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

Patnaik Hongray Howrelia,Sang Min Kyu,Park Jie Eun,Song Dae Kwon,Jeong Jun Yang,Hong Chan Eui,Kim Yong Tae,Shin Hyeon Jun,Ziwei Liu,Hwang Hee Ju,Park So Young,Kang Se Won,Ko Jung Ho,Lee Jun Sang,Park 한국유전학회 2023 Genes & Genomics Vol.45 No.8

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.

Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, <i>Tenebrio molitor</i>

Patnaik, Bharat Bhusan,Kang, Seong Min,Seo, Gi Won,Lee, Hyo Jeong,Patnaik, Hongray Howrelia,Jo, Yong Hun,Tindwa, Hamisi,Lee, Yong Seok,Lee, Bok Luel,Kim, Nam Jung,Bang, In Seok,Han, Yeon Soo Molecular Diversity Preservation International (MD 2013 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.14 No.10

<P>CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, <I>Tenebrio molitor.</I> The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. <I>In silico</I> analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to <I>T. molitor</I> immune response against various microbial pathogens.</P>

Silencing of apolipophorin‐III causes abnormal adult morphological phenotype and susceptibility to Listeria monocytogenes infection in Tenebrio molitor

Bharat Bhusan Patnaik,Hongray Howrelia PATNAIK,박기범,조용훈,이용석,한연수 한국곤충학회 2015 Entomological Research Vol.45 No.2

Insect apolipophorin‐III is an exchangeable protein that is abundantly found in the hemolymph, and serves an important role in lipid transport, development, and innate immunity. In this study, we examined the role of apolipophorin‐III (TmapoLp‐III) during the adult eclosion stages of Tenebrio molitor by RNA interference (RNAi) analysis. After silencing of the mRNA transcripts at both larval and pupal stages, adult phenotypic defects were noticed. Defects included the incomplete shedding of pupal skin, shorter extension of the elytra, and improper folding of the hind wings. Most of the adults were malformed and died possibly due to dehydration. We also showed the involvement of TmapoLp‐III in conferring resistance to T. molitor larvae against Listeria monocytogenes infection. Mortality was found to be lower in non‐silenced intoxicated larvae while the TmapoLp‐III silenced larvae showed a significant susceptibility after 7 days post‐injection with a dose of 106 cfu/larvae.

Molecular cloning, sequence characterization and expression analysis of a CD63 homologue from the coleopteran beetle, Tenebrio molitor

Bharat Bhusan Patnaik,Seong Min Kang,Gi Won Seo,Hyo Jeong Lee,Hongray Howrelia Patnaik,Yong Hun Jo,Hamisi Tindwa,Yong Seok Lee,Bok Luel Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

CD63, a member of tetraspanin membrane protein family, plays pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic ‘Cys-Cys-Gly’ motif and ‘Cys188’ residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcript was upregulated the maximum 4.5 fold in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also made significant increase in the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Transcriptome sequencing and de novo characterization of Korean endemic land snail, Koreanohadra kurodana for functional transcripts and SSR markers

Kang, Se Won,Patnaik, Bharat Bhusan,Hwang, Hee-Ju,Park, So Young,Chung, Jong Min,Song, Dae Kwon,Patnaik, Hongray Howrelia,Lee, Jae Bong,Kim, Changmu,Kim, Soonok,Park, Hong Seog,Han, Yeon Soo,Lee, Jun Springer-Verlag 2016 Molecular genetics and genomics Vol.291 No.5

