Metformin monotherapy versus predominantly older non-metformin antidiabetic medications for cerebrovascular risk in early type 2 diabetes management.

AIM: Choosing the initial treatment for type 2 diabetes (T2D) is pivotal, requiring consideration of solid clinical evidence and patient characteristics. Despite metformin's historical preference, its efficacy in preventing cerebrovascular events lacked empirical validation. This study aimed to evaluate the associations between first-line monotherapy (metformin or non-metformin antidiabetic medications) and cerebrovascular complications in patients with T2D without diabetic complications. METHODS: We analysed 9090 patients with T2D without complications who were prescribed either metformin or non-metformin medications as initial therapy. Propensity score matching ensured group comparability. Cox regression analyses, stratified by initial metformin use, assessed cerebrovascular disease risk, adjusting for multiple covariates and using competing risk analysis. Metformin exposure was measured using cumulative defined daily doses. RESULTS: Metformin users had a significantly lower crude incidence of cerebrovascular diseases compared with non-users (p < .0001). Adjusted hazard ratios (aHRs) consistently showed an association between metformin use and a lower risk of overall cerebrovascular diseases (aHRs: 0.67-0.69) and severe events (aHRs: 0.67-0.69). The association with reduced risk of mild cerebrovascular diseases was significant across all models (aHRs: 0.73-0.74). Higher cumulative defined daily doses of metformin correlated with reduced cerebrovascular risk (incidence rate ratio: 0.62-0.94, p < .0001), indicating a dose-dependent effect. CONCLUSION: Metformin monotherapy is associated with a reduced risk of cerebrovascular diseases in early-stage T2D, highlighting its dose-dependent efficacy. However, the observed benefits might also be influenced by baseline differences and the increased risks associated with other medications, such as sulphonylureas. These findings emphasize the need for personalized diabetes management, particularly in mitigating cerebrovascular risk in early T2D stages.

Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

Physiological Measurements and Transcriptomics Reveal the Fitness Costs of Monochamus saltuarius to Bursaphelenchus xylophilus.

The pine wood nematode (PWN) uses several Monochamus species as vehicles, through a temporary hitchhiking process known as phoresy, enabling it to access new host plant resources. Monochamus saltuarius acts as a new and major vector of the PWN in Northeastern China, showing lower PWN carrying capacity and a shorter transmission cycle compared to established vectors. The apparently altered symbiotic relationship offers an interesting area for researching the costs and adaptions involved in nematode-beetle, a specialized phoresy. We analyzed the response and fitness costs of M. saltuarius through physiological measurements and transcriptomics. The PWN exerted adverse repercussions on the growth and development of M. saltuarius. The PWN accelerated larval development into pupae, while beetle adults carrying the PWN exhibited an elevated abnormality rate and mortality, and reduced starvation resistance. During the pupal stage, the expression of growth-related genes, including ecdysone-inducible genes (E74EA), cuticle proteins, and chitin genes (CHTs), markedly increased. Meanwhile, the induced immune response, mainly by the IMD and Toll signaling pathways, could be a contributing factor to adult abnormality and mortality. Adult gonads and trachea exhibited enrichment in pathways related to fatty acid elongation, biosynthesis, and metabolism. FASN, ELOVL, and SCD possibly contributed to resistance against PWN. Our research indicated that phoretic interactions between vector beetles and PWN vary throughout the vector's lifespan, particularly before and after entry into the trachea. This study highlighted the fitness costs of immunity and metabolism on the vector beetle, indicating the adaptation mechanisms and evolutionary trade-offs to PWN.

Functional and Compositional Changes in Sirex noctilio Gut Microbiome in Different Habitats: Unraveling the Complexity of Invasive Adaptation.

