LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

The melanin pigment gene black mediates body pigmentation and courtship behaviour in the German cockroach Blattella germanica.

Genes involved in melanin production directly impact insect pigmentation and can affect diverse physiology and behaviours. The role these genes have on sex behaviour, however, is unclear. In the present study, the crucial melanin pigment gene black was functionally characterised in an urban pest, the German cockroach, Blattella germanica. RNAi knockdown of B. germanica black (Bgblack) had no effect on survival, but did result in black pigmentation of the thoraxes, abdomens, heads, wings, legs, antennae, and cerci due to cuticular accumulation of melanin. Sex-specific variation in the pigmentation pattern was apparent, with females exhibiting darker coloration on the abdomen and thorax than males. Bgblack knockdown also resulted in wing deformation and negatively impacted the contact sex pheromone-based courtship behaviour of males. This study provides evidence for black function in multiple aspects of B. germanica biology and opens new avenues of exploration for novel pest control strategies.

The microRNA-7322-5p/p38/Hsp19 axis modulates Chilo suppressalis cell-defences against Cry1Ca: an effective target for a stacked transgenic rice approach.

Bacillus thuringiensis (Bt)-secreted crystal (Cry) toxins form oligomeric pores in host cell membranes and are a common element in generating insect-resistant transgenic crops. Although Cry toxin function has been well documented, cellular defences against pore-formation have not been as well developed. Elucidation of the processes underlying this defence, however, could contribute to the development of enhanced Bt crops. Here, we demonstrate that Cry1Ca-mediated downregulation of microRNA-7322-5p (miR-7322-5p), which binds to the 3' untranslated region of p38, negatively regulates the susceptibility of Chilo suppressalis to Cry1Ca. Moreover, Cry1Ca exposure enhanced phosphorylation of Hsp19, and hsp19 downregulation increased susceptibility to Cry1Ca. Further, Hsp19 phosphorylation occurs downstream of p38, and pull-down assays confirmed the interactions between Hsp19 and Cry1Ca, suggesting that activation of Hsp19 by the miR-7322-5p/p38/Hsp19 pathway promotes Cry1Ca sequestration. To assess the efficacy of targeting this pathway in planta, double-stranded RNA (dsRNA) targeting C. suppressalis p38 (dsp38) was introduced into a previously generated cry1Ca-expressing rice line (1CH1-2) to yield a single-copy cry1Ca/dsp38 rice line (p38-rice). Feeding on this rice line triggered a significant reduction in C. suppressalis p38 expression and the line was more resistant to C. suppressalis than 1CH1-2 in both short term (7-day) and continuous feeding bioassays as well as field trials. These findings provide new insights into invertebrate epithelium cellular defences and demonstrate a potential new pyramiding strategy for Bt crops.

Isoforms of the neuropeptide myosuppressin differentially modulate the cardiac neuromuscular system of the American lobster, Homarus americanus.

Post-translational modifications (PTMs) diversify peptide structure and allow for greater flexibility within signaling networks. The cardiac neuromuscular system of the American lobster, Homarus americanus, is made up of a central pattern generator, the cardiac ganglion (CG), and peripheral cardiac muscle. Together, these components produce flexible output in response to peptidergic modulation. Here, we examined the role of PTMs in determining the effects of a cardioactive neuropeptide, myosuppressin (pQDLDHVFLRFamide), on the whole heart, the neuromuscular junction/muscle, the isolated CG, and the neurons of the CG. Mature myosuppressin and noncyclized myosuppressin (QDLDHVFLRFamide) elicited similar and significant changes in whole heart contraction amplitude and frequency, stimulated muscle contraction amplitude and the bursting pattern of the intact and ligatured neurons of the ganglion. In the whole heart, nonamidated myosuppressin (pQDLDHVFLRFG) elicited only a small decrease in frequency and amplitude. In the absence of motor neuron input, nonamidated myosuppressin did not cause any significant changes in the amplitude of stimulated contractions. In the intact CG, nonamidated myosuppressin elicited a small but significant decrease in burst duration. Further analysis revealed a correlation between the extent of modulation elicited by nonamidated myosuppressin in the whole heart and the isolated, intact CG. When the neurons of the CG were physically decoupled, nonamidated myosuppressin elicited highly variable responses. Taken together, these data suggest that amidation, but not cyclization, is critical in enabling this peptide to exert its effects on the cardiac neuromuscular system.NEW & NOTEWORTHY Myosuppressin (pQDLDHVFLRFamide), a well-characterized crustacean neuropeptide, and its noncyclized (QDLDHVFLRFamide) and nonamidated (pQDLDHVFLRFG) isoforms alter the output of the cardiac neuromuscular system of the American lobster, Homarus americanus. Mature myosuppressin and noncyclized myosuppressin elicited similar and significant changes across all levels of the isolated system, whereas responses to nonamidated myosuppressin were significantly different from other isoforms and were highly variable. These data support the diversity of peptide action as a function of peptide structure.

