Altered gene expression in human brain microvascular endothelial cells in response to the infection of influenza H1N1 virus.

Influenza viruses not only cause respiratory illness, but also have been reported to elicit neurological manifestations following acute viral infection. The central nervous system (CNS) has a specific defense mechanism against pathogens structured by cerebral microvasculature lined with brain endothelial cells to form the blood-brain barrier (BBB). To investigate the response of human brain microvascular endothelial cells (hBMECs) to the Influenza A virus (IAV), we inoculated the cells with the A/WSN/33 (H1N1) virus. We then conducted an RNAseq experiment to determine the changes in gene expression levels and the activated disease pathways following infection. The analysis revealed an effective activation of the innate immune defense by inducing the pattern recognition receptors (PRRs). Along with the production of proinflammatory cytokines, we detected an upregulation of interferons and interferon-stimulated genes, such as IFN-ß/λ, ISG15, CXCL11, CXCL3 and IL-6, etc. Moreover, infected hBMECs exhibited a disruption in the cytoskeletal structure both on the transcriptomic and cytological levels. The RNAseq analysis showed different pathways and candidate genes associated with the neuroactive ligand-receptor interaction, neuroinflammation, and neurodegenerative diseases, together with a predicted activation of the neuroglia. Likewise, some genes linked with the mitochondrial structure and function displayed a significantly altered expression. En masse, this data supports that hBMECs could be infected by the IAV, which induces the innate and inflammatory immune response. The results suggest that the influenza virus infection could potentially induce a subsequent aggravation of neurological disorders. Supplementary Information: The online version contains supplementary material available at 10.1186/s44149-022-00053-9.

Argonaute1 and Gawky Are Required for the Development and Reproduction of Melon fly, Zeugodacus cucurbitae.

Argonaute family genes encode a highly conserved group of proteins that have been associated with RNA silencing in both animals and plants. This study investigates the importance of microRNA biogenesis key regulators Argonaute1 (Ago1) and Gawky genes in the post-embryonic and ovarian development of the melon fly, Zeugodacus cucurbitae. The expression levels of these genes were mapped in all developmental stages and different adult tissues. Their roles in development were investigated using RNA interference (RNAi) via two different dsRNA delivery techniques. Embryo microinjection and oral feeding of third instar larvae successfully knocked down and greatly reduced the expression level of the target genes. Additionally, ex vivo essays revealed the stability of dsRNA in food was sufficient for gene silencing, although its integrity was affected in midgut. A wide range of phenotypes were observed on pupation, segmentation, pigmentation, and ovarian development. RNAi-mediated silencing of Gawky caused high mortality and loss of body segmentation, while Ago1 knockdown affected ovarian development and pigmentation. Developmental abnormalities and ovarian malformations caused by silencing these genes suggest that these genes are crucial for viability and reproductive capacity of Z. cucurbitae, and may be used as potential target genes in pest management.

CRISPR/Cas9-Mediated Genome Editing System in Insect Genomics and Pest Management.

The CRISPR/Cas9 is being developed as an invaluable system that allows rapid and site-specific genome editing in a wide variety of organisms, including diverse insects. It has been successfully used for gene function annotations of RNAi pathway in insect genomics and will facilitate research on RNAi mechanism. Here, we describe a streamlined method to generate and detect somatic and germline knockout mutations of desired target genes in tephritid pests by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed single guide RNA (sgRNA) into embryos. Target site selection, sgRNA synthesis, Cas9 synthesis, microinjection, and mutation identification are presented in detail.

A Novel Solid Artificial Diet for Zeugodacus cucurbitae (Diptera: Tephritidae) Larvae With Fitness Parameters Assessed by Two-Sex Life Table.

The melon fly, Zeugodacus cucurbitae (Coquillett), is a serious pest of many fruits and vegetables throughout the world. Here we have developed an easy and quick-to-prepare solid medium with multiple benefits including reductions in post-rearing waste, storage space, and labor for rearing Z. cucurbitae larvae. The development time from egg to pupa was 19.11 d when larvae were reared on the artificial diet, slightly longer than 17.73 d on pumpkin and 17.13 d on cucumber. Zeugodacus cucurbitae achieved higher values of pupal weight, length, and width on the artificial diet than two natural diet controls. The rates of pupation and adult emergence of Z. cucurbitae grown on the solid medium were comparable with those on pumpkin and cucumber. Furthermore, determined by age-specific two-sex life table method, the age-specific survival rate of Z. cucurbitae was higher on the artificial diet than cucumber but lower than pumpkin. The reproductive ability and population dynamics of Z. cucurbitae were not significantly affected on the solid medium compared with those on the two natural diets. The results suggest that our solid artificial diet is excellent for rearing Z. cucurbitae larvae in laboratory and may be used for its mass rearing, therefore facilitating its research and control.

TGF-ß and BMP signals regulate insect diapause through Smad1-POU-TFAM pathway.

The transforming growth factor-ß (TGF-ß) superfamily signaling pathway contains two general branches, known as TGF-ß and bone morphogenetic protein (BMP), that regulate development in animals. It is well known that TGF-ß superfamily signaling participates in the regulation of dauer (lifespan extension) in Caenorhabditis elegans, but little is known about the molecular mechanisms of lifespan extension in the pathway. Diapause, a programmed developmental arrest in insects, is similar to dauer in C. elegans. In this study, we find that TGF-ß superfamily signaling regulates Helicoverpa armigera diapause via a novel mechanism. Both TGF-ß and BMP signals are weaker in the brains of diapause-destined pupae than in nondiapause-destined pupae, and the levels of p-Smad1, POU, TFAM, and mitochondrial activity are decreased in diapause pupae. Development in nondiapause pupae is delayed by an injection of TGF-ß or BMP receptor inhibitors. Both TGF-ß and BMP signals can activate a common target, Smad1. ChIP and EMSA assays indicate that Smad1 can bind to the POU promoter to regulate its expression. POU can improve the transcription of TFAM, which regulates mitochondrial activity. This is the first report showing that both TGF-ß and BMP signals regulate development or diapause through the Smad1-POU-TFAM-mitochondrial activity in insects.

Reactive oxygen species extend insect life span using components of the insulin-signaling pathway.

Reactive oxygen species (ROS) are well-known accelerants of aging, but, paradoxically, we show that physiological levels of ROS extend life span in pupae of the moth Helicoverpa armigera, resulting in the dormant state of diapause. This developmental switch appears to operate through a variant of the conventional insulin-signaling pathway, as evidenced by the facts that Akt, p-Akt, and PRMT1 are elevated by ROS, but not insulin, and that high levels of p-Akt fail to phosphorylate FoxO through PRMT1-mediated methylation. These results suggest a distinct signaling pathway culminating in the elevation of FoxO, which in turn promotes the extension of life span characteristic of diapause.

TLR7 Deficiency Leads to TLR8 Compensative Regulation of Immune Response against JEV in Mice.

Japanese encephalitis virus (JEV) is a highly fatal pathogen to human beings. Toll-like receptor 7 (TLR7) plays a role as the first host defense against most single-stranded RNA flaviviruses. This study aims to investigate the role of TLR7 in inducing adaptive immune response in mice against JEV. In vitro and in vivo studies were conducted to examine the expression of toll-like receptors (TLRs) in mice. After JEV infection, physical parameters of mice (survival rate and body weight) were evaluated, and organs or cells were collected for further analysis. The expression of TLR7 was increased significantly as compare to other TLR molecules post-JEV infection. The expression of CD80, CD86, and CD273 on bone marrow-derived dendritic cells was increased significantly in TLR7-/- mice. Furthermore, viral load was also increased significantly in TLR7-/- mice as compare to C57BL/6 mice. But there was no significant difference among survival rate and body weight in TLR7-/- mice as compare to C57BL/6. Interestingly, we also found that TLR8 was upregulated in TLR7-/- mice. The study concluded that TLR8 was upregulated in TLR7-deficient mice, and it might play a compensatory role in the immune response in TLR7-/- mice.

Cathepsin L participates in the remodeling of the midgut through dissociation of midgut cells and activation of apoptosis via caspase-1.

The larval midgut in holometabolous insects must undergo a remodeling process during metamorphosis to form the pupal-adult midgut. However, the molecular mechanism of larval midgut cell dissociation remains unknown. Here, we show that the expression and activity of Helicoverpa armigera cathepsin L (Har-CatL) are high in the midgut at the mid-late stage of the 6th-instar larvae and are responsive to the upstream hormone ecdysone. Immunocytochemistry shows that signals for Har-CatL-like are localized in midgut cells, and an inhibitor experiment demonstrates that Har-CatL functions in the dissociation of midgut epithelial cells. Mechanistically, Har-CatL can cleave pro-caspase-1 into the mature peptide, thereby increasing the activity of caspase-1, which plays a key role in apoptosis, indicating that Har-CatL is also involved in the apoptosis of midgut cells by activating caspase-1. We believe that this is the first report that Har-CatL regulates the dissociation and apoptosis of the larval midgut epithelium for midgut remodeling.

HIF-1 regulates insect lifespan extension by inhibiting c-Myc-TFAM signaling and mitochondrial biogenesis.

Diapause (developmental arrest) is characterized by dramatic depression of metabolic activity and profoundly extends insect lifespan, similar to the Caenorhabditis elegans dauer stage and Drosophila longevity; however, the molecular mechanism of low metabolism in insect diapause is unclear. Here, we show that HIF-1α expression is significantly increased in diapause-destined pupal brains compared to nondiapause-destined pupal brains and that HIF-1α negatively regulates mitochondrial biogenesis. HIF-1α mediates this effect by inhibiting c-Myc activity via proteasome-dependent degradation of c-Myc. The mitochondrial transcription factor A (TFAM), which encodes a key factor involved in mitochondrial transcription and mitochondrial DNA replication, is activated by the binding of c-Myc to the TFAM promoter, thereby inducing transcription. Loss of TFAM expression is a major factor contributing to reducing the mitochondrial activity. Thus, the HIF-1α-c-Myc-TFAM signaling pathway participates in the regulation of mitochondrial activity for insect diapause or lifespan extension.

Hexokinase is a key regulator of energy metabolism and ROS activity in insect lifespan extension.

Developmental arrest (diapause) is a 'non-aging' state that is similar to the Caenorhabditis elegans dauer stage and Drosophila lifespan extension. Diapause results in low metabolic activity and a profound extension of insect lifespan. Here, we cloned the Helicoverpa armigera Hexokinase (HK) gene, a gene that is critical for the developmental arrest of this species. HK expression and activity levels were significantly increased in nondiapause-destined pupae compared with those of diapause-destined pupae. Downregulation of HK activity reduced cell viability and delayed pupal development by reducing metabolic activity and increasing ROS activity, which suggests that HK is a key regulator of insect development. We then identified the transcription factors Har-CREB, -c-Myc, and -POU as specifically binding the Har-HK promoter and regulating its activity. Intriguingly, Har-POU and -c-Myc are specific transcription factors for HK expression, whereas Har-CREB is nonspecific. Furthermore, Har-POU and -c-Myc could respond to ecdysone, which is an upstream hormone. Therefore, low ecdysone levels in diapause-destined individuals lead to low Har-POU and -c-Myc expression levels, ultimately repressing Har-HK expression and inducing entry into diapause or lifespan extension.