The mutualistic symbiosis relationship between the gut microbiome and their insect hosts has attracted much scientific attention. The native woodwasp, Sirex nitobei, and the invasive European woodwasp, Sirex noctilio, are two pests that infest pines in northeastern China. Following its encounter with the native species, however, there is a lack of research on whether the gut microbiome of S. noctilio changed, what causes contributed to these alterations, and whether these changes were more conducive to invasive colonization. We used high-throughput and metatranscriptomic sequencing to investigate S. noctilio larval gut and frass from four sites where only S. noctilio and both two Sirex species and investigated the effects of environmental factors, biological interactions, and ecological processes on S. noctilio gut microbial community assembly. Amplicon sequencing of two Sirex species revealed differential patterns of bacterial and fungal composition and functional prediction. S. noctilio larval gut bacterial and fungal diversity was essentially higher in coexistence sites than in separate existence sites, and most of the larval gut bacterial and fungal community functional predictions were significantly different as well. Moreover, temperature and precipitation positively correlate with most of the highly abundant bacterial and fungal genera. Source-tracking analysis showed that S. noctilio larvae at coexistence sites remain dependent on adult gut transmission (vertical transmission) or recruitment to frass (horizontal transmission). Meanwhile, stochastic processes of drift and dispersal limitation also have important impacts on the assembly of S. noctilio larval gut microbiome, especially at coexistence sites. In summary, our results reveal the potential role of changes in S. noctilio larval gut microbiome in the successful colonization and better adaptation of the environment.

Dysregulation of iron homeostasis and ferroptosis in sevoflurane and isoflurane associated perioperative neurocognitive disorders.

In recent years, sevoflurane and isoflurane are the most popular anesthetics in general anesthesia for their safe, rapid onset, and well tolerant. Nevertheless, many studies reported their neurotoxicity among pediatric and aged populations. This effect is usually manifested as cognitive impairment such as perioperative neurocognitive disorders. The wide application of sevoflurane and isoflurane during general anesthesia makes their safety a major health concern. Evidence indicates that iron dyshomeostasis and ferroptosis may establish a role in neurotoxicity of sevoflurane and isoflurane. However, the mechanisms of sevoflurane- and isoflurane-induced neuronal injury were not fully understood, which poses a barrier to the treatment of its neurotoxicity. We, therefore, reviewed the current knowledge on mechanisms of iron dyshomeostasis and ferroptosis and aimed to promote a better understanding of their roles in sevoflurane- and isoflurane-induced neurotoxicity.

Cyanidin-3-O-glucoside Contributes to Leaf Color Change by Regulating Two bHLH Transcription Factors in Phoebe bournei.

Anthocyanins produce different-colored pigments in plant organs, which provide ornamental value. Thus, this study was conducted to understand the mechanism of anthocyanin synthesis in ornamental plants. Phoebe bournei, a Chinese specialty tree, has high ornamental and economic value due to its rich leaf color and diverse metabolic products. Here, the metabolic data and gene expression of red P. bournei leaves at the three developmental stages were evaluated to elucidate the color-production mechanism in the red-leaved P. bournei species. First, metabolomic analysis identified 34 anthocyanin metabolites showing high levels of cyanidin-3-O-glucoside (cya-3-O-glu) in the S1 stage, which may suggest that it is a characteristic metabolite associated with the red coloration of the leaves. Second, transcriptome analysis showed that 94 structural genes were involved in anthocyanin biosynthesis, especially flavanone 3'-hydroxy-lase (PbF3'H), and were significantly correlated with the cya-3-O-glu level. Third, K-means clustering analysis and phylogenetic analyses identified PbbHLH1 and PbbHLH2, which shared the same expression pattern as most structural genes, indicating that these two PbbHLH genes may be regulators of anthocyanin biosynthesis in P. bournei. Finally, overexpression of PbbHLH1 and PbbHLH2 in Nicotiana tabacum leaves triggered anthocyanin accumulation. These findings provide a basis for cultivating P. bournei varieties that have high ornamental value.

Bicuculline and Bumetanide Attenuate Sevoflurane-Induced Impairment of Myelination and Cognition in Young Mice.