Reproductive capacity in Adelphocoris suturalis (Hemiptera: Miridae) is regulated by the insulin signaling pathway.

The peptide hormone insulin has essential roles in regulating insect metabolism, growth, and reproduction. There are, however, few studies assessing the effects of insulin signaling on reproduction in Miridae (Hemiptera). Here, we used RNA interference (RNAi)-mediated knockdown to examine the role of three critical insulin signaling pathway components (insulin receptor, InR; insulin receptor substrate 1, IRS1; and forkhead box O, FOXO) on reproductive capacity in the mirid Adelphocoris suturalis. Knockdown of AsIRS1 led to a significant reduction in egg maturation in unmated females. To further verify the role of AsIRS1, we examined several reproductive parameters following knockdown. Suppression of AsIRS1 transcript levels throughout the reproductive period resulted in reduced lifetime fecundity, egg hatch rate, and oviposition capacity as well as statistically significant reductions in female survival rate and longevity. These findings demonstrate that the insulin signaling pathway plays a key role in the reproductive development of A. suturalis, and that IRS1 is a key regulatory factor. These findings provide an important theoretical basis for the regulation of insect reproduction by insulin and introduce a new target for potential development is A. suturalis control.

Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning.

Peptides are the largest and most diverse class of molecules modulating physiology and behavior. Previously, we predicted a peptidome for the western tarnished plant bug, Lygus hesperus, using transcriptomic data produced from whole individuals. A potential limitation of that analysis was the masking of underrepresented genes, in particular tissue-specific transcripts. Here, we reassessed the L. hesperus peptidome using a more comprehensive dataset comprised of the previous transcriptomic data as well as tissue-specific reads produced from heads and accessory glands. This augmented assembly significantly improves coverage depth providing confirmatory transcripts for essentially all of the previously identified families and new transcripts encoding a number of new peptide precursors corresponding to 14 peptide families. Several families not targeted in our initial study were identified in the expanded assembly, including agatoxin-like peptide, CNMamide, neuropeptide-like precursor 1, and periviscerokinin. To increase confidence in the in silico data, open reading frames of a subset of the newly identified transcripts were amplified using RT-PCR and sequence validated. Further PCR-based profiling of the putative L. hesperus agatoxin-like peptide precursor revealed evidence of alternative splicing with near ubiquitous expression across L. hesperus development, suggesting the peptide serves functional roles beyond that of a toxin. The peptides predicted here, in combination with those identified in our earlier study, expand the L. hesperus peptidome to 42 family members and provide an improved platform for initiating molecular and physiological investigations into peptidergic functionality in this non-model agricultural pest.

Cloning and RNAi-mediated three lethal genes that can be potentially used for Chilo suppressalis (Lepidoptera: Crambidae) management.

RNA interference (RNAi) has gained attention in recent years as a viable pest control strategy. Here, RNAi assays were performed to screen the potential functionality of genes in Chilo suppressalis, a serious pest of rice, and to determine their potential for developing a highly targeted molecular control approach. Potential homologs of NADH dehydrogenase (ND), glycerol 3-phosphate dehydrogenase (GPDH) and male specific lethal 3 (MSL3) were cloned from C. suppressalis, and their spatiotemporal gene expression evaluated. The expression of all three genes was higher in the pupal and adult stages than the larval stages and largely higher in the larval head compared to other tissues. Newly hatched larvae exhibited high mortalities and suppressed growth when fed bacteria producing double-stranded RNAs (dsRNAs) corresponding to the three target genes. This study provides insights into the function of ND, GPDH and MSL3 during C. suppressalis larval development and suggests that all may be candidate gene targets for C. suppressalis pest management.

