Mi Young Noh

Apolipophorin-III Mediates Antiplasmodial Epithelial Responses in Anopheles gambiae (G3) Mosquitoes

Background Apolipophorin-III (ApoLp-III) is known to play an important role in lipid transport and innate immunity in lepidopteran insects. However, there is no evidence of involvement of ApoLp-IIIs in the immune responses of dipteran insects such as Drosophila and mosquitoes. Methodology/Principal Findings We report the molecular and functional characterization of An. gambiae apolipophorin-III (AgApoLp-III). Mosquito ApoLp-IIIs have diverged extensively from those of lepidopteran insects; however, the predicted tertiary structure of AgApoLp-III is similar to that of Manduca sexta (tobacco hornworm). We found that AgApoLp-III mRNA expression is strongly induced in the midgut of An. gambiae (G3 strain) mosquitoes in response to Plasmodium berghei infection. Furthermore, immunofluorescence stainings revealed that high levels of AgApoLp-III protein accumulate in the cytoplasm of Plasmodium-invaded cells and AgApoLp-III silencing increases the intensity of P. berghei infection by five fold. Conclusion There are broad differences in the midgut epithelial responses to Plasmodium invasion between An. gambiae strains. In the G3 strain of An. gambiae AgApoLp-III participates in midgut epithelial defense responses that limit Plasmodium infection.

Tribolium castaneum RR-1 Cuticular Protein TcCPR4 Is Required for Formation of Pore Canals in Rigid Cuticle

Insect cuticle is composed mainly of structural proteins and the polysaccharide chitin. The CPR family is the largest family of cuticle proteins (CPs), which can be further divided into three subgroups based on the presence of one of the three presumptive chitin-binding sequence motifs denoted as Rebers-Riddiford (R&R) consensus sequence motifs RR-1, RR-2 and RR-3. The TcCPR27 protein containing the RR-2 motif is one of the most abundant CPs present both in the horizontal laminae and in vertical pore canals in the procuticle of rigid cuticle found in the elytron of the red flour beetle, Tribolium castaneum. Depletion of TcCPR27 by RNA interference (RNAi) causes both unorganized laminae and pore canals, resulting in malformation and weakening of the elytron. In this study, we investigated the function(s) of another CP, TcCPR4, which contains the RR-1 motif and is easily extractable from elytra after RNAi to deplete the level of TcCPR27. Transcript levels of the TcCPR4 gene are dramatically increased in 3 d-old pupae when adult cuticle synthesis begins. Immunohistochemical studies revealed that TcCPR4 protein is present in the rigid cuticles of the dorsal elytron, ventral abdomen and leg but not in the flexible cuticles of the hindwing and dorsal abdomen of adult T. castaneum. Immunogold labeling and transmission electron microscopic analyses revealed that TcCPR4 is predominantly localized in pore canals and regions around the apical plasma membrane protrusions into the procuticle of rigid adult cuticles. RNAi for TcCPR4 resulted in an abnormal shape of the pore canals with amorphous pore canal fibers (PCFs) in their lumen. These results support the hypothesis that TcCPR4 is required for achieving proper morphology of the vertical pore canals and PCFs that contribute to the assembly of a cuticle that is both lightweight and rigid.

Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt

In the insect cuticle, structural proteins (CPs) and the polysaccharide chitin are the major components. It has been hypothesized that CPs are cross-linked to other CPs and possibly to chitin by quinones or quinone methides produced by the laccase2-mediated oxidation of N-acylcatechols. In this study we investigated functions of TcCP30, the third most abundant CP in protein extracts of elytra (wing covers) from Tribolium castaneum adults. The mature TcCP30 protein has a low complexity and highly polar amino acid sequence. TcCP30 is localized with chitin in horizontal laminae and vertically oriented columnar structures in rigid cuticles, but not in soft and membranous cuticles. Immunoblot analysis revealed that TcCP30 undergoes laccase2-mediated cross-linking during cuticle maturation in vivo, a process confirmed in vitro using recombinant rTcCP30. We identified TcCPR27 and TcCPR18, the two most abundant proteins in the elytra, as putative cross-linking partners of TcCP30. RNAi for the TcCP30 gene had no effect on larval and pupal growth and development. However, during adult eclosion, ~70% of the adults were unable to shed their exuvium and died. These results support the hypothesis that TcCP30 plays an integral role as a cross-linked structural protein in the formation of lightweight rigid cuticle of the beetle.

Loss of function of the yellow-e gene causes dehydration-induced mortality of adult Tribolium castaneum.