Sevoflurane (Sevo) is one of the most commonly used general anesthetics for infants and young children. We investigated whether Sevo impairs neurological functions, myelination, and cognition via the γ-aminobutyric acid A receptor (GABAAR) and Na+-K+-2Cl- cotransporter (NKCC1) in neonatal mice. On postnatal days 5-7, mice were exposed to 3% Sevo for 2 h. On postnatal day 14, mouse brains were dissected, and oligodendrocyte precursor cell line level lentivirus knockdown of GABRB3, immunofluorescence, and transwell migration assays were performed. Finally, behavioral tests were conducted. Multiple Sevo exposure groups exhibited increased neuronal apoptosis levels and decreased neurofilament protein levels in the mouse cortex compared with the control group. Sevo exposure inhibited the proliferation, differentiation, and migration of the oligodendrocyte precursor cells, thereby affecting their maturation process. Electron microscopy revealed that Sevo exposure reduced myelin sheath thickness. The behavioral tests showed that multiple Sevo exposures induced cognitive impairment. GABAAR and NKCC1 inhibition provided protection against Sevo-induced neurotoxicity and cognitive dysfunction. Thus, bicuculline and bumetanide can protect against Sevo-induced neuronal injury, myelination impairment, and cognitive dysfunction in neonatal mice. Furthermore, GABAAR and NKCC1 may be mediators of Sevo-induced myelination impairment and cognitive dysfunction.

Association between vitamin D concentration and delirium in hospitalized patients: A meta-analysis.

BACKGROUND: Now the occurrence of delirium is more concerning to clinicians and psychiatrists. It has been reported that vitamin D deficiency may be a relevant factor in the development of delirium in hospitalized patients. STUDY OBJECTIVE: To investigate the association between vitamin D concentration and delirium in hospitalized patients. DESIGN: Meta-analysis. METHODS: A systematic literature search was conducted using PubMed, EMBASE, and the Cochrane Library. The primary outcome was the occurrence of delirium in the inpatient setting. Odds ratios (OR) were calculated with random or fixed effects models. RESULTS: In this article, we define the normal range of vitamin D concentrations as greater than 75 nmol / L, 50-75 nmol / L as vitamin D insufficiency, 25-50 nmol / L as vitamin D deficiency, and less than 25 nmol / L as vitamin D severe deficiency. The Results showed that severe vitamin D deficiency (OR: 1.98 [1.41-2.79], P<0.001) and vitamin D deficiency (OR: 1.50 [1.12-2.00], P = 0.006) were more likely to develop delirium than normal vitamin D levels. Subgroup analysis also revealed that low vitamin D concentrations were associated with a higher incidence of delirium, whether the cutoff point was 25 nmol/L (OR: 1.52 [1.40-1.64], P<0.001), 50 nmol/L (OR: 1.47 [1.19-1.82], P<0.001), or 75 nmol/L (OR: 1.54 [1.21-1.96], P<0.001). The included studies scored medium and high on the Newcastle-Ottawa quality assessment scale. CONCLUSION: Compared with normal vitamin D levels, severe vitamin D deficiency and vitamin D deficiency, but not vitamin D insufficiency, are associated with a higher incidence of delirium in hospitalized patients. TRIAL REGISTRATION: This review was registered in the PROSPERO database under identifier CRD42021271347. https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021271347.

Geopolitical Risk Evolution and Obstacle Factors of Countries along the Belt and Road and Its Types Classification.

As a great practice of building a community of shared future for mankind, the Belt and Road Initiative is facing geopolitical risk brought by great power games, regional conflicts and terrorism. It is an important mission of geopolitical research to scientifically deal with the geopolitical risk along the Belt and Road. This study systematically constructs the geopolitical risk assessment index system and analyzes the spatiotemporal evolution, obstacle factors and risk types of geopolitical risk of countries along the Belt and Road by using the entropy weight TOPSIS model, obstacle degree model and minimum variance method. The research results showed that: (1) From 2005 to 2020, the polarization of geopolitical risk in countries along the Belt and Road was very significant, and the overall trend of geopolitical risk tended to deteriorate. (2) The Middle East and Eastern Europe were the most important geopolitical risk zones along the Belt and Road, and Afghanistan, Iraq, Russia and Ukraine were the main high geopolitical risk centers, with significant risk spillover effects from these centers. (3) Terrorism and close relations with the United States were the most important obstacle factors for geopolitical risk in countries along the Belt and Road, and military intervention politics, trade dependence degree and foreign debt burden were important obstacle factors for geopolitical risk in countries along the Belt and Road. (4) Geopolitical risk along the Belt and Road can be divided into sovereign risk dominant type, sovereign and military risk dominant type, sovereign and major power intervention risk dominant type, and sovereign and military and major power intervention risk jointly dominated type, among which sovereign and military and major power intervention risk jointly dominated type was the most important geopolitical risk type. In order to scientifically deal with geopolitical risk in countries along the Belt and Road, it is necessary to strengthen geopolitical risk awareness, pay attention to the dominant geopolitical risk factors, strengthen the control of regional geopolitical risk spillover and formulate reasonable risk prevention and control scheme based on geopolitical risk types.

