more possibilities toward personalized cancer medicine.

Expression of L-amino acid oxidase of Trichoderma harzianum in tobacco confers resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a flavoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.

The Neural Mechanism of the Social Framing Effect: Evidence from fMRI and tDCS Studies.

As an important cognitive bias, the framing effect shows that our decision preferences are sensitive to the verbal description (i.e., frame) of options. This study focuses on the neural underpinnings of the social framing effect, which is based on decision-making regarding other people. A novel paradigm was used in which participants made a trade-off between economic benefits and the feelings of others. This decision was described as either a "harm" to, or "not helping," other persons in two conditions (Harm frame vs Help frame). Both human males and females were recruited. Participants behaved more prosocially for Harm frame compared with Help frame, resulting in a significant social framing effect. Using functional magnetic resonance imaging, Experiment 1 showed that the social framing effect was associated with stronger activation in the temporoparietal junction (TPJ), especially its right part. The functional connectivity between the right TPJ (rTPJ) and medial prefrontal cortex predicted the social framing effect on the group level. In Experiment 2, we used transcranial direct current stimulation to modulate the activity of the rTPJ and found that the social framing effect became more prominent under anodal (excitatory) stimulation, while the nonsocial framing effect elicited by the economic gain/loss gambling frame remained unaffected. The rTPJ results might be associated with moral conflicts modulated by the social consequences of an action or different levels of mentalizing with others under different frame conditions, but alternative interpretations are also worth noting. These findings could help elucidate the psychological mechanisms of the social framing effect.SIGNIFICANCE STATEMENT Previous studies have suggested that the framing effect is generated from an interaction between the amygdala and anterior cingulate cortex. This opinion, however, is based on findings from nonsocial framing tasks. Recent research has highlighted the importance of distinguishing between the social and nonsocial framing effects. The current study focuses on the social framing effect and finds out that the temporoparietal junction and its functional connectivity with the medial prefrontal cortex play a significant role. Additionally, modulating the activity of this region leads to changes in social (but not nonsocial) framing effect. Broadly speaking, these findings help understand the difference in neural mechanisms between social and nonsocial decision-making. Meanwhile, they might be illuminating to promote helping behavior in society.

Down-regulation of FOXR2 inhibits hypoxia-driven ROS-induced migration and invasion of thyroid cancer cells via regulation of the hedgehog pathway.

Forkhead box R2 (FOXR2), a new member of the FOX family, is involved in a wide range of biological processes such as embryogenesis, differentiation, transformation and metabolic homeostasis. Recently, FOXR2 has been reported to be aberrantly expressed in a variety of cancers and correlated with cancer development. However, the specific role of FOXR2 in thyroid cancer (TC) remains unclear. In this study, we showed that FOXR2 was highly expressed in TC tissues and cell lines. Moreover, down-regulation of FOXR2 inhibited hypoxia-induced reactive oxygen species (ROS) production and migration/invasion of TC cells. We also found that the hedgehog pathway was responsible for the partial mechanisms underlying the inhibitory effect. Taken together, these findings indicated that down-regulation of FOXR2 inhibits hypoxia-driven ROS-induced migration and invasion of TC cells via regulation of the hedgehog pathway. Thus, FOXR2 may hold great potential for TC treatment.

The stereotype of professional roles influences neural responses to moral transgressions: ERP evidence.

The person-centered account of moral judgments is important since immoral behaviors are diagnostic of an individual's character. The present study explored how the professional stereotypes associated with the agents shaped the way people perceiving moral/immoral behaviors. The behavioral ratings and neural responses (i.e., P200, N2, LPC event-related potentials (ERPs)) to moral/immoral behaviors done by agents with respectable or ordinary professional roles were recorded and compared. Behaviorally, we found that participants rated the agent with a respectable professional role behaving immorally as more dislikable comparing to the agent with an ordinary professional role. For ERPs, we found that: 1) the agents with respectable professional roles elicited larger P200 than agents with ordinary professional roles did; 2) immoral behavior elicited larger LPC than moral behaviors did; 3) for agents with respectable professional roles, the immoral behaviors elicited significantly more positive N2 than the moral behaviors did whereas this difference was not significant for the agents with ordinary professional roles. The immoral behaviors done by agents with respectable professional roles elicited more positive N2 than the immoral behavior done by agents with ordinary professional roles. Moreover, this effect was correlated with the participants' subjective rating of the professional roles' respectable level. These results suggest that 1) the more the agents with respectable professional roles are respected, the more dislikable they became when behaving immorally; 2) the moral stereotype associated with professional roles can influence the early processing stage reflected in N2 but not the later evaluative process reflected in LPC.