Yellow protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway that significantly accelerates pigmentation reactions in insects. Recent studies have suggested that the insect yellow genes represent a rapidly evolving gene family generating functionally diverse paralogs, but the exact physiological functions of several yellow genes are still not understood. To study the function(s) of one of the yellow genes, yellow-e (TcY-e), in the red flour beetle, Tribolium castaneum, we performed real-time PCR to analyze its developmental and tissue-specific expression, and utilized immunohistochemistry to identify the localization of the TcY-e protein in adult cuticle. Injection of double-stranded RNA for TcY-e (dsTcY-e) into late instar larvae had no effect on larval-pupal molting or pupal development. The pupal cuticle, including that lining the setae, gin traps and urogomphi, underwent normal tanning. Adult cuticle tanning including that of the head, mandibles and legs viewed through the translucent pupal cuticle was initiated on schedule (pupal days 4-5), indicating that TcY-e is not required for pupal or pharate adult cuticle pigmentation in T. castaneum. The subsequent pupal-adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (~25%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became highly desiccated. Interestingly, both the failure of the pupal-adult molt and desiccation-induced mortality were prevented by maintaining the dsTcY-e-treated insects at 100% relative humidity (rh). However, when the high humidity-rescued adults were removed from 100% rh and transferred to 50% rh, they rapidly dehydrated and died, whereas untreated beetles thrived throughout development at 50% rh. We also observed that the body color of the high humidity-rescued dsTcY-e-adults was slightly darker than that of control animals. These results support the hypothesis that TcY-e has a role not only in normal body pigmentation in T. castaneum adults but also has a vital waterproofing function.

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and β-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.

Cuticle formation and pigmentation in beetles

Adult beetles (Coleoptera) are covered primarily by a hard exoskeleton or cuticle. For example, the beetle elytron is a cuticle-rich highly modified forewing structure that shields the underlying hindwing and dorsal body surface from a variety of harmful environmental factors by acting as an armor plate. The elytron comes in a variety of colors and shapes depending on the coleopteran species. As in many other insect species, the cuticular tanning pathway begins with tyrosine and is responsible for production of a variety of melanin-like and other types of pigments. Tanning metabolism involves quinones and quinone methides, which also act as protein cross-linking agents for cuticle sclerotization. Electron microscopic analyses of rigid cuticles of the red flour beetle, Tribolium castaneum, have revealed not only numerous horizontal chitin-protein laminae but also vertically oriented columnar structures called pore canal fibers. This structural architecture together with tyrosine metabolism for cuticle tanning is likely to contribute to the rigidity and coloration of the beetle exoskeleton.

RNAi‐based functional genomics in Tribolium castaneum and possible application for controlling insect pests

RNA interference or RNAi has been observed in eukaryotes and plays a critical role not only in messenger RNA degradation but also in fine‐tuning of gene activation, transcription and translation. In insects, double‐stranded RNA (dsRNA)‐mediated gene silencing is a simple and powerful reverse genetics tool for analyzing gene functions in both model and non‐model organisms. The red flour beetle, Tribolium castaneum, is an excellent genetic model for pest insect species. Since the completion of the genome sequence of T. castaneum, numerous studies of gene function have been reported. Here we review recent RNAi‐based functional genomics research and systemic RNAi in T. castaneum, and we discuss potential applications of RNAi technology to pest insect control.

Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model.

Transcriptome Analysis of the Tadpole Shrimp (Triops longicaudatus) by Illumina Paired-End Sequencing: Assembly, Annotation, and Marker Discovery

The tadpole shrimp (Triops longicaudatus) is an aquatic crustacean that helps control pest populations. It inhabits freshwater ponds and pools and has been described as a living fossil. T. longicaudatus was officially declared an endangered species South Korea in 2005; however, through subsequent protection and conservation management, it was removed from the endangered species list in 2012. The limited number of available genetic resources on T. longicaudatus makes it difficult to obtain valuable genetic information for marker-aided selection programs. In this study, whole-transcriptome sequencing of T. longicaudatus generated 39.74 GB of clean data and a total of 269,822 contigs using the Illumina HiSeq 2500 platform. After clustering, a total of 208,813 unigenes with an N50 length of 1089 bp were generated. A total of 95,105 unigenes were successfully annotated against Protostome (PANM), Unigene, Eukaryotic Orthologous Groups (KOG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using BLASTX with a cut-off of 1E−5. A total of 57,731 unigenes were assigned to GO terms, and 7247 unigenes were mapped to 129 KEGG pathways. Furthermore, 1595 simple sequence repeats (SSRs) were detected from the unigenes with 1387 potential SSR markers. This is the first report of high-throughput transcriptome analysis of T. longicaudatus, and it provides valuable insights for genetic research and molecular-assisted breeding of this important species.

Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus

The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.