In silico identification and expression analysis of superoxide dismutases in Tenebrio molitor.

BACKGROUND: Insects encounter various environmental stresses, in response to which they generate reactive oxygen species (ROS). Superoxide dismutase (SOD) is an antioxidant metalloenzyme that scavenges superoxide radicals to prevent oxidative damage. OBJECTIVE: To investigate expressions of SODs under oxidative stress in Tenebrio molitor. METHODS: Here, we investigated the transcriptional expression of SODs by pesticide and heavy metals in Tenebrio moltior. First, we searched an RNA-Seq database for T. molitor SOD (TmSOD) genes and identified two SOD isoforms (TmSOD1-iso1 and iso2). We examined their activities under developmental stage, tissue-specific, and various types (pesticide and heavy metal) of oxidative stress by using qPCR. RESULTS: Our results revealed two novel forms of TmSODs. These TmSODs had a copper/zinc superoxide dismutase domain, active site, Cu2+ binding site, Zn2+ binding site, E-class dimer interface, and P-class dimer interface. TmSODs (TmSOD1-iso1 and iso2) were expressed in diverse developmental phases and tissues. Pesticides and heavy metals caused an upregulation of these TmSODs. CONCLUSION: Our findings suggest that the two TmSODs have different functions in T. molitor, providing insights into the detoxification ability of T. molitor.

Phytometabolites from coral jasmine flower extracts: Toxic effects on Spodoptera litura and enzyme inhibition in nontarget earthworm Eisenia fetida as an alternative approach.

Green pesticides, derived from natural sources, have gained wider attention as an alternative to synthetic pesticides for managing polyphagous pests, such as Spodoptera litura. In this study, the methanolic flower extract of Nyctanthes arbor-tristis (Mx-Na-t) was subjected to chemical screening, and 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (3H-dp) and tyrosol (Ty-ol) were identified as the major derivatives. The toxic effects of Mx-Na-t (500 ppm) were highest in third-instar S. litura larvae (96.4%), while those of 3H-dp and Ty-ol (5 ppm) were highest in second-instar larvae (76.5% and 81.4%, respectively). The growth and development of S. litura larvae and pupae were significantly reduced by all three treatments. Fecundity rates were also reduced by all treatments [from 1020 eggs (control) to 540 eggs by Mx-Na-t treatment, 741 eggs by 3H-dp treatment, and 721 eggs by Ty-ol treatment]. The extract and its active constituents decreased adult emergence and slowed total larval development in a dose-dependent manner. A decrease was noted in the major gut enzymes of young S. litura larvae exposed to Mx-Na-t, 3H-dp, and Ty-ol. Moreover, midgut tissues of fourth-instar larvae were severely damaged by Mx-Na-t (250 ppm), 3H-dp (2.5 ppm), and Ty-ol (2.5 ppm); the treatments induced structural damage to the epithelial cells and gut lumen. The earthworm Eisenia fetida was used to assess nontarget toxicity. Compared with cypermethrin, the phytochemicals exhibited minimal effects on the earthworm's detoxifying enzymes superoxide dismutase and catalase after 14 days of treatment. Moreover, in silico predictions using BeeTox and ProTox-II indicated little or no toxicity of 3H-dp and Ty-ol toward honey bees and other nontarget species.

In silico identification and expression analyses of peroxidases in Tenebrio molitor.

