Evidence of Bombyx mori (Lepidoptera: Bombycidae) odorant receptors related to oviposition behavior

Local and cis Effects of the H Element on Expression of Odorant Receptor Genes in Mouse

BackgroundAedes aegypti is one of the most important vectors of zoonotic diseases worldwide, and its survival and reproductive processes depend heavily on its olfactory system. In this study, the expression levels of all odorant receptor (OR) genes of Ae. aegypti were explored during different physiological periods to identify olfactory genes that may be associated with mosquito blood-feeding and the search for oviposition sites.MethodsFour experimental groups, consisting of Ae. aegypti males, pre-blood-feeding females, post-blood-feeding females and post-oviposition females, were established. A total of 114 pairs of primers targeting all messenger RNA encoded by OR genes were designed based on the whole genome of Ae. aegypti. The expression of OR genes was evaluated by real-time fluorescence quantitative PCR for relative quantification and the comparison of differences between groups.ResultsA total of 53 differentially expressed OR genes were identified between males and females in Ae. aegypti antennae. Also, eight, eight and 13 differentially expressed OR genes were identified in pre- versus post-blood-feeding females, in pre- versus post-oviposition females and in post-blood-feeding versus post-oviposition females, respectively. In addition, 16 OR genes were significantly differentially expressed in multiple physiological periods of the mosquitoes.ConclusionsA large number of ORs with significant intergroup differences and high expression levels were screened in this study. Some of these genes are reported for the first time, providing possible targets for the development of mosquito control pathways based on the olfactory system.Graphical

Transmembrane emp24 domain (TMED) proteins have been extensively studied in mammalian embryonic development, immune regulation, and signal transduction. However, their role in insects, apart from Drosophila melanogaster, remains largely unexplored. Our previous study demonstrated the abundant expression of BmTMED6 across all stages and tissues of the silkworm. In this study, we investigate the function of BmTMED6 in reproduction. We observe significant differences in the expression of BmTMED6 between male and female silkworms, particularly in the head and fatboby, during the larval stage. Furthermore, qRT-PCR and WB analysis reveal substantial variation in BmTMED6 levels in the ovaries during pupal development, suggesting a potential association with silkworm female reproduction. We find that reducing TMED6 expression significantly decreases the number of eggs laid by female moths, leading to an accumulation of unlaid eggs in the abdomen. Moreover, downregulation of BmTMED6 leads to a decrease in the expression of BmDop2R1 and BmDop2R2, while overexpression of BmTMED6 in vitro has the opposite effect. These indicate that BmTMED6 plays a role in oviposition in female moths, potentially through the dopamine signaling pathway. This study provides a new regulatory mechanism for female reproduction in insects.

Purpose: Paratrioza sinica is one of the important pests on Lycium barbarum . Adults and nymphs suck in the juice of leaves and tender shoots, causing leaves to turn yellow, even tree death. It results in a decrease in the yield and quality of fruits. The odorant receptor’s receptors (ORs) genes of Paratrioza sinica were cloned and analyzed by bioinformatics methods in this study. Method: Based on the previous transcriptome sequencing data of Paratrioza sinica , the transcript sequences of PsinOr50, PsinOr15, and PsinOrco were obtained. The CDS sequences of three odorant receptors (ORs) genes in Paratrioza sinica were cloned using rapid amplification of cDNA ends (RACE) cloning technology. The expression levels of these three genes in adult insects of different ages were determined by qRT-PCR. Physical and chemical properties, characteristics of sequences, proteins structure, and evolutionary relationships of three genes and their encoded proteins were analyzed systematically. Result: The CDS sequences of three ORs genes in Paratrioza sinica were obtained, and their accession numbers in the NCBI database were PP556383 (PsinOr15), PP556384 (PsinOr50), and PP556385 (PsinOrco). Three genes all achieved significantly higher expression levels within 1- to 3-day-old adults of Paratrioza sinica . PsinOr15 is a hydrophilic stable protein that does not contain signal peptides but has a transmembrane domain. It is subcellularly located in the nucleus. PsinOr50 belongs to hydrophobic unstable proteins, with signal peptides and transmembrane domain, and subcellular localization in the cytoplasm. PsinOrco is a hydrophobic stable protein which does not contain signal peptides but has a transmembrane domain. It is subcellularly located in the endoplasmic reticulum. The prediction of the second and tertiary protein structure shows that the proteins encoded by the three ORs genes are mainly alpha helices and random curls. Amino acid sequence alignment revealed that they all contain four conserved amino acid sequence sites. Phylogenetic analysis showed that three ORs genes differentiated into three branches, consistent with the sequence comparison results. Conclusion: The gene sequences and bioinformatic results of the PsinOr15, PsinOr50, and PsinOrco genes in Paratrioza sinica were obtained in this study. It provides a theoretical basis for studying the role of ORs genes and their encoded proteins in the feeding, mating, and egg laying processes of Paratrioza sinica .

BackgroundChemosensory receptors, which are all G-protein-coupled receptors (GPCRs), come in four types: odorant receptors (ORs), vomeronasal receptors, trace-amine associated receptors and formyl peptide receptor-like proteins. The ORs are the most important receptors for detecting a wide range of environmental chemicals in daily life. Most fish OR genes have been identified from genome databases following the completion of the genome sequencing projects of many fishes. However, it remains unclear whether these OR genes from the genome databases are actually expressed in the fish olfactory epithelium. Thus, it is necessary to clone the OR mRNAs directly from the olfactory epithelium and to examine their expression status.ResultsEighty-nine full-length and 22 partial OR cDNA sequences were isolated from the olfactory epithelium of the large yellow croaker, Larimichthys crocea. Bayesian phylogenetic analysis classified the vertebrate OR genes into two types, with several clades within each type, and showed that the L. crocea OR genes of each type are more closely related to those of fugu, pufferfish and stickleback than they are to those of medaka, zebrafish and frog. The reconciled tree showed 178 duplications and 129 losses. The evolutionary relationships among OR genes in these fishes accords with their evolutionary history. The fish OR genes have experienced functional divergence, and the different clades of OR genes have evolved different functions. The result of real-time PCR shows that different clades of ORs have distinct expression levels.ConclusionWe have shown about 100 OR genes to be expressed in the olfactory epithelial tissues of L. crocea. The OR genes of modern fishes duplicated from their common ancestor, and were expanded over evolutionary time. The OR genes of L. crocea are closely related to those of fugu, pufferfish and stickleback, which is consistent with its evolutionary position. The different expression levels of OR genes of large yellow croaker may suggest varying roles of ORs in olfactory function.

Sericulture, often known as silk farming is the practice of rearing silkworms for production of raw silk. One of the major impediments faced by the sericulture community is the silkworm diseases that cause significant crop losses. In the present study, the efficacy of phototrophic bacteria as feed supplements in improving the economic traits as well as disease resistance in mulberry silkworm, Bombyx mori L. was assessed. A total of three phototrophic bacteria were isolated and identified as belonging to the genera Marichromatium, Rhodobacter and Rhodopseudomonas based on 16S rRNA gene sequencing analysis. Their morphological, physiological and biochemical characters were studied, grown under ambient conditions and biomass was harvested. Mulberry fortified with the aforementioned phototrophic bacteria in solitude and combinations at varying concentrations (0.5, 1, 2 and 5%) were found to be innocuous to the mulberry silkworm. The best results were observed when mulberry leaf was supplemented with Rhodopseudomonas sp. at 2% concentration which improved survival, cocoon weight, shell weight, shell%, filament length and non-breakable filament length by 5.18, 0.27, 3.86, 3.5, 1.5 and 1.2%, respectively under normal rearing conditions. Under Staphylococcus sp. infected conditions, the aforesaid diet also enhanced survival in silkworms by 16%. On the other hand, the same diet didn’t exhibit any discernible influence on survival against fungal, viral, or microsporidian infections in silkworm.

BackgroundThe olfactory system plays a crucial role in regulating insect behaviors. The detection of odorants is mainly mediated by various odorant receptors (ORs) that are expressed in the dendrites of olfactory neurons of chemosensilla. Anophelessinensis is a major malaria vector in Eastern Asia and its genome has recently been successfully sequenced and annotated. In this study, we present genome-wide identification and expression profiling of OR genes in different chemosensory tissues of An.sinensis.MethodsThe OR genes were identified using the available genome sequences of An.sinensis. A series of bioinformatics analyses were conducted to investigate the structure, genome distribution, selective pressure and phylogenetic relationships of OR genes, the conserved domains and specific functional sites in the OR amino acid sequences. The expression levels of OR genes were analyzed from transcriptomic data from An.sinensis antennae, proboscis and maxillary palps of both sexes.ResultsA total of 59 putative OR genes have been identified and characterized in An.sinensis. This number is significantly less than that in An.gambiae. Whether this difference is caused by the contraction or expansion of OR genes after divergence of the two species remains unknown. The RNA-seq analysis showed that AsORs have obvious tissue- and sex-specific expression patterns. Most AsORs are highly expressed in the antennae and the expression pattern and number of AsORs expressed in antennae are similar in males and females. However, the relative levels of AsOR transcripts are much higher in female antennae than in male antennae, which indicates that the odor sensitivity is likely to be increased in female mosquitoes. Based on the expression patterns and previous studies, we have speculated on the functions of some OR genes but this needs to be validated by further behavioral, molecular and electrophysiological studies. Further studies are necessary to compare the olfactory-driven behaviors and identify receptors that respond strongly to components of human odors that may act in the process of human recognition.ConclusionsThis is the first genome-wide analysis of the entire repertoire of OR genes in An.sinensis. Characterized features and profiled expression patterns of ORs suggest their involvement in the odorous reception of this species. Our findings provide a basis for further research on the functions of OR genes and additional genetic and behavioral targets for more sustainable management of An.sinensis in the future.Graphical

Olfactory neurons have a complex phenotype characterized by their expression of a specific odor receptor (OR) gene and their targeting of an equally specific locus in the olfactory bulb. In the adult fish, olfactory neurons expressing specific ORs are broadly distributed in the epithelium, intermingling with neurons expressing other OR phenotypes. This distributed adult pattern has led to the suggestion that olfactory neuron phenotype is determined by a stochastic process, independent of external positional cues. However, when the fish olfactory system is established during embryogenesis it is simple in its organization, with few olfactory neurons and an olfactory epithelium that has not yet folded into the adult morphology. It is possible that positional cues might act in the embryo to establish an initial population and pattern of olfactory neuron phenotypes and that subsequent morphogenesis and neuronal addition lead to the randomized distribution of neurons. To test this possibility, we examined the spatial patterns of olfactory neurons expressing specific OR genes in 48 h embryos, a time of relative simplicity in the developing olfactory epithelium. Three-dimensional plots of neuron distributions were made, and comparison of OR expression patterns were made between right and left epithelia, between individual animals and between different OR genes. The patterns of OR gene expression were not conserved in these comparison. Mathematical analysis of 21 epithelia for the degree of order in the distribution of olfactory neurons argued strongly that the neurons expressing given ORs are randomly distributed in the 48 h embryos. These results are consistent with those observed from adult tissue and support models suggesting that extrinsic positional cues do not have a major role in specifying olfactory neuron phenotypes.

In mammals, perception of pheromones is based on the expression in each vomeronasal sensory neuron of a limited set of receptor genes, chosen among a large repertoire. Here, we report an extremely tight control of the monogenic and monoallelic transcription of the V1rb2 receptor gene. Combining genetic and electrophysiological approaches, we show that the transcription of a non-functional V1r allele leads to the coexpression of another, functional V1r gene. The choice of this coexpressed gene surprisingly includes genes located on the cluster homologous to the one from which the mutant allele is transcribed. However, V1r genes located in cis relative to the transcribed mutant allele are excluded from the coexpression choice. Our observations strongly suggest a monogenic regulatory mechanism acting (a) at a general level, via the expression of the V1r receptor itself, and (b) at a more local level, defined by the V1r gene cluster.

The morphological characterization among domesticated insect like silkworm (Bombyx mori L.) is an important aspect for selection of suitable parents for breeding programme. The present study was conducted on twenty bivoltine silkworm breeds to estimate the extent relationship by morphological characterization. Eighteen morphological traits both qualitative and quantitative viz., egg shape, chorion colour, colour of the newly hatched larva, larval marking, cocoon colour, cocoon shape, pupa colour and shape, colour of male and female moth, fecundity, hatching, larva weight 5th day, –th instar duration, total larval period, percentage of malformed cocoons, pupation rate, single cocoon weight, single shell weight and shell percent were studied and data revealed significant variability among the breeds. Maximum Euclidean distance was observed between NB4D2 and CSR19 (163.25) followed by CSR19 and UDHEY-5 (145.13) and minimum between UDHEY-1 and UDHEY-3 (4.20). The dendrogram of phenotypic data grouped all breeds in two clusters; Cluster A and B. Cluster A was sub divided into two sub-clusters cluster AI and cluster A II. Cluster A I contained eight breeds and Cluster AII eleven breeds. Cluster B comprised of one breed CSR19. In PCA the genetic divergence was maximum between NB4D2 and CSR19, whereas it was minimum in UDHEY-7 and ND2. PCoA results depicted CSR19 and SPO having greater divergence, whereas it was minimum between UDHEY-7 and ND2. On the basis of Euclidean distance metric the performance of the breeds; CSR19, NB4D2, PO3, UDHEY-8, SPO, UDHEY-4, ND2, CC1, UDHEY-2, UDHEY-5, UDHEY-7, UDHEY-6 have been identified as promising breeds and are recommended for further breeding to boost bivoltine silk production.

Sericulture has been serving the humanity by providing natural animal silk for several centuries. The quality as well as the quantity of mulberry leaves along with environmental factors have a direct influence on the production of raw silk spun by Bombyx mori L. Larvae before pupation in the form of cocoons. Study on the nutritional aspect of the silkworm is an essential prerequisite for its proper commercial exploitation. Silkworm nutrition is the sole factor which has almost individually augmented the quality and quantity of the silkworm cocoon production and productivity. Despite the balancing of the silkworm nutrients by the mulberry leaf, the quantity available is not sufficient for the larval growth owing to variation in mulberry plant and in its management. This created a requirement for the dietary fortification with good quality mulberry leaves in optimum quantity for successful cocoon production. In this study, mulberry leaves were fortified with Honey, Glucose, B- complex, Lemon and Calciferol and the growth and dietary efficiency of the larvae fed with the fortified diet were evaluated. The results indicated improvement in terms of growth as well as cocoon parameters. Our studies support the concept of dietary fortification the Mulberry leaves fed to silkworm for netter production of silk.

BackgroundSilkworm pupae is the main by-product of the sericulture industry with an interesting nutritional profile, especially in terms of proteins. In consideration of its possible use as a food or food ingredient in Western countries, a comparative proteomic experiment has been performed to investigate the differences of the protein profile of male and female silkworm pupae reared on mulberry leaves or on an artificial diet.MethodsThe nutritional profile of lyophilized silkworm pupae in terms of dry matter and ash was evaluated according to the AOAC procedures, the total nitrogen content was determined by a nitrogen analyzer and the silkworm pupae gross energy value was measured using an adiabatic calorimetric bomb. The comparative proteomic analysis was performed on male and female silkworm pupae reared on mulberry leaves or on the artificial diet. Proteins were separated by two-dimensional electrophoresis and, after a multivariate statistical analysis, the differentially expressed proteins were identified by LC-MS/MS.ResultsThe comparative proteomic approach highlighted 47 silkworm pupae proteins differentially expressed comparing diet and gender. PCA analysis showed that seven proteins were more effective in discriminating the sex and five were more effective in discriminating the diet type. In spite of the above-mentioned differences in the silkworm pupae protein profile, no strong alteration of the pupa physiological traits have been demonstrated, suggesting a general silkworm pupae flexibility to adapt to a well-balanced artificial diet. Differences in lipid transport and metabolism were found among the experimental groups, that might have a relevant effect on the timing and on hormone secretion. This aspect may also affect silk production, as univoltine strains are the most productive. The proteomic data provided in this work, may offer a contribution in understanding also the influence of gender and farming strategy on the allergen profile of Bombyx mori, when used as food or as a food ingredient. Female silkworm pupae reared on mulberry leaves seemed to contain lower levels of known allergens than those reared in the other experimental conditions; these findings will have to be taken into account when farming B. mori for food production purposes. However, our results need to be supported by further characterization of the allergenic potential of B. mori.

