Identification of genes associated with the silk gland size using multi-omics in silkworm (Bombyx mori).

Silk gland size in silkworms (Bombyx mori) affects silk output. However, the molecular mechanisms by which genes regulate silk gland size remain unclear. In this study, silk glands from three pure silkworm strains (A798, A306 and XH) with different silk gland weight phenotypes were compared using transcriptomics and proteomics to identify differentially expressed genes (DEGs) and proteins (DEPs). When comparing A798 to A306 and A798 to XH, 830 and 469 DEGs were up-regulated, respectively. These genes were related to the gene ontology terms, metabolic process, transport activity and biosynthesis process. In addition, 372 and 302 up-regulated differentially expressed proteins were detected in A798 to A306 and A798 to XH, respectively, related to the gene ontology terms, ribosome and protein export, ribosome and polypeptide biosynthesis processes. Moreover, combined transcriptomics, proteomics and weighted correlation network analyses showed that five genes (BGIBMGA002524, BGIBMGA002629, BGIBMGA005659, BGIBMGA005711 and BGIBMGA010889) were significantly associated with the silk gland weight. Reverse Transcription-quantitative real-time Polymerase Chain Reaction (RT-qPCR) and Enzyme linked immunosorbent assay (ELISA) were used to verify the mRNA and protein expression of five genes in the silk glands and tissues of 18 silkworm strains. The results showed that four genes have higher expression levels in heavier silk glands. These genes are associated with glycogen metabolism, fatty acid synthesis and branched chain amino acid metabolism, thus potentially promoting growth and silk protein synthesis. These findings provide valuable insights into the molecular mechanisms underlying the relationship between silk gland weight and silk yield in silkworms.

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.

Characterization of the UDP-glycosyltransferase UGT33D1 in silkworm Bombyx mori.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT-interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms.

Recent advances in the biosensors application for reviving infectious disease management in silkworm model: a new way to combat microbial pathogens.

Silkworms are an essential economic insect but are susceptible to diseases during rearing, leading to yearly losses in cocoon production. While chemical control is currently the primary method to reduce disease incidences, its frequent use can result in loss of susceptibility to pathogens and, ultimately, antibiotic resistance. To effectively prevent or control disease, growers must accurately, sensitively, and quickly detect causal pathogens to determine the best management strategies. Accurate recognition of diseased silkworms can prevent pathogen transmission and reduce cocoon loss. Different pathogen detection methods have been developed to achieve this objective, but they need more precision, specificity, consistency, and promptness and are generally unsuitable for in-situ analysis. Therefore, detecting silkworm diseases under rearing conditions is still an unsolved problem. As a consequence of this, there is an enormous interest in the development of biosensing systems for the early and precise identification of pathogens. There is also significant room for improvement in translating novel biosensor techniques to identify silkworm pathogens. This study explores the types of silkworm diseases, their symptoms, and their causal microorganisms. Moreover, we compare the traditional approaches used in silkworm disease diagnostics along with the latest sensing technologies, with a precise emphasis on lateral flow assay-based biosensors that can detect and manage silkworm pathogens.

Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2.

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.

Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Alteration of ß-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence.

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as ß-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of ß-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface ß-glucan while low pH triggers an increase in ß-glucan. There is no inverse pattern between exposure levels of ß-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in ß-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in ß-glucan triggers an increase in the virulence of C. auris. This study demonstrates that ß-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

Contrasting gut bacteriomes unveiled between wild Antheraea assamensis Helfer (Lepidoptera: Saturniidae) and domesticated Bombyx mori L. (Lepidoptera: Bombycidae) silkworms.