Genome-Wide Identification and Expression Analysis of Calmodulin and Calmodulin-like Genes, Revealing CaM3 and CML13 Participating in Drought Stress in Phoebe bournei.

Calmodulin (CaM) and calmodulin-like (CML) proteins are major Ca2+ sensors involved in the regulation of plant development and stress responses by converting Ca2+ signals into appropriate cellular responses. However, characterization and expression analyses of CaM/CML genes in the precious species, Phoebe bournei, remain limited. In this study, five PbCaM and sixty PbCML genes were identified that only had EF-hand motifs with no other functional domains. The phylogenetic tree was clustered into 11 subgroups, including a unique clade of PbCaMs. The PbCaMs were intron-rich with four EF-hand motifs, whereas PbCMLs had two to four EF-hands and were mostly intronless. PbCaMs/CMLs were unevenly distributed across the 12 chromosomes of P. bournei and underwent purifying selection. Fragment duplication was the main driving force for the evolution of the PbCaM/CML gene family. Cis-acting element analysis indicated that PbCaMs/CMLs might be related to hormones, growth and development, and stress response. Expression analysis showed that PbCaMs were generally highly expressed in five different tissues and under drought stress, whereas PbCMLs showed specific expression patterns. The expression levels of 11 candidate PbCaMs/CMLs were responsive to ABA and MeJA, suggesting that these genes might act through multiple signaling pathways. The overexpression of PbCaM3/CML13 genes significantly increased the tolerance of yeast cells to drought stress. The identification and characterization of the CaM/CML gene family in P. bournei laid the foundation for future functional studies of these genes.

Identification and Validation of Ferroptosis-Related Genes in Sevoflurane-Induced Hippocampal Neurotoxicity.

Background: Sevoflurane is one of the most popular inhalational anesthetics during perioperative period but presenting neurotoxicity among pediatric and aged populations. Recent experiments in vivo and in vitro have indicated that ferroptosis may contribute to the neurotoxicity of sevoflurane anesthesia. However, the exact mechanism is still unclear. Methods: In current study, we explored the differential expressed genes (DEGs) in HT-22 mouse hippocampal neuronal cells after sevoflurane anesthesia using RNA-seq. Differential expressed ferroptosis-related genes (DEFRGs) were screened and analyzed by Gene Ontology (GO) and pathway enrichment analysis. Protein-to-protein interaction (PPI) network was constructed by the Search Tool for the Retrieval of Interacting Genes (STRING). Significant modules and the hub genes were identified by using Cytoscape. The Connectivity Map (cMAP) was used for screening drug candidates targeting the identified DEFRGs. Potential TF-gene network and drug-gene pairs were established towards the hub genes. In final, we validated these results in experiments. Results: A total of 37 ferroptosis-related genes (18 upregulated and 19 downregulated) after sevoflurane exposure in hippocampal neuronal cells were finally identified. These differentially expressed genes were mainly involved into the biological processes of cellular response to oxidative stress. Pathway analysis indicated that these genes were involved in ferroptosis, mTOR signaling pathway, and longevity-regulating pathway. PPI network was constructed. 10 hub genes including Prkaa2, Chac1, Arntl, Tfrc, Slc7a11, Atf4, Mgst1, Lpin1, Atf3, and Sesn2 were found. Top 10 drug candidates, gene-drug networks, and TFs targeting these genes were finally identified. These results were validated in experiments. Conclusion: Our results suggested that ferroptosis-related genes play roles in sevoflurane anesthesia-related hippocampal neuron injury and offered the hub genes and potential therapeutic agents for investigating and treatment of this neurotoxicity after sevoflurane exposure. Finally, therapeutic effect of these drug candidates and function of potential ferroptosis targets should be further investigated for treatment and clarifying mechanisms of sevoflurane anesthesia-induced neuron injury in future research.

Sevoflurane exposure induces neurotoxicity by regulating mitochondrial function of microglia due to NAD insufficiency.

Developmental neurons received with sevoflurane, the commonly used inhalational anesthetic agent in clinical surgery, several times tend to be destroyed. Microglia, the resident immune cells of the central nervous system (CNS), are activated after sevoflurane exposure, accompanied by releasing proinflammatory cytokines that damage developing neurons. The sevoflurane-induced neurotoxicity could be attributed to activated microglia presenting proinflammatory and anti-inflammatory functions. Proinflammatory microglia release cytokines to impair the CNS, while anti-inflammatory microglia engulf damaged neurons to maintain CNS homeostasis. Sevoflurane exposure promotes the secretion of proinflammatory cytokines by microglia, inhibiting the microglial phagocytic function. Microglia with poor phagocytic function cannot engulf damaged neurons, leading to the accumulation of damaged neurons. The mechanism underlying poor phagocytic function may be attributed to mitochondrial dysfunction of microglia induced by sevoflurane exposure, in which affected mitochondria cannot generate adequate ATP and NAD to satisfy the energy demand. We discovered that sevoflurane treatment impaired the mitochondrial metabolism of microglia, which resulted in NAD deficiency and couldn't produce sufficient energy to clear damaged neurons to maintain CNS development. Our findings provide an explanation of a new mechanism underlying sevoflurane-induced neurotoxicity.

Role of GABAA receptor depolarization-mediated VGCC activation in sevoflurane-induced cognitive impairment in neonatal mice.

Background: In neonatal mice, anesthesia with sevoflurane depolarizes the GABA Type A receptor (GABAAR), which leads to cognitive impairment. Calcium accumulation in neurons can lead to neurotoxicity. Voltage-gated calcium channels (VGCCs) can increase intracellular calcium concentration under isoflurane and hypoxic conditions. The underlying mechanisms remain largely unknown. Methods: Six-day-old mice were anesthetized with 3% sevoflurane for 2 h/day for 3 days. The Y-Maze, new object recognition (NOR) test, the Barnes maze test, immunoassay, immunoblotting, the TUNEL test, and Golgi-Cox staining were used to assess cognition, calcium concentration, inflammatory response, GABAAR activation, VGCC expression, apoptosis, and proliferation of hippocampal nerve cells in mice and HT22 cells. Results: Compared with the control group, mice in the sevoflurane group had impaired cognitive function. In the sevoflurane group, the expression of Gabrb3 and Cav1.2 in the hippocampal neurons increased (p < 0.01), the concentration of calcium ions increased (p < 0.01), inflammatory reaction and apoptosis of neurons increased (p < 0.01), the proliferation of neurons in the DG area decreased (p < 0.01), and dendritic spine density decreased (p < 0.05). However, the inhibition of Gabrb3 and Cav1.2 alleviated cognitive impairment and reduced neurotoxicity. Conclusions: Sevoflurane activates VGCCs by inducing GABAAR depolarization, resulting in cognitive impairment. Activated VGCCs cause an increase in intracellular calcium concentration and an inflammatory response, resulting in neurotoxicity and cognitive impairment.

Chromosome-level genome assembly of Monochamus saltuarius reveals its adaptation and interaction mechanism with pine wood nematode.

Monochamus saltuarius (Coleoptera: Cerambycidae) was reported as the vector beetle of the pine wood nematode (PWN, Bursaphelenchus xylophilus) in Japan and Europe. It was first reported to transmitted the PWN to native Pinus species in 2018 in Liaoning Province, China. However, the lack of genomic resources has limited the in-depth understanding of its interspecific relationship with PWN. Here, we obtained a chromosome-level reference genome of M. saltuarius combining Illumina, Nanopore and Hi-C sequencing technologies. We assembled the scaffolds into ten chromosomes (including an X chromosome) and obtained a 682.23 Mb chromosome-level genome with a N50 of 73.69 Mb. In total, 427.67 Mb (62.69 %) repeat sequences were identified and 14, 492 protein-coding genes were predicted, of which 93.06 % were annotated. We described the mth/mthl, P450, OBP and OR gene families associated with the vector beetle's development and resistance, as well as the host selection and adaptation, which serve as a valuable resource for understanding the host adaptation in insects during evolution. This high quality reference genome of M. saltuarius also provide new avenues for researching the mechanism of this synergistic damage between vector beetles and PWN.

The role of depolarizing activation of Na+-Ca2+ exchanger by oligodendrocyte progenitor cells in the effect of sevoflurane on myelination.

AIMS: The aim of this study was to investigate the role of depolarizing activation of Na+-Ca2+ exchanger (NCX) by oligodendrocyte progenitor cells (OPC) in the effect of sevoflurane on myelination. MAIN METHODS: On postnatal days 7, 8, and 9, mice were exposed to 3 % sevoflurane for 2 h per day. The proliferation, differentiation, and myelin sheath of OPC were observed with immunofluorescence, quantitative real-time polymerase chain reaction (QRT-PCR), and transmission electron microscopy (TEM) at various time points. The open field, Y maze, and new object recognition tests were used to measure spatial learning and memory. siRNA was used for the knockdown NCX1 in human OPC (HOPC) before sevoflurane exposure; the Transwell migration assay was used to measure cell migration ability and Fluo 4-AM was used to measure intracellular Ca2+ concentration. KEY FINDINGS: Pretreatment with an NCX inhibitor attenuated the proliferation and differentiation of OPC induced by sevoflurane and induced a remarkable increase in platelet-derived growth factor receptor-alpha (PDGFRα), 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase), oligodendrocyte transcription factor 2 (Olig2), and homeodomain protein NK2 homeobox 2 (NKX2.2) levels. Pretreatment with an NCX inhibitor alleviated the sevoflurane-induced myelination disorder and cognitive impairment. The decreased cell migration and increased intracellular Ca2+ concentration observed in the siRNA-negative control group was reversed in the sevoflurane plus siRNA-NCX1 group. SIGNIFICANCE: This study suggests that repeated sevoflurane exposure in newborn mice leads to depolarization of OPC, which leads to Ca2+ influx through NCX and affects OPC proliferation, migration, differentiation, and myelination, ultimately leading to cognitive impairment.

Effect of Anesthesia on Oligodendrocyte Development in the Brain.

Oligodendrocytes (OLs) participate in the formation of myelin, promoting the propagation of action potentials, and disruption of their proliferation and differentiation leads to central nervous system (CNS) damage. As surgical techniques have advanced, there is an increasing number of children who undergo multiple procedures early in life, and recent experiments have demonstrated effects on brain development after a single or multiple anesthetics. An increasing number of clinical studies showing the effects of anesthetic drugs on the development of the nervous system may mainly reside in the connections between neurons, where myelin development will receive more research attention. In this article, we review the relationship between anesthesia exposure and the brain and OLs, provide new insights into the development of the relationship between anesthesia exposure and OLs, and provide a theoretical basis for clinical prevention of neurodevelopmental risks of general anesthesia drugs.

Identification and Validation of Reference Genes for Gene Expression Analysis in Monochamus saltuarius Under Bursaphelenchus xylophilus Treatment.

A special mutual relationship exists between the pine wood nematode (PWN) Bursaphelenchus xylophilus and its vector beetles of genus Monochamus, which enables PWN to spread, at the same time provides longhorned beetles with more weak hosts. PWN are attracted to the pupal chambers and then carried inside the trachea of beetle adults, which is a necessary part to complete the B. xylophilus infection cycle. The growth and immune responses of the vector beetle will affect this carrying process, however, they were rarely studied in Monochamus saltuarius. Real-time quantitative polymerase chain reaction (RT-qPCR), one of the most common methods for quantitative gene expression analysis, was performed to explore the key genes and pathways involved in the growth, development and immune responses of M. saltuarius at different developmental stages associated with infection of PWN and PWN treatment conditions. To enhance the accuracy of RT-qPCR data, the expression of target genes needs to be normalized with reference genes, which are stably expressed under varied experimental conditions. In our study, the stability of 14 candidate reference genes in M. saltuarius samples at different developmental stages associated with infection of PWN or PWN treatment conditions was evaluated using delta Ct, geNorm, NormFinder, BestKeeper and RefFinder algorithms. Moreover, KLF gene was used to validate the stability of the selected reference genes. Under experimental conditions of this study, RPL7 and TER were suitable reference genes at different developmental stages associated with infection of PWN. RPL7 and RPS5 were considered the most stable reference genes in the pupae treated with PWN. RPS5 and SNX6 could be used as reference genes in the adults treated with PWN. RPL7, EF1-γ, and RPS5 could be used as stable reference genes in all the samples. This work is the first to evaluate reference genes in M. saltuarius, laying a foundation for further gene expression experimental procedures and understanding the phoretic relationship between M. saltuarius and B. xylophilus.