Moral contagion: Devaluation effect of immorality on hypothetical judgments of economic value.

Moral contagion is a phenomenon in which individuals or objects take on the moral essence of the people who are associated with them. Previous studies have found that individuals value objects associated with moral and likable people more than those associated with immoral and dislikable people. However, the neural mechanisms underlying this "moral contagion effect" have not yet been explored. In the present study, we combined a novel "Second-hand Goods Pricing" paradigm with functional magnetic resonance imaging to (a) confirm the existence of the moral contagion effect on the hypothetical economic valuation of objects and (b) determine the neural substrates underlying it. Participants were shown second-hand goods, information regarding the moral valence of the previous owner, and an initial price assigned to the object by computer. The participants were then asked to adjust the initial price to one they deemed most reasonable. Behaviorally, we found a significant devaluation effect for immoral owners and a weaker reverse effect for moral owners. Imaging data showed that the devaluation effect was primarily driven by neural responses in the dorsal striatum (mainly the caudate nucleus) that were triggered by high initial prices assigned to the "contaminated" objects. Dynamic causal modeling revealed that the high initial price assigned to "contaminated" objects led to increased effective connectivity from the caudate nucleus to the ventromedial prefrontal cortex-the brain area that integrates values during decision making.

Social Mindfulness Shown by Individuals With Higher Status Is More Pronounced in Our Brain: ERP Evidence.

"Social mindfulness" refers to being thoughtful of others and considering their needs before making decisions, and can be characterized by low-cost and subtle gestures. The present study compared the behavioral and neural responses triggered by observing others' socially mindful/unmindful choices and how these responses were modulated by the social status of the agency. At the behavioral level, observing socially mindful choices made observers feel better, rate the actors as more likable, and behave more cooperatively than did observing socially unmindful choices. Analysis of event-related potentials in the brain revealed that compared with socially unmindful choices, mindful choices elicited more negative feedback-related negativity (FRN). Notably, while this effect of social mindfulness was only significant when the actor's social status was medium and high, it was undetectable when the actor's social status was low. These results demonstrate that the social mindfulness of others can be rapidly detected and processed, as reflected by FRN, even though it does not seem to receive further, more elaborate evaluation. These findings indicated that low-cost cooperative behaviors such as social mindfulness can also be detected and appreciated by our brain, which may result in better mood and more cooperative behaviors in the perceivers. Besides, the perception of social mindfulness is sensitive to important social information, such as social status.

Magnetic-Based Microfluidic Device for On-Chip Isolation and Detection of Tumor-Derived Exosomes.

Exosomes are membrane-enclosed phospholipid extracellular vesicles, which can act as mediators of intercellular communication. Although the original features endow tumor-derived exosomes great potential as biomarkers, efficient isolation and detection methods remain challenging. Here, we presented a two-stage microfluidic platform (ExoPCD-chip), which integrates on-chip isolation and in situ electrochemical analysis of exosomes from serum. To promote exosomes capture efficiency, an improved staggered Y-shaped micropillars mixing pattern was designed to create anisotropic flow without any surface modification. By combining magnetic enrichment based on specific phosphatidylserine-Tim4 protein recognition with a new signal transduction strategy in a chip for the first time, the proposed platform enables highly sensitive detection for CD63 positive exosomes as low as 4.39 × 103 particles/mL with a linear range spanning 5 orders of magnitude, which is substantially better than the existing methods. The reduced volume of sample (30 µL) and simple affinity method also make it ideal for rapid downstream analysis of complex biofluids within 3.5 h. As a proof-of-concept, we performed exosomes analysis in human serum and liver cancer patients can be well discriminated from the healthy controls by the ExoPCD-chip. These results demonstrate that this proposed ExoPCD-chip may serve as a comprehensive exosome analysis tool and potential noninvasive diagnostic platform.

Predictability and probability modulate the neural responses to other's pain: An event-related potential investigation.