Human advancements in agriculture, urbanization, and industrialization have led to various forms of environmental pollution, including heavy metal pollution. Insects, as highly adaptable organisms, can survive under various environmental stresses, which induce oxidative damage and impair antioxidant systems. To investigate the peroxidase (POX) family in Tenebrio molitor, we characterized two POXs, namely TmPOX-iso1 and TmPOX-iso2. The full-length cDNA sequences of TmPox-iso1 and TmPox-iso2 respectively consisted of an open reading frame of 1815 bp encoding 605 amino acids and an open reading frame of 2229 bp encoding 743 amino acids. TmPOX-iso1 and TmPOX-iso2 homologs were found in five distinct insect orders. In the phylogenetic tree analysis, TmPOX-iso1 was clustered with the predicted POX protein of T. castaneum, and TmPOX-iso2 was clustered with the POX precursor protein of T. castaneum. During development, the highest expression level of TmPox-iso1 was observed in the pre-pupal stage, while that of TmPox-iso2 expression were observed in the pre-pupal and 4-day pupal stages. TmPox-iso1 was primarily expressed in the early and late larval gut, while TmPox-iso2 mRNA expression was higher in the fat bodies and Malpighian tubules. In response to cadmium chloride treatment, TmPox-iso1 expression increased at 3 hours and then declined until 24 hours, while in the zinc chloride-treated group, TmPox-iso1 expression peaked 24 hours after the treatment. Both treated groups showed increases in TmPox-iso2 expression 24 hours after the treatments.

Chemical composition and toxicity of commercial Mentha spicata and Eucalyptus citriodora essential oils on Culex quinquefasciatus and non-target insects.

Current vector control strategies based on synthetic chemicals are not eco-friendly against non-target organisms; hence, alternative approaches are highly required. Commercially purchased oil of Mentha spicata (Spearmint) and Eucalyptus citriodora (Citriodora) were examined against the medical pest Cx. quinquefasciatus (Say) and their non-toxicity on the aquatic species was evaluated. Chemical screening with gas chromatography coupled with mass spectrometry (GC-MS) analysis revealed a total of 14 and 11 compounds in Citriodora and Spearmint oils, respectively, with the highest peak (%) at carvone (70.44%) and isopulegol (30.4%). The larvicidal activity on the fourth instar larvae of Cx. quinquefasciatus showed dose-dependent mortality and significance at a 100 ppm concentration 48 h post-treatment with Citriodora (76.4%, P ≤ 0.001) and Spearmint (100%, P ≤ 0.001). Additionally, the photomicrograph of the fourth instar larvae revealed significant physical abnormalities in the head and midgut tissues post-exposure to Spearmint and Citriodora oils. Moreover, the histological assay revealed severe damage in the epithelial cells and gut lumen 2 to 24 h post-treatment. The repellency percentage of adult Culex mosquitoes was prominent across both oils at 150 ppm 210 min post-exposure. Non-target toxicity on the aquatic predator showed both essential oils (Spearmint oil (17.2%) and Citriodora oil (15.2%)) are safer at the maximum treatment (200 ppm) compared to temephos (75.4% at 1 ppm). The in silico screening of phyto-compounds derived by both essential oils with BeeTox (online server) showed no contact toxicity to the honey bee Apis mellifera. Overall, the present research revealed that Spearmint and Citriodora essential oils and their active phyto-compounds were toxic to Cx. quinquefasciatus and harmless to the aquatic predator and honey bee.

Effects of TmTak1 silencing on AMP production as an Imd pathway component in Tenebrio molitor.

Mealworms beetles, Tenebrio molitor, are the limelight next-generation food for humans due to their high nutrient contents. Since Tenebrio molitor is used as feed for pets and livestock in addition to their ability to decompose polystyrene and plastic waste, it is recognized as an insect with an industrial core value. Therefore, it is important to study the immune mechanism related to the development and infection of mealworms for mass breeding purposes. The immune deficiency (Imd) signaling is one of the main pathways with pivotal roles in the production of antimicrobial peptides (AMPs). Transforming growth factor-ß activated kinase (TAK1) is one of the Imd pathway components, forms a complex with TAK1 binding protein 2 (TAB2) to ultimately help activate the transcription factor Relish and eventually induce host to produce AMPs. Relatively, little has been revealed about TAK1 in insect models, especially in the T. molitor. Therefore, this study was conducted to elucidate the function of TmTak1 in T. molitor. Our results showed that the highest and lowest mRNA expression of TmTak1 were found in egg and young larvae respectively. The tissue-specific expression patterns were reported in the gut of T. molitor larvae and the fat bodies of adults. Systemic microbial challenge illustrated TmTak1 high expression following the fungal infection in all dissected tissues except for the whole body. However, silencing TmTak1 experiments showed that the survivability of T. molitor larvae affected significantly following Escherichia coli infection. Accordingly, AMP induction after TmTak1 knock down was mainly reported in the integument and the fat bodies.

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.

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.

Innate Immune Response of TmToll-3 Following Systemic Microbial Infection in Tenebrio molitor.

Although Toll-like receptors have been widely identified and functionally characterized in mammalian models and Drosophila, the immunological function of these receptors in other insects remains unclear. Here, we explored the relevant innate immune response of Tenebrio molitor (T. molitor) Toll-3 against Gram-negative bacteria, Gram-positive bacteria, and fungal infections. Our findings indicated that TmToll-3 expression was mainly induced by Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of young T. molitor larvae. Surprisingly, Escherichia coli systemic infection caused mortality after TmToll-3 knockdown via RNA interference (RNAi) injection, which was not observed in the control group. Further analyses indicated that in the absence of TmToll-3, the final effector of the Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated after E. coli challenge. Our results indicated that the expression of almost all AMP genes was suppressed in silenced individuals, whereas the expression of relevant genes was positively regulated after fungal injection. Therefore, this study revealed the immunological involvement of TmToll-3 in T. molitor in response to systematic infections.

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.

Recent trends in insect gut immunity.

The gut is a crucial organ in insect defense against various pathogens and harmful substances in their environment and diet. Distinct insect gut compartments possess unique functionalities contributing to their physiological processes, including immunity. The insect gut's cellular composition is vital for cellular and humoral immunity. The peritrophic membrane, mucus layer, lumen, microvilli, and various gut cells provide essential support for activating and regulating immune defense mechanisms. These components also secrete molecules and enzymes that are imperative in physiological activities. Additionally, the gut microbiota initiates various signaling pathways and produces vitamins and minerals that help maintain gut homeostasis. Distinct immune signaling pathways are activated within the gut when insects ingest pathogens or hazardous materials. The pathway induced depends on the infection or pathogen type; include immune deficiency (imd), Toll, JAK/STAT, Duox-ROS, and JNK/FOXO regulatory pathways. These pathways produce different antimicrobial peptides (AMPs) and maintain gut homeostasis. Furthermore, various signaling mechanisms within gut cells regulate insect gut recovery following infection. Although some questions regarding insect gut immunity in different species require additional study, this review provides insights into the insect gut's structure and composition, commensal microorganism roles in Drosophila melanogaster and Tenebrio molitor life cycles, different signaling pathways involved in gut immune systems, and the insect gut post-infection recovery through various signaling mechanisms.

Immunological Roles of TmToll-2 in Response to Escherichia coli Systemic Infection in Tenebrio molitor.

The antimicrobial roles of Toll-like receptors have been mainly identified in mammalian models and Drosophila. However, its immunological function in other insects has yet to be fully clarified. Here, we determined the innate immune response involvement of TmToll-2 encountering Gram-negative, Gram-positive, and fungal infection. Our data revealed that TmToll-2 expression could be induced by Escherichia coli, Staphylococcus aureus, and Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of Tenebrio molitor young larvae. However, TmToll-2 silencing via RNAi technology revealed that sole E. coli systemic infection caused mortality in the double-strand RNA TmToll-2-injected group compared with that in the control group. Further investigation indicated that in the absence of TmToll-2, the final effector of Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated, mainly E. coli post-insult. We showed that the expression of all AMP genes was suppressed in the main immune organ of insects, namely, fat bodies, in silenced individuals, while the relevant expressions were not affected after fungal infection. Thus, our research revealed the immunological roles of TmToll-2 in different organs of T. molitor in response to pathogenic insults.

First report of alstroemeria mosaic virus infecting Alstroemeria in Korea.

Alstroemeria, a member of the Alstroemriaceae family, is a popular cut flower plant with a long-base life and a wide variety of flower colors. It is widely cultivated in many countries, especially in Central and South America. However, numerous viruses such as alstroemeria carlavirus (AlCV), alstroemeria mosaic virus (AlMV), cucumber mosaic virus (CMV), tomato spotted wilt virus (TSWV), alstroemeria streak virus (AlSV), and impatiens necrotic virus (INSV) can infect Alstroemeria and significantly decrease its yield (Kim, 2020). Among these viruses, AlMV is well known to cause an endemic viral disease in the Netherlands (Corine M. et al. 1992). AlMV is a member of the genus potyvirus in the family Potyviridae, one of the most widely distributed families of plant viruses. In 2021, symptomatic alstroemeria plants showing interveinal leaf streaking with elongated light green and chlorosis of leaves were identified from farms in a greenhouse in Gwangju, South Korea. Potyvirus-like particles (approximately 750-800 nm in length) were observed from sap of the symptomatic plants by electron microscope (Supplementary Fig. 1). To confirm virus infection, total RNA was extracted from an alstroemeria leaf using a Beniprep® Super Plant RNA extraction kit (IVT7005, Invirustech Co., Korea). A cDNA library was synthesized and analyzed by high throughput sequencing (HTS) using an Illumina NovaSeq6000 S4 sequencer. A total of 48,072,240 raw reads were obtained after quality filtering with FastQC. Remaining sequences were de novo assembled into contigs with a Trinity assembler. Nucleotide blast analysis of contigs against NCBI viral reference database revealed that 24 assembled contigs (> 1,000 bp) were sequences of AlMV. To confirm AlMV detection, raw reads were mapped to known AlMV complete genome (9,774 bp) using Bowtie2 program. Results showed that a total of 4,698,112 reads were mapped. A consensus sequence (9,778 bp, accession no. LC709275) was then obtained. To verify the presence of AlMV, RT-PCR assay was conducted with AlMV's CP gene-specific primers: AlMV-F (5'-CACGAGGCTGTGAAACAAGC -3') and AlMV-R (5'- CCAGGCGACACGGCTAAATA-3'). PCR products of the expected size (538 bp) were cloned, sequenced, and subjected to GenBank BLASTn search. A 538 bp partial CP sequence was used for BLAST analysis which revealed that it shared 100% identities with the consensus sequence (LC709275) and 96.99~98.76% nucleotide identities with four AlMV isolates (MK440140, NC043135, MT892648, DQ295032). Phylogenetic analysis based on partial CP sequences of representative members of potyviruses (family Potyviridae) using 1,000 bootstrap replicates based on either neighbor-joining or Kimura 2 parameter methods in MEGA-X revealed that AlMV isolate JNU-2 was grouped together with the four known AlMV isolates (Supplementary Fig. 2). To determine the incidence of AlMV in a greenhouse, 30 alstroemeria samples were collected and tested by RT-PCR. Results showed that 23 samples were positive for AlMV by PCR-gel electrophoresis and Sanger sequencing, suggesting a high incidence of AlMV infection. To the best of our knowledge, this is the first report of natural infection with AlMV in Alstroemeria in Korea. Further surveys of AlMV infection in greenhouses will help us prevent the spread of this viral disease in Alstroemeria.

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.

Development and application of reverse transcription droplet digital PCR assay for sensitive detection of apple scar skin viroid during in vitro propagation of apple plantlets.

Apple scar skin viroid (ASSVd), of the genus Apscaviroid, causes serious pome fruit diseases, such as apple scar skin, dapple apple, pear rusty skin, pear fruit crinkle, and pear dimple fruit. This study aimed at establishing a sensitive and accurate method for quantification of ASSVd in apple leaves and plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity was analyzed using other apple viruses, and the negative amplification of the cross-reaction assay demonstrated the high specificity of RT-ddPCR. The detection limit of ASSVd by RT-ddPCR was 1.75 × 102 copies/µL (0.14 concentration), and the sensitivity was ten-fold higher than that of RT-qPCR. Similarly, positive detection in apple plantlet samples by RT-ddPCR was higher than that by RT-qPCR. The RT-ddPCR assay represents a promising alternative for accurate quantitative detection and diagnosis of ASSVd infection in ASSVd-free certification programs.

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.