HPE1, an Effector from Zebra Chip Pathogen Interacts with Tomato Proteins and Perturbs Ubiquitinated Protein Accumulation.

The gram-negative bacterial genus Liberibacter includes economically important pathogens, such as 'Candidatus Liberibacter asiaticus' that cause citrus greening disease (or Huanglongbing, HLB) and 'Ca. Liberibacter solanacearum' (Lso) that cause zebra chip disease in potato. Liberibacter pathogens are fastidious bacteria transmitted by psyllids. Pathogen manipulation of the host' and vector's immune system for successful colonization is hypothesized to be achieved by Sec translocon-dependent effectors (SDE). In previous work, we identified hypothetical protein effector 1 (HPE1), an SDE from Lso, that acts as a suppressor of the plant's effector-triggered immunity (ETI)-like response. In this study, using a yeast two-hybrid system, we identify binding interactions between tomato RAD23 proteins and HPE1. We further show that HPE1 interacts with RAD23 in both nuclear and cytoplasmic compartments in planta. Immunoblot assays show that HPE1 is not ubiquitinated in the plant cell, but rather the expression of HPE1 induced the accumulation of other ubiquitinated proteins. A similar accumulation of ubiquitinated proteins is also observed in Lso infected tomato plants. Finally, earlier colonization and symptom development following Lso haplotype B infection are observed in HPE1 overexpressing plants compared to wild-type plants. Overall, our results suggest that HPE1 plays a role in virulence in Lso pathogenesis, possibly by perturbing the ubiquitin-proteasome system via direct interaction with the ubiquitin-like domain of RAD23 proteins.

Does Differential Receptor Distribution Underlie Variable Responses to a Neuropeptide in the Lobster Cardiac System?

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.

Differential neuropeptide modulation of premotor and motor neurons in the lobster cardiac ganglion.

The American lobster, Homarus americanus, cardiac neuromuscular system is controlled by the cardiac ganglion (CG), a central pattern generator consisting of four premotor and five motor neurons. Here, we show that the premotor and motor neurons can establish independent bursting patterns when decoupled by a physical ligature. We also show that mRNA encoding myosuppressin, a cardioactive neuropeptide, is produced within the CG. We thus asked whether myosuppressin modulates the decoupled premotor and motor neurons, and if so, how this modulation might underlie the role(s) that these neurons play in myosuppressin's effects on ganglionic output. Although myosuppressin exerted dose-dependent effects on burst frequency and duration in both premotor and motor neurons in the intact CG, its effects on the ligatured ganglion were more complex, with different effects and thresholds on the two types of neurons. These data suggest that the motor neurons are more important in determining the changes in frequency of the CG elicited by low concentrations of myosuppressin, whereas the premotor neurons have a greater impact on changes elicited in burst duration. A single putative myosuppressin receptor (MSR-I) was previously described from the Homarus nervous system. We identified four additional putative MSRs (MSR-II-V) and investigated their individual distributions in the CG premotor and motor neurons using RT-PCR. Transcripts for only three receptors (MSR-II-IV) were amplified from the CG. Potential differential distributions of the receptors were observed between the premotor and motor neurons; these differences may contribute to the distinct physiological responses of the two neuron types to myosuppressin.NEW & NOTEWORTHY Premotor and motor neurons of the Homarus americanus cardiac ganglion (CG) are normally electrically and chemically coupled, and generate rhythmic bursting that drives cardiac contractions; we show that they can establish independent bursting patterns when physically decoupled by a ligature. The neuropeptide myosuppressin modulates different aspects of the bursting pattern in these neuron types to determine the overall modulation of the intact CG. Differential distribution of myosuppressin receptors may underlie the observed responses to myosuppressin.

Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans.

Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eyestalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification.

AMGSEFLamide, a member of a broadly conserved peptide family, modulates multiple neural networks in Homarus americanus.

