A pheromone bouquet controls the reproductive behaviour of the male shore crab, Carcinus maenas

The reproduction of many brachyuran crustaceans involves the formation of mating pairs often around the time of the female moult with attraction of a sexual partner and mating behaviour controlled by sex pheromones. In shore crabs, Carcinus maenas, females produce sex pheromones that are released in the urine. High Performance Liquid Chromatography analysis (HPLC) of female urine shows that the pheromone, identified as the nucleotide uridine diphosphate (UDP), elutes as an unresolved peak with structurally related nucleotides. We examined female urine samples over the moult cycle and detected UDP as well as uridine triphosphate (UTP). Bioassays were conducted to establish the possibility of a blend of nucleotides forming a sex pheromone bouquet in C. maenas. Whilst UDP induced the male mate guarding behaviour (cradling), a mixture of the two nucleotides at a ratio of 4:1 UDP:UTP elicited an even stronger mating response than either UDP or UTP individually. The urine concentration and composition of these nucleotides changes over the moult period pre and post ecdysis, providing evidence that a pheromone bouquet composition is not always constant. The change of the bouquet is related to the physiological state of the sender, here the moult cycle. Our study unravels the functionality of reaction-specific molecules in a pheromone bouquet. Whilst UDP is the mating signal, UTP acts as an attractant and combined they maximise the reproductive response. The use of bouquets provides species-specificity, potentially enabling reproductive isolation of sympatric species, and contains valuable information on the physiological state of the sender.

MATING DISRUPTION OF GRAPHOLITA MOLESTA BUSCK. TO IMPROVE QUALITY AND HEALTHY OF PEACH FRUITS

In Bulgarian fruit growing agriculture mainly chemical plant protection is applied. Applications with organophosphates and pyrethroids are carried out to control the economically most important peach pest - the oriental fruit moth (Grapholita molesta Busck.). Annually during the vegetation season 7-8 insecticide treatments are applied. This leads to contamination of fruit products and the environment, which is a prerequisite for seeking other approaches. Regarding to fruit moth, sex pheromones are one of the most widely used environmental control methods, but they are most commonly used in apples. Their introduction in the monitoring of the main peach pest can lead to a reduction of chemical treatments and production with less pesticide residues. A promising direction is the use of pheromones to control the pests through sexual disorientation of males. The trial of mating disruption in the present study were carried out with ISOMATE® OFM TT dispensers in 0.9 ha peach orchard in 2020 and 2021. Pheromone traps type "Delta" of the Hungarian company CSALOMON® were used. The ISOMATE OFM TT dispensers, installed before the first flight of OFM at the rate of 250 units per ha, efficiently reduced fruit damages - down to 0.1-0.2% at harvest. In the reference orchard with 6 insecticide treatment against OFM, the damages reached 3.0-3.6%. The results indicate that mating disruption for control of Grapholita molesta Busck. can be used in production of quality and healthy peach fruits.

Transcriptome analysis identifies candidate genes in the biosynthetic pathway of sex pheromones from a zygaenid moth, Achelura yunnanensis (Lepidoptera: Zygaenidae)

In most moth species, sex pheromones responsible for mating and communication of both sexes are primarily produced by the pheromone glands (PGs) of female moths. Although the PG transcriptomes and pheromone production related genes from 24 moth species have been characterized, studies on the related information remain unknown in the Zygaenidae family. Here, we sequenced the PG transcriptome of a zygaenid moth, Achelura yunnanensis. Such the sequencing resulted in the yields of 47,632,610 clean reads that were assembled into 54,297 unigenes, coupled with RNA sequencing data from 12 other tissues. Based on the transcriptome, a total of 191 genes encoding pheromone biosynthesis and degradation enzymes were identified, 161 of which were predicted to have full-length sequences. A comparative analysis among 24 moth species of nine families indicated that the numbers of the genes were variable, ranging from 14 in two Grapholita species to 191 in A. yunnanensis. Phylogenetic analysis in parallel with the expression data highlighted some key genes, including three △9 and four △11 desaturases, four fatty acyl-CoA reductases (FARs) clustering in the pgFAR clade, and three significantly antennae-enriched aldehyde oxidases. An extensive tissue- and sex- expression profile revealed a broad distribution of the genes, in which 128 relatives were detected in the PGs and 127 in the antennae. This study reports, for the first time, the gene repertoires associated with the pheromone production in Zygaenidae, and provides a valuable resource for exploring putative roles of the PG-enriched genes in A. yunnanensis.

Computational Interaction Analysis of Sirex noctilio Odorant-Binding Protein (SnocOBP7) Combined with Female Sex Pheromones and Symbiotic Fungal Volatiles

Sirex noctilio, a major forestry quarantine pest, has spread rapidly and caused serious harm. However, existing methods still need to be improved because its olfactory interaction mechanisms are poorly understood. In order to study the role of male-specific protein SnocOBP7 in the protein–ligand interactions, we selected it as the object of computational simulation and analysis. By docking it with 11 ligands and evaluating free binding energy decomposition, the three best binding ligands were found to be female sex pheromones ((Z)-7-heptacosene and (Z)-7-nonacosene) and symbiotic fungal volatiles ((−)-globulol). Binding mode analysis and computational alanine scanning suggested that five residues play key roles in the binding of each female sex pheromone to SnocOBP7, whereas two residues play key roles in (−)-globulol binding. Phe108 and Leu36 may be the crucial sites via which SnocOBP7 binds female sex pheromones, whereas Met40 may regulate the courtship behavior of males, and Leu61 may be related to mating and host finding. Our studies predicted the function of SnocOBP7 and found that the interaction between SnocOBP7 and pheromone is a complex process, and we successfully predicted its binding key amino-acid sites, providing a basis for the development of new prevention and control methods relying on female sex pheromones and symbiotic fungi.

