Study on the characteristics of genetic diversity of different populations of Guizhou endemic plant Rhododendron pudingense based on microsatellite markers.

BACKGROUND: Rhododendron pudingense, firstly discovered in Puding county of Guizhou province in 2020, have adapted to living in rocky fissure habitat, which has important ornamental and economic values. However, the genetic diversity and population structure of this species have been rarely described, which seriously affects the collection and protection of wild germplasm resources. RESULTS: In the present study, 13 pairs of primers for polymorphic microsatellite were used to investigate the genetic diversity of 65 R. pudingense accessions from six different geographic populations. A total of 254 alleles (Na) were obtained with an average of 19.5 alleles per locus. The average values of polymorphic information content (PIC), observed heterozygosity (Ho), and expected heterozygosity (He) were 0.8826, 0.4501, and 0.8993, respectively, These results indicate that the microsatellite primers adopted demonstrate good polymorphism, and the R. pudingense exhibits a high level of genetic diversity at the species level. The average genetic differentiation coefficient (Fst) was 0.1325, suggested that moderate divergence occurred in R. pudingense populations. The average values of genetic differentiation coefficient and gene flow among populations were 0.1165 and 3.1281, respectively. The analysis of molecular variance (AMOVA) indicated that most of the population differences (88%) were attributed to within-population variation. The PCoA results are consistent with the findings of the UPGMA clustering analysis, supporting the conclusion that the six populations of R. pudingense can be clearly grouped into two separate clusters. Based on Mantel analysis, we speculate that the PD population may have migrated from WM-1 and WM-2. Therefore, it is advised to protect the natural habitat of R. pudingense in situ as much as possible, in order to maximize the preservation of its genetic diversity. CONCLUSIONS: This is the first comprehensive analysis of genetic diversity and population structure of R. pudingense in Guizhou province. The research results revealed the high genetic diversity and moderate population diferentiation in this horticulture plant. This study provide a theoretical basis for the conservation of wild resources of the R. pudingense and lay the foundation for the breeding or cultivation of this new species.

Adenoviruses vectored hepatitis C virus vaccine cocktails induce broadly specific immune responses against multi-genotypic HCV in mice.

BACKGROUND: Hepatitis C virus (HCV) vaccines are an urgent need to prevent hepatitis C and its further progression of hepatocellular carcinoma. Since the promising T cell based chimpanzee adenovirus and modified vaccinia virus Ankara vectorial HCV vaccines were failed in clinical phase II trial, the vaccine designs to improve protection efficacy in combination of cellular and humoral immunity have been hypothesized against multi-genotypic HCV. METHODS: Eight HCV vaccine strains were constructed with two novel adenovirus vectors (Sad23L and Ad49L) encoding E1E2 or NS3-5B proteins of HCV genotype (Gt) 1b and 6a isolates, covering 80 % HCV strains prevalent in south China and south-east Asia. Eight HCV vaccine strains were grouped into Sad23L-based vaccine cocktail-1 and Ad49L-based vaccine cocktail-2 for vaccinating mice, respectively. RESULTS: The immunogenicity of a single dose of 107-1010 PFU HCV individual vaccines was evaluated in mice, showing weak specific antibody to E1 and E2 protein but a dose-dependent T cell response to E1E2/NS3-5B peptides, which could be significantly enhanced by boosting with an alternative vector vaccine carrying homologous antigen. Prime-boost vaccinations with vaccine cocktail-1 and cocktail-2 induced significantly higher cross-reactive antibody and stronger T cell responses to HCV Gt-1b/6a. The high frequency of intrasplenic and intrahepatic NS31629-1637 CD8+ T cell responses were identified, in which the high proportion of TRM and TEM cells might play an important role against HCV infection in liver. CONCLUSIONS: Prime-boost regimens with HCV vaccine cocktails elicited the broad cross-reactive antibody and robust T cell responses against multi-genotypic HCV in mice.

Lightweight Deep Learning Models for High-Precision Rice Seedling Segmentation from UAV-Based Multispectral Images.

Accurate segmentation and detection of rice seedlings is essential for precision agriculture and high-yield cultivation. However, current methods suffer from high computational complexity and poor robustness to different rice varieties and densities. This article proposes 2 lightweight neural network architectures, LW-Segnet and LW-Unet, for high-precision rice seedling segmentation. The networks adopt an encoder-decoder structure with hybrid lightweight convolutions and spatial pyramid dilated convolutions, achieving accurate segmentation while reducing model parameters. Multispectral imagery acquired by unmanned aerial vehicle (UAV) was used to train and test the models covering 3 rice varieties and different planting densities. Experimental results demonstrate that the proposed LW-Segnet and LW-Unet models achieve higher F1-scores and intersection over union values for seedling detection and row segmentation across varieties, indicating improved segmentation accuracy. Furthermore, the models exhibit stable performance when handling different varieties and densities, showing strong robustness. In terms of efficiency, the networks have lower graphics processing unit memory usage, complexity, and parameters but faster inference speeds, reflecting higher computational efficiency. In particular, the fast speed of LW-Unet indicates potential for real-time applications. The study presents lightweight yet effective neural network architectures for agricultural tasks. By handling multiple rice varieties and densities with high accuracy, efficiency, and robustness, the models show promise for use in edge devices and UAVs to assist precision farming and crop management. The findings provide valuable insights into designing lightweight deep learning models to tackle complex agricultural problems.

Characterization of Pre-Existing Neutralizing Antibody to Human Adenovirus Types 5 and 49 and Simian Type 23 in Chinese Population.

Recombinant adenovirus vector has been widely used in vaccine development. Due to the pre-existing immunity of human adenovirus type 5 (HAd5) in humans, a range of rare human and chimpanzee adenovirus vectors have been developed. In the previous study, we constructed novel adenovirus vector Sad23L and Ad49L based on simian adenovirus type 23 (SAd23) and human adenovirus type 49 (HAd49), which were used in the development of ZIKV and COVID-19 vaccines. However, the levels of pre-existing neutralizing antibody (NAb) of HAd49 and SAd23 remain unclear in China. In this study, we measured NAbs titers of HAd5, HAd49, and SAd23 in 600 healthy blood donors from 6 regions across China. NAb titer of HAd49 or SAd23 was significantly lower than that of HAd5 (p < 0.001). There was no significant difference in seroprevalence and NAb titers of three adenoviruses between male and female donors. The seropositive rates of HAd5 and SAd23 increased with age growth in a positive correlation (p < 0.01), while in contrast to HAd5, HAd49, and SAd23 had a low level of pre-existing immunity in Chinese population, which suggested that Ad49L and Sad23L vectors could be used in vaccine development for humans.

A novel simian adenovirus-vectored COVID-19 vaccine elicits effective mucosal and systemic immunity in mice by intranasal and intramuscular vaccination regimens.

IMPORTANCE: The essential goal of vaccination is to generate potent and long-term protection against diseases. Several factors including vaccine vector, delivery route, and boosting regimen influence the outcome of prime-boost immunization approaches. The immunization regimens by constructing a novel simian adenovirus-vectored COVID-19 vaccine and employing combination of intranasal and intramuscular inoculations could elicit mucosal neutralizing antibodies against five mutant strains in the respiratory tract and strong systemic immunity. Immune protection could last for more than 32 weeks. Vectored vaccine construction and immunization regimens have positively impacted respiratory disease prevention.

Two Novel Adenovirus Vectors Mediated Differential Antibody Responses via Interferon-α and Natural Killer Cells.

Recombinant adenovirus vectors have been widely used in vaccine development. To overcome the preexisting immunity of human adenovirus type 5 (Ad5) in populations, a range of chimpanzee or rare human adenovirus vectors have been generated. However, these novel adenovirus vectors mediate the diverse immune responses in the hosts. In this study, we explored the immune mechanism of differential antibody responses to SARS-CoV-2 S protein in mice immunized by our previously developed two novel simian adenovirus type 23 (Sad23L) and human adenovirus type 49 (Ad49L), and Ad5 vectored COVID-19 vaccines. Sad23L-nCoV-S and Ad5-nCoV-S vaccines induced the low level of interferon-α (IFN-α) and the high level of antigen-specific antibody responses in wild-type and IFN-α/ß receptor defective (IFNAR-/-) C57 mice, while Ad49L-nCoV-S vaccine induced the high IFN-α and low antibody responses in C57 mice but the high antibody response in IFNAR-/- mice. In addition, the high antibody response was detected in natural killer (NK) cells-blocked but the low in follicular helper T (TFH) cells -blocked C57 mice immunized with Ad49L-nCoV-S vaccine. These results showed that Ad49L vectored vaccine stimulated IFN-α secretion to activate NK cells, and then reduced the number of TFH cells, generation center (GC) B cells and plasma cells, and subsequently reduced antigen-specific antibody production. The different novel adenovirus vectors could be selected for vaccine development according to the need for either humoral or cellular or both immune protections against a particular disease. IMPORTANCE Novel adenovirus vectors are an important antigen delivery platform for vaccine development. Understanding the immune diversity between different adenoviral vectors is critical to design the proper vaccine against an aim disease. In this study, we described the immune mechanism of Sad23L and Ad49L vectored vaccines for raising the equally high specific T cell response but the different level of specific antibody responses in mice. We found that Ad49L-vectored vaccine initiated the high IFN-α and activated NK cells to inhibit antibody response via downregulating the number of CD4+ TFH cells leading to the decline of GC B cells and plasma cells.

Occult Hepatitis B Virus Infection and Liver Fibrosis in Chinese Patients.

BACKGROUND: The impact of hepatitis B surface antigen (HBsAg)-negative/hepatitis B virus (HBV) DNA-positive occult HBV infection (OBI) on the severity of liver fibrosis remains unclear. METHODS: A total of 1772 patients negative for HBsAg but positive for antibody to hepatitis B core antigen (HBcAg), stratified by the presence or absence of OBI, were selected for long-term carriage leading to elevation of ≥2 of 4 liver fibrosis indexes-hyaluronic acid (HA), laminin, type III procollagen peptide (PCIII), and type IV collagen (CIV)-at testing in a Chinese hospital. Patients were tested for serum viral load, HBV markers, and histopathological changes in liver biopsy specimens. RESULTS: OBI was identified in 148 patients with liver fibrosis (8.4%), who had significantly higher levels of HA, laminin, PCIII, and CIV than 1624 fibrotic patients without OBI (P < .05). In 36 patients with OBI who underwent liver biopsy, significant correlations were observed between OBI viral load and serum HA levels (P = .01), PCIII levels (P = .01), and pathological histological activity index (HAI) scores (P < .001), respectively; HAI scores and PCIII levels (P = .04); HBcAg immunohistochemical scores and HA levels (P < .001); and HBcAg immunohistochemical scores and PCIII levels (P = .03). Positive fluorescent in situ hybridization results were significantly more frequent in patients with OBIs (80.6% vs 37.5% in those without OBIs). Among patients with OBIs, HBcAg was detected in the liver tissue in 52.8% and HBsAg in 5.6%. CONCLUSIONS: OBI status appears to be associated with liver fibrosis severity.

Pre-existing immunity to SARS-CoV-2 associates with strong T cell responses induced by inactivated COVID-19 vaccines.

Individuals with a recent common cold coronavirus infection, which leads to pre-existing immunity against SARS-CoV-2, displayed a less severe course of COVID-19. However, the relationship between pre-existing immunity against SARS-CoV-2 and the inactivated-vaccine-induced immune response is still unknown. Here, 31 healthcare workers who received standard two doses of inactivated COVID-19 vaccines (Weeks 0 and 4, respectively) were enrolled, vaccine-induced neutralization and T cell responses were detected, and the correlation between the pre-existing SARS-CoV-2-specific immunity was analyzed. We found the SARS-CoV-2-specific antibodies, pseudovirus neutralization test (pVNT) titers, and spike-specific interferon gamma (IFN-γ) production in CD4+ and CD8+ T cells were significantly elevated after two doses of inactivated vaccines. Interestingly, the pVNT titers after the second dose of vaccination displayed no significant correlation with the pre-existing SARS-CoV-2-specific antibodies or B cells, nor the pre-existing spike-specific CD4+ T cells. Notably, the spike-specific T cell response after the second dose of vaccination was positively correlated with the pre-existing receptor binding domain (RBD)-specific B cells and CD4+ T cells, which were documented by the frequencies of RBD-binding B cells, the breadth of RBD-specific B cell epitopes, and the frequency of IFN-γ-expressing RBD-specific CD4+ T cells. Overall, the inactivated-vaccine-induced T cell responses, not the inactivated-vaccine-induced neutralization, closely correlated with pre-existing immunity to SARS-CoV-2. Our results provide a better understanding of inactivated-vaccine-induced immunity and help predict the immunogenicity induced by inactivated vaccines in individuals.

Vaccination of cats with Sad23L-nCoV-S vaccine candidate against major variants of SARS-CoV-2.

Cats are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and risk transmitting viruses to naive cats or humans. Here, based on our novel adenovirus-vectored COVID-19 vaccine, the immunogenicity of Sad23L-nCoV-S vaccine was evaluated in cats by prime-boost vaccinations. Five cats were primed with a dose of 108 plaque-forming units (PFUs) Sad23L-nCoV-S vaccine and then boosted with an equal dose of same vaccine at a 4-week interval. Cat serum neutralizing antibody (NAb) titers (the sample dilution at which 50% inhibitory concentration [IC50]) were measured as IC50 15,849 to wild-type strain, IC50 6,591 to Alpha, IC50 2,315 to Beta, IC50 2,744 to Gamma, IC50 1,848 to Delta, and IC50 318 to Omicron variants of pseudotyped SARS-CoV-2 viruses at week 6 post-prime vaccination. All NAb levels to these five variants were ≥IC50 49 from vaccinated cats at week 10, while 48.8% to Delta and 100% to Omicron variants were

A Self-Biomineralized Novel Adenovirus Vectored COVID-19 Vaccine for Boosting Immunization of Mice.

SARS-CoV-2 has caused more than 3.8 million deaths worldwide, and several types of COVID-19 vaccines are urgently approved for use, including adenovirus vectored vaccines. However, the thermal instability and pre-existing immunity have limited its wide applications. To circumvent these obstacles, we constructed a self-biomineralized adenovirus vectored COVID-19 vaccine (Sad23L-nCoV-S-CaP) by generating a calcium phosphate mineral exterior (CaP) based on Sad23L vector carrying the full-length gene of SARS-CoV-2 spike protein (S) under physiological condition. This Sad23L-nCoV-S-CaP vaccine was examined for its characteristics of structure, thermostability, immunogenicity and avoiding the problem of preexisting immunity. In thermostability test, Sad23L-nCoV-S-CaP could be stored at 4 °C for over 45 days, 26 °C for more than 8 days and 37 °C for approximately 2 days. Furthermore, Sad23L-nCoV-S-CaP induced higher level of S-specific antibody and T cell responses, and was not affected by the pre-existing anti-Sad23L immunity, suggesting it could be used as boosting immunization on Sad23L-nCoV-S priming vaccination. The boosting with Sad23L-nCoV-S-CaP vaccine induced high titers of 105.01 anti-S1, 104.77 anti-S2 binding antibody, 103.04 pseudovirus neutralizing antibody (IC50), and robust T-cell response of IFN-γ (1466.16 SFCs/106 cells) to S peptides, respectively. In summary, the self-biomineralization of the COVID-19 vaccine Sad23L-nCoV-S-CaP improved vaccine efficacy, which could be used in prime-boost regimen for prevention of SARS-CoV-2 infection in humans.

Track-etched membrane microplate and smartphone immunosensing for SARS-CoV-2 neutralizing antibody.

The level of neutralizing antibody (NAb) to SARS-CoV-2 could be used to evaluate the acquired protective immunity of COVID-19 patients or vaccinees. Here we develop a track-etched microporous membrane filtration microplate (TEM) and optical fibers transmitted immunosensing smartphone platform (TEMFIS) based surrogate virus neutralization test (TEMFIS-sVNT) for rapid one-step testing of NAb to SARS-CoV-2. Coefficient variation (CV) of intra-assay and inter-assay precisions of TEMFIS-sVNT varied below 9% or 14%, respectively. By agreement with pseudovirus neutralization test (pVNT) and ELISA-sVNT for testing of serum samples from 41 COVID-19 patients, 50 COVID-19 vaccinees and 320 healthy blood donors (P = 0.895), TEMFIS-sVNT detected the NAb positivity (sensitivity) in 92.68% COVID-19 patients and 76% vaccinees, but the NAb negativity (specificity) in 100% blood donors. In conclusion, TEMFIS-sVNT can be used for quantitatively point-of-care testing of neutralizing antibody to SARS-CoV-2 in blood samples from COVID-19 patients and vaccinees.

Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates.

ABSTRACTCOVID-19 vaccines are being developed urgently worldwide. Here, we constructed two adenovirus vectored COVID-19 vaccine candidates of Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length gene of SARS-CoV-2 spike protein. The immunogenicity of two vaccines was individually evaluated in mice. Specific immune responses were observed by priming in a dose-dependent manner, and stronger responses were obtained by boosting. Furthermore, five rhesus macaques were primed with 5 × 109 PFU Sad23L-nCoV-S, followed by boosting with 5 × 109 PFU Ad49L-nCoV-S at 4-week interval. Both mice and macaques well tolerated the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost regimen induced high titers of 103.16 anti-S, 102.75 anti-RBD binding antibody and 102.38 pseudovirus neutralizing antibody (pNAb) at 2 months, while pNAb decreased gradually to 101.45 at 7 months post-priming. Robust T-cell response of IFN-γ (712.6 SFCs/106 cells), IL-2 (334 SFCs/106 cells) and intracellular IFN-γ in CD4+/CD8+ T cell (0.39%/0.55%) to S peptides were detected in vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.

A high infectious simian adenovirus type 23 vector based vaccine efficiently protects common marmosets against Zika virus infection.

Zika virus (ZIKV) has spread in many countries or territories causing severe neurologic complications with potential fatal outcomes. The small primate common marmosets are susceptible to ZIKV, mimicking key features of human infection. Here, a novel simian adenovirus type 23 vector-based vaccine expressing ZIKV pre-membrane-envelope proteins (Sad23L-prM-E) was produced in high infectious titer. Due to determination of immunogenicity in mice, a single-dose of 3×108 PFU Sad23L-prM-E vaccine was intramuscularly inoculated to marmosets. This vaccine raised antibody titers of 104.07 E-specific and 103.13 neutralizing antibody (NAb), as well as robust specific IFN-γ secreting T-cell response (1,219 SFCs/106 cells) to E peptides. The vaccinated marmosets, upon challenge with a high dose of ZIKV (105 PFU) six weeks post prime immunization, reduced viremia by more than 100 folds, and the low level of detectable viral RNA (<103 copies/ml) in blood, saliva, urine and feces was promptly eliminated when the secondary NAb (titer >103.66) and T-cell response (>726 SFCs/106 PBMCs) were acquired 1-2 weeks post exposure to ZIKV, while non-vaccinated control marmosets developed long-term high titer of ZIKV (105.73 copies/ml) (P<0.05). No significant pathological lesions were observed in marmoset tissues. Sad23L-prM-E vaccine was detectable in spleen, liver and PBMCs at least 4 months post challenge. In conclusion, a prime immunization with Sad23L-prM-E vaccine was able to protect marmosets against ZIKV infection when exposed to a high dose of ZIKV. This Sad23L-prM-E vaccine is a promising vaccine candidate for prevention of ZIKV infection in humans.

Occurrence of occult hepatitis B virus infection associated with envelope protein mutations according to anti-HBs carriage in blood donors.

OBJECTIVES: Occult hepatitis B virus infection (OBI) carries a risk of hepatitis B virus (HBV) transmission and hepatocellular carcinoma. As previous studies have had a limited sample size, the characteristics of OBI with genotype B and C (OBIB and OBIC) mutations relating to hepatitis B surface antibody (anti-HBs) elicited by vaccination or a limited host immune response to HBV have not been fully explored. METHODS: In this study, the occurrence of OBIB or OBIC strains associated with envelope protein (pre-S/S) amino acid substitutions obtained from 99 blood donors stratified according to anti-HBs carriage were characterized extensively. RESULTS: According to the presence of anti-HBs within each genotype, the number and frequency of substitution sites specific for anti-HBs(-) OBIB were higher than those specific for anti-HBs(+) OBIB strains (67 vs 31; 117 vs 41), but the reverse pattern was found in OBIC strains (3 vs 24; 3 vs 26). Mutations pre-s1T68I and sQ129R/L were found uniquely in 15-25% of anti-HBs(+) OBIB carriers and mutation pre-s1A54E was found preferentially in anti-HBs(+) OBIC, while 17 substitutions were found preferentially in 11-38% of anti-HBs(-) OBIB strains. In the major hydrophilic region (MHR) region, mutations sS167 in OBIB, sT118 in OBIC, and sA166 in both genotypes were possibly immune-induced escape mutation sites. CONCLUSIONS: Several mutations in pre-S/S of OBI appeared to be associated with carrier anti-HBs pressure, which might be risk factors for potential reactivation of viruses under anti-HBs selection in OBI carriers.

A rapid strategy for constructing novel simian adenovirus vectors with high viral titer and expressing highly antigenic proteins applicable for vaccine development.

Adenoviral vectors have been widely used for the development of infectious disease vaccines. However, the challenge of human adenoviral vector rooted from the predominant adenovirus serotype 5 strain limiting its usefulness by the widespread pre-existing neutralizing antibodies in recipients. To circumvent this obstacle, we generated an ad-hoc adenovirus vector in human or primates. Here, a chimeric simian adenoviral vector Sad23 was constructed consisting in deleting of E1 and E3 regions of the full-length simian adenovirus serotype 23 genome (SAdV23) by Gibson assembly. To improve Sad23 virus propagating efficiency, the E4 region open reading frame 6 (orf6) was replaced by the corresponding element of human adenovirus type 5 (Ad5), designated Sad23L. The procedure for cloning this novel vector took a single week, and recombinant adenovirus was packaged with high titer in HEK293 cells. To verify the ability of this novel adenoviral vector to deliver foreign genes, Zika virus (ZIKV) prM-E genes were used as target genes for antigen expression. Recombinant adenoviruses Sad23L-prM-E, Sad23-prM-E and Ad5-prM-E were intramuscularly inoculated into Ad5-eGFP none pre-exposed or pre-exposed mice, and the immune response to ZIKV prM-E was compared between vectors. Sad23L-prM-E induced a fairly robust immune response and maintained immunogenicity in Ad5 pre-exposed mice, which suggested that Ad5 pre-existing immunity did not affect Sad23L-prM-E immunization. These preliminary results suggest that the proposed rapid strategy was effective in constructing a new adenoviral vector platform (Sad23 L) usable for the development of human vaccines.

YAP Aggravates Inflammatory Bowel Disease by Regulating M1/M2 Macrophage Polarization and Gut Microbial Homeostasis.

Inflammation, epithelial cell regeneration, macrophage polarization, and gut microbial homeostasis are critical for the pathological processes associated with inflammatory bowel disease (IBD). YAP (Yes-associated protein) is a key component of the Hippo pathway and was recently suggested to promote epithelial cell regeneration for IBD recovery. However, it is unclear how YAP regulates macrophage polarization, inflammation, and gut microbial homeostasis. Although YAP has been shown to promote epithelial regeneration and alleviate IBD, here we show that YAP in macrophages aggravates IBD, accompanied by the production of antimicrobial peptides and changes in gut microbiota. YAP impairs interleukin-4 (IL-4)/IL-13-induced M2 macrophage polarization while promoting lipopolysaccharide (LPS)/interferon γ (IFN-γ)-triggered M1 macrophage activation for IL-6 production. In addition, YAP expression is differently regulated during the induction of M2 versus M1 macrophages. This study suggests that fully understanding the multiple functions of YAP in different cell types is crucial for IBD therapy.

YorkieCA overexpression in the posterior silk gland improves silk yield in Bombyx mori.

The traditional hybrid breeding techniques can no longer meet the increasing demands for silk production by the silkworm, Bombyx mori, and further improvement of the silk yield will depend on modern molecular breeding techniques. Here, we report improved silk yield in transgenic silkworms overexpressing the oncogene YorkieCA specifically in the posterior silk gland (PSG). The YorkieCA cDNA was ligated downstream of the hr3 enhancer and the fibroin L-chain (Fil) promoter, then inserted into a piggyBac vector for transgene. Overexpression of YorkieCA in the PSG significantly increased the weight of the PSG, and also increased the weight of the cocoon, larval body, and pupal body to decreasing degrees. Overexpression of YorkieCA up-regulated the Yorkie target genes resulting in increased cell size, endomitosis, the number of protein synthesis organelles, the expression of fibroin genes in the PSG, and eventually silk yield. Additionally, as we reported previously using the binary GAL4/UAS system, transgenic silkworms overexpressing Ras1CA with the hr3 enhancer and the Fil promoter also showed improved silk yield. Unfortunately, the hybrid progeny of YorkieCA-overexpressing silkworms and Ras1CA-overexpressing silkworms did not show overlapping improved silk yield due to the failure to increase expression of both Yorkie and Ras1.

Yorkie Facilitates Organ Growth and Metamorphosis in Bombyx.

The Hippo pathway, which was identified from genetic screens in the fruit fly, Drosophila melanogaster, has a major size-control function in animals. All key components of the Hippo pathway, including the transcriptional coactivator Yorkie that is the most critical substrate and downstream effector of the Hippo kinase cassette, are found in the silkworm, Bombyx mori. As revealed by microarray and quantitative real-time PCR, expression of Hippo pathway genes is particularly enriched in several mitotic tissues, including the ovary, testis, and wing disc. Developmental profiles of Hippo pathway genes are generally similar (with the exception of Yorkie) within each organ, but vary greatly in different tissues showing nearly opposing expression patterns in the wing disc and the posterior silk gland (PSG) on day 2 of the prepupal stage. Importantly, the reduction of Yorkie expression by RNAi downregulated Yorkie target genes in the ovary, decreased egg number, and delayed larval-pupal-adult metamorphosis. In contrast, baculovirus-mediated Yorkie(CA) overexpression upregulated Yorkie target genes in the PSG, increased PSG size, and accelerated larval-pupal metamorphosis. Together the results show that Yorkie potentially facilitates organ growth and metamorphosis, and suggest that the evolutionarily conserved Hippo pathway is critical for size control, particularly for PSG growth, in the silkworm.