BACKGROUND: Insect gut microbiomes play a fundamental role in various aspects of insect physiology, including digestion, nutrient metabolism, detoxification, immunity, growth and development. The wild Muga silkworm, Antheraea assamensis Helfer holds significant economic importance, as it produces golden silk. METHODS AND RESULTS: In the current investigation, we deciphered its intricate gut bacteriome through high-throughput 16S rRNA amplicon sequencing. Further, to understand bacterial community dynamics among silkworms raised under outdoor environmental conditions, we compared its gut bacteriomes with those of the domesticated mulberry silkworm, Bombyx mori L. Most abundant bacterial phyla identified in the gut of A. assamensis were Proteobacteria (78.1%), Bacteroidetes (8.0%) and Firmicutes (6.6%), whereas the most-abundant phyla in B. mori were Firmicutes (49-86%) and Actinobacteria (10-36%). Further, Gammaproteobacteria (57.1%), Alphaproteobacteria (10.47%) and Betaproteobacteria (8.28%) were the dominant bacterial classes found in the gut of A. assamensis. The predominant bacterial families in A. assamensis gut were Enterobacteriaceae (27.7%), Comamonadaceae (9.13%), Pseudomonadaceae (9.08%) Flavobacteriaceae (7.59%) Moraxellaceae (7.38%) Alteromonadaceae (6.8%) and Enterococcaceae (4.46%). In B. mori, the most-abundant bacterial families were Peptostreptococcaceae, Enterococcaceae, Lactobacillaceae and Bifidobacteriaceae, though all showed great variability among the samples. The core gut bacteriome of A. assamensis consisted of Pseudomonas, Acinetobacter, Variovorax, Myroides, Alteromonas, Enterobacter, Enterococcus, Sphingomonas, Brevundimonas, Oleispira, Comamonas, Oleibacter Vagococcus, Aminobacter, Marinobacter, Cupriavidus, Aeromonas, and Bacillus. Comparative gut bacteriome analysis revealed a more complex gut bacterial diversity in wild A. assamensis silkworms than in domesticated B. mori silkworms, which contained a relatively simple gut bacteriome as estimated by OTU richness. Predictive functional profiling of the gut bacteriome suggested that gut bacteria in A. assamensis were associated with a wide range of physiological, nutritional, and metabolic functions, including biodegradation of xenobiotics, lipid, amino acid, carbohydrate metabolism, and biosynthesis of secondary metabolites and amino acids. CONCLUSIONS: These results showed great differences in the composition and diversity of gut bacteria between the two silkworm species. Both insect species harbored core bacterial taxa commonly found in insects, but the relative abundance and composition of these taxa varied markedly.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Establishment of diabetes mellitus model using Bombyx mori silkworms in a low-temperature environment.

Due to the high prevalence of diabetes mellitus, researchers have conducted numerous experimental animal studies. However, the mammalian diabetes model is cumbersome and expensive to operate, while the cheap and simple common silkworm diabetes model has the disadvantage of a short cycle time. Since the growth of silkworms is greatly affected by environmental factors, we extended the five-age cycle of silkworms by lowering the ambient temperature to establish a novel low-temperature silkworm diabetes model. Our goal was to determine whether the low-temperature feeding of a high-sugar diet to silkworms could serve as an effective animal model for diabetes. Also, we aimed to resolve certain issues concerning the normal temperature silkworm diabetes model, such as the short time frame for experiments and erratic fluctuations in blood sugar levels. Silkworms weighing between 0.9 and 1.0 g at the beginning of the fifth instar were selected, and we created diabetic silkworms by feeding mulberry leaves containing 4% glucose daily in a 16-20°C environment. When the silkworms were kept at a cooler temperature, the fifth instar stage lasted for an additional 9-11 days. In the model group, 83.3% of the silkworms had blood glucose levels greater than 7.8 mmol/L, while the total prevalence of diabetic silkworms was 89.8%. Moreover, JNK phosphorylation expression rose in the model group, while PI3K expression fell. Additionally, the JNK and PI3K signaling pathway expressions matched diabetic signals. Therefore, using silkworms to create a diabetes model in a cool environment is a straightforward and cost-effective approach to studying diabetes in animals.

Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

Physiological and transcriptomic responses of silkworms to graphene oxide exposure.

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis-Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO.

Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.