Two ERP experiments were designed to explore the effect of predictability (Exp1) and probability (Exp2) on the perception of others' pain, respectively. In Exp1, we compared the ERP responses to painful and non-painful pictures when they were fully predictable and fully unpredictable. Results revealed that when the valence of the pictures (painful or non-painful) was fully predictable, the amplitudes of N2 and P3 components triggered by the painful pictures were significantly more positive than the amplitudes of N2 and P3 components triggered by the non-painful ones. When the valence of the pictures was fully unpredictable, the amplitudes of N2 and P3 triggered by the painful and the non-painful pictures were comparable. Besides, the P3 amplitude was positively correlated with the scores of empathy trait (i.e., personal distress). In Exp2, the probability of the presentation of a painful picture was manipulated as low (30%), medium (60%) and high (90%). Results showed that with the increase in the probability of a painful picture's presentation, the amplitude of N2 elicited decreased. No significant effect was observed on P3. These findings indicated that the early perceptual processing stage reflected in N2 was more likely to be a threat-detection stage, while the later stage reflected in P3 may actually be the stage in which participant empathized other's pain. Moreover, the more expected pain of others induced stronger empathic responses reflected in the P3. To the best of our knowledge, this study offers the very first psychophysical evidence of the predictability and probability's effect on pain empathy.

Antioxidant Role of PcGSTd1 in Fenpropathrin Resistant Population of the Citrus Red Mite, Panonychus citri (McGregor).

The citrus red mite, Panonychus citri, a major citrus pest distributed worldwide, has evolved severe resistance to various classes of chemical acaricides/insecticides including pyrethroids. It is well known that the resistance to pyrethroids is mainly caused by point mutations of voltage-gated sodium channel gene in a wide range of pests. However, increasing number of evidences support that pyrethroids resistance might also be resulted from the integrated mechanisms including metabolic mechanisms. In this study, firstly, comparative analysis of RNA-seq data showed that multiple detoxification genes, including a GSTs gene PcGSTd1, were up-regulated in a fenpropathrin-resistant population compared with the susceptible strain (SS). Quantitative real time-PCR results showed that the exposure of fenpropathrin had an induction effect on the transcription of PcGSTd1 in a time-dependent manner. In vitro inhibition and metabolic assay of recombinant PcGSTd1 found that fenpropathrin might not be metabolized directly by this protein. However, its antioxidant role in alleviating the oxidative stress caused by fenpropathrin was demonstrated via the reversely genetic experiment. Our results provide a list of candidate genes which may contribute to a multiple metabolic mechanisms implicated in the evolution of fenpropathrin resistance in the field population of P. citri. Furthermore, during the detoxification process, PcGSTd1 plays an antioxidant role by detoxifying lipid peroxidation products induced by fenpropathrin.

Iron Phthalocyanine Decorated Nitrogen-Doped Graphene Biosensing Platform for Real-Time Detection of Nitric Oxide Released from Living Cells.

Nitric oxide (NO) is a transcellular messenger involved in many physiological and pathological processes, but the real-time detection of NO in biological systems is still challenging due to its rapid diffusion, low concentration, and short half-life. A novel electrochemical sensing platform based on iron phthalocyanine (FePc) functionalized nitrogen-doped graphene (N-G) nanocomposites was constructed to achieve in situ monitoring of NO released from living cells on the sensing layer. By taking advantage of the synergetic effect of N-G and FePc nanocomposites, the N-G/FePc sensor displays excellent electrocatalytic activity toward NO with a high sensitivity of 0.21 µA µM-1 cm-2 and a low detection limit of 180 nmol L-1. The following layer-by-layer assembly of poly-l-lysine (PLL) and Nafion further improved the capacity of resisting disturbance as well as the biocompatibility of the sensing interface. The flexible design of the ITO substrate based electrode provides a more controlled cellular biosensing system which could capture molecular signals immediately after NO released from human umbilical vein endothelial cells (HUVECs). The exhibited additional features of high sensitivity, rapid response, and ease of operation implies that the proposed N-G/FePc/Nafion/PLL ITO biosensor is a promising powerful platform in various complex biological systems.

Transcranial direct current stimulation of the medial prefrontal cortex modulates the propensity to help in costly helping behavior.

