Transcriptome sequencing of the endangered land snail Karaftohelix adamsi from the Island Ulleung: De novo assembly, annotation, valuation of fitness genes and SSR markers.

BACKGROUND: The Bradybaenidae snail Karaftohelix adamsi is endemic to Korea, with the species tracked from Island Ulleung in North Gyeongsang Province of South Korea. K. adamsi has been classified under the Endangered Wildlife Class II species of Korea and poses a severe risk of extinction following habitat disturbances. With no available information at the DNA (genome) or mRNA (transcriptome) level for the species, conservation by utilizing informed molecular resources seems difficult. OBJECTIVE: In this study, we used the Illumina short-read sequencing and Trinity de novo assembly to draft the reference transcriptome of K. adamsi. RESULTS: After assembly, 13,753 unigenes were obtained of which 10,511 were annotated to public databases (a maximum of 10,165 unigenes found homologs in PANM DB). A total of 6,351, 3,535, 358, and 3,407 unigenes were ascribed to the functional categories under KOG, GO, KEGG, and IPS, respectively. The transcripts such as the HSP 70, aquaporin, TLR, and MAPK, among others, were screened as putative functional resources for adaptation. DNA transposons were found to be thickly populated in comparison to retrotransposons in the assembled unigenes. Further, 2,164 SSRs were screened with the promiscuous presence of dinucleotide repeats such as AC/GT and AG/CT. CONCLUSION: The transcriptome-guided discovery of molecular resources in K. adamsi will not only serve as a basis for functional genomics studies but also provide sustainable tools to be utilized for the protection of the species in the wild. Moreover, the development of polymorphic SSRs is valuable for the identification of species from newer habitats and cross-species genotyping.

Transcriptome analysis of Haemaphysalis flava female using Illumina HiSeq 4000 sequencing: de novo assembly, functional annotation and discovery of SSR markers.

BACKGROUND: Ticks are ectoparasites capable of directly damaging their hosts and transmitting vector-borne diseases. The ixodid tick Haemaphysalis flava has a broad distribution that extends from East to South Asia. This tick is a reservoir of severe fever with thrombocytopenia syndrome virus (SFTSV) that causes severe hemorrhagic disease, with cases reported from China, Japan and South Korea. Recently, the distribution of H. flava in South Korea was found to overlap with the occurrence of SFTSV. METHODS: This study was undertaken to discover the molecular resources of H. flava female ticks using the Illumina HiSeq 4000 system, the Trinity de novo sequence assembler and annotation against public databases. The locally curated Protostome database (PANM-DB) was used to screen the putative adaptation-related transcripts classified to gene families, such as angiotensin-converting enzyme, aquaporin, adenylate cyclase, AMP-activated protein kinase, glutamate receptors, heat shock proteins, molecular chaperones, insulin receptor, mitogen-activated protein kinase and solute carrier family proteins. Also, the repeats and simple sequence repeats (SSRs) were screened from the unigenes using RepeatMasker (v4.0.6) and MISA (v1.0) software tools, followed by the designing of SSRs flanking primers using BatchPrimer 3 (v1.0) software. RESULTS: The transcriptome produced a total of 69,822 unigenes, of which 46,175 annotated to the homologous proteins in the PANM-DB. The unigenes were also mapped to the EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) specializations. Promiscuous presence of protein kinase, zinc finger (C2H2-type), reverse transcriptase, and RNA recognition motif domains was observed in the unigenes. A total of 3480 SSRs were screened, of which 1907 and 1274 were found as tri- and dinucleotide repeats, respectively. A list of primer sequences flanking the SSR motifs was detailed for validation of polymorphism in H. flava and the related tick species. CONCLUSIONS: The reference transcriptome information on H. flava female ticks will be useful for an enriched understanding of tick biology, its competency to act as a vector and the study of species diversity related to disease transmission.

Isolation and characterization of a lectin-like chitinase from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii.