Recent genomic/transcriptomic studies have identified a novel peptide family whose members share the carboxyl terminal sequence -GSEFLamide. However, the presence/identity of the predicted isoforms of this peptide group have yet to be confirmed biochemically, and no physiological function has yet been ascribed to any member of this peptide family. To determine the extent to which GSEFLamides are conserved within the Arthropoda, we searched publicly accessible databases for genomic/transcriptomic evidence of their presence. GSEFLamides appear to be highly conserved within the Arthropoda, with the possible exception of the Insecta, in which sequence evidence was limited to the more basal orders. One crustacean in which GSEFLamides have been predicted using transcriptomics is the lobster, Homarus americanus Expression of the previously published transcriptome-derived sequences was confirmed by reverse transcription (RT)-PCR of brain and eyestalk ganglia cDNAs; mass spectral analyses confirmed the presence of all six of the predicted GSEFLamide isoforms - IGSEFLamide, MGSEFLamide, AMGSEFLamide, VMGSEFLamide, ALGSEFLamide and AVGSEFLamide - in H. americanus brain extracts. AMGSEFLamide, of which there are multiple copies in the cloned transcripts, was the most abundant isoform detected in the brain. Because the GSEFLamides are present in the lobster nervous system, we hypothesized that they might function as neuromodulators, as is common for neuropeptides. We thus asked whether AMGSEFLamide modulates the rhythmic outputs of the cardiac ganglion and the stomatogastric ganglion. Physiological recordings showed that AMGSEFLamide potently modulates the motor patterns produced by both ganglia, suggesting that the GSEFLamides may serve as important and conserved modulators of rhythmic motor activity in arthropods.

RNA interference-mediated knockdown of eye coloration genes in the western tarnished plant bug (Lygus hesperus Knight).

Insect eye coloration arises from the accumulation of various pigments. A number of genes that function in the biosynthesis (vermilion, cinnabar, and cardinal) and importation (karmoisin, white, scarlet, and brown) of these pigments, and their precursors, have been identified in diverse species and used as markers for transgenesis and gene editing. To examine their suitability as visible markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for all seven genes were identified. Bioinformatic-based sequence and phylogenetic analyses supported initial annotations as eye coloration genes. Consistent with their proposed role, each of the genes was expressed in adult heads as well as throughout nymphal and adult development. Adult eyes of those injected with double-stranded RNAs (dsRNAs) for karmoisin, vermilion, cinnabar, cardinal, and scarlet were characterized by a red band along the medial margin extending from the rostral terminus to the antenna. In contrast, eyes of insects injected with dsRNAs for both white and brown were a uniform light brown. White knockdown also produced cuticular and behavioral defects. Based on its expression profile and robust visible phenotype, cardinal would likely prove to be the most suitable marker for developing gene editing methods in Lygus species.

What can transcriptomics reveal about the phylogenetic/structural conservation, tissue localization, and possible functions of CNMamide peptides in decapod crustaceans?

Over the past several years, in silico analyses of arthropod genomes/transcriptomes have led to the identification of several previously unknown peptide families. The CNMamides are one such peptide group, having been discovered via computational analyses of the fruit fly, Drosophila melanogaster, genome; both a CNMamide precursor and receptor were identified. Recently, a CNMamide family member, VMCHFKICNLamide (disulfide bridging between the cysteine residues), was predicted via in silico mining of a crayfish, Procambarus clarkii, transcriptome, suggesting the presence of this peptide group in members of the Decapoda. Here, using publically accessible transcriptomic data, the phylogenetic/structural conservation, tissue localization, and possible functions of the CNMamide family in decapods were explored. Evidence for CNMamide precursors was found for members of each decapod infraorder for which significant sequence data are available, suggesting a ubiquitous conservation of the CNMamide family in the Decapoda. For the Penaeoidea, Caridea, Astacidea and Achelata, the isoform of CNMamide originally identified from P. clarkii appears to be ubiquitously conserved; in members of the Brachyura, VMCHFKICNMamide (disulfide bridging between the cysteine residues) is the native isoform. Interestingly, the decapod CNMamide gene appears to also have a splice variant in which the carboxy-terminal portion of the preprohormone containing the CNMamide peptide is replaced by one containing a different disulfide bridged peptide that is structurally unrelated to it; this second peptide shows considerable conservation within, but variation among, decapod infraorders. A highly conserved putative CNMamide receptor was identified from members of the Penaeoidea, Astacidea and Brachyura. Phylogenetic analyses support the annotation of the decapod receptor as a true member of the CNMamide receptor family. The presence of precursor and receptor transcripts in both nervous system- and reproductive tissue-specific transcriptomes suggests CNMamides serve as modulators of decapod neural and reproductive control systems.