Use of formula feed (FF) for silkworms for all instars, has promoted transformation and progress in traditional sericulture. However, the cocoon yield of FF silkworms has failed to reach that of silkworms fed mulberry leaves (ML). The biological mechanisms underlying this phenomenon have not been well described. This study aimed to identify metabolic mechanisms and potential biomarkers relating to the poor cocoon yield of FF silkworms. In this study, silkworms received treatments of either ML (ML group) or FF (FF group) for all instars. At the 3rd day of the 5th instar, the midgut (MG), hemolymph (HL) and posterior silk gland (PSG) were collected for the metabolome profiles detection. The remaining silkworms were fed ML or FF until cocooning for investigation. The whole cocoon yield (WCY) was significantly higher in the FF group than the ML group (p < 0.05), whereas the cocoon shell weight (CSW) and cocoon shell rate (CSR) were significantly lower in the FF group (p < 0.05). A total of 845, 867 and 831 metabolites were qualified and quantified in the MG, HL and PSG of the FF silkworms, respectively. Correspondingly, 789, 833 and 730 metabolites were quantified in above three tissues of the ML group. Further, 230, 249 and 304 significantly different metabolites (SDMs) were identified in the MG, HL and PSG between the FF and ML group, respectively. Eleven metabolic pathways enriched by the SDMs were mutual among the three tissues. Among them, cysteine and methionine metabolism, arginine biosynthesis, and arginine and proline metabolism were the top three pathways with the highest impact value in the PSG. Six biomarkers were obtained through biomarker analysis and Pearson correlation calculation. Among them, homocitrulline, glycitein, valyl-threonine, propyl gallate and 3-amino-2,3-dihydrobenzoic acid were positively correlated with WCY, but negatively correlated with CSW and CSR (p < 0.05). An opposite correlation pattern was observed between 3-dimethylallyl-4-hydroxyphenylpyruvate and the three cocoon performance traits. Overall, three key metabolic pathways and six biomarkers associated with cocoon yield were interpreted, and should provide directions for formula feed optimization in factory-raised silkworms.

The new technology of silkworm (Bombyx mori L.) artificial feed breeding has many characteristics and advantages. This study assessed silkworm rearing with mulberry leaf at all instars (MF) as the control, and used metabolomics to explore the differences in haemolymph metabolism of fifth instar silkworms under two modes of rearing with an artificial diet at all instars (AF) and rearing with an artificial diet during first to third instars and mulberry leaf during the fourth and fifth instars (AMF). The results show that, compared with silkworms of the MF group, the amount and fold change of various metabolites were higher in the haemolymph of AF group silkworms, and the metabolism of amino acids and uric acid, carbohydrates, lipids, and vitamins were changed. These changes may be the reasons for the poor performance of the AF silkworms. However, the amount and fold change of the various metabolites of silkworms in the AMF group were lower, and some metabolic pathways were more active. The amount of material and energy supply were greater. These changes could explain the high efficiency growth of body weight of silkworms after the conversion from artificial diet rearing to mulberry leaf rearing. These findings provide an important theoretical basis for the optimisation of artificial diet rearing technology for silkworms.

Sericulture is one of the great inventions of the Chinese people and has become an important cultural feature of China. As China is the long-lasting center of silk production, genetic breeding of silkworm was highly developed historically, and has formed a comprehensive system for breeding and preservation of new varieties. However, silkworm breeding reached a bottleneck recently, because most of the traditional genetic resources have been utilized and silkworm strains have become homogeneous. Meanwhile, sericulture in China meets huge challenges in the 21st century. In recent years, with the development and rapid application of molecular biology, genomics, transgene and genome editing, silkworm genetic breeding has entered a new era. In this review, we summarize the development of silkworm genetic breeding, especially the progress and perspective of transgene and genome editing in genetic engineering of silkworms. We also discuss the future development of silkworm genetic breeding.