No-tillage enhances soil water storage, grain yield and water use efficiency in dryland wheat (Triticum aestivum) and maize (Zea mays) cropping systems: a global meta-analysis.

Climate change significantly affects crop production and is a threat to global food security. Conventional tillage (CT) is the primary tillage practice in rain-fed areas to conserve soil moisture. Despite previous research on the effect of tillage methods on different cropping systems, a comparison of tillage methods on soil water storage, crop yield and crop water use in wheat (Triticum aestivum ) and maize (Zea mays ) under different soil textures, precipitation and temperature patterns is needed. We reviewed 119 published articles and used meta-analysis to assess the effects of three conservation tillage practices (NT, no-tillage; RT, reduced tillage; ST, subsoil tillage), on precipitation storage efficiency (PSE), soil water storage at crop planting (SWSp), grain yield, evapotranspiration (ET) and water use efficiency (WUE) under varying precipitation and temperature patterns and soil textures in dryland wheat and maize, with CT as the control treatment. Conservation tillage methods increased PSE, SWSp, grain yield, ET and WUE in both winter wheat-fallow and spring maize cropping systems. More precipitation water was conserved in fine-textured soils than in medium-textured and coarse-textured soils, which improved ET. Conservation tillage increased soil water conservation and yield under high mean annual precipitation (MAP) and moderate mean annual temperature (MAT) conditions in winter wheat. However, soil water conservation and yield were greater under MAP <400mm and moderate MAT. We conclude that conservation tillage could be promising for increasing precipitation storage, soil water conservation and crop yield in regions with medium to low MAPs and medium to high MATs.

Molecular characterization, immune functions and DNA protective effects of peroxiredoxin-1 gene in Antheraea pernyi.

Peroxiredoxins are antioxidant proteins that detoxify peroxynitrite, hydrogen peroxide, and organic hydroperoxides, impacting various physiological processes such as immune responses, apoptosis, cellular homeostasis, and so on. In the present study, we identified and characterized peroxiredoxin 1 from Antheraea pernyi (thereafter designated as ApPrx-1) that encodes a predicted 195 amino acid residue protein with a 21.8 kDa molecular weight. Quantitative real-time PCR analysis revealed that the mRNA level of ApPrx-1 was highest in the hemocyte, fat body, and midgut. Immune-challenged larval fat bodies and hemocytes showed increased ApPrx-1 transcript. Moreover, ApPrx-1 expression was induced in hemocytes and the whole body of A. pernyi following exogenous H2O2 administration. A DNA cleavage assay performed using recombinant ApPrx-1 protein showed that rApPrx-1 protein manifests the ability to protect supercoiled DNA damage from oxidative stress. To test the rApPrx-1 protein antioxidant activity, the ability of the rApPrx-1 protein to remove H2O2 was assessed in vitro using rApPrx-1 protein and DTT, while BSA + DDT served as a control group. The results revealed that ApPrx-1 can efficiently remove H2O2 in vitro. In the loss of function analysis, we found that ApPrx-1 significantly increased the levels of H2O2 in ApPrx-1-depleted larvae compared to the control group. We also found a significantly lower survival rate in the larvae in which ApPrx-1 was knocked down. Interestingly, the antibacterial activity was significantly higher in the ApPrx-1 depleted larvae, compared to the control. Collectively, evidence strongly suggests that ApPrx-1 may regulate physiological activities and provides a reference for further studies to validate the utility of the key genes involved in reliving oxidative stress conditions and regulating the immune responses of insects.

Peroxiredoxin-2 gene in Antheraea pernyi modulates immune functions and protect DNA damage.