Genome-Wide Analysis of Cotton miRNAs During Whitefly Infestation Offers New Insights into Plant-Herbivore Interaction.

Although the regulatory function of miRNAs and their targets have been characterized in model plants, a possible underlying role in the cotton response to herbivore infestation has not been determined. To investigate this, we performed small RNA and degradome sequencing between resistant and susceptible cotton cultivar following infestation with the generalist herbivore whitefly. In total, the 260 miRNA families and 241 targets were identified. Quantitative-PCR analysis revealed that several miRNAs and their corresponding targets exhibited dynamic spatio-temporal expression patterns. Moreover, 17 miRNA precursors were generated from 29 long intergenic non-coding RNA (lincRNA) transcripts. The genome-wide analysis also led to the identification of 85 phased small interfering RNA (phasiRNA) loci. Among these, nine PHAS genes were triggered by miR167, miR390, miR482a, and two novel miRNAs, including those encoding a leucine-rich repeat (LRR) disease resistance protein, an auxin response factor (ARF) and MYB transcription factors. Through combined modeling and experimental data, we explored and expanded the miR390-tasiARF cascade during the cotton response to whitefly. Virus-induced gene silencing (VIGS) of ARF8 from miR390 target in whitefly-resistant cotton plants increased auxin and jasmonic acid (JA) accumulation, resulting in increased tolerance to whitefly infestation. These results highlight the provides a useful transcriptomic resource for plant-herbivore interaction.

Identification and functional characterization of the pheromone biosynthesis activating neuropeptide receptor isoforms from Mamestra brassicae.

In most moth species, including Mamestra brassicae, pheromone biosynthesis activating neuropeptide (PBAN) regulates pheromone production. Generally, PBAN acts directly on the pheromone gland (PG) cells via its specific G protein-coupled receptor (i.e. PBANR) with Ca2+ as a second messenger. In this study, we identified cDNAs encoding three variants (A, B and C) of the M. brassicae PBANR (Mambr-PBANR). The full-length coding sequences were transiently expressed in cultured Trichoplusia ni cells and Sf9 cells for functional characterization. All three isoforms dose-dependently mobilized extracellular Ca2+ in response to PBAN analogs with Mambr-PBANR-C exhibiting the greatest sensitivity. Fluorescent confocal microscopy imaging studies demonstrated binding of a rhodamine red-labeled ligand (RR10CPBAN) to all three Mambr-PBANR isoforms. RR10CPBAN binding did not trigger ligand-induced internalization in cells expressing PBANR-A, but did in cells expressing the PBANR-B and -C isoforms. Furthermore, activation of the PBANR-B and -C isoforms with the 18 amino acid Mambr-pheromonotropin resulted in co-localization with a Drosophila melanogaster arrestin homolog (Kurtz), whereas stimulation with an unrelated peptide had no effect. PCR-based profiling of the three transcripts revealed a basal level of expression throughout development with a dramatic increase in PG transcripts from the day of adult emergence with PBANR-C being the most abundant.

A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability.

The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1-16h in the presence of 0-2.0% (w/v) n-dodecyl-ß-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.

Prediction of a peptidome for the western tarnished plant bug Lygus hesperus.