A lectin was isolated from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii by affinity chromatography using mucin-sepharose matrix. The purity of the isolated lectin was confirmed in native gradient PAGE that showed a single protein band of ∼37.9 kDa. In SDS-PAGE also one band of ∼43.3 kDa molecular weight was observed that indicated the protein to be a monomer. The band from the SDS-PAGE gel was identified through mass spectrometry as chitinase 1. The purified chitinase (50 µg/ml) hemagglutinated rabbit RBCs and, mucin and glucose inhibited hemagglutination with minimum concentrations of 0.1 mg/ml and 100 mM, respectively. Bacterial agglutination with Gram -ve Vibrio harveyi, Aeromonas sobria and Escherichia coli was also observed by this protein. Thus, chitinase 1 showed lectin-like properties besides its chitin hydrolytic activity. In western blot with hepatopancreas sample, rabbit antiserum against chitinase 1 cross-reacted to two additional proteins namely, chitinase 1C and obstructor E (a chitin-binding protein, CBP), besides its specific reactivity. An indirect ELISA was developed with the antiserum to quantify chitinases/CBP in hepatopancreas and serum samples of M. rosenbergii. The assay was used in samples from juvenile prawns following V. harveyi challenge. At 72 h post-challenge, significantly higher levels of chitinases/CBP were quantified in the hepatopancreas of the challenged group (1.8 ± 0.2 mg/g tissue) compared to the control (1.2 ± 0.1 mg/g tissue). This study suggests that the chitinase 1 protein with lectin-like properties is possibly induced at the protein level and can be putatively involved in the innate immune response of M. rosenbergii.

A review of the endangered mollusks transcriptome under the threatened species initiative of Korea.

Transcriptome studies for conservation of endangered mollusks is a proactive approach towards managing threats and uncertainties facing these species in natural environments. The population of these species is declining due to habitat destruction, illicit wildlife trade, and global climate change. These activities risk the free movement of species across the wild landscape, loss of breeding grounds, and restrictions in displaying the physiological attributes so crucial for faunal welfare. Gastropods face the most negative ecological effects and have been enlisted under Korea's protective species consortium based on their population dynamics in the last few years. Moreover, with the genetic resources restricted for such species, conservation by informed planning is not possible. This review provides insights into the activities under the threatened species initiative of Korea with special reference to the transcriptome assemblies of endangered mollusks. The gastropods such as Ellobium chinense, Aegista chejuensis, Aegista quelpartensis, Incilaria fruhstorferi, Koreanohadra kurodana, Satsuma myomphala, and Clithon retropictus have been represented. Moreover, the transcriptome summary of bivalve Cristaria plicata and Caenogastropoda Charonia lampas sauliae is also discussed. Sequencing, de novo assembly, and annotation identified transcripts or homologs for the species and, based on an understanding of the biochemical and molecular pathways, were ascribed to predictive gene function. Mining for simple sequence repeats from the transcriptome have successfully assisted genetic polymorphism studies. A comparison of the transcriptome scheme of Korean endangered mollusks with the genomic resources of other endangered mollusks have been discussed with homologies and analogies for dictating future research.

IKKß regulates antimicrobial innate immune responses in the yellow mealworm, Tenebrio molitor.

