RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring freshwater snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remains unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes of 4 snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus and Lymnaea stagnalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all 4 species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for double-stranded RNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2 and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in these snails as an experimental tool and an alternative control method to CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based approaches.

This study investigates germination transcriptome changes in seeds that aged during cool, dry storage, searching for signals of the alive-to-dead transition. Signals may reflect the activity of uncharacteristic metabolic pathways or the effects of accumulated small injuries that impair recovery from life in the dry state. Cohorts of soybean (Glycine max cv. ‘Williams 82’) seeds were stored for 3, 19 and 22 years, stored in years 1996, 1999 and 2015, respectively. These cohorts showed < 2, 40 and ∼99% mortality, respectively, in response to storage and ageing. Transcriptomes of dry embryonic axes and axes after 24 hours of imbibition were sequenced. Imbibed axes had 19,340 genes significantly differentially expressed (SDE) compared to dry axes. Imbibed axis transcriptomes clustered into three groups, comprised of axes with high, low, or no germination potential (GP). There were 17,360 SDE genes in high-GP axes and 4,892 SDE genes, mostly up-regulated, in no-GP axes. Transcriptomes of no-GP axes imbibed for 24 hours were similar to healthy axes imbibed for 3 hours. Slow transcription, rather than transcription errors or novel expression pathways, signals failure to transition from seed to seedling. We conclude that the death of dry-aged seeds during imbibition arises from metabolism that is ‘too little, too late’.

Heat shock proteins (HSPs) maintain cellular homeostasis and regulate immune responses across species. HSP90 stabilises and activates regulatory proteins, while HSP70 facilitates protein folding and prevents aggregation. In this study, the complete cDNA sequences of Dastarcus helophoroides – HSP70, HSP90, and β-actin (used as a reference for real-time quantitative polymerase chain reaction) – were obtained using rapid amplification of cDNA ends-polymerase chain reaction, and the expression of HSP70 and HSP90 under cold stress was analysed. The full-length cDNA of HSP90 contains a 2346-bp open reading frame (ORF) encoding 781 amino acids with a molecular weight (MW) of 89.6 kDa. The HSP70 cDNA contains a 1911-bp ORF encoding 636 amino acids (MW: 69.7 kDa), and the β-actin cDNA contains a 1131-bp ORF encoding 376 amino acids (MW: 41.7 kDa). Cold stress significantly affected HSP expression: HSP90 expression peaked at −15 °C, with a 9.45-fold increase compared to the control (P < 0.05), whereas HSP70 expression increased markedly at −10 °C, with a 60.42-fold increase compared to the control (P < 0.05). These findings have important biological implications for predicting insect performance under fluctuating thermal environments and for optimising low-temperature storage and release strategies of D. helophoroides in biological control programmes.

The successful freezing of rat spermatozoa and the development of innovative cryoprotectant formulations are of great importance in the field of reproductive biotechnology. In the present study, 4 mg/mL Ascorbic acid and 20 nM Biotin were added to cryopreservation medium containing 8% lactose-monohydrate, 23% egg yolk, and 10% tris-aminomethane to investigate the freezability of rat sperm. After cryopreservation, motility, viable spermatozoon ratio, plasma membrane integrity, abnormal acrosome ratio, and mRNA levels of certain heat shock proteins that play a critical role in the sperm cryopreservation process were evaluated. After thawing, the highest motility rate was found in the ascorbic acid group (17.50 ± 3.27), which was significantly higher compared with the control group (10.13 ± 1.16) (p < 0.05). Likewise, the highest viability rate was found in the ascorbic acid group (41.75 ± 3.69), while it was found to be significantly higher compared with the control group (19.63 ± 1.44) (p < 0.05). When the cells were evaluated in terms of plasma membrane integrity and abnormal acrosome rate, no statistical difference was found between the groups (p > 0.05). The expression levels of heat shock protein-related genes were determined using the RT-qPCR technique. Addition of biotin and ascorbic acid significantly reduced Hsp40, Hsp60, and Hsp70 mRNA levels compared with the control group (p < 0.05). In conclusion, based on the evaluated parameters, it was revealed that the addition of the antioxidant Ascorbic acid and biotin to rat sperm extender provides effective protection against cryodamage during the cryopreservation process. These findings provide a valuable foundation for research into the development of next-generation mediums for sperm cryopreservation from a molecular biotechnology perspective.

High-energy, sugar-rich diets are associated with obesity and pancreatic disorders. We investigated the effects of consumption of a high-fat and high-fructose diet (HFFrD) on gene expression related to insulin synthesis, inflammation and apoptosis in pancreatic β-cells. Weaned Wistar rats were fed either a control diet (CD; 11 % kcal from fat and 0 % from fructose) or an HFFrD (48 % kcal from fat and 33 % from fructose) for 22 weeks; after a 6-h fast, animals were euthanised. Body weight and total fat were recorded. Serum analyses included: glucose, insulin, triacylglycerides (TAG), malondialdehyde (MDA), TNFα, IL-6, IL-10 and advanced glycation end-products (AGE). Pancreatic islets were analysed for gene expression linked to insulin synthesis, inflammation and apoptosis. Pancreatic assessments included TUNEL assay and immunohistochemistry. HFFrD increased body weight, total fat, MDA, insulin, homeostatic model assessment for insulin resistance, AGE, TAG and IL-6 concentrations in both sexes. Gene expression revealed sex-dependent differences: Glut2, Gck, Khk, Ins2 and the transcription factor Mafa were downregulated in males but upregulated in females. Pdx1 expression increased in females, whereas NeuroD1 increased in males. Pro-inflammatory markers (Il-1β, Il-6) increased in both sexes, whereas Il-10 decreased in males and increased in females. Bax/Bcl2 ratio decreased in males but increased in females; antioxidant and apoptotic markers Nrf2 and Casp3 increased in females. Endocannabinoid receptors (Cnr1, Cnr2) increased in both sexes. HFFrD altered the expression of genes involved in insulin synthesis, inflammation and apoptosis in a sex-specific manner. Compared with males, females showed lower vulnerability, possibly because of adaptive responses in insulin synthesis signalling influenced by sex hormones.

Global demand for pork, the most consumed meat worldwide, continues to rise, driving producers to adopt early weaning practices to increase pigs born each year. However, early weaning disrupts intestinal development and function, which can limit growth, therefore, understanding the mechanisms associated with maintenance of intestinal health are central to maximizing growth performance and productivity of pigs. The stressors of the post-weaning period induce changes to the complex microbiota–host–immune interactions that are further influenced by dietary nutrients. Central to these interactions is the recognition of changes in the intestinal microbiota by pattern recognition receptors of enterocytes and immune cells. Recognition of conserved microbial structures activates signaling cascades that regulate cytokine production, epithelial barrier function and redox balance. Bacterial influence on these pathways differs, with some bacteria promoting tolerance and anti-inflammatory signaling, whereas others induce pro-inflammatory responses. Dietary composition, including protein and non-starch polysaccharides content, and select feed additives, can influence the composition of the microbiota, thereby modulating microbiota–host–immune interactions. By utilizing nutriomics, an integrative framework that combines nutritional interventions with multi-omics technologies to connect dietary inputs to changes in the microbiota–host–immune interactions, this review highlights the complexity and context-dependent nature of these interactions and the role of nutrition to optimize intestinal health and enhance growth performance in nursery pigs.

Obsessive-compulsive disorder (OCD) is a complex psychiatric disorder. While existing studies have revealed abnormalities in brain structure and function associated with OCD, there is a paucity of research integrating these two aspects, and the transcriptional patterns underlying these abnormalities remain unclear. This study is a multiscale, exploratory investigation designed to generate hypotheses rather than to test causal mechanisms. We aimed to investigate aberrations in brain structure–function coupling (SFC) in OCD patients and, by integrating gene expression profiles and neurotransmitter maps, to explore the potential molecular and genetic bases of these changes. We recruited 100 medication-free OCD patients and 90 healthy controls, and employed multimodal imaging techniques to systematically analyze abnormalities in static SFC in OCD patients. Subsequently, we conducted transcriptomic analysis to identify genes associated with SFC abnormalities and performed spatial correlation analysis with neurotransmitter atlases to investigate potential links between SFC dysregulation and transcriptional patterns. Our findings demonstrated that OCD patients exhibit significant SFC abnormalities in the right temporoparietal junction (rTPJ). These SFC abnormalities are significantly associated with 2,421 gene expression profiles and the serotonin neurotransmitter system. Gene enrichment analysis revealed that these aberrant genes are primarily involved in key biological processes, such as brain development, synaptic signaling, cell projection development, and regulation of neuronal processes. By integrating multimodal imaging, transcriptomic, and neurotransmitter data, this study provides multiscale evidence for the potential molecular basis of SFC abnormalities in the rTPJ of OCD patients, offering preliminary insights into a possible pathological pathway of OCD.

The immunomodulatory effects of Ziziphus jujuba Mill. fruit extracts were investigated using Galleria mellonella as an insect immune model. The expression of antimicrobial peptides, hemolin, phenoloxidase genes (PO-I and PO-II), and enzyme activity was measured in response to Candida albicans, silica beads, and Z. jujuba fruit extract. Responses were found to be stimulus- and time-dependent. Gallerimycin and galiomycin, key antimicrobial peptides, exhibited distinct expression patterns, with gallerimycin showing a more pronounced response to pathogens and beads. The Z. jujuba extract stimulated an early but balanced immune activation, likely due to its bioactive compounds. Hemolin expression varied between larvae and haemocytes depending on the type and duration of the challenge, supporting its role in immune recognition and opsonisation. Phenoloxidase activity and gene expression were also enhanced, supporting their role in promoting melanisation processes. Docking analyses suggested that hemolin contributes to phenoloxidase activation by stabilising PO-I and interacting with the phenoloxidase-activating factor-1-like protein (PAP1). The findings suggest that Z. jujuba extract effectively modulates immune responses, promoting enhanced protection while maintaining immune balance. GC-MS analysis revealed multiple bioactive compounds potentially contributing to immune modulation. This study highlights the significant immunomodulatory effects of Z. jujuba fruit extract on the immune system of G. mellonella. The findings suggest its potential as a natural immunostimulant and warrant further investigation of the underlying molecular mechanisms and bioactive components.

Schistosoma mansoni is a parasitic helminth that is vectored through freshwater snails. While the anti-schistosome defense of the South American snail, Biomphalaria glabrata, is well studied, little is known about the immune response of the African snail, Biomphalaria sudanica. We measured expression of five candidate immune genes in B. sudanica 8, 24, and 72 hours post-exposure to S. mansoni using reverse transcription quantitative PCR. Expression patterns of incompatible snails were compared to compatible snails and those sham exposed. We also assessed how diet (lettuce vs. pellet) affected expression of three genes, given prior findings that pellet-fed snails were more susceptible to S. mansoni. Results indicated that incompatible snails constitutively expressed higher levels of superoxide dismutase 1 (SOD1) than compatible snails at multiple time points, and of toll-like receptor (TLR) and granulin (GRN) at a single time point. Parasite-induced expression occurred at 8 hours in SOD1, biomphalysin 2, thioester protein 1 (TEP1), and granulin (GRN); however, for biomphalysin 2 and TEP1, induced expression was only detected for susceptible snails. At 24 hours, biomphalysin 2 expression increased in exposed resistant snails, and at 72 hours, all exposed snails decreased biomphalysin 2 expression compared to controls. Parasite-induced expression of SOD1, biomphalysin 2, TEP1, and GRN supports the hypothesis that these genes play a role in B. sudanica anti-schistosome defense; however, increased expression does not necessarily yield clearance of S. mansoni. SOD1 expression was higher in lettuce-fed snails at 8 and 24 hours, consistent with their greater resistance. Together, these results demonstrate the conserved and unique aspects of the B. sudanica anti-schistosome response.

Gene expression can be quantified using the sensitive technique of quantitative reverse transcription real-time polymerase chain reaction. Inter-sample variances can be minimised through normalisation with an appropriate reference gene. Bemisia tabaci, a significant insect vector of the Begomovirus family, transmits the Tomato Leaf Curl Bangalore Virus, for which there is a dearth of information regarding appropriate reference genes for autophagy. The viral load surpasses the vector’s capacity when autophagy is activated, which is also detrimental to whiteflies, particularly concerning virus translocation. To mitigate this vector using a double-stranded RNA approach, a precise measurement of gene silencing is required. For this investigation, normalisation of housekeeping or internal control genes is necessary. The present work utilised software tools such as geNorm, NormFinder, and BestKeeper to assess the suitability of five reference genes, namely, α-tubulin, β-tubulin, elongation factor, actin, and sucrose synthase, for gene expression studies in viruliferous and non-viruliferous B. tabaci. The analysis of the data showed that β-tubulin, which exhibits more stable expression, is the best-ranked reference gene. Furthermore, the reference genes were verified using the target gene expression of atg3 (an autophagy gene). The current findings enable precise measurement of gene expression in begomovirus-induced autophagy conditions of B. tabaci.

Cathepsin B (CTSB) is a cysteine protease that is widely found in eukaryotes and plays a role in insect growth, development, digestion, metamorphosis, and immunity. In the present study, we examined the role of CTSB in response to environmental stresses in Myzus persicae Sulzer (Hemiptera: Aphididae). Six MpCTSB genes, namely MpCTSB-N, MpCTSB-16D1, MpCTSB-3098, MpCTSB-10270, MpCTSB-mp2, and MpCTSB-16, were identified and cloned from M. persicae. The putative proteins encoded by these genes contained three conserved active site residues, i.e. Cys, His, and Asn. A phylogenetic tree analysis revealed that the six MpCTSB proteins of M. persicae were highly homologous to other Hemipteran insects. Real-time polymerase chain reaction revealed that the MpCTSB genes were expressed at different stages of M. persicae and highly expressed in winged adults or first-instar nymphs. The expression of nearly all MpCTSB genes was significantly upregulated under different environmental stresses (38°C, 4°C, and ultraviolet-B). This study shows that MpCTSB plays an important role in the growth and development of M. persicae and its resistance to environmental stress.

Throughout all the domains of life, and even among the co-existing viruses, RNA molecules play key roles in regulating the rates, duration, and intensity of the expression of genetic information. RNA acts at many different levels in playing these roles. Trans-acting regulatory RNAs can modulate the lifetime and translational efficiency of transcripts with which they pair to achieve speedy and highly specific recognition using only a few components. Cis-acting recognition elements, covalent modifications, and changes to the termini of RNA molecules encode signals that impact transcript lifetime, translation efficiency, and other functional aspects. RNA can provide an allosteric function to signal state changes through the binding of small ligands or interactions with other macromolecules. In either cis or trans, RNA can act in conjunction with multi-enzyme assemblies that function in RNA turnover, processing and surveillance for faulty transcripts. These enzymatic machineries have likely evolved independently in diverse life forms but nonetheless share analogous functional roles, implicating the biological importance of cooperative assemblies to meet the exact demands of RNA metabolism. Underpinning all the RNA-mediated processes are two key aspects: specificity, which avoids misrecognition, and speedy action, which confers timely responses to signals. How these processes work and how aberrant RNA species are recognised and responded to by the degradative machines are intriguing puzzles. We review the biophysical basis for these processes. Kinetics of assembly and multivalency of interacting components provide windows of opportunity for recognition and action that are required for the key regulatory events. The thermodynamic irreversibility of RNA-mediated regulation is one emergent feature of biological systems that may help to account for the apparent specificity and optimal rates.

Juvenile hormone (JH) regulates multiple physiological functions in insects including growth, metamorphosis, and reproduction. Juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone esterase (JHE) are degradative enzymes that metabolise JH, and JH receptor (methoprene-tolerant, Met) functions in the regulation of female reproduction and vitellogenesis. In this study, JH titres in Coccinella septempunctata adult females were determined using ultra high-performance liquid chromatography and tandem mass spectrometry; the JH titres ranged from 0.03 to 0.16 ng g−1 in 5- to 30-day-old female adults. JHEH, JHE, and Met expression were studied in different reproductive stages of C. septempunctata females by quantitative real-time PCR. JHEH transcription levels were highest in 25-day-old female adults and were 1.93-fold higher than expression levels in 5-day-old adults. JHEH and JHE expression levels were inhibited by the addition of JH to the artificial diet. Met expression in C. septempunctata supplied with 3 μl JH in artificial diet was similar to Met transcription in females supplied with an aphid diet, and the results showed that supplementation with 3 μl JH in 582.2 g of artificial diet was the most suitable for reproductive regulation of C. septempunctata. The results of this study provide important insights for the improvement of C. septempunctata artificial diets.