Knickkopf (LmKnk) is required for chitin organization in the foregut of Locusta migratoria.

The foregut, located at the front of the digestive tract, serves a vital role in insects by storing and grinding food into small particles. The innermost layer of the foregut known as the chitinous intima, comes into direct contact with the food and acts as a protective barrier against abrasive particles. Knickkopf (Knk) is required for chitin organization in the chitinous exoskeleton, tracheae and wings. Despite its significance, little is known about the biological function of Knk in the foregut. In this study, we found that LmKnk was stably expressed in the foregut, and highly expressed before molting in Locusta migratoria. To ascertain the biological function of LmKnk in the foregut, we synthesized specific double-stranded LmKnk (dsLmKnk) and injected it into locusts. Our findings showed a significant decrease in the foregut size, along with reduced food intake and accumulation of residues in the foregut after dsLmKnk injection. Morphological observations revealed that newly formed intima became thinner and lacked chitin lamella. Furthermore, fluorescence immunohistochemistry revealed that LmKnk was located in the apical region of new intima and epithelial cells. Taken together, this study provides insights into the biological function of LmKnk in the foregut, and identifies the potential target gene for exploring biological pest management strategies.

Bone Marrow Stromal Cell-Secreted Extracellular Vesicles Containing miR-34c-5p Alleviate Lung Injury and Inflammation in Bronchopulmonary Dysplasia Through Promotion of PTEN Degradation by Targeting OTUD3.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is the predominant chronic disorder in preterm neonates. This study explored impacts of miR-34c-5p carried by bone marrow stromal cells-secreted extracellular vesicles (BMSC-EVs) on BPD progression. METHODS: A BPD mouse model was established, followed by measurement of miR-34c-5p, OTUD3, and PTEN expression. EVs were isolated from BMSCs transfected with miR-34c-5p mimic or mimic NC and intratracheally injected into mice. CD31 and Ki67 expression was detected and the pathological changes of lung tissues and lung function indexes were observed for mice. A neonatal human pulmonary microvascular endothelial cell (HPMEC) model was developed with hyperoxia, followed by co-culture with extracted EVs and ectopic experiments for measurement of cell viability, migration, and angiogenesis. IL-4, IL-13, IL-1ß, and IL-6 levels were measured in cell supernatants and lung tissues. Dual-luciferase reporter, ubiquitination, Co-IP, and RIP assays were adopted to determine the relationship among miR-34c-5p, OTUD3, and PTEN. RESULTS: Lung tissues of BPD mice had downregulated miR-34c-5p expression and upregulated OTUD3 and PTEN expression. BMSC-EVs and BMSC-EVs-miR-34c-5p treatment improved lung injury and alveolar structure, decreased lung resistance and IL-4, IL-13, IL-1ß, and IL-6 levels, and elevated dynamic lung compliance in BPD mice, as well as enhanced proliferation, angiogenesis, and migration and restrained inflammation in HPMECs. Mechanistically, miR-34c-5p negatively targeted OTUD3 which restrained ubiquitination to promote PTEN protein stabilization. Upregulation of OTUD3 or PTEN negated the changes in the proliferation, angiogenesis, migration, and inflammation of hyperoxia-treated HPMECs induced by BMSC-EVs-miR-34c-5p. CONCLUSION: BMSC-EVs-miR-34c-5p alleviated lung injury and inflammation in hyperoxia-induced BPD by blocking the OTUD3/PTEN axis.

A retrospective analysis of MS/MS screening for IEM in high-risk areas.

Inborn errors of metabolism (IEM) can lead to severe motor and neurological developmental disorders and even disability and death in children due to untimely treatment. In this study, we used tandem mass spectrometry (MS/MS) for primary screening and recall of those with positive primary screening for rescreening. Further diagnosis was based on biochemical tests, imaging and clinical presentation as well as accurate genetic testing using multi-gene panel with high-throughput sequencing of 130 IEM-related genes. The screening population was 16,207 newborns born between July 1, 2019, and December 31, 2021. Based on the results, 8 newborns were diagnosed with IEM, constituting a detection rate of 1:2,026. Phenylketonuria was the most common form of IEM. In addition, seven genes associated with IEM were detected in these eight patients. All eight patients received standardized treatment starting in the neonatal period, and the follow-up results showed good growth and development. Therefore, our study suggests that MS/MS rescreening for IEM pathogenic variants in high-risk areas, combined with a sequencing validation strategy, can be highly effective in the early detection of affected children. This strategy, combined with early intervention, can be effective in preventing neonatal morbidity and improving population quality.

Clathrin heavy chain is essential for the development and reproduction of Locusta migratoria.

Clathrin heavy chain (Chc) is a constituent of clathrin-coated vesicles and serves important functions in endocytosis and intracellular membrane trafficking but appears to have physiological roles also at the organismal level. Most of what we know about Chc functions originates from studies performed in fungal or vertebrate cells. However, the physiological functions of Chc in insects remain poorly understood. Here, we identified a Chc ortholog from a Locusta migratoria transcriptome database. RT-qPCR revealed that LmChc was constitutively expressed in fifth-instar nymphs. In this developmental stage, LmChc showed the highest expression in the ovary followed by hemolymph, testis, hindgut, midgut, and foregut. In isolated hemocytes, we detected the Chc protein in patches at the plasma membrane. To examine the role of LmChc in L. migratoria during development, RNA interference was performed by injecting dsRNA into the early fifth-instar nymphs. Silencing of LmChc caused a lethal phenotype with molting defect from nymph to adult. In addition, silencing of LmChc resulted in abnormal development of the ovaries, the size of which was significantly smaller than that in controls. Taken together, our results suggest that LmChc is a vital gene in L. migratoria that plays an important role in growth, development, and reproduction. LmChc may be used as an efficient RNAi target gene for developing dsRNA-based biological insecticides to manage insect pests.

Lethal giant larvae gene is required for normal nymphal development and midgut morphogenesis in Locusta migratoria.

Disruption of morphogenesis, an essential process in organismal development, can lead to disruption of biological processes, reduction in fitness, or even death of an organism. The roles of lethal giant larvae (Lgl) protein in maintaining tissue organization have been studied extensively in mammals, but little is known about this gene's roles in promoting correct tissue morphogenesis in insects. In this study, we identified an Lgl ortholog in Locusta migratoria. RT-qPCR results revealed that LmLgl was constitutively expressed during third, fourth, and fifth instar nymphs. Furthermore, LmLgl showed highest expression in the ovary followed by wing pads, midgut, hindgut, Malpighian tubules, and foregut of the third-instar nymphs. To examine the role of LmLgl in L. migratoria development, RNA interference was performed during nymphal stages. Silencing of LmLgl increased body size but decreased bodyweight by 9.0%. Histological sections of the midgut revealed abnormal large masses of disordered epithelial cells in dsLmLgl-injected nymphs. In addition, downregulation of LmLgl transcript levels significantly altered the morphological structure in midgut, resulting in the formation of tumor-like structures. Our results indicated that LmLgl may act as a tumor-suppressor gene, which plays an essential role in maintaining a normal morphological structure in the midgut of L. migratoria. Our results also suggest that LmLgl may be explored as a potential target for developing dsRNA-based biological pesticides for managing insect pests.

Vacuolar (H+ )-ATPase subunit c is essential for the survival and systemic RNA interference response in Locusta migratoria.

BACKGROUND: Vacuolar (H+ )-ATPase (V-ATPase) is a multi-subunit enzyme that hydrolyzes adenosine triphosphate (ATP) to transport protons across a cellular membrane, and it plays an important role in numerous biological processes, including in growth, development and immune responses. The c subunit of V-ATPase is a highly conserved subunit of the rotatory proteolipid ring that is required for binding and transporting protons. To date, there are only a few published reports on V-ATPase-c functions in insects. RESULTS: We identified and characterized the V-ATPase-c gene in Locusta migratoria, one of the most destructive agricultural insect pests in the world. LmV-ATPase-c was predominately expressed in Malpighian tubules of nymphs, followed by the hindgut and ovary, while the other tissues showed relatively low expression levels. Silencing of LmV-ATPase-c caused severe molting defects in nymphs and a high mortality rate of > 90%. Histological staining and microscopic examination of sections from the abdominal cuticle revealed the absence of newly formed cuticle in nymphs that were injected with dsLmV-ATPase-c. In addition, silencing of LmV-ATPase-c transcript levels significantly impaired RNA interference (RNAi) efficiency of a reporter gene. By quantifying double-stranded RNA (dsRNA) amounts by quantitative polymerase chain reaction (PCR), we found that RNAi against LmV-ATPase-c provoked a dramatic accumulation of dsRNA in the endosomes of epidermal and midgut cells of Locusta migratoria. CONCLUSION: Our results indicate that LmV-ATPase-c is indispensable for the formation of new cuticle during the molting process and has pivotal functions in dsRNA escape from endosomes. LmV-ATPase-c might be a valuable target for developing new strategies for insect pest management. © 2022 Society of Chemical Industry.

Identification of Rab family genes and functional analyses of LmRab5 and LmRab11A in the development and RNA interference of Locusta migratoria.

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double-stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria.

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults.

High specificity for silencing target genes and single-copy target genes that yield clear phenotypes are two important factors for the success of RNA interference (RNAi). The lethal giant larvae (Lgl) gene appears to be an ideal gene for RNAi because RNAi can effectively suppress its expression and results in molting defects and mortality in Tribolium castaneum. To investigate the suitability of this gene for RNAi in other insects, we identified and characterized DvLgl from the western corn rootworm, Diabrotica virgifera virgifera, a species exhibiting high RNAi efficiency. DvLgl was expressed in all developmental stages and tissues investigated. The deduced DvLgl protein showed high amino-acid sequence identities and similar domain architecture to Lgls from other insect species. Despite many similarities among insect Lgls, RNAi-mediated suppression of DvLgl failed to produce a phenotype in D. v. virgifera adults. The difference in developing phenotypes could be attributed greatly to the level of gene suppression and the insect developmental stages for RNAi. These results highlight the variability in RNAi response among insects and showcase the importance of screening multiple target genes when conducting RNAi studies. Our findings are expected to help the design of future RNAi studies and future investigations of Lgl in insects.

Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis.

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis.

BACKGROUND: Targeting insect-specific genes through post-transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double-stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS: Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan-based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION: These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from Ostrinia nubilalis.

Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), Ostrinia nubilalis, due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases (OndsRNase1, 2, 3, and 4) and one REase (OnREase) were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.

Transcriptome analysis of antennal cytochrome P450s and their transcriptional responses to plant and locust volatiles in Locusta migratoria.

Cytochrome P450 monooxygenases (P450s) constitute a large superfamily of heme-thiolate proteins that are involved in the biosynthesis or degradation of endogenous compounds and detoxification of exogenous chemicals. It has been reported that P450s could serve as odorant-degrading enzymes (ODEs) to inactivate odorants to avoid saturating the antennae. However, there is little information about P450s in the antennae of Locusta migratoria. In the current work, we conducted an antenna transcriptome analysis and identified 92 P450s, including 68 full-length and 24 partial sequences. Phylogenetic analysis showed that 68 full-length P450s were grouped into four clans: CYP2, CYP3, CYP4, and mitochondria clans. Tissue, stage, and sex-dependent expressions of these 68 P450s were investigated. The results showed that 4 P450s were antenna-specific, whereas others were antenna-rich but also expressed in other tissues, implying their various potential roles in the antennae. In addition, the responses of seven selected P450s to five gramineous plant volatiles and four locust volatiles were determined. CYP6MU1 could be induced by almost all compounds tested, suggesting its important roles in odorant processing. Different P450s exhibited diverse responses to odorants, indicating that specific regulation of P450 expression by odorants might modulate the sensitivity of the olfactory responses to various chemicals.

Overexpression of Mn-superoxide dismutase in Oxya chinensis mediates increased malathion tolerance.

Superoxide dismutase (SOD) is the first line of defense against oxidative damage. Malathion is an organophosphate insecticide and can induce the production of reactive oxygen species (ROS) and cause the intracellular oxidative stress. The present study was undertaken to examine the effects of malathion on SODs activity and their transcriptional levels in Oxya chinensis (Thunberg) (Orthoptera: Acrididae). The results showed that total SOD and MnSOD activities increased as a dose-dependent manner while CuZnSOD activity has no significant changes after malathion treatments. Total SOD and MnSOD activities were the highest at the concentration of 0.8 µg µL-1 malathion treatment and increased significantly about 1.81- and 2.48-fold compared with the control, respectively. Increased mRNA expression of MnSOD, ecCuZnSOD1, and ecCuZnSOD2 were observed after malathion treatments. Moreover, the alteration of MnSOD transcript was similar to the profiles of MnSOD activity. These results suggested that the up-regulation expression of MnSOD transcript led to the increase of MnSOD activity in order to eliminate the excessive ROS caused by malathion. In addition, we evaluated the role of individual SOD gene in malathion stress by using RNAi and recombinant SOD proteins. The results showed that ROS contents increased significantly after the silencing of MnSOD and ecCuZnSOD1 genes. The OD values of the E. coli cells transformed with pET-28a-OcMnSOD plasmid were 1.13-1.31-fold and 1.08-1.33-fold higher than those of cells with pET-28a plasmids under 0.4 and 0.8 µg µL-1 malathion treatments, respectively. These findings indicated that MnSOD exerted an important role in defense oxidative stress caused by malathion.