Establishment of a Faba Bean Banker Plant System with Predator Orius strigicollis for the Control of Thrips Dendrothrips minowai on Tea Plants under Laboratory Conditions.

The stick tea thrip Dendrothrips minowai (Priesner) (Thysanoptera: Thripidae) is a destructive pest in tea plantations in south and southwest China. To control this pest, a non-crop banker plant system was developed using a polyphagous predator Orius strigicollis (Poppius) (Heteroptera: Anthocoridae) with the black bean aphid Aphis fabae (Scopoli) (Hemiptera: Aphididae) as an alternative prey and the faba bean Vicia faba as the banker plant to support the predator in targeting the pest. The fitness of A. fabae on tea plants and faba bean was evaluated to determine its host specificity. Moreover, the control efficacy of the banker plant system on D. minowai on tea plants was tested in the laboratory and compared with that of direct release of O. strigicollis. The experiments showed that faba bean was an excellent non-crop host for A. fabae because, while the aphid population increased quickly on faba bean, it could only survive for up to 9 days on tea plants. Compared with direct release of O. strigicollis, lower densities of pest were observed when introducing the banker plant system. Our results indicate that this banker plant system has the potential to be implemented in the field to improve the control of the pest thrips.

Orius similis (Hemiptera: Anthocoridae): A Promising Candidate Predator of Spodoptera frugiperda (Lepidoptera: Noctuidae).

The bug Orius similis Zheng is a native generalist predator of insect pests in southern China. The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a notorious defoliator that is now an economically important insect pest throughout China. To investigate the ability of O. similis to control FAW, we evaluated the predatory capacity, behavior, and functional response of O. similis with respect to FAW and their olfactory response. Both females and males successfully preyed on FAW eggs and first-instar larvae but not on second-instar or older larvae. Adult O. similis only attacked and killed one egg or one larva at a time before sucking the prey, and similar predatory behavior was also observed with regard to FAW egg masses. Both female and male O. similis exhibited type II functional response when preying on FAW eggs and first-instar larvae. Maximum estimated prey consumed per day was 23.7 eggs and 26.2 larvae for adult females and 22.5 eggs and 19.6 larvae for adult males. Moreover, in a Y-tube olfactometer experiment, both female and male O. similis exhibited a significant preference for maize (Zea mays L.) seedlings damaged by FAW over clean air, healthy seedlings, FAW feces, and FAW, suggesting that seedlings damaged by FAW may releases specific volatiles attracting the predator. Collectively, the results of the study suggest that O. similis is a promising candidate for the biological control of FAW eggs and first-instar larvae, particularly given its attraction to FAW-damaged leaves, which may enable it to locate the target prey rapidly.

Efficacy of Tong-Xie-Yao-Fang granule and its impact on whole transcriptome profiling in diarrhea-predominant irritable bowel syndrome patients: study protocol for a randomized controlled trial.

BACKGROUND: Irritable bowel syndrome (IBS) is one kind of common functional bowel disease with obscure pathogenesis, and exploration about whole transcriptome profiling in IBS-D is still negligible. Conventional medications have limited effects, which makes focus shifted to traditional Chinese medicine (TCM). Tong-Xie-Yao-Fang, as a classic herbal formula in TCM, is pretty effective and safe for the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D), but the underlying therapeutic mechanism remains unknown. We aim to verify the efficacy and safety of TXYF granule (the formula particles mixed together) in IBS-D and elucidate the gene-level mechanism of IBS-D and therapeutic targets of TXYF granule based on whole transcriptome analysis. METHODS/DESIGN: This is a randomized, double-blind, and placebo-controlled clinical trial consisting of 2 weeks of run-in period, 12 weeks of treatment period, and 8 weeks of follow-up period. We will enroll 120 participants with IBS-D, who will be randomly assigned to the TXYF granule group and the placebo group, and recruit additional 10 healthy individuals as controls for mechanistic outcome. The two groups respectively take TXYF granule or placebo orally for treatment. The primary outcome is the response rate of IBS-Symptom Severity Score (IBS-SSS). The secondary outcomes include adequate relief (AR), IBS-Quality of Life Questionnaire (IBS-QOL), and long-term efficacy. Mechanistic outcome is the whole transcriptome profiling of the intestinal mucosae from IBS participants before and after the treatment and healthy individuals. DISCUSSION: This trial will prove the effectiveness and safety of TXYF granule with high-quality evidence and provide a penetrating and comprehensive perspective on the molecular mechanism of IBS-D by whole transcriptome analysis, which makes us pinpoint specific biomarkers of IBS-D and therapeutic targets of TXYF. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR-IOR-1900021785 . Registered on 9 March 2019.