<P>The Korean endemic land snail Koreanohadra kurodana (Gastropoda: Bradybaenidae) found in humid areas of broadleaf forests and shrubs have been considered vulnerable as the number of individuals are declining in recent years. The species is poorly characterized at the genomic level that limits the understanding of functions at the molecular and genetics level. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset of visceral mass tissue of K. kurodana by the Illumina paired-end sequencing technology. Over 234 million quality reads were assembled to a total of 315,924 contigs and 191,071 unigenes, with an average and N50 length of 585.6 and 715 bp and 678 and 927 bp, respectively. Overall, 36.32 % of the unigenes found matches to known protein/nucleotide sequences in the public databases. The direction of the unigenes to functional categories was determined using COG, GO, KEGG, and InterProScan protein domain search. The GO analysis search resulted in 22,967 unigenes (12.02 %) being categorized into 40 functional groups. The KEGG annotation revealed that metabolism pathway genes were enriched. The most prominent protein motifs include the zinc finger, ribonuclease H, reverse transcriptase, and ankyrin repeat domains. The simple sequence repeats (SSRs) identified from > 1 kb length of unigenes show a dominancy of dinucleotide repeat motifs followed with tri- and tetranucleotide motifs. A number of unigenes were putatively assessed to belong to adaptation and defense mechanisms including heat shock proteins 70, Toll-like receptor 4, AMP-activated protein kinase, aquaporin-2, etc. Our data provide a rich source for the identification and functional characterization of new genes and candidate polymorphic SSR markers in K. kurodana. The availability of transcriptome information would promote the utilization of the resources for phylogenetics study and genetic diversity assessment.</P>

Current trends in large‐scale viral surveillance methods in mosquitoes

Park Ki Beom,Patnaik Hongray Howrelia,Kim Tae‐Yun,Jo Yong Hun,Kim Nam‐Yeon,Yang Sung‐Chan,Lee Wook‐Gyo,Lee Hee‐Il,Cho Shin‐Hyeong,Han Yeon Soo 한국곤충학회 2020 Entomological Research Vol.50 No.6

Vector-borne and zoonotic infectious diseases are serious public health concerns that affect approximately half of the world’s population. In particular, arthropod-borne viruses (arboviruses) have contributed to more mortality and morbidity worldwide with the emergence of dengue, chikungunya, yellow fever, and Zika virus diseases. The infections have scaled up due to urbanization, globalization, and international mobility. Traditionally, the spread of mosquito-borne viral diseases to humans was considered a low health priority concern. However, their categorization as emerging infectious diseases and public health emergencies of international concern has heightened the attention given by the government, academia, research, and industry for the development of timely, cost-efficient, and sustainable solutions. The urgency has increased in the wake of global climate change. The focus on effective interventions includes epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants. In this review, we discuss the etiology and predisposition of mosquito-borne viruses that are detrimental to public health and economically damaging when disseminated as epidemics. We focus on the large-scale virus surveillance methods with special reference to innovations and interventions in molecular detection science and technologies that include viral nucleic acid isolation, polymerase chain reaction (PCR)-based diagnostics, and high-throughput sequencing technologies. In addition, we discuss the development of a viral RNA extraction and PCR-based diagnostic kit (Invirustech) that can extract viral RNA from mosquitoes with verified applications in PCR-based molecular diagnostics of Pan-flavivirus.

<i>De novo</i> Transcriptome Generation and Annotation for Two Korean Endemic Land Snails, <i>Aegista chejuensis</i> and <i>Aegista quelpartensis</i> , Using Illumina Paired-End Sequencing Technology

Kang, Se Won,Patnaik, Bharat Bhusan,Hwang, Hee-Ju,Park, So Young,Wang, Tae Hun,Park, Eun Bi,Chung, Jong Min,Song, Dae Kwon,Patnaik, Hongray Howrelia,Lee, Jae Bong,Kim, Changmu,Kim, Soonok,Park, Hong S MDPI 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.3