Peroxiredoxins have been shown to protect insects from oxidative damage and to play a role in the immune system. In the present study, we cloned and characterized the Antheraea pernyi peroxiredoxin 2 (ApPrx-2) gene, then assessed its functional roles. The ApPrx-2 gene has a 687 bp open reading frame that encodes a protein with 288 amino acid residues. Quantitative real-time PCR analysis revealed that the mRNA levels of ApPrx-2 were highest in the hemocytes. Immune challenge assay revealed that ApPrx-2 transcription could be induced after microbial challenge. A DNA cleavage assay employing recombinant ApPrx-2 protein and a metal-catalyzed oxidation system showed that rApPrx-2 protein could protect supercoiled DNA against oxidative stress. The protein antioxidant activity of rApPrx-2 was examined, and it was found that rApPrx-2 exhibited a high level of antioxidant activity by removing H2O2. In addition, ApPrx-2 knockdown larvae had higher H2O2 levels and a lower survival rate when compared to controls. Interestingly, the antibacterial activity was significantly higher in ApPrx-2 depleted larvae compared with control. Overall, our findings indicate that ApPrx-2 may be involved in a range of physiological functions of A. pernyi, as it protects supercoiled DNA from oxidative stress and regulates antibacterial activity.

Insight into crustacean cathepsins: Structure-evolutionary relationships and functional roles in physiological processes.

Cathepsins belong to a group of proteins that are present in both prokaryotic and eukaryotic organisms and have an extremely high degree of evolutionary conservation. These proteins are functionally active in extracellular environments as soluble enzymatic proteins or attached to plasma membrane receptors. In addition, they occur in cellular secretory vesicles, mitochondria, the cytosol, and within the nuclei of eukaryotic cells. Cathepsins are classified into various groups based on their sequence variations, leading to their structural and functional diversification. The molecular understanding of the physiology of crustaceans has shown that proteases, including cathepsins, are expressed ubiquitously. They also contain one of the central regulatory systems for crustacean reproduction, growth, and immune responses. This review focuses on various aspects of the crustaceans cathepsins and emphasizes their biological roles in different physiological processes such as reproduction, growth, development, and immune responses. We also describe the bioactivity of crustaceans cathepsins. Because of the vital biological roles that cathepsins play as cellular proteases in physiological processes, they have been proposed as potential novel targets for the development of management strategies for the aquaculture industries.

The Potential Biological Roles of Circular RNAs in the Immune Systems of Insects to Pathogen Invasion.

Circular RNAs (circRNAs) are a newly discovered class of endogenously expressed non-coding RNAs (ncRNAs). They are highly stable, covalently closed molecules that frequently exhibit tissue-specific expression in eukaryotes. A small number of circRNAs are abundant and have been remarkably conserved throughout evolution. Numerous circRNAs are known to play important biological roles by acting as microRNAs (miRNAs) or protein inhibitors ('sponges'), by regulating the function of proteins, or by being translated themselves. CircRNAs have distinct cellular functions due to structural and production differences from mRNAs. Recent advances highlight the importance of characterizing circRNAs and their targets in a variety of insect species in order to fully understand how they contribute to the immune responses of these insects. Here, we focus on the recent advances in our understanding of the biogenesis of circRNAs, regulation of their abundance, and biological roles, such as serving as templates for translation and in the regulation of signaling pathways. We also discuss the emerging roles of circRNAs in regulating immune responses to various microbial pathogens. Furthermore, we describe the functions of circRNAs encoded by microbial pathogens that play in their hosts.

Antheraea pernyi Suppressor of Cytokine Signaling 2 Negatively Modulates the JAK/STAT Pathway to Attenuate Microbial Infection.

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway has been shown to govern various physiological processes, including immune responses, hematopoiesis, cell growth, and differentiation. Recent studies show that suppressors of cytokine signaling (SOCS) proteins attenuate JAK-STAT signaling in mammals; however, their functions are less clear in lepidopteran insects. Here, we report a full-length sequence of SOCS-2 from the Chinese oak silkworm Antheraea pernyi (designated as ApSOCS-2) and study its biological role in immune responses via the JAK-STAT pathway. ApSOCS-2 expression was high in the fat bodies and hemocytes of A. pernyi fifth instar larvae. After pathogen infection with nucleopolyhedrovirus, Beauveria bassiana, Escherichia coli, and Microccus luteus, ApSOCS-2 mRNA was strongly increased compared to the control group. To elucidate the possible involvement in innate immunity, we measured antimicrobial peptide genes expression profiles in the fat body of A. pernyi. In contrast, recombinant ApSOCS-2 protein administration significantly reduced the AMPs transcription, while the depletion of ApSOCS-2 by RNAi increased their expression. Furthermore, we observed higher antibacterial activity and lower bacterial replication in dsApSOCS-2-treated larvae. The ApSOCS-2 transcription level was reduced in STAT depleted A. pernyi larvae challenged by M. luteus. The ApSOCS-2 RNAi data sets were also subjected to transcriptomic analysis, which suggests that ApSOCS-2 is a key regulator of immune function. Taken together, our data suggest that ApSOCS-2 is required for the negative regulation of AMPs transcripts via the JAK-STAT pathway in the insect.