Social decision-making engages traditional decision-making processes (e.g. valuation), as well as social cognition processes (e.g. inferring the affective and mental states of another person). Neuroimaging and neuro-stimulation studies have suggested the involvement of the medial prefrontal cortex (mPFC) in a variety of social decision-making tasks. Yet no study has investigated the effect of the cortical excitability of mPFC in the decision-making of costly helping behavior. Here, we used tDCS to demonstrate the causal relationship between the cortical excitability of mPFC and costly helping decision-making. Subjects assigned to the anodal, cathodal and sham groups were required to decide whether they would like to cost their own money to relieve another subject (a confederate actually) from painful electrical shocks with a certain probability of success. Results showed that the subjects receiving anodal stimulation acted more prosaically than the subjects receiving cathodal stimulation. And this effect was only significant when the probability of success was high. We proposed that tDCS induced modulation of the cortical excitability, targeting the mPFC, can affect the prosocial propensity in costly helping behavior, and the possible underlying mechanisms were discussed.

Characterization and expression patterns of key ecdysteroid biosynthesis and signaling genes in a spider mite (Panonychus citri).

Ecdysteroids play a crucial role in regulating molting in the phylum of Arthropoda and much is known with members of the subphylum of Hexapoda including the Insecta. However, this is still unclear in key pests as spider mites belonging to the subphylum of Chelicerata that originated earlier in the Cambrian period. In this study, we investigated 14 key genes of ecdysteroid biosynthesis and signaling and their expression over the different developmental stages in the citrus red mite, Panonychus citri (Acari: Stigmaeidae). P. citri is an economically important and widespread pest of citrus crops and it has five developmental stages of egg, larva, protonymph, deutonymph and adult. Typically, the expression of the ecdysteroid-synthesizing Halloween gene Spook (PcSpo) followed a positive zigzag-like pattern with a peak in the first half of each developmental stage and a drop in the second half prior to the molting to the next stage. Similar to PcSpo, PcDib, PcSad, PcRXR2, PcE75 and PcHR38 showed a positive zigzag-like expression pattern, while that of PcE78, PcHR3 and PcFTZ-F1 was opposite that we called a negative zigzag-like pattern. Silencing of the PcSpo gene by RNAi showed that molting was inhibited. Interestingly, we could rescue these RNAi effects by supplementing ponasterone A (PonA) and not by 20E, which is indicative that mites use PonA rather than 20E as ecdysteroid hormone. Modeling of the ecdysteroid receptor (PcEcR) hormone binding cavity also predicted binding of PonA, but showed a steric hindrance for 20E. We believe our data provide insight into the evolution and expression patterns of key ecdysteroid biosynthesis and signaling genes in a distant, non-insect species, and can become a foundation to develop new targets for controlling important agricultural pests such as spider mites.

Identification of responsive proteins in Panonychus citri exposed to abamectin by a proteomic approach.

Abamectin is a microbial-derived pesticide widely used for control of agricultural pests. However, sustained use of abamectin has led to the development of resistance in some target species. Previous studies on arthropod resistance to abamectin have mainly used traditional biochemical and molecular approaches. To understand the responses of citrus red mite, Panonychus citri, exposed to abamectin, comparative proteomic analysis was conducted using two-dimensional electrophoresis (2-DE). A total of 26 distinct protein spots were present in response to abamectin exposure. Tandem mass spectrometry (MS/MS) identified 16 proteins that were mainly involved in energy metabolism and detoxification. Some remaining proteins were not identifiable, suggesting that they may be novel. The expression levels of transcripts associated with proteins were analyzed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, to validate the proteomic data obtained in the present study, Western-blot experiment was performed and the expression of sHsp and PcE1 proteins were confirmed, respectively. BIOLOGICAL SIGNIFICANCE: The citrus red mite has developed resistance to many acaricides, including abamectin. In the current study, we used the proteomic approaches involving 2-DE, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and MS/MS to document changes in adult P. citri during 24h of abamectin exposure. Abamectin stress induced a total of 16 differentially regulated proteins. The proteomic results were validated in mRNA expression patterns using qRT-PCR. This is the first analysis of differentially expressed proteins in P. citri exposed to abamectin. The results help clarify the physiological mechanisms of P. citri responses to abamectin exposure.

Up-regulation of golgi α-mannosidase IA and down-regulation of golgi α-mannosidase IC activates unfolded protein response during hepatocarcinogenesis.