Gross saponin of Tribulus terrestris improves erectile dysfunction in type 2 diabetic rats by repairing the endothelial function of the penile corpus cavernosum.

OBJECTIVE: To investigate the effect of gross saponins of Tribulus terrestris (GSTT) on erectile function in rats resulting from type 2 diabetes mellitus (T2DMED). METHODS: The T2DMED model was constructed by high-fat and high-sugar feeding and streptozotocin injection. After 4 weeks of GSTT intervention. Intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured in each group. The level of nitric oxide (NO) in the cavernous tissue was detected using the nitrate reductase method. The production of reactive oxygen species (ROS) was detected using DHE fluorescent probe detection. Cyclic adenosine monophosphate (cGMP) level was detected by enzyme-linked immunosorbent assay, and endothelial nitric oxide synthase (eNOS) was detected using immunohistochemistry. Masson staining was used to detect the cavernosal smooth muscle/collagen ratio. Apoptosis in endothelial cells was measured using TUNEL. Western blotting method to detect the protein expression level of eNOS, TIMP-1, cleaved caspase 3, and cleaved caspase 9. RESULTS: After treatment, the ICP and ICP/MAP values of the GSTT were significantly higher than those of the T2DMED group (P<0.05). Unlike the T2DMED group, the GSTT group showed significantly increased NO levels (P<0.05) and decreased ROS levels (P<0.05). There was no significant difference between the GSTT group and the sildenafil group in increasing cGMP levels (P>0.05), and the mixed group had higher levels than these two groups (P<0.05). Immunohistochemistry and Western blotting showed that the expression of eNOS in the GSTT was significantly higher than that in the T2DMED groups (P<0.05). Masson staining showed that the smooth muscle/collagen ratio of the GSTT group was significantly higher than that of the T2DMED groups (P<0.05), the expression of TIMP-1 was lower than that of T2DMED group (P<0.05). TUNEL assay showed that the apoptotic index and cleaved caspase 3 and cleaved caspase 9 expression level of GSTT group were lower than that of the T2DMED group (P<0.05). CONCLUSION: GSTT can protect T2DMED rats' erectile function by improving penile endothelial function and inhibiting cavernosum fibrosis, inhibiting apoptosis, and is synergistic with sildenafil.

Screening potential reference genes for quantitative real-time PCR analysis in the oriental armyworm, Mythimna separata.

The oriental armyworm, Mythimna separata, is a major insect pest in China and other Asian countries. Unfortunately, suitable reference genes for quantitative real-time PCR (qRT-PCR) have not been previously identified in M. separata for normalizing target gene expression. In this study, we evaluated the expression stability of eight candidate genes (18S, ACT, EF1-α, GAPDH, RPS7, RPS13, RPL32 and TUB) in M. separata using the comparative ΔCt method, BestKeeper, Normfinder geNorm and ReFinder, a comprehensive software platform. The results indicated that the appropriate reference gene varied depending on the experimental conditions. We found that ACTIN, EF1-α and TUB were optimal for different developmental stages; TUB, RPS13 and EF1-α showed the most stable expresssion in different tissues; RPS13 and 18S were the best reference genes for monitoring expression under high temperature conditions; TUB, RPS13 and RPS7 exhibited the most stable expression under larval-crowding conditions; RPS7, EF1-α, RPL32 and GAPDH were the best for pesticide exposure experiments. This study provides tools for reliable normalization of qRT-PCR data and forms a foundation for functional studies of target gene expression in M. separata.

Differential tolerance capacity to unfavourable low and high temperatures between two invasive whiteflies.

Thermal response and tolerance to ambient temperature play important roles in determining the geographic distribution and seasonal abundance of insects. We examined the survival and performance, as well as expression of three heat shock protein related genes, of two species of invasive whiteflies, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), of the Bemisia tabaci species complex following exposure to a range of low and high temperatures. Our data demonstrated that the MED species was more tolerant to high temperatures than the MEAM1 species, especially in the adult stage, and this difference in thermal responses may be related to the heat shock protein related genes hsp90 and hsp70. These findings may assist in understanding and predicting the distribution and abundance of the two invasive whiteflies in the field.