Many strategies for controlling insect pests require an understanding of their hormonal signaling agents, peptides being the largest and most diverse single class of these molecules. Lygus hesperus is a pest species of particular concern, as it is responsible for significant damage to a wide variety of commercially important plant crops. At present, little is known about the peptide hormones of L. hesperus. Here, transcriptomic data were used to predict a peptidome for L. hesperus. Fifty-three L. hesperus transcripts encoding peptide precursors were identified, with a subset amplified by PCR for sequence verification. The proteins deduced from these transcripts allowed for the prediction of a 119-sequence peptidome for L. hesperus. The predicted peptides include isoforms of allatostatin A, allatostatin B (AST-B), allatostatin C, allatotropin, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, GSEFLamide, insulin-like peptide, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pyrokinin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide. Of note were several isoforms of AST-B that possess -WX7Wamide carboxyl-termini rather than the stereotypical -WX6Wamide (e.g., KWQDMQNPGWamide), an allatotropin ending in -SARGFamide rather than -TARGFamide (GLKNGPLNSARGFamide), a GSEFLamide ending in -GTEFLamide (TVGTEFLamide), several orcokinins with PMDEIDR- rather than NFDEIDR- amino-termini (e.g., PMDEIDRAGFTHFV), and an eight rather than 12 amino acid long isoform of SIFamide (PPFNGSIFamide). Collectively, the L. hesperus peptidome predicted here provides a resource for initiating physiological investigations of peptidergic signaling in this species, including studies directed at the biological control of this agricultural pest.

Transcriptome analysis reveals a comprehensive insect resistance response mechanism in cotton to infestation by the phloem feeding insect Bemisia tabaci (whitefly).

The whitefly (Bemisia tabaci) causes tremendous damage to cotton production worldwide. However, very limited information is available about how plants perceive and defend themselves from this destructive pest. In this study, the transcriptomic differences between two cotton cultivars that exhibit either strong resistance (HR) or sensitivity (ZS) to whitefly were compared at different time points (0, 12, 24 and 48 h after infection) using RNA-Seq. Approximately one billion paired-end reads were obtained by Illumina sequencing technology. Gene ontology and KEGG pathway analysis indicated that the cotton transcriptional response to whitefly infestation involves genes encoding protein kinases, transcription factors, metabolite synthesis, and phytohormone signalling. Furthermore, a weighted gene co-expression network constructed from RNA-Seq datasets showed that WRKY40 and copper transport protein are hub genes that may regulate cotton defenses to whitefly infestation. Silencing GhMPK3 by virus-induced gene silencing (VIGS) resulted in suppression of the MPK-WRKY-JA and ET pathways and lead to enhanced whitefly susceptibility, suggesting that the candidate insect resistant genes identified in this RNA-Seq analysis are credible and offer significant utility. Taken together, this study provides comprehensive insights into the cotton defense system to whitefly infestation and has identified several candidate genes for control of phloem-feeding pests.

ECDYSTEROID AND CHITINASE FLUCTUATIONS IN THE WESTERN TARNISHED PLANT BUG (Lygus hesperus) PRIOR TO MOLT INDICATE ROLES IN DEVELOPMENT.

Vital physiological processes that drive the insect molt represent areas of interest for the development of alternative control strategies. The western tarnished plant bug (Lygus hesperus Knight) is a pest of numerous agronomic and horticultural crops but the development of novel control approaches is impeded by limited knowledge of the mechanisms regulating its molt. To address this deficiency, we examined the fundamental relationship underlying the hormonal and molecular components of ecdysis. At 27°C L. hesperus exhibits a temporally controlled nymph-adult molt that occurs about 4 days after the final nymph-nymph molt with ecdysteroid levels peaking 2 days prior to the final molt. Application of exogenous ecdysteroids when endogenous levels had decreased disrupted the nymphal-adult molt, with treated animals exhibiting an inability to escape the old exoskeleton and resulting in mortality compared to controls. Using accessible transcriptomic data, we identified 10 chitinase-like sequences (LhCht), eight of which had protein motifs consistent with chitinases. Phylogenetic analyses revealed orthologous relationships to chitinases critical to molting in other insects. RT-PCR based transcript profiling revealed that expression changes to four of the LhChts was coordinated with the molt period and ecdysteroid levels. Collectively, our results support a role for ecdysteroid regulation of the L. hesperus molt and suggest that cuticle clearance is mediated by LhCht orthologs of chitinases that are essential to the molt process. These results provide the initial hormonal and molecular basis for future studies to investigate the specific roles of these components in molting.