α-1,2 mannosidases, key enzymes in N-glycosylation, are required for the formation of mature glycoproteins in eukaryotes. Aberrant regulation of α-1,2 mannosidases can result in cancer, although the underlying mechanisms are unclear. Here, we report the distinct roles of α-1,2 mannosidase subtypes (MAN1A, MAN1B, ERMAN1, MAN1C) in the formation of hepatocellular carcinoma (HCC). Clinicopathological analyses revealed that the clinical stage, tumor size, α-fetoprotein level, and invasion status were positively correlated with the expression levels of MAN1A1, MAN1B1, and MAN1A2. In contrast, the expression of MAN1C1 was decreased as early as stage I of HCC. Survival analyses showed that high MAN1A1, MAN1A2, and MAN1B1 expression levels combined with low MAN1C1 expression levels were significantly correlated with shorter overall survival rates. Functionally, the overexpression of MAN1A1 promoted proliferation, migration, and transformation as well as in vivo migration in zebrafish. Conversely, overexpression of MAN1C1 reduced the migration ability both in vitro and in vivo, decreased the colony formation ability, and shortened the S phase of the cell cycle. Furthermore, the expression of genes involved in cell cycle/proliferation and migration was increased in MAN1A1-overexpressing cells but decreased in MAN1C1-overexpressing cells. MAN1A1 activated the expression of key regulators of the unfolded protein response (UPR), while treatment with endoplasmic reticulum stress inhibitors blocked the expression of MAN1A1-activated genes. Using the MAN1A1 liver-specific overexpression zebrafish model, we observed steatosis and inflammation at earlier stages and HCC formation at a later stage accompanied by the increased expression of the UPR modulator binding immunoglobulin protein (BiP). These data suggest that the up-regulation of MAN1A1 activates the UPR and might initiate metastasis. Conclusion: MAN1A1 represents a novel oncogene while MAN1C1 plays a role in tumor suppression in hepatocarcinogenesis. (Hepatology Communications 2017;1:230-247).

Involvement of Three Esterase Genes from Panonychus citri (McGregor) in Fenpropathrin Resistance.

The citrus red mite, Panonychus citri (McGregor), is a major citrus pest with a worldwide distribution and an extensive record of pesticide resistance. However, the underlying molecular mechanism associated with fenpropathrin resistance in this species have not yet been reported. In this study, synergist triphenyl phosphate (TPP) dramatically increased the toxicity of fenpropathrin, suggesting involvement of carboxylesterases (CarEs) in the metabolic detoxification of this insecticide. The subsequent spatiotemporal expression pattern analysis of PcE1, PcE7 and PcE9 showed that three CarEs genes were all over-expressed after insecticide exposure and higher transcripts levels were observed in different field resistant strains of P. citri. Heterologous expression combined with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) cytotoxicity assay in Spodoptera frugiperda (Sf9) cells revealed that PcE1-, PcE7- or PcE9-expressing cells showed significantly higher cytoprotective capability than parental Sf9 cells against fenpropathrin, demonstrating that PcEs probably detoxify fenpropathrin. Moreover, gene silencing through the method of leaf-mediated dsRNA feeding followed by insecticide bioassay increased the mortalities of fenpropathrin-treated mites by 31% (PcE1), 27% (PcE7) and 22% (PcE9), respectively, after individual PcE gene dsRNA treatment. In conclusion, this study provides evidence that PcE1, PcE7 and PcE9 are functional genes mediated in fenpropathrin resistance in P. citri and enrich molecular understanding of CarEs during the resistance development of the mite.

Characterization and functional analysis of a novel glutathione S-transferase gene potentially associated with the abamectin resistance in Panonychus citri (McGregor).

The citrus red mite, Panonychus citri (McGregor), a major citrus pest distributed worldwide, has been found to be resistant to various insecticides and acaricides used in China. However, the molecular mechanisms associated with the abamectin resistance in this species have not yet been reported. In this study, results showed over-expression of a novel glutathione S-transferases (GSTs) gene (PcGSTm5) in abamectin-resistant P. citri. Quantitative real-time PCR analysis showed that the transcripts of PcGSTm5 were also significantly up-regulated after exposure to abamectin and the maximum mRNA expression level at nymphal stage. The recombinant protein of PcGSTm5-pET-28a produced by Escherichia coli showed a pronounced activity toward the conjugates of 1-chloro-2,4 dinitrobenzene (CDNB) and glutathione (GSH). The kinetics of CDNB and GSH and its optimal pH and thermal stability were also determined. Reverse genetic study through a new method of leaf-mediated dsRNA feeding further support a link between the expression of PcGSTm5 and abamectin resistance. However, no direct evidence was found in metabolism or inhibition assays to confirm the hypothesis that PcGSTm5 can metabolize abamectin. Finally, it is here speculated that PcGSTm5 may play a role in abamectin detoxification through other pathway such as the antioxidant protection.