Temporal changes of symbiont density and host fitness after rifampicin treatment in a whitefly of the Bemisia tabaci species complex.

Microbial symbionts are essential or important partners to phloem-feeding insects. Antibiotics have been used to selectively eliminate symbionts from their host insects and establish host lines with or without certain symbionts for investigating functions of the symbionts. In this study, using the antibiotic rifampicin we attempted to selectively eliminate certain symbionts from a population of the Middle East-Asia Minor 1 whitefly of the Bemisia tabaci species complex, which harbors the primary symbiont "Candidatus Portiera aleyrodidarum" and two secondary symbionts "Candidatus Hamiltonella defensa" and Rickettsia. Neither the primary nor the secondary symbionts were completely depleted in the adults (F0) that fed for 48 h on a diet treated with rifampicin at concentrations of 1-100 µg/mL. However, both the primary and secondary symbionts were nearly completely depleted in the offspring (F1) of the rifampicin-treated adults. Although the F1 adults produced some eggs (F2), most of the eggs failed to hatch and none of them reached the second instar, and consequently the rifampicin-treated whitefly colony vanished at the F2 generation. Interestingly, quantitative polymerase chain reaction assays showed that in the rifampicin-treated whiteflies, the density of the primary symbiont was reduced at an obviously slower pace than the secondary symbionts. Mating experiments between rifampicin-treated and untreated adults demonstrated that the negative effects of rifampicin on host fitness were expressed when the females were treated by the antibiotic, and whether males were treated or not by the antibiotic had little contribution to the negative effects. These observations indicate that with this whitefly population it is not feasible to selectively eliminate the secondary symbionts using rifampicin without affecting the primary symbiont and establish host lines for experimental studies. However, the extinction of the whitefly colony at the second generation after rifampicin treatment indicates the potential of the antibiotic as a control agent of the whitefly pest.

Differential temporal changes of primary and secondary bacterial symbionts and whitefly host fitness following antibiotic treatments.

Where multiple symbionts coexist in the same host, the selective elimination of a specific symbiont may enable the roles of a given symbiont to be investigated. We treated the Mediterranean species of the whitefly Bemisia tabaci complex by oral delivery of the antibiotic rifampicin, and then examined the temporal changes of its primary symbiont "Candidatus Portiera aleyrodidarum" and secondary symbiont "Ca. Hamiltonella defensa" as well as host fitness for three generations. In adults treated with rifampicin (F0), the secondary symbiont was rapidly reduced, approaching complete disappearance as adults aged. In contrast, the primary symbiont was little affected until later in the adult life. In the offspring of these adults (F1), both symbionts were significantly reduced and barely detectable when the hosts reached the adult stage. The F1 adults laid few eggs (F2), all of which failed to hatch. Mating experiments illustrated that the negative effects of rifampicin on host fitness were exerted via female hosts but not males. This study provides the first evidence of differential temporal reductions of primary and secondary symbionts in whiteflies following an antibiotic treatment. Studies that disrupt functions of bacterial symbionts must consider their temporal changes.

Polymorphic microsatellite markers for Allium mongolicum Regel (Amaryllidaceae).

We developed polymorphic microsatellite markers in Allium mongolicum Regel, a desert plant widely distributed in western China. To better conserve this species, we need to investigate its genetic diversity and population genetic structure, as well as its evolutionary history. Using the combined biotin capture technique, we isolated 13 novel polymorphic microsatellite loci and evaluated their characteristics using 60 individuals from two populations of A. mongolicum. Two to fourteen alleles per locus were identified for these microsatellites. The observed and expected heterozygosities ranged from 0.132 to 0.875 and 0.447 to 0.986, respectively. These microsatellite markers can be used to assess the genetic variation and genetic structure of A. mongolicum.

The immune strategy and stress response of the Mediterranean species of the Bemisia tabaci complex to an orally delivered bacterial pathogen.