Toll and IMD pathways regulate antimicrobial innate immune responses in insect model systems. The transcriptional activation of antimicrobial peptides (AMPs) confers humoral immunity in the host against invaded pathogens. The IKK kinase complex (IKKα, IKKß, and the regulatory subunit IKKγ/NEMO) centrally regulates the NF-κB response to various stimuli. It triggers an appropriate antimicrobial immune response in the host. In this study, a TmIKKß (or TmIrd5) homolog was screened from the RNA-seq database of the coleopteran beetle, Tenebrio molitor. A single exon characterizes the TmIKKß gene, and the open reading frame (ORF) comprises of 2112 bp that putatively encodes a polypeptide of 703 amino acid residues. TmIKKß contains a serine/threonine kinase domain and is phylogenetically close to Tribolium castaneum IKKß homolog (TcIKKß). TmIKKß transcripts were highly expressed in the early pupal (P1) and adult (A5) stages. Among the tissues, TmIKKß showed higher expression in the integument of the last instar larvae and the fat body and hemocytes of 5-day-old adults. TmIKKß mRNA was upregulated post-E. coli challenge to the host. Moreover, RNAi-based TmIKKß mRNA silencing increased host larvae' susceptibility against E. coli, S. aureus and C. albicans. TmIKKß RNAi in the fat body led to a downregulation in mRNA expression of ten out of fourteen AMP genes, including TmTenecin1, -2, and -4; TmDefensin, and -like; TmColeoptericinA, and -B; and TmAttacin1a, -1b, and -2, suggesting the requirement of the gene in antimicrobial innate immune responses. Further, a decrease in the mRNA expression of NF-κB factors such as TmRelish, TmDorsal1, and TmDorsal2 in the fat body of T. molitor larvae was observed post-microorganisms challenge. Thus, TmIKKß regulates antimicrobial innate immune responses in T. molitor.

Transcriptome analysis of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) and characterization of genes associated to immunity, growth, and reproduction.

BACKGROUND: Dung beetles recycle organic matter through the decomposition of feces and support ecological balance. However, these insects are threatened by the indiscriminate use of agrochemicals and habitat destruction. Copris tripartitus Waterhouse (Coleoptera: Scarabaeidae), a dung beetle, is listed as a class-II Korean endangered species. Although the genetic diversity of C. tripartitus populations has been investigated through analysis of mitochondrial genes, genomic resources for this species remain limited. In this study, we analyzed the transcriptome of C. tripartitus to elucidate functions related to growth, immunity and reproduction for the purpose of informed conservation planning. RESULTS: The transcriptome of C. tripartitus was generated using next-generation Illumina sequencing and assembled de novo using a Trinity-based platform. In total, 98.59% of the raw sequence reads were processed as clean reads. These reads were assembled into 151,177 contigs, 101,352 transcripts, and 25,106 unigenes. A total of 23,450 unigenes (93.40%) were annotated to at least one database. The largest proportion of unigenes (92.76%) were annotated to the locally curated PANM-DB. A maximum of 5,512 unigenes had homologous sequences in Tribolium castaneum. Gene Ontology (GO) analysis revealed a maximum of 5,174 unigenes in the Molecular function category. Further, in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, a total of 462 enzymes were associated with established biological pathways. Based on sequence homology to known proteins in PANM-DB, representative immunity, growth, and reproduction-related genes were screened. Potential immunity-related genes were categorized into pattern recognition receptors (PRRs), the Toll-like receptor signaling pathway, the MyD88- dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related transcripts. Among PRRs, we conducted detailed in silico characterization of TLR-2, CTL, and PGRP_SC2-like. Repetitive elements such as long terminal repeats, short interspersed nuclear elements, long interspersed nuclear elements and DNA elements were enriched in the unigene sequences. A total of 1,493 SSRs were identified among all unigenes of C. tripartitus. CONCLUSIONS: This study provides a comprehensive resource for analysis of the genomic topography of the beetle C. tripartitus. The data presented here clarify the fitness phenotypes of this species in the wild and provide insight to support informed conservation planning.

Characterization of a Lipopolysaccharide- and Beta-1,3-Glucan Binding Protein (LGBP) from the Hepatopancreas of Freshwater Prawn, Macrobrachium rosenbergii, Possessing Lectin-Like Activity.

The study focuses on the isolation, characterization, and expression analysis of a lectin from the hepatopancreas of Macrobrachium rosenbergii. The protein was isolated by affinity chromatography on a melibiose-agarose column. The molecular weight of the native protein was found to be ~120 kDa which consists of a single polypeptide of ~39.5 kDa. On mass spectrometric analysis, the protein was identified as lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP). LGBP showed hemagglutination with rabbit RBC like a lectin and its carbohydrate-binding specificity was determined by the hemagglutination inhibition test. The protein also showed antibacterial activity against two Gram-negative bacteria Vibrio harveyi and Aeromonas sobria, and one Gram positive bacteria Bacillus cereus in the disc diffusion test. Rabbit antiserum was raised against the purified LGBP and used to develop a sandwich ELISA system for quantitation of the protein in hepatopancreas and serum samples of M. rosenbergii. The expression of the LGBP transcripts in muscle, hepatopancreas, and gill tissues from M. rosenbergii juveniles at 72 h post-challenge of V. harveyi was not modulated as noticed in qPCR analysis. However, significant increases in the concentrations of LGBP protein in hepatopancreas (5.23 ± 0.45 against 3.43 ± 0.43 mg/g tissue in control) and serum (1.08 ± 0.14 against 0.61 ± 0.08 µg/ml in control) were observed in the challenged group of prawns in ELISA suggesting its putative role against bacterial infections. The study for the first time characterized the native LGBP of M. rosenbergii showing a multifunctional role in immunity.

Current Status of Immune Deficiency Pathway in Tenebrio molitor Innate Immunity.

Yellow mealworm (Tenebrio molitor) is a highly beneficial beetle that serves as an excellent source of edible protein as well as a practical study model. Therefore, studying its immune system is important. Like in other insects, the innate immune response effected through antimicrobial peptides production provides the most critical defense armory in T. molitor. Immune deficiency (Imd) signaling is one of the major pathways involved in the humoral innate immune response in this beetle. However, the nature of the molecules involved in the signaling cascade of the Imd pathway, from recognition to the production of final effectors, and their mechanism of action are yet to be elucidated in T. molitor model. In this review, we present a general overview of the current literature available on the Imd signaling pathway and its identified interaction partners in T. molitor.

Molecular cloning, sequence characterization, and expression analysis of C-type lectin (CTL) and ER-Golgi intermediate compartment 53-kDa protein (ERGIC-53) homologs from the freshwater prawn, Macrobrachium rosenbergii.

Lectin protein families are diverse and multi-functional in crustaceans. The carbohydrate-binding domains (CRDs) of lectins recognize the molecular patterns associated with pathogens and orchestrate important roles in crustacean defense. In this study, two lectin homologs, a single CRD containing C-type lectin (CTL) and an L-type lectin (LTL) domain containing endoplasmic reticulum Golgi intermediate compartment 53 kDa protein (ERGIC-53) were identified from the freshwater prawn, Macrobrachium rosenbergii. The open reading frames of MrCTL and MrERGIC-53 were 654 and 1,515 bp, encoding polypeptides of 217 and 504 amino acids, respectively. Further, MrCTL showed a 20-amino acid transmembrane helix region and 10 carbohydrate-binding residues within the CRD. MrERGIC-53 showed a signal peptide region, a type-I transmembrane region, and a coiled-coil region at the C-terminus. Phylogenetic analysis revealed a close relationship between MrCTL and MrLectin and M. nipponense CTL (MnCTL), whereas MrERGIC-53 shared high sequence identity with Eriocheir sinensis ERGIC-53 and Penaeus vannamei MBL-1. A homology-based model predicted small carbohydrate-combining sites with a metal-binding site for ligand binding (Ca2+ binding site) in MrCTL and beta-sheets connected by short loops and beta-bends forming a dome-shaped beta-barrel structure representing the LTL domain of MrERGIC-53. Quantitative real-time polymerase chain reaction detected MrCTL and MrERGIC-53 transcripts in all examined tissues, with particularly high levels observed in hemocytes, hepatopancreas, and mucosal-associated tissues, such as the stomach and intestine. Further, the expression levels of MrCTL and MrERGIC-53 transcripts were remarkably altered after V. harveyi challenge, suggesting putative function in host innate immunity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10499-022-00845-3.

Current knowledge of immune priming in invertebrates, emphasizing studies on Tenebrio molitor.