Molecular Identification of Two DNA Methyltransferase Genes and Their Functional Characterization in the Anti-Bacterial Immunity of Antheraea pernyi.

Under different physiological conditions, such as microbial infection, epigenetic mechanisms regulate genes at the transcription level in living organisms. DNA methylation is a type of epigenetic mechanism in which DNA methyltransferases modify the expression of target genes. Here, we identified a full-length sequence of DNMT-1 and DNMT-2 from the Chinese oak silkworm, A. pernyi, which was highly similar to the homologous sequences of Bombyx mori. ApDNMT-1 and ApDNMT-2 have unique domain architectures of insect DNMTs, highlighting their conserved functions in A. pernyi. ApDNMT-1 and ApDNMT-2 were found to be widely expressed in various tissues, with the highest levels of expression in hemocytes, the ovary, testis, and fat bodies. To understand the biological role of these genes in microbial resistance, we challenged the fifth instar larvae of A. pernyi by administrating Gram-positive and Gram-negative bacteria and fungi. The results revealed that transcript levels of ApDNMT-1 and ApDNMT-2 were increased compared to the control group. The inhibition of these genes by a DNMTs inhibitor [5-azacytidine (5-AZA)] significantly reduced bacterial replication and larvae mortality. In addition, 5-AZA treatment modified the expression patterns of antimicrobial peptides (AMPs) in the A. pernyi larvae. Our results suggest that ApDNMT-1 and ApDNMT-2 seem to have a crucial role in innate immunity, mediating antimicrobial peptide responses against bacterial infection in A. pernyi.

Integrins in the Immunity of Insects: A Review.

Integrins are a large group of cell-surface proteins that are classified as transmembrane proteins. Integrins are classified into different types based on sequence variations, leading to structural and functional diversity. They are broadly distributed in animals and have a wide range of biological functions such as cell-to-cell communication, intracellular cytoskeleton organization, cellular signaling, immune responses, etc. Integrins are among the most abundant cell surface proteins in insects, exhibiting their indispensability in insect physiology. Because of their critical biological involvement in physiological processes, they appear to be a novel target for designing effective pest control strategies. In the current literature review, we first discuss the discovery and expression responses of integrins against various types of pathogens. Secondly, we examine the specific biological roles of integrins in controlling microbial pathogens, such as phagocytosis, encapsulation, nodulation, immune signaling, and so on. Finally, we describe the possible uses of integrins to control agricultural insect pests.

Molecular Characterization of Two Genes Encoding Novel Ca2+-Independent Phospholipase A2s from the Silkworm, Bombyx mori.

Eicosanoids are crucial downstream signals in the insect immune responses. Phospholipase A2 (PLA2) catalyzes phospholipids, the initial step in eicosanoid biosynthesis. In mammals, the biological roles of Ca2+-independent Phospholipase A2 (iPLA2) have been extensively studied; however, only a few studies have attempted to explore iPLA2 functions in insects. In this study, we identified two iPLA2 genes (designated as BmiPLA2A and BmiPLA2B) in the silkworm, Bombyx mori. BmiPLA2A had a 2427 base pair (bp) open reading frame (ORF) that coded for a protein with 808 amino acids. In contrast, BmiPLA2B had a 1731 bp ORF that coded for a protein with 576 amino acids. Domain analysis revealed that BmiPLA2A had six ankyrin repeat domains, but BmiPLA2B lacks these domains. BmiPLA2A and BmiPLA2B were transcribed widely in various tissues and developmental stages with different expression patterns. The administration of 20-hydroxyecdysone increased their expression levels in the epidermis and hemocytes. Furthermore, challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria induced the expression of BmiPLA2A and BmiPLA2B with variable degrees along with different time points. Our findings imply that BmiPLA2A and BmiPLA2B may have important biological roles in the development and innate immunity of B. mori.

Transcriptome Sequencing Highlights the Regulatory Role of DNA Methylation in Immune-Related Genes' Expression of Chinese Oak Silkworm, Antheraea pernyi.

Antheraea pernyi is an important lepidopteran used as a model insect species to investigate immune responses, development, and metabolism modulation. DNA methylation has recently been found to control various physiological processes throughout the life of animals; however, DNA methylation and its effect on the physiology of insects have been poorly investigated so far. In the present study, to better understand DNA methylation and its biological role in the immune system, we analyzed transcriptome profiles of A. pernyi pupae following DNA methylation inhibitor injection and Gram-positive bacteria stimulation. We then compared the profiles with a control group. We identified a total of 55,131 unigenes from the RNA sequence data. A comparison of unigene expression profiles showed that a total of 680 were up-regulated and 631 unigenes were down-regulated in the DNA-methylation-inhibition-bacteria-infected group compared to the control group (only bacteria-injected pupae), respectively. Here, we focused on the immune-related differentially expressed genes (DEGs) and screened 10 genes that contribute to immune responses with an up-regulation trend, suggesting that microbial pathogens evade host immunity by increasing DNA methylation of the host genome. Furthermore, several other unigenes related to other pathways were also changed, as shown in the KEGG analysis. Taken together, our data revealed that DNA methylation seems to play a crucial biological role in the regulation of gene expression in insects, and that infection may enhance the host genome DNA methylation by a yet-unknown mechanism.

Identification and the immunological role of two Nimrod family genes in the silkworm, Bombyx mori.

In animals, immune signaling pathways and effector molecules participate in attenuating microbial infection. Recent work has shown that the Nimrod family proteins can directly bind to bacteria, and this binding leads to bacterial phagocytosis. Although the Nimrod gene family has been reported in many non-drosophilids, their functions remain unexplored in most insect species. Here, we report two members (Nimrod-B and Draper) of the Nimrod gene family from Bombyx mori and analyzed their role in immunity. The two genes were ubiquitously expressed in the tested tissues; but, they transcribed preferentially in immune tissues. The developmental profiles showed that BmNimrod-B and BmDraper transcription levels were highest in the pupal stages. Challenge with microbial pathogens induced the transcription levels of all two genes at different time points. Knockdown of BmDraper decreased the bacterial clearance and increased their replication relative to the control group, whereas, BmNimrod-B suppression had a non-significant effect on them. Furthermore, the mortality rate was increased after BmDraper silencing. The knockdown of these genes did not significantly affect the production of antimicrobial peptides following E. coli infection. Taken together, the Nimrod family genes play a crucial role in host defense by positively regulating the antibacterial immune response in silkworm B. mori.

Suppressor of cytokine signalling 6 is a potential regulator of antimicrobial peptides in the Chinese oak silkworm, Antheraea pernyi.

The SOCS/CIS is a family of intracellular proteins distributed widely among living organisms. The members of this family have extensively been studied in mammals and have been shown to regulate various physiological processes. In contrast, the functional roles of SOCS/CIS family proteins are unknown in most invertebrates, including insects. Here, we retrieved a full-length open reading frame (ORF) of SOCS-6 from Chines oak silkworm, Antheraea pernyi (Designated as ApSOCS-6), using the RNA-seq database. The predicted ApSOCS-6 amino acid sequence comprised an N-terminal SH2 domain and a C-terminal SOCS-box domain. It shared the highly conserved structures of the SOCS proteins with other lepidopteran species. ApSOCS-6 mRNA transcript was detected in all the tested tissues of the A. pernyi larvae; however, the highest mRNA levels were found in the larval hemocytes, fat bodies, and integuments. The mRNA transcript levels of ApSOCS-6 were increased in the A. pernyi larval hemocytes and fat bodies after a challenge by the Gram-positive bacteria, M. luteus, Gram-negative bacteria, Escherichia coli, Virus, ApNPV, and Fungus, B. bassiana. After the knockdown of ApSOCS-6, we found a significant increase in bacterial clearance and a decrease in the relative replication of bacteria. To evaluate the possible cause of enhanced antibacterial activity, we measured antimicrobial peptides expression in the fat body of A. pernyi larvae. The production of AMPs was strongly increased in the B. cereus infected larval fat bodies following silencing of ApSOCS-6. Our data indicate that ApSOCS-6 negatively regulates the expression of AMPs in immune tissues in response to the B. cereus challenge.

The identification of nuclear factor Akirin with immune defense role in silkworm, Bombyx mori.

Akirins, highly conserved nuclear factors, regulate diverse physiological processes such as innate immunity. The biological functions of Akirins have extensively been studied in vertebrates and many invertebrates; however, there is no report so far on lepidopteran insects. In the present study, we identified and characterized a novel Akirin from the silkworm, Bombyx mori (designated as BmAkirin), and explored its potential roles in innate immunity. The expression analysis revealed the unequal mRNA levels of BmAkirin in all the tested tissues; however, the gene's transcription level was highest in testis, followed by ovaries and hemocytes. It also had significant expression levels at the early stages of embryonic development. Expression of BmAkirin in fat bodies and hemocytes exhibited an increase in various degrees when challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria. Immunofluorescence analysis showed BmAkirin protein was prominently localized in the nucleus. Knockdown of BmAkirin strongly reduced the expression of AMPs and decreased the survival ability of larva upon immune stimulation. Moreover, the bacterial clearance ability of larvae was also decreased following the depletion of BmAkirin. Collectively, our results demonstrate that BmAkirin plays an indispensable role in the innate immunity of Bombyx mori (B. mori) by positively modulating AMPs expression in vivo.

Molecular characterization of a heat shock protein 21 (Hsp21) from red swamp crayfish, Procambarus clarkii in response to immune stimulation.

Small heat shock proteins are a molecular chaperone and implicated in various physiological and stress processes in animals. However, the immunological functions of Hsp genes remain to elucidate in the crustaceans, particularly in red swamp crayfish, Procambarus clarkii. Here we report the cloning of heat shock protein 21 from the P. clarkii (hereafter Pc-Hsp21). The open reading frame of Pc-Hsp21 was 555 base pairs, encoding a protein of 184 amino acid residues with an alpha-crystallin family domain. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive transcript expression of Pc-Hsp21 in the tested tissue, with the highest in hepatopancreas. The transcript abundance for this gene enhanced in hepatopancreas following immune challenge with the lipopolysaccharide, peptidoglycan, and poly I:C compared to the control group. The depletion of Pc-Hsp21 by double-stranded RNA altered transcript expression profiles of several genes in hepatopancreas, genes involved in the crucial immunological pathways of P. clarkii. These results suggest that Pc-Hsp21 plays an essential biological role in the microbial stress response by modulating the expression of immune-related genes in P. clarkii.

Characterization of the cathepsin D in Procambarus clarkii and its biological role in innate immune responses.

Cathepsin D belongs to aspartic protease family, produced in the rough endoplasmic reticulum, and then transported to lysosomes, where it participates in various physiological processes. Despite its importance, only a few reports available on the functional role of cathepsin D in crustaceans. Herein, we cloned a cDNA fragment of cathepsin D from the hepatopancreas of the red swamp crayfish, Procambarus clarkii (Pc-cathepsin D) for the first time. It included 1158 base pairs open reading frame, encoding a protein of 385 amino acids. Multiple alignment analysis confirmed the presence of aspartic proteinase active sites and N glycosylation sites. Pc-cathepsin D mRNA expression was high in the gills followed by gut, heart, hepatopancreas of P. clarkii. At different time points post-infection with lipopolysaccharides, peptidoglycan, or polyinosinic polycytidylic acid, Pc-cathepsin D mRNA expression significantly enhanced compared with the control group. Knockdown of the Pc-cathepsin D by double-stranded RNA, strikingly, changed the expression of all the tested P. clarkii immune-associated genes, including Pc-Toll, Pc-lectin, Pc-cactus, Pc-anti-lipopolysaccharide factor, Pc-phospholipase, and Pc-sptzale. Altogether, these results suggest that Pc-cathepsin D is needed to confer innate immunity against microbial pathogens by modulating the expression of crucial transcripts that encode immune-associated genes.

The red swamp crayfish, Procambarus clarkii cathepsin C, participates in the innate immune response to the viral and bacterial pathogens.

The cathepsin C, a lysosomal cysteine protease, involves the modulation of immune and inflammatory responses in living organisms. However, the knowledge on cathepsin C in red swamp crayfish (Procambarus clarkii), a freshwater crustacean with economic values, remained unclear. In the present study, we provide identification and molecular characterization of cathepsin C from P. clarkii. (Hereafter Pc-cathepsin C). The Pc-cathepsin C cDNA contained a 1356 bp open reading frame that encoded a protein of 451 amino acid residues. The deduced amino acid sequence comprised of cathepsin C exclusion domain and pept_C1 domain, and also catalytic residues (Cys248, His395 and Asn417). Analysis of the transcriptional patterns of the Pc-cathepsin C gene revealed that it was broadly distributed in various tissues of P. clarkii, and it was more abundant in the hepatopancreas and gut. Following a challenge with viral and bacterial pathogen-associated molecular patterns, the expression of Pc-cathepsin C was strongly enhanced at different time points. The knockdown of Pc-cathepsin C, altered the expression of immune-responsive genes, suggesting its immunoregulatory role in P. clarkii. This study has identified and provided the immunoregulatory function of Pc-cathepsin C, which will contribute to further investigation of the molecular mechanism of cathepsin C in crustaceans.

Niemann-Pick type C1 regulates cholesterol transport and metamorphosis in silkworm, Bombyx mori (Dazao).

NPC1 is a large glycoprotein with 13 transmembrane-spanning domains, which plays a crucial biological role in cholesterol transport and metamorphosis of animals. However, the physiological functions of this gene have rarely been elucidated in insects. Here, we isolated the NPC1 gene from Bombyx mori (BmNPC1), sequenced and evaluated its physiological functions. BmNPC1 comprised of 3702 bp open reading frame, encoding a protein of 1233 amino acid residues. The recombinant protein was expressed, and anti-BmNPC1 antibodies were synthesized. Immunofluorescence assay revealed that BmNPC1 protein localized in the cytoplasm of the cells. The qRT-PCR analysis showed that BmNPC1 expression was most significant in the testis, followed by the malpighian tubules, hemocytes, and ovary. The knockdown of BmNPC1 by double-stranded RNA caused the accumulation of cholesterol in the cells. Furthermore, suppression of this gene influenced the expression of ecdysone-responsive genes and also prevented the molting in B. mori (Dazao) larvae. Overall, BmNPC1 may have different biological roles in the physiology of silkworm, B. mori (Dazao), since it regulates the cholesterol transport and molting process.

Heat shock protein 20 from Procambarus clarkii is involved in the innate immune responses against microbial infection.

Small heat shock proteins (shsps) are conserved across invertebrate species. They are implicated in the modulation of various biological processes, such as immune responses, abiotic stress tolerance metamorphosis, and embryonic development. Herein, we identified a heat shock protein 20 from the red swamp crayfish, Procambarus clarkii (named as Pc-Hsp20), and performed in vivo studies to elucidate its physiological functions in the innate immunity. The open reading frame of Pc-Hsp20 was 609 base pair, encoding a protein of 202 amino acid residues with a hsp20/alpha crystallin family domain. Pc-Hsp20 was ubiquitously expressed in various tissues; however, it was highest in the hepatopancreas. The challenge with immune elicitors remarkably enhanced the transcript level of Pc-Hsp20 in the hepatopancreas when compared with the control. Administration of double-stranded RNA could significantly reduce expression of the Pc-Hsp20 mRNAs, and most of the immune-related genes expression enhanced with a variable concentration in the hepatopancreas. Altogether, these results suggest that Pc-Hsp20 may participate in innate immunity against microbial pathogens.