BACKGROUND: The whitefly, Bemisia tabaci, a notorious agricultural pest, has complex relationships with diverse microbes. The interactions of the whitefly with entomopathogens as well as its endosymbionts have received great attention, because of their potential importance in developing novel whitefly control technologies. To this end, a comprehensive understanding on the whitefly defense system is needed to further decipher those interactions. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a comprehensive investigation of the whitefly's defense responses to infection, via oral ingestion, of the pathogen, Pseudomonas aeruginosa, using RNA-seq technology. Compared to uninfected whiteflies, 6 and 24 hours post-infected whiteflies showed 1,348 and 1,888 differentially expressed genes, respectively. Functional analysis of the differentially expressed genes revealed that the mitogen associated protein kinase (MAPK) pathway was activated after P. aeruginosa infection. Three knottin-like antimicrobial peptide genes and several components of the humoral and cellular immune responses were also activated, indicating that key immune elements recognized in other insect species are also important for the response of B. tabaci to pathogens. Our data also suggest that intestinal stem cell mediated epithelium renewal might be an important component of the whitefly's defense against oral bacterial infection. In addition, we show stress responses to be an essential component of the defense system. CONCLUSIONS/SIGNIFICANCE: We identified for the first time the key immune-response elements utilized by B. tabaci against bacterial infection. This study provides a framework for future research into the complex interactions between whiteflies and microbes.

Analysis of whitefly transcriptional responses to Beauveria bassiana infection reveals new insights into insect-fungus interactions.

BACKGROUND: The fungal pathogen, Beauveria bassiana, is an efficient biocontrol agent against a variety of agricultural pests. A thorough understanding of the basic principles of insect-fungus interactions may enable the genetic modification of Beauveria bassiana to enhance its virulence. However, the molecular mechanism of insect response to Beauveria bassiana infection is poorly understood, let alone the identification of fungal virulent factors involved in pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here, next generation sequencing technology was applied to examine the expression of whitefly (Bemisia tabaci) genes in response to the infection of Beauveria bassiana. Results showed that, compared to control, 654 and 1,681genes were differentially expressed at 48 hours and 72 hours post-infected whiteflies, respectively. Functional and enrichment analyses indicated that the DNA damage stimulus response and drug metabolism were important anti-fungi strategies of the whitefly. Mitogen-activated protein kinase (MAPK) pathway was also likely involved in the whitefly defense responses. Furthermore, the notable suppression of general metabolism and ion transport genes observed in 72 hours post-infected B. tabaci might be manipulated by fungal secreted effectors. By mapping the sequencing tags to B. bassiana genome, we also identified a number of differentially expressed fungal genes between the early and late infection stages. These genes are generally associated with fungal cell wall synthesis and energy metabolism. The expression of fungal cell wall protein genes might play an important role in fungal pathogenesis and the dramatically up-regulated enzymes of carbon metabolism indicate the increasing usage of energy during the fungal infection. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first report on the molecular mechanism of fungus-whitefly interactions. Our results provide a road map for future investigations on insect-pathogen interactions and genetically modifying the fungus to enhance its efficiency in whitefly control.

Cloning and functional characterization of c-Jun NH2-terminal kinase from the Mediterranean species of the Whitefly Bemisia tabaci complex.

c-Jun NH2-terminal kinase (JNK) signaling is a highly conserved pathway that controls gene transcription in response to a wide variety of biological and environmental stresses. In this study, a JNK from the invasive Mediterranean (MED) species of the whitefly Bemisia tabaci complex was cloned and characterized. The full-length JNK cDNA of MED consists of 1565 bp, with an 1176 bp open reading frame encoding 392 amino acids. Comparison of JNK amino acid sequences among different species showed that the sequences of JNKs are highly conserved. To reveal its biological function, the gene expression and functional activation of JNK were analyzed during various stress conditions. Quantitative RT-PCR analysis showed that the relative expression level of JNK remained hardly unchanged when the insects were transferred from cotton (a suitable host plant) to tobacco (an unsuitable host plant), infected with bacteria and treated with high and low temperatures. However, the mRNA level of JNK significantly increased when treated with fungal pathogens. Furthermore, we found that the amount of phosphorylated JNK increased significantly after fungal infection, while there is no obvious change for phosphorylated p38 and ERK. Our results indicate that the whitefly JNK plays an important role in whitefly's immune responses to fungal infection.

Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2013-31 March 2013.

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross-tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii.