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Additional file 5: Figure S2. Expression pattern (RT-PCR) and relative expression levels (RT-qPCR) of Orco and SlitOR40 across different instars. (A) RT-PCR products obtained from SlitOrco and SlitOR40 amplification from the cDNA of first, second, third and fourth instar larval heads and adult antennae. (B) Quantitative RT-qPCR expression levels (relative fold-change) of Orco in first instar larval heads, fourth instar larval heads and adult male antennae using ��-actin, L13A and Ef1A as reference genes. Male antennae samples expressed significantly higher levels of Orco compared to samples from first and fourth instar larvae (Df = 28, t= -22.622, P

Additional file 11: Table S5. List of chemical compounds used in the electrophysiological recordings.

Additional file 2: Table S2. Expression profile of Spodoptera littoralis ORs (SlitORs) in the first and fourth instar larval heads. Expression levels of ORs are displayed as FPKM values.

Additional file 7: Figure S4. Attraction of Spodoptera littoralis wild type and SlitOR40-knockout larvae to positive control odors: Attraction of first and fourth instar WT and KO genotypes to positive control odors (CLD, cotton leaf discs) in both arms of the olfactometer (first instar WT: n = 44; fourth instar WT: n = 24; first instar KO: n = 59; fourth instar KO: n = 43) (top bar in Fig. S4A, S4B, S4C and S4D, respectively). Similarly, behavioral responses of WT and KO genotypes of S. littoralis first and fourth instar to the odor of cotton leaf discs versus clean air (control blank) (first instar WT: n = 50; fourth instar WT: n = 48; first instar KO: n = 21; fourth instar KO: n = 32) (bottom bar in Fig. S4A, S4B, S4C and S4D, respectively).

Additional file 4: Figure S1. RT-PCR gel images of PCR-amplified ORs at different developmental stages of Spodoptera littoralis. Comparison included negative-control (control), first instar heads (Slit_1), fourth instar heads (Slit_4), male antennae (Slit_���) and female antennae (Slit_���) with 1kb gene ruler ladder (Thermo Fischer Scientific). Red rectangles around SlitOR4 and SlitOR40 represent differential expression in the first and fourth instar, respectively, supported by RT-PCR and transcriptomic analysis.

Additional file 9: Figure S6. Maximum-likelihood phylogenetic tree of ORs expressed in larvae and adults of Spodoptera littoralis: The analyses included all described ORs from S. littoralis (purple), B. mori (blue) and H. armigera (green). Highlighted clades: Orco (cadet blue); SlitOR40 and SlitOR70 along with ORs from B. mori and H. armigera (lilac). The unrooted phylogenetic tree was built using online tool PhyML 3.0.

Additional file 6: Figure S3. SlitOR40 CRISPR/Cas9 induced indels (insertion and deletion). (A) Wild Type (WT) cDNA sequence including exon1 and exon2. An asterisk separates the two exons��� sequences. Blue letters indicate the primer sequences used for genotyping. Orange and purple boxes surround sequence regions where the Cas9 cleaved SlitOR40 and correspond to the same color boxes in B. (B) The three most prominent double mutational events obtained are shown around each of the two CRISPR guides. Indels��� produced between the two guides are described (NA means not applied) and insertion sequences are indicated in red letters. The number of nucleotide differences (��) between the WT line and the three mutant obtained lines are given between brackets. In both A and B, green letters show the CRISPR guide sequences with their PAM highlighted in yellow. (C) Electrophoregram traces for each of the three main double mutations obtained (BioEdit). Line 10 mutants were chosen for further crossing and phenotypic analysis.

Additional file 3: Table S3. Newly identified SlitOR70 amino acid sequence.

Additional file 8: Figure S5. EAG responses of Spodoptera littoralis wild type and SlitOr40-knockout moth antennae to positive control odors: The EAG responses of WT and KO line adult moth antennae to positive control stimuli (guaiacol and (��)-linalool) were tested at 10 ��g dose (minimum n = 9 per sex). There was no statistical difference in responses to positive control stimuli, guaiacol (ANOVA: Sex: Df = 1; F = 1.63; P = 0.2; Genotype: Df = 1; F = 3.55; P = 0.066; Interaction: Df = 1; F = 0.13; P = 0.72; Residual Df = 47) and (��)-linalool (ANOVA: Sex: Df = 1; F = 0.66; P = 0.42; Genotype: Df = 1; F = 0.812; P = 0.372; Interaction: Df = 1; F = 1.68; P = 0.202; Residual Df = 47) in the antennae from both genotypes. The boxplots represent mean values along with the minimum and maximum values.

Background: The detection of environmental cues and signals via the sensory system directs behavioral choices in diverse organisms. Insect larvae rely on input from the chemosensory system, mainly olfaction, for locating food sources. In several lepidopteran species, foraging behavior and food preferences change across larval instars; however, the molecular mechanisms underlying such behavioral plasticity during larval development are not fully understood. Here, we hypothesize that expression patterns of odorant receptors (ORs) change during development, as a possible mechanism influencing instar-specific olfactory-guided behavior and food preferences.Results: We investigated the expression patterns of ORs in larvae of the cotton leafworm Spodoptera littoralis between the first and fourth instar and revealed that some of the ORs show instar-specific expression. We functionally characterized one OR expressed in the first instar, SlitOR40, as responding to the plant volatile, beta-caryophyllene and its isomer alpha-humulene. In agreement with the proposed hypothesis, we showed that first but not fourth instar larvae responded behaviorally to beta-caryophyllene and alpha-humulene. Moreover, knocking out this odorant receptor via CRISPR-Cas9, we confirmed that instar-specific responses towards its cognate ligands rely on the expression of SlitOR40.Conclusion: Our results provide evidence that larvae of S. littoralis change their peripheral olfactory system during development. Furthermore, our data demonstrate an unprecedented instar-specific behavioral plasticity mediated by an OR, and knocking out this OR disrupts larval behavioral plasticity. The ecological relevance of such behavioral plasticity for S. littoralis remains to be elucidated, but our results demonstrate an olfactory mechanism underlying this plasticity in foraging behavior during larval development.

Background Deciphering the molecular mechanisms mediating the chemical senses, taste, and smell has been of vital importance for understanding the nature of how insects interact with their chemical environment. Several gene families are implicated in the uptake, recognition, and termination of chemical signaling, including binding proteins, chemosensory receptors and degrading enzymes. The cotton leafworm, Spodoptera littoralis, is a phytophagous pest and current focal species for insect chemical ecology and neuroethology. Results We produced male and female Illumina-based transcriptomes from chemosensory and non-chemosensory tissues of S. littoralis, including the antennae, proboscis, brain and body carcass. We have annotated 306 gene transcripts from eight gene families with known chemosensory function, including 114 novel candidate genes. Odorant receptors responsive to floral compounds are expressed in the proboscis and may play a role in guiding proboscis probing behavior. In both males and females, expression of gene transcripts with known chemosensory function, including odorant receptors and pheromone-binding proteins, has been observed in brain tissue, suggesting internal, non-sensory function for these genes. Conclusions A well-curated set of annotated gene transcripts with putative chemosensory function is provided. This will serve as a resource for future chemosensory and transcriptomic studies in S. littoralis and closely related species. Collectively, our results expand current understanding of the expression patterns of genes with putative chemosensory function in insect sensory and non-sensory tissues. When coupled with functional data, such as the deorphanization of odorant receptors, the gene expression data can facilitate hypothesis generation, serving as a substrate for future studies. Electronic supplementary material The online version of this article (10.1186/s12864-019-5815-x) contains supplementary material, which is available to authorized users.

A group of relatively small moths in the family Tortricidae is commonly the most destructive insect pests of deciduous tree fruits throughout the world (van der Geest and Evenhuis 1991). Within each geographical area there is typically a defined group of tortricids. This group can include key species whose larvae feed on fruit, externally or internally, and which drives the entire seasonal pest management programme, and any number of external feeders whose larvae may cause sporadic fruit injury and require occasional management. Successful control of tortricids can have a significant impact on the global trade of tree fruits and various phytosanitary regulations are in place to counter their accidental introduction into new areas. This chapter summarizes the recent literature associated with several applied aspects of tortricid management.

Examination of closely related species pairs is suggested for evolutionary comparisons of different degrees of polyphagy, which we did here with three taxa of lepidopteran herbivores, Spodoptera spp (S. littoralis, S. frugiperda maize (C) and rice (R) strains) for a RNAseq analysis of the midguts from the 3rd instar insect larvae for differential metabolic responses after feeding on pinto bean based artificial diet vs maize leaves. Paired-end (2×100 bp) Illumina HiSeq2500 sequencing resulted in a total of 24, 23, 24, and 21 million reads for the SF-C-Maize, SF-C-Pinto, SF-R-Maize, SF-R Pinto, and a total of 35 and 36 million reads for the SL-Maize and SL-Pinto samples, respectively. After quality control measures, a total of 62.2 million reads from SL and 71.7 million reads from SF were used for transcriptome assembly (TA). The resulting final de novo reference TA (backbone) for the SF taxa contained 37,985 contigs with a N50 contig size of 1030 bp and a maximum contig length of 17,093 bp, while for SL, 28,329 contigs were generated with a N50 contig size of 1980 bp and a maximum contig length of 18,267 bp. The data presented herein contains supporting information related to our research article Roy et al. (2016) http://dx.doi.org/10.1016/j.ibmb.2016.02.006[1]., Graphical abstract fx1

Olfaction plays a dominant role in the mate-finding and host selection behaviors of the codling moth (Cydia pomonella), an important pest of apple, pear and walnut orchards. Antennal transcriptome analysis (Bengtsson et al. 2012, Walker et al. 2016) revealed a number of abundantly expressed genes related to the moth olfactory system, including those encoding the olfactory receptors (ORs) CpomOR1, CpomOR3 and CpomOR6a, which belong to the putative pheromone receptor (PR) lineage, and the co-receptor (CpomOrco). Using heterologous expression, in both human embryonic kidney (HEK293T) cells and in Drosophila olfactory sensory neurons, coupled with calcium imaging and electrophysiological recording, respectively, we characterize the basic physiological and pharmacological properties of these receptors and demonstrate that they form functional ionotropic receptor channels.

Background Mate finding and recognition in animals evolves during niche adaptation and involves social signals and habitat cues. Drosophila melanogaster and related species are known to be attracted to fermenting fruit for feeding and egg-laying, which poses the question of whether species-specific fly odours contribute to long-range premating communication. Results We have discovered an olfactory channel in D. melanogaster with a dual affinity to sex and food odorants. Female flies release a pheromone, (Z)-4-undecenal (Z4-11Al), that elicits flight attraction in both sexes. Its biosynthetic precursor is the cuticular hydrocarbon (Z,Z)-7,11-heptacosadiene (7,11-HD), which is known to afford reproductive isolation between the sibling species D. melanogaster and D. simulans during courtship. Twin olfactory receptors, Or69aB and Or69aA, are tuned to Z4-11Al and food odorants, respectively. They are co-expressed in the same olfactory sensory neurons, and feed into a neural circuit mediating species-specific, long-range communication; however, the close relative D. simulans, which shares food resources with D. melanogaster, does not respond to Z4-11Al. Conclusion The Or69aA and Or69aB isoforms have adopted dual olfactory traits. The underlying gene yields a collaboration between natural and sexual selection, which has the potential to drive speciation.

WOS: 000346034600019, PubMed ID: 24668906, BACKGROUND: Acetamiprid is a neonicotinoid insecticide that is effective against both soil and plant insects, including insects of the orders Lepidoptera, Coleoptera, Homoptera and Thysanoptera. In order to estimate the effects associated with insecticide exposure and devise better pest management tactics, a field population of Phenacoccus solenopsis was exposed to acetamiprid in the laboratory. Subsequently, cross-resistance and the effects of acetamiprid on the biological parameters of P. solenopsis were investigated. RESULTS: Following five rounds of selection with acetamiprid, P. solenopsis developed a 315-fold greater resistance to this chemical compared with an unexposed control population. The selected population also demonstrated very high to moderate cross-resistance to other tested insecticides. Furthermore, acetamiprid resistance remained unstable when the acetamiprid-selected population was not exposed for a further five generations. The acetamiprid-selected population had a relative fitness of 0.22, with significantly lower survival rate, pupal weight, fecundity, percentage hatching, net reproductive rate, intrinsic rate of natural increase, biotic potential and mean relative growth rate, with prolonged male and female nymphal duration, developmental time from egg to female adult and male and female longevity compared with the control population. CONCLUSION: P. solenopsis biological parameters are greatly affected by acetamiprid, and it is of significant cost for the insects to counter these effects. This study will be a valuable source of information for further understanding of acetamiprid resistance and for assisting the development of resistance management programmes. (C) 2014 Society of Chemical Industry

Discusses the classification of materials in art libraries using the Library of Congress and Dewey Decimal Systems. (PF)