Exploring the Terminal Pathway of Sex Pheromone Biosynthesis and Metabolism in the Silkworm

Sex pheromones are vital to sexual communication and reproduction in insects. Although some key enzymes in pheromone production have been well studied, information on genes involved in the terminal pathway is limited. The domestic silkworm employs a pheromone blend containing (E,Z)-10,12-hexadecadienol (bombykol) and analogous (E,Z)-10,12-hexadecadienal (bombykal); whereas, its wild ancestor B. mandarina uses only bombykol. The two closely related moths might be a good model for exploring the genes involved in aldehyde pheromone synthesis and metabolism. By deep sequencing and analyzing the sex pheromone gland (PG) transcriptomes; we identified 116 candidate genes that may be related to pheromone biosynthesis, metabolism, and chemoreception. Spatiotemporal expression profiles and differentially expressed analysis revealed that four alcohol oxidases (BmorAO1; 2; 3; and 4); one aldehyde reductase (BmorAR1); and one aldehyde oxidase (BmorAOX5) might be involved in the terminal pathway. Phylogenetic analysis showed that, except for BmorAO3 and MsexAO3, AOs did not show a conversed orthologous relationship among moths; whereas, ARs and AOXs were phylogenetically conserved. This study provides crucial candidates for further functional elucidation, and which may be utilized as potential targets to disrupt sexual communication in other moth pests.

CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt

Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.

Male and Female Cloacal Anatomy of the Fischer’s Clawed Salamander, Onychodactylus fischeri (Caudata, Hynobiidae)

The cloaca of salamanders is a complex organ with exocrine glands involved in the production of sex pheromones, spermatophores, and storage of sperm. Since the cloaca provides reproductive functions, its signs are important for phylogenetic analysis in the evolutionary biology of tailed amphibians. For clarification of intrafamilial variation of cloacal characteristics in hynobiids, we studied the anatomy of male and female cloacae of Onychodactylus fischeri via histological, histochemical, and 3D-reconstruction methods. Males and females had ciliated cloacal linings and with sexual dimorphism in cloacal conformation and cloacal glands. As in other males and females of hynobiids, females of O. fischeri possessed only ventral glands, secreting neutral glycoproteins. In contrast, males of this species had three types of the cloacal glands. Glands «B» were like ventral glands of females and other hynobiids, while glands «A» and «C» had different histochemical and morphological characteristics. As our results are generally consistent with the data for the related species O. japonicus, these characteristics of the male and female cloacal anatomy may be common to all species of the genus Onychodactylus. The presence of three types of unique cloacal glands in males distinguishes Onychodactylus from all other hynobiids and salamanders.

Transcriptome Analysis and Characterization of Chemosensory Genes in the Forest Pest, Dioryctria abietella (Lepidoptera: Pyralidae)

In Lepidoptera, RNA sequencing has become a useful tool in identifying chemosensory genes from antennal transcriptomes, but little attention is paid to non-antennal tissues. Though the antennae are primarily responsible for olfaction, studies have found that a certain number of chemosensory genes are exclusively or highly expressed in the non-antennal tissues, such as proboscises, legs and abdomens. In this study, we report a global transcriptome of 16 tissues from Dioryctria abietella, including chemosensory and non-chemosensory tissues. Through Illumina sequencing, totally 952,658,466 clean reads were generated, summing to 142.90 gigabases of data. Based on the transcriptome, 235 chemosensory-related genes were identified, comprising 42 odorant binding proteins (OBPs), 23 chemosensory proteins (CSPs), 75 odorant receptors (ORs), 62 gustatory receptors (GRs), 30 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs). Compared to a previous study in this species, 140 novel genes were found. A transcriptome-wide analysis combined with PCR results revealed that except for GRs, the majority of other five chemosensory gene families in Lepidoptera were expressed in the antennae, including 160 chemosensory genes in D. abietella. Using phylogenetic and expression profiling analyses, members of the six chemosensory gene repertoires were characterized, in which 11 DabiORs were candidates for detecting female sex pheromones in D. abietella, and DabiOR23 may be involved in the sensing of plant-derived phenylacetaldehyde. Intriguingly, more than half of the genes were detected in the proboscises, and one fourth of the genes were found to have the expression in the legs. Our study not only greatly extends and improves the description of chemosensory genes in D. abietella, but also identifies potential molecular targets involved in olfaction, gustation and non-chemosensory functions for control of this pest.

Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8 μg g-1 FW) and high levels of Z11-16OH (111.4 μg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements.