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BACKGROUND: Bursicon is a heterodimeric neuropeptide that is involved in many physiological activities such as cuticle tanning, wing expansion, reproduction and immunity in insects. In this study, the role of bursicon in the wing expansion was investigated in Bactrocera dorsalis, an important invasive insect pest in agriculture. RESULTS: The cDNA sequences and deduced amino acids of bursicon genes (named BdBurs‐α and BdBurs‐β) were determined, and two proteins typically contained 11 cysteine residues in conserved positions that were highly conserved in other insect species. The spatiotemporal expressions of bursicon genes showed that higher expression occurred at the pupal, early adult stage and ovaries, and lower expression at the late larval stage and in wing tissue (8‐day‐old pupae). Dysfunction of bursicon genes by dsRNA microinjection into 5‐day‐old pupae reduced PKA (a downstream component of the bursicon pathway) activity and resulted in malformed adult wings. PKA inhibitor injection into 5‐day‐old pupae also resulted in similar phenotypes. Hematoxylin & eosin staining of the adult wing showed that RNAi and PKA inhibitor treatment reduced the thickness of the wing cuticle, which wing cuticle thickness were ≈50% thinner than in the control. Furthermore, the expression of hedgehog (Bdhh) (one of 10 tested genes related to wing development) was significantly upregulated after RNAi and PKA inhibitor application. CONCLUSION: The results indicate that bursicon plays a crucial role in the wing expansion of B. dorsalis, suggesting bursicon genes have potential to be the targets for B. dorsalis control. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Diapause is an environmentally preprogrammed period of arrested development, and characterized by metabolic depression that can occur during any development stage of insect. The insect steroid hormone 20‐hydroxyecdysone (20E), is converted from ecdysone by the cytochrome P450 enzyme shade (CYP314A1), and it exerts a potent effect on the induction and maintenance of diapause in obligatory diapause insects. However, the regulatory mechanism of 20E in obligatory diapause development remains unclear. In this study, the function of 20E in the pupal diapause of Bactrocera minax was investigated. RESULTS: We determined the expression pattern of Halloween P450 genes from larval to adult B. minax, and found differential expression of CYP314A1 from other P450 genes, with a high level in larvae and a low level in pupae. Dysfunction of CYP314A1 by dsCYP314A1 microinjection in third‐instar larvae caused significant larval mortality or abnormal pupae. Compared with dsGFP and DEPC‐water, dsCYP314A1‐injected larvae had significantly reduced 20E titer and altered energy metabolism, and many individuals failed to pupate. Exogenous 20E microinjected into late third‐instar larvae or 20E fed to early third‐instar larvae both caused similar energy metabolism changes. The 20E‐treated larvae of B. minax had reduced total lipids and increased amounts of trehalose and glycogen. Furthermore, 20E‐treated diapause individuals showed rapid pupal development. CONCLUSION: The 20E biosynthesis was regulated by the expression of CYP314A1, and was involved in the induction and termination phase of obligate diapause by regulating energy metabolism in B. minax. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

As an essential environmental factor that affects the economic benefits of aquaculture, hypoxia is one of the urgent problems to be solved in the aquaculture fish breeding industry. Common carp (Cyprinus carpio) is a critical economic fish in China, and at present, there are many breeding strains of common carp with different character advantages in China, including Hebao red carp (C. carpio var wuyuanesis) and Songpu mirror carp (C. carpio var specularis). Even if the environmental adaptation of common carp is generally strong, the genetic background of hypoxia tolerance in different strains of common carp is unclear yet. This study tested the hypoxia tolerance of Songpu minor carp, Hebao red carp, and their hybrid F1 population by an acute hypoxia treatment. Muscle and liver tissues were used for transcriptome sequencing analysis to identify the key factors for hypoxia tolerance and explore the potential genetic mechanism for breeding high hypoxia tolerance in common carp. The comparative transcriptomic analysis revealed abundant hypoxia response-related genes and their differential regulation mechanism in these two tissues of different common carp strains under acute hypoxia, including immune response, cellular stress response, HIFs (hypoxia-inducible factors), MAP kinase, iron ion binding, and heme binding. Our findings will facilitate future investigation on the hypoxia response mechanism and provide a solid theoretical basis for breeding projects in common carp. [ABSTRACT FROM AUTHOR]

: During DNA double-strand break repair, two structure-specific DNA nucleases are controlled by the same regulator Pxd1, but in opposite manners. [ABSTRACT FROM AUTHOR]

Aim: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is the key of the development of diabetic retinopathy (DR), and lncRNA NEAT1 could accelerate EMT in diabetic nephropathy. Meanwhile, as a diabetes susceptibility gene, whether sex-determining region Y-related (SRY) high-mobility group box 4 (SOX4) has relationship with lncRNA NEAT1 in DR remains unclear., Methods: Firstly, NEAT1, SOX4 and miR-204 were evaluated by qRT-PCR (quantitative reverse-transcriptase PCR) under high glucose condition. Then, cell viability, proliferation, migration and invasion were respectively detected by MTT, BrdU staining, wound healing and transwell assay after NEAT1 knockdown or miR-204 overexpression. Also, the EMT-related proteins were examined by western blot and cell immunofluorescence assay. In order to confirm the relationship between miR-204 and NEAT1 or SOX4, dual luciferase reporter gene assay was conducted. At the same time, the protein levels of SOX4 and EMT-related proteins were investigated by immunohistochemistry in vivo ., Results: High glucose upregulated NEAT1 and SOX4 and downregulated miR-204 in ARPE19 cells. NEAT1 knockdown or miR-204 overexpression inhibited the proliferation and EMT progression of ARPE19 cells induced by high glucose. NEAT1 was identified as a molecular sponge of miR-204 to increase the level of SOX4. The effect of NEAT1 knockdown on the progression of EMT under high glucose condition in ARPE19 cells could be reversed by miR-204 inhibitor. Also, NEAT1 knockdown inhibited retinal EMT in diabetic mice., Conclusion: NEAT1 regulated the development of EMT in DR through miR-204/SOX4 pathway, which could provide reference for clinical prevention and treatment., Competing Interests: The authors declare there are no competing interests., (©2021 Yang et al.)

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication. Ribonucleotides are incorporated into the pol2-16 genome in patterns consistent with leading-strand replication by Pol δ when Pol ε is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.

Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)-associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions. SPI-seq faithfully detected, in fission yeast, Rad52 enrichment at artificially induced double-strand breaks (DSBs) as well as endogenously programmed DSBs for mating-type switching. Applying Rad52 SPI-seq to fission yeast mutants defective in DNA helicase Pfh1 or histone H3K56 deacetylase Hst4, led to global views of DNA lesion hotspots emerging in these mutants. We also found serendipitously that histone dosage aberration can activate retrotransposon Tf2 and cause the accumulation of a Tf2 cDNA species bound by Rad52. SPI-seq should be widely applicable for mapping sites of DNA damage and uncovering the causes of genome instability.