RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

<p>Abstract</p> <p>Background</p> <p>Bed bugs (<it>Cimex lectularius</it>) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of <it>C. lectularius </it>has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance.</p> <p>Results</p> <p>We performed a next-generation RNA sequencing (RNA-<it>Seq</it>) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of <it>C. lectularius</it>. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from <it>de novo </it>assembled mRNA-<it>Seq </it>tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione <it>S</it>-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid.</p> <p>Conclusions</p> <p>We developed significant molecular resources for <it>C. lectularius </it>putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-<it>Seq </it>profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in <it>C. lectularius</it>. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in <it>C. lectularius</it>.</p

Transcriptomic Signatures of Ash (Fraxinus spp.) Phloem

Ash (Fraxinus spp.) is a dominant tree species throughout urban and forested landscapes of North America (NA). The rapid invasion of NA by emerald ash borer (Agrilus planipennis), a wood-boring beetle endemic to Eastern Asia, has resulted in the death of millions of ash trees and threatens billions more. Larvae feed primarily on phloem tissue, which girdles and kills the tree. While NA ash species including black (F. nigra), green (F. pennsylvannica) and white (F. americana) are highly susceptible, the Asian species Manchurian ash (F. mandshurica) is resistant to A. planipennis perhaps due to their co-evolutionary history. Little is known about the molecular genetics of ash. Hence, we undertook a functional genomics approach to identify the repertoire of genes expressed in ash phloem.Using 454 pyrosequencing we obtained 58,673 high quality ash sequences from pooled phloem samples of green, white, black, blue and Manchurian ash. Intriguingly, 45% of the deduced proteins were not significantly similar to any sequences in the GenBank non-redundant database. KEGG analysis of the ash sequences revealed a high occurrence of defense related genes. Expression analysis of early regulators potentially involved in plant defense (i.e. transcription factors, calcium dependent protein kinases and a lipoxygenase 3) revealed higher mRNA levels in resistant ash compared to susceptible ash species. Lastly, we predicted a total of 1,272 single nucleotide polymorphisms and 980 microsatellite loci, among which seven microsatellite loci showed polymorphism between different ash species.The current transcriptomic data provide an invaluable resource for understanding the genetic make-up of ash phloem, the target tissue of A. planipennis. These data along with future functional studies could lead to the identification/characterization of defense genes involved in resistance of ash to A. planipennis, and in future ash breeding programs for marker development

Lessons from the Far End: Caterpillar FRASS-Induced Defenses in Maize, Rice, Cabbage, and Tomato

Plant defenses to insect herbivores have been studied in response to several insect behaviors on plants such as feeding, crawling, and oviposition. However, we have only scratched the surface about how insect feces induce plant defenses. In this study, we measured frass-induced plant defenses in maize, rice, cabbage, and tomato by chewing herbivores such as European corn borer (ECB), fall armyworm (FAW), cabbage looper (CL), and tomato fruit worm (TFW). We observed that caterpillar frass induced plant defenses are specific to each host-herbivore system, and they may induce herbivore or pathogen defense responses in the host plant depending on the composition of the frass deposited on the plant, the plant organ where it is deposited, and the species of insect. This study adds another layer of complexity in plant-insect interactions where analysis of frass-induced defenses has been neglected even in host-herbivore systems where naturally frass accumulates in enclosed feeding sites over extended periods of time

Allelopathic effects of glucosinolate breakdown products in Hanza (Boscia senegalensis (Pers.) Lam.) processing waste water

Boscia senegalensis is a drought resistant shrub whose seeds are used in West Africa as food. However, the seeds, or hanza, taste bitter which can be cured by soaking them in water for 4-7 days. The waste water resulting from the processing takes up the bitter taste, which makes it unsuitable for consumption. When used for irrigation, allelopathic effects were observed. Glucosinolates and their breakdown products are the potential causes for both the bitter taste and the allelopathic effects. The objectives of this study are to identify and quantify the glucosinolates present in processed and unprocessed hanza as well as different organs of B. senegalensis, to analyze the chemical composition of the processing water, and to pinpoint the causal agent for the allelopathic properties of the waste water. Hanza (seeds without testa), leaves, branches, unripe and ripe fruits were collected in three populations and subjected to glucosinolate analyses. Methylglucosinolates were identified in all plant parts and populations, with the highest concentrations being found in the hanza. The levels of methylglucosinolates in the hanza reduced significantly during the soaking process. Waste water was collected for 6 days and contained large amounts of macro- and micronutrients, methylglucosinolate as well as methylisothiocyanate, resulting from the conversion of glucosinolates. Waste water from days 1-3 (High) and 4-6 (Low) was pooled and used to water seeds from 11 different crops and weeds. The High treatment significantly delayed or reduced germination of all the plant species tested. Using similar levels of methylisothiocyanate as detected in the waste water, we found that germination of a subset of the plant species was inhibited equally to the waste water treatments. This confirmed that the levels of methylisiothiocyanate in the waste water were sufficient to cause the allelopathic effect. This leads to the possibility of using hanza waste water in weed control programs