Full-Length Transcriptome Sequencing-Based Analysis of Pinus sylvestris var. mongolica in Response to Sirex noctilio Venom.

Sirex noctilio is a major international quarantine pest that recently emerged in northeast China to specifically invade conifers. During female oviposition, venom is injected into the host together with its symbiotic fungus to alter the normal Pinus physiology and weaken or even kill the tree. In China, the Mongolian pine (Pinus sylvestris var. mongolica), an important wind-proof and sand-fixing species, is the unique host of S. noctilio. To explore the interplay between S. noctilio venom and Mongolian pine, we performed a transcriptome comparative analysis of a 10-year-old Mongolian pine after wounding and inoculation with S. noctilio venom. The analysis was performed at 12 h, 24 h and 72 h. PacBio ISO-seq was used and integrated with RNA-seq to construct an accurate full-length transcriptomic database. We obtained 52,963 high-precision unigenes, consisting of 48,654 (91.86%) unigenes that were BLASTed to known sequences in the public database and 4309 unigenes without any annotation information, which were presumed to be new genes. The number of differentially expressed genes (DEGs) increased with the treatment time, and the DEGs were most abundant at 72 h. A total of 706 inoculation-specific DEGs (475 upregulated and 231 downregulated) and 387 wounding-specific DEGs (183 upregulated and 204 downregulated) were identified compared with the control. Under venom stress, we identified 6 DEGs associated with reactive oxygen species (ROS) and 20 resistance genes in Mongolian pine. Overall, 52 transcription factors (TFs) were found under venom stress, 45 of which belonged to the AP2/ERF TF family and were upregulated. A total of 13 genes related to the photosystem, 3 genes related photo-regulation, and 9 TFs were identified under wounding stress. In conclusion, several novel putative genes were found in Mongolian pine by PacBio ISO seq. Meanwhile, we also identified various genes that were resistant to S. noctilio venom, such as GAPDH, GPX, CAT, FL2, CERK1, and HSP83A, etc.

Multilocus Genotyping and Intergenic Spacer Single Nucleotide Polymorphisms of Amylostereum areolatum (Russulales: Amylostereacea) Symbionts of Native and Non-Native Sirex Species.

Sirex noctilio along with its mutualistic fungal symbiont, Amylostereum areolatum (a white rot fungus), is an invasive pest that causes excessive damage to Pinus plantations in Northeast China. In 2015, S. noctilio were found to attack Pinus sylvestris var. mongolica, and often share larval habitat with the native woodwasp, S. nitobei. The objective of this study was to determine the possible origin(s) of the introduced pest complex in China and analyse the genetic diversity between A. areolatum isolated from invasive S. noctilio, native S. nitobei and other woodwasps collected from Europe (native range) and other countries. Phylogenetic analyses were performed using the intergenic spacer (IGS) dataset and the combined 4-locus dataset (the internal transcribed spacer region (ITS), translation elongation factor alpha 1 (tef1), DNA-directed ribosomal polymerase II (RPB2), and mitochondrial small subunit (mtSSU)) of three Amylostereum taxa. The multilocus genotyping of nuclear ribosomal regions and protein coding genes revealed at least three distinct multilocus genotypes (MLGs) of the fungus associated with invasive S. noctilio populations in Northeast China, which may have come from North America or Europe. The IGS region of A. areolatum carried by S. noctilio from China was designated type B1D2. Our results showed a lack of fidelity (the paradigm of obligate fidelity to a single fungus per wasp species) between woodwasp hosts and A. areolatum. We found that the native S. nitobei predominantly carried A. areolatum IGS-D2, but a low percentage of females instead carried A. areolatum IGS-B1D2 (MLG A13), which was presumably due to horizontal transmission from S. noctilio, during the sequential use of the same wood for larval development. The precise identification of the A. areolatum genotypes provides valuable insight into co-evolution between Siricidae and their symbionts, as well as understanding of the geographical origin and history of both Sirex species and their associated fungi.