Domestication Gene Mlx and Its Partner Mondo Are Involved in Controlling the Larval Body Size and Cocoon Shell Weight of Bombyx mori.

Bombyx mori was domesticated from Bombyx mandarina. The long-term domestication of the silkworm has brought about many remarkable changes to its body size and cocoon shell weight. However, the molecular mechanism underlying the improvement in the economic characteristics of this species during domestication remains unclear. In this study, we found that a transposable element (TE)-Bm1-was present in the upstream regulatory region of the Mlx (Max-like protein X) gene in wild silkworms but not in all domesticated silkworms. The absence of Bm1 caused an increase in the promoter activity and mRNA content of Mlx. Mlx and its partner Mondo belong to the bHLHZ transcription factors family and regulate nutrient metabolism. RNAi of Mlx and Mondo decreased the expression and promoter activity of glucose metabolism-related genes (trehalose transport (Tret), phosphofructokinase (PFK), and pyruvate kinase (PK)), lipogenic genes (Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)), and glutamine synthesis gene (Glutamine synthase 2, (GS2)). Furthermore, the transgenic overexpression of Mlx and Mondo in the fat body of silkworms increased the larval body size, cocoon shell weight, and egg number, but the silencing of the two genes resulted in the opposite phenotypes. Our results reveal the molecular mechanism of Mlx selection during domestication and its successful use in the molecular breeding of Bombyx mori.

Isolation and Structure Analysis of Chitin Obtained from Different Developmental Stages of the Mulberry Silkworm (Bombyx mori).

Chitin, a ubiquitous biopolymer, holds paramount scientific and economic significance. Historically, it has been primarily isolated from marine crustaceans. However, the surge in demand for chitin and the burgeoning interest in biopolymers have necessitated the exploration of alternative sources. Among these methods, the mulberry silkworm (Bombyx mori) has emerged as a particularly intriguing prospect. To isolate chitin from Bombyx mori, a chemical extraction methodology was employed. This process involved a series of meticulously orchestrated steps, including Folch extraction, demineralization, deproteinization, and decolorization. The resultant chitin was subjected to comprehensive analysis utilizing techniques such as attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), 13C nuclear magnetic resonance (NMR) spectroscopy, and wide-angle X-ray scattering (WAXS). The obtained results allow us to conclude that the Bombyx mori represents an attractive alternative source of α-chitin.

Revealing the synergistic effect of hydration and pulsed ultrasound on the emulsifying properties of silkworm pupa protein and its stabilized emulsion.

BACKGROUND: Silkworm (Bombyx moil L.) Pupa protein (SPP) is a high-quality insect protein and is considered a sustainable alternative source for traditional animal food protein. However, the utilization of SPP is limited because of its low solubility and emulsifying ability. In the present study, the synergistic effect of hydration and pulsed ultrasound on the physicochemical properties of SPP and SPP-stabilized Pickering emulsions was evaluated. RESULTS: Pulsed ultrasound changed the particle size of SPP and its conformation. As the pulsed ultrasound increased from 0 s to 5 s, the α-helix and SS contents of SPP decreased, whereas the ß-sheet and SH contents increased, which in turn improved its solubility and amphiphilicity. As a result, the SPP treated by a combination of 12 h of hydration and 3 s of ultrasound exhibited a contact angle of 74.95°, hydrophobicity of 904.83, EAI of 6.66 m2 g-1 and ESI of 190.69 min. Compared with the combination of 1 h of hydration and 5 s of ultrasound, the combination of 12 h of hydration and 3 s of ultrasound exerted more soluble and hydrophobic SPP, whereas the EAI and ESI of the samples were higher. Notably, the ultrasound-treated SPP can form a stable gel-like emulsion (oil fraction ranging from 70% to 80%). CONCLUSION: The combination of hydration and ultrasound can effectively improve the physicochemical characteristics of SPP as well as its emulsion stability. Sufficient hydration is a cost-effective method for facilitating the modification of proteins by ultrasound treatment. © 2024 Society of Chemical Industry.