Vertebrates rely on the most sophisticated adaptive immunity to defend themselves against various pathogens. This includes immunologic memory cells, which mount a stronger and more effective immune response against an antigen after its first encounter. Unlike vertebrates, invertebrates' defense completely depends on the innate immunity mechanisms including humoral and cell-mediated immunity. Furthermore, the invertebrate equivalent of the memory cells was discovered only recently. Since the discovery of transgenerational immune priming (TGIP) in crustaceans, numerous findings have proven the IP in invertebrate classes such as insects. TGIP can be induced through maternal priming pathways such as transcriptional regulation of antimicrobial peptides, and also paternal IP including the induction of proPO system activity. We appraise the diversity and specificity of IP agents to provide sustained immunologic memory in insects, particularly T. molitor in the review. An understanding of IP (more so TGIP) response in T. molitor will deepen our knowledge of invertebrate immunity, and boost the mass-rearing industry by reducing pathogen infection rates.

Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides.

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

Autophagy in Tenebrio molitor Immunity: Conserved Antimicrobial Functions in Insect Defenses.

The yellow mealworm beetle (Tenebrio molitor) has been exploited as an experimental model to unravel the intricacies of cellular and humoral immunity against pathogenic infections. Studies on this insect model have provided valuable insights into the phenotypic plasticity of immune defenses against parasites and pathogens. It has thus been possible to characterize the hemocoelic defenses of T. molitor that rely on the recognition of non-self-components of pathogens by pattern recognition receptors (PRRs). The subsequent signaling cascade activating pathways such as the NF-κB controlled by Toll and IMD pathways lead to the synthesis of antimicrobial peptides (AMPs), onset of hemocyte-driven phagocytosis, and activation of the prophenoloxidase cascade regulating the process of melanization. Nevertheless, the activation of autophagy-mediated defenses of T. molitor against the facultative intracellular gram-positive bacterium Listeria monocytogenes provides clear evidence of the existence of a cross-talk between autophagy and the IMD pathway. Moreover, the identification of several autophagy-related genes (Atgs) in T. molitor transcriptome and expressed sequence tag (EST) databases has contributed to the understanding of the autophagy-signaling cascade triggered by L. monocytogenes challenge. Providing further evidence of the cross-talk hypothesis, TmRelish has been shown to be required not only for regulating the synthesis of AMPs through the PGRP-LE/IMD pathway activation but also for the expression of Atgs in T. molitor larvae following L. monocytogenes challenge. Notably, L. monocytogenes can stimulate the T. molitor innate immune system by producing molecules recognized by the multifunctional PRR (TmPGRP-LE), which stimulates intracellular activation of the IMD pathway and autophagy. Considering the conservation of autophagy components involved in combating intracellular pathogens, it will be interesting to extrapolate a dynamic cross-talk model of immune activation. This review summarizes the most significant findings on the regulation of autophagy in T. molitor during L. monocytogenes infection and on the role of the innate immunity machinery, including the NF-κB pathway, in the control of pathogenic load.

De novo transcriptome sequencing of triton shell Charonia lampas sauliae: Identification of genes related to neurotoxins and discovery of genetic markers.

Charonia lampas sauliae (triton snails, triton shells or tritons; Mollusca, Caenogastropoda, Littorinimorpha, Ranellidae) is a marine species with a wide distribution. In Korea, this species is listed as vulnerable and is regionally protected as an endangered species. Here, we report the first comprehensive transcriptome dataset of C. lampas sauliae obtained using the Illumina HiSeq 2500 platform. In total, 97.68% of raw read sequences were processed as clean reads. Of the 577,478 contigs obtained, 146,026 sequences were predicted to contain coding regions. About 89.34% of all annotated unigene sequences showed homologous matches to protein sequences in PANM DB (Protostome database). Further, about one-third of the unigene sequences were annotated using the UniGene, Swiss-Prot, Clusters of Orthologous Groups (COG) and Gene Ontology (GO) databases. In total, 190 enzymes were predicted under key metabolic pathways under stood through Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotation. Repetitive elements such as long terminal repeats (LTRs), short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), and DNA elements were enriched in the unigene sequences. Among the identified transcripts were the channel proteins, some of which were blocked by tetrodotoxin, which is thought to be synthesized by symbiotic bacteria inhabiting the shells. In addition, conotoxin superfamily peptides, such as B-conotoxin, conotoxin superfamily T and alpha-conotoxin, were identified, which may have relevance to biomedical and evolutionary research. A transcriptome-wide search for polymorphic loci identified 21,568 simple sequence repeats (SSRs) in the unigene sequences. Most SSRs were dinucleotides, among which AC/GT was the dominant SSR type. The molecular and genetic resources revealed in this study could be utilized for investigations on the fitness of the species in the marine environment and sustainability in a changing habitat.

Gene expression analysis of inflammation-related genes in macrophages treated with α-(1 → 3, 1 → 6)-D-glucan extracted from Streptococcus mutans.

Streptococcus mutans is a gram-positive bacterium that causes tooth decay. The exopolyssacharides, mostly glucans synthesized by the bacterium are responsible for establishing pathogenic bio-films associated with dental caries disease. The regulatory immune and inflammatory reactions implicated by the synthesized glucans are still not clearly understood. In this study, a water-soluble exopolyssacharide (WSP) was extracted from culture of Str. mutans. The structural properties of WSP, [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] were confirmed using Fourier-transform infrared spectroscopy and 13C-nuclear magnetic resonance spectroscopy. Furthermore, the effects of WSP on the global gene expression of the macrophage-like RAW 264.7 cells were analyzed using mRNA-seq analysis. Using Gene Ontology analysis, we compiled a total of 24,421 genes that were upregulated or downregulated by more than 5.0-fold and 0.3-fold, respectively. Most of the transcripts were grouped under immune response and inflammation-related gene categories. Among the 802 immunity-related genes analyzed, chemokine ligand 7 (Ccl7), interleukin-1ß (IL-1ß), interleukin-1α (IL-1α) and interleukin-6 (IL-6) were upregulated after WSP exposure. In addition, among a total of 344 genes related to inflammation, Ccl7, IL-1α and IL-6 were upregulated. These results suggest that [α-(1 â†’ 3, 1 â†’ 6)-D-glucan] from Str. mutans produces activates macrophages and may contribute to the immune and inflammatory response to periodontal disease.

TmIKKε Is Required to Confer Protection Against Gram-Negative Bacteria, E. coli by the Regulation of Antimicrobial Peptide Production in the Tenebrio molitor Fat Body.

The inhibitor of nuclear factor-kappa B (NF-κB) kinase (IKK) is the core regulator of the NF-κB pathway against pathogenic invasion in vertebrates or invertebrates. IKKß, -ε and -γ have pivotal roles in the Toll and immune deficiency (IMD) pathways. In this study, a homolog of IKKε (TmIKKε) was identified from Tenebrio molitor RNA sequence database and functionally characterized for its role in regulating immune signaling pathways in insects. The TmIKKε gene is characterized by two exons and one intron comprising an open reading frame (ORF) of 2,196 bp that putatively encodes a polypeptide of 731 amino acid residues. TmIKKε contains a serine/threonine protein kinases catalytic domain. Phylogenetic analysis established the close homology of TmIKKε to Tribolium castaneum IKKε (TcIKKε) and its proximity with other IKK-related kinases. The expression of TmIKKε mRNA was elevated in the gut, integument, and hemocytes of the last-instar larva and the fat body, Malpighian tubules, and testis of 5-day-old adults. TmIKKε expression was significantly induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenge in whole larvae and tissues, such as hemocytes, gut, and fat body. The knockdown of the TmIKKε messenger RNA (mRNA) expression significantly reduced the survival of the larvae against microbial challenges. Further, we investigated the induction patterns of 14 T. molitor antimicrobial peptides (AMPs) genes in TmIKKε gene-silencing model after microbial challenges. While in hemocytes, the transcriptional regulation of most AMPs was negatively regulated in the gut and fat body tissue of T. molitor, AMPs, such as TmTenecin 1, TmTenecin 4, TmDefensin, TmColeoptericin A, TmColeoptericin B, TmAttacin 1a, and TmAttacin 2, were positively regulated in TmIKKε-silenced individuals after microbial challenge. Collectively, the results implicate TmIKKε as an important factor in antimicrobial innate immune responses in T. molitor.

IKKγ/NEMO Is Required to Confer Antimicrobial Innate Immune Responses in the Yellow Mealworm, Tenebrio Molitor.

IKKγ/NEMO is the regulatory subunit of the IκB kinase (IKK) complex, which regulates the NF-κB signaling pathway. Within the IKK complex, IKKγ/NEMO is the non-catalytic subunit, whereas IKKα and IKKß are the structurally related catalytic subunits. In this study, TmIKKγ was screened from the Tenebrio molitor RNA-Seq database and functionally characterized using RNAi screening for its role in regulating T. molitor antimicrobial peptide (AMP) genes after microbial challenges. The TmIKKγ transcript is 1521 bp that putatively encodes a polypeptide of 506 amino acid residues. TmIKKγ contains a NF-κB essential modulator (NEMO) and a leucine zipper domain of coiled coil region 2 (LZCC2). A phylogenetic analysis confirmed its homology to the red flour beetle, Tribolium castaneum IKKγ (TcIKKγ). The expression of TmIKKγ mRNA showed that it might function in diverse tissues of the insect, with a higher expression in the hemocytes and the fat body of the late-instar larvae. TmIKKγ mRNA expression was induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenges in the whole larvae and in tissues such as the hemocytes, gut and fat body. The knockdown of TmIKKγ mRNA significantly reduced the survival of the larvae after microbial challenges. Furthermore, we investigated the tissue-specific induction patterns of fourteen T. molitor AMP genes in TmIKKγ mRNA-silenced individuals after microbial challenges. In general, the mRNA expression of TmTenecin1, -2, and -4; TmDefensin1 and -2; TmColeoptericin1 and 2; and TmAttacin1a, 1b, and 2 were found to be downregulated in the hemocytes, gut, and fat body tissues in the TmIKKγ-silenced individuals after microbial challenges. Under similar conditions, TmRelish (NF-κB transcription factor) mRNA was also found to be downregulated. Thus, TmIKKγ is an important factor in the antimicrobial innate immune response of T. molitor.

Lectin-Like Activity of Hemocyanin in Freshwater Prawn, Macrobrachium rosenbergii.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0-10.0 and temperature range of 10-40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

Aedes albopictus Autophagy-Related Gene 8 (AaAtg8) Is Required to Confer Anti-Bacterial Gut Immunity.

Autophagy is an important process by which pathogens and damaged or unused organelles are eliminated. The role of autophagy in development and the immune response to pathogens is well established. Autophagy-related protein 8 (Atg8) is involved in the formation of the autophagosome and, with the help of the serine protease Atg4, mediates the delivery of both vesicles and the autophagosome to the vacuole. Here, we cloned the Aedes albopictus autophagy-related protein 8 (AaAtg8) gene and characterized its role in the innate immunity of the mosquito against microbial infections. AaAtg8 is comprised of an open reading frame (ORF) region of 357 bp encoding a polypeptide of 118 amino acid residues. A domain analysis of AaAtg8 revealed an Atg8 ubiquitin-like domain, Atg7/Atg4 interaction sites, and peptide binding sites. The AaAtg8 mRNA expression was high in the Malpighian tubules and heads of both sugar-fed and blood-fed adult female mosquitoes. The expression level of AaAtg8 mRNA increased in the midgut and abdominal carcass following being challenged with Listeria monocytogenes. To investigate the role of AaAtg8 in the innate immune responses of Ae. albopictus, AaAtg8 gene-silenced adult mosquitoes were challenged by injection or by being fed microorganisms in blood. High mortality rates were observed in mosquitoes in which AaAtg8 was silenced after challenges of microorganisms to the host by blood feeding. This suggests that Atg8-autophagy plays a critical role in the gut immunity in Ae. albopictus.