Host plant driven transcriptome plasticity in the salivary glands of the cabbage looper (<i>Trichoplusia ni</i>)

<div><p>Generalist herbivores feed on a wide array of plants and need to adapt to varying host qualities and defenses. One of the first insect derived secretions to come in contact with the plant is the saliva. Insect saliva is potentially involved in both the pre-digestion of the host plant as well as induction/suppression of plant defenses, yet how the salivary glands respond to changes in host plant at the transcriptional level is largely unknown. The objective of this study was to determine how the labial salivary gland transcriptome varies according to the host plant on which the insect is feeding. In order to determine this, cabbage looper (<i>Trichoplusia ni</i>) larvae were reared on cabbage, tomato, and pinto bean artificial diet. Labial glands were dissected from fifth instar larvae and used to extract RNA for RNASeq analysis. Assembly of the resulting sequencing reads resulted in a transcriptome library for <i>T</i>. <i>ni</i> salivary glands consisting of 14,037 expressed genes. Feeding on different host plant diets resulted in substantial remodeling of the gland transcriptomes, with 4,501 transcripts significantly differentially expressed across the three treatment groups. Gene expression profiles were most similar between cabbage and artificial diet, which corresponded to the two diets on which larvae perform best. Expression of several transcripts involved in detoxification processes were differentially expressed, and transcripts involved in the spliceosome pathway were significantly downregulated in tomato-reared larvae. Overall, this study demonstrates that the transcriptomes of the salivary glands of the cabbage looper are strongly responsive to diet. It also provides a foundation for future functional studies that can help us understand the role of saliva of chewing insects in plant-herbivore interactions.</p></div

Differential expression (DE) of genes involved in the detoxification of cabbage and tomato specific defenses.

<p>A. List of defensive compounds associated with different hosts (cabbage and tomato) and insect-related detoxification genes. B. Heatmap of detoxification genes in three comparisons–Tomato vs. Cabbage, Tomato vs. Artificial Diet, and Cabbage vs. Artificial Diet. Darker colors (purple) indicate upregulation in each comparison, light blue indicates no significant differential expression and white indicates downregulation. Only transcripts differentially expressed in at least one of the comparisons were included [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182636#pone.0182636.ref026" target="_blank">26</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182636#pone.0182636.ref040" target="_blank">40</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182636#pone.0182636.ref065" target="_blank">65</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182636#pone.0182636.ref069" target="_blank">69</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182636#pone.0182636.ref086" target="_blank">86</a>].</p

A Beneficial Plant-Associated Fungus Shifts the Balance toward Plant Growth over Resistance, Increasing Cucumber Tolerance to Root Herbivory

Plants allocate their limited resources toward different physiological processes, dynamically adjusting their resource allocation in response to environmental changes. How beneficial plant-associated microbes influence this allocation is a topic that continues to interest plant biologists. In this study, we examined the effect of a beneficial fungus, Phialemonium inflatum, on investment in growth and anti-herbivore resistance traits in cucumber plants (Cucumis sativus). We inoculated cucumber seeds with P. inflatum spores and measured several growth parameters, including germination rate, above and belowground biomass, and number of flowers. We also examined plant resistance to adult and larval striped cucumber beetles (Acalymma vitattum), and quantified levels of defense hormones in leaves and roots. Our results indicate that P. inflatum strongly enhances cucumber plant growth and reproductive potential. Although fungus treatment did not improve plant resistance to cucumber beetles, inoculated plants were more tolerant to root herbivory, experiencing less biomass reduction. Together, these findings document how a beneficial plant-associated fungus shifts plant investment in growth over herbivore resistance, highlighting the importance of microbes in mediating plant-herbivore interactions. These findings also have important implications for agricultural systems, where beneficial microbes are often introduced or managed to promote plant growth or enhance resistance

Gene expression of detoxification genes in salivary glands of cabbage looper feeding on wildtype and mutant (def-1) tomatoes.

<p>Salivary glands were dissected from 5^th instar larvae fed on wildtype or mutant tomatoes and used for real time qPCR analyses. Grey and white bars indicate expression of detoxification genes on salivary glands of larvae fed on wildtype and mutant tomatoes, respectively. Bars indicate standard error. Differences were analyzed using the nonparametric test Mann-Whitney U test. * indicates significance at p<0.05.</p