<P><I>Aegista chejuensis</I> and <I>Aegista quelpartensis</I> (Family-Bradybaenidae) are endemic to Korea, and are considered vulnerable due to declines in their population. The limited genetic resources for these species restricts the ability to prioritize conservation efforts. We sequenced the transcriptomes of these species using Illumina paired-end technology. Approximately 257 and 240 million reads were obtained and assembled into 198,531 and 230,497 unigenes for <I>A. chejuensis</I> and <I>A. quelpartensis</I>, respectively. The average and N50 unigene lengths were 735.4 and 1073 bp, respectively, for <I>A. chejuensis</I>, and 705.6 and 1001 bp, respectively, for <I>A. quelpartensis</I>. In total, 68,484 (34.5%) and 77,745 (33.73%) unigenes for <I>A. chejuensis</I> and <I>A. quelpartensis</I>, respectively, were annotated to databases. Gene Ontology terms were assigned to 23,778 (11.98%) and 26,396 (11.45) unigenes, for <I>A. chejuensis</I> and <I>A. quelpartensis</I>, respectively, while 5050 and 5838 unigenes were mapped to 117 and 124 pathways in the Kyoto Encyclopedia of Genes and Genomes database. In addition, we identified and annotated 9542 and 10,395 putative simple sequence repeats (SSRs) in unigenes from <I>A. chejuensis</I> and <I>A. quelpartensis</I>, respectively. We designed a list of PCR primers flanking the putative SSR regions. These microsatellites may be utilized for future phylogenetics and conservation initiatives.</P>

RNA sequencing, de novo assembly, and functional annotation of an endangered Nymphalid butterfly, Fabriciana nerippe Felder, 1862

황희주,Bharat Bhusan Patnaik,강세원,박소영,왕태훈,박은비,정종민,송대권,Hongray Howrelia PATNAIK,김창무,김순옥,이재봉,정헌천,박홍석,한연수,이용석 한국곤충학회 2016 Entomological Research Vol.46 No.2

Grassland butterflies are considered representative indicators of biodiversity and ecosystem health. Their dramatic decline caused by habitat destruction, intensifying agriculture and global warming has prompted concern for conservation. While ecological indices of butterflies have been documented, there is a lack of genomic resources relative to the biological importance of this group. Here, we report, first whole‐transcriptomic resource for Fabriciana nerippe, a brush‐footed butterfly member that is endangered in Korea. Approximately, 241.3 million clean reads were obtained from paired‐end Illumina sequencing of adult whole‐body RNA. The de novo assembly resulted in 114 405 unigenes with length ranging from 133 to 33 218 bp. We found 41 868 assembled unigenes homologous to sequences in locally curated PANM‐DB (Protostome DB). We assigned gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology terms to 18 085 and 5779 unigenes, respectively. InterProScan predicted 6691 protein domains for the assembled unigene sequences, with the reverse transcriptase and zinc finger domains found to be the most predominant. A total of 315 282 simple sequence repeats (SSRs) were identified from the assembled unigenes, when considering dinucleotide to octanucleotide repeats with a minimum of three repeat units. AT/AT, AAT/ATT, and AAAT/ATTT represented the most prominent SSR repeat types in the transcriptome. The unigene annotation profile and microsatellites generated in the study can serve as a reference resource for closely related species and gene functional analysis studies. Our data can be utilized to study ecological drift and loss of the species, consequently leading to better protection of the population.

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes

Ju Young Noh,Bharat Bhusan Patnaik,Hamisi Tindwa,Gi Won Seo,Dong Hyun Kim,Hongray Howrelia Patnaik,Yong Hun Jo,Yong Seok Lee,Bok Ruel Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

PANM DB ver 5.0 : An update of the PANM database for invertebrate NGS data analysis

정준양,박지은,송대권,홍찬의,김용태,신현준,류자미,상민규,패트나익 (Patnaik, Bharat Bhusan),패트나익 (Patnaik, Hongray Howrelia),강세원,박소영,이준상,한연수,박홍석,이용석 한국패류학회 2022 The Korean Journal of Malacology Vol.38 No.3

The PANM database (Protostome DB) is a public database platform established in 2015 for the efficient annotation of next-generation sequencing (NGS) data arising from genome sequencing projects involving the Invertebrates (include Arthropoda, Mollusca, and Nematoda). The database has been updated on a routine basis to further increase the accuracy and speed of NGS data annotation. In PANM DB version 5.0 release a total of 21,276,123 protein sequences belonging to the Protostomes have been made available (registered sequences of NCBI). The PANM DB version 5.0 contains about 4% of the total NCBI-nr data shortening the time for overall annotation of large-scale NGS data of the Protostomes. PANM DB version 5.0 can be downloaded for free from http://panm.sch.ac.kr/ for annotation based on local BLAST analysis.