The Mechanisms of Silkworm Resistance to the Baculovirus and Antiviral Breeding.

Silkworm (Bombyx mori) is not only an economic insect but also a model organism for life science research. Bombyx mori nucleopolyhedrovirus (BmNPV) disease is a major infectious disease in the world's sericulture industry. The cocoon loss caused by this disease accounts for more than 60% of the total loss caused by all silkworm diseases. To date, there has been no effective solution for preventing and treating this disease. The most effective measure is to breed disease-resistant varieties. The quickest way to breed disease-resistant varieties is to apply genetic modification. However, this requires that we obtain disease resistance genes and know the mechanism of disease resistance. Since the discovery of disease-resistant resources in 1989, scholars in the sericulture industry around the world have been inspired to search for resistance genes. In the past two decades, with the help of multi-omics technologies, screening of resistance genes, gene localization, protein modification, virus-host interactions, etc., researchers have found some candidate genes that have been proposed to function at the cellular or individual level. Several disease-resistant varieties have been obtained and used in production through hybrid breeding, RNA interference, and genetic modification. This article summarizes and reviews the discovery of and research advances related to silkworm resistance to BmNPV. It is anticipated that the review will inspire scientific researchers to continue searching for disease resistance genes, clarify the molecular mechanism of silkworm disease resistance, and promote disease-resistant silkworm breeding.

Structural model for ligand binding and channel opening of an insect gustatory receptor.

Insect gustatory receptors play roles in sensing tastants, such as sugars and bitter substances. We previously demonstrated that the BmGr9 silkworm gustatory receptor is a d-fructose-gated ion channel receptor. However, the molecular mechanism of how d-fructose could initiate channel opening were unclear. Herein, we present a structural model for a channel pore and a d-fructose-binding site in BmGr9. Since the membrane topology and oligomeric state of BmGr9 appeared to be similar to those of an insect odorant receptor coreceptor, Orco, we constructed a structural model of BmGr9 based on the cryo-EM Orco structure. Our site-directed mutagenesis data suggested that the transmembrane region 7 forms channel pore and controls channel gating. This model also suggested that a pocket formed by transmembrane helices 2 to 4 and 6 binds d-fructose. Using mutagenesis experiments in combination with docking simulations, we were able to determine the potent binding mode of d-fructose. Finally, based on these data, we propose a conformational change that leads to channel opening upon d-fructose binding. Taken together, these findings detail the molecular mechanism by which an insect gustatory receptor can be activated by its ligand molecule.

Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase.

Broad evolutionary expansion of polymerase families has enabled specialization of their activities for distinct cellular roles. In addition to template-complementary synthesis, many polymerases extend their duplex products by nontemplated nucleotide addition (NTA). This activity is exploited for laboratory strategies of cloning and sequencing nucleic acids and could have important biological function, although the latter has been challenging to test without separation-of-function mutations. Several retroelement and retroviral reverse transcriptases (RTs) support NTA and also template jumping, by which the RT performs continuous complementary DNA (cDNA) synthesis using physically separate templates. Previous studies that aimed to dissect the relationship between NTA and template jumping leave open questions about structural requirements for each activity and their interdependence. Here, we characterize the structural requirements for cDNA synthesis, NTA, template jumping, and the unique terminal transferase activity of Bombyx mori R2 non-long terminal repeat retroelement RT. With sequence alignments and structure modeling to guide mutagenesis, we generated enzyme variants across motifs generally conserved or specific to RT subgroups. Enzyme variants had diverse NTA profiles not correlated with other changes in cDNA synthesis activity or template jumping. Using these enzyme variants and panels of activity assay conditions, we show that template jumping requires NTA. However, template jumping by NTA-deficient enzymes can be rescued using primer duplex with a specific length of 3' overhang. Our findings clarify the relationship between NTA and template jumping as well as additional activities of non-long terminal repeat RTs, with implications for the specialization of RT biological functions and laboratory applications.