애기뿔소똥구리 (Copris tripartitus)는 배설물 분해를 통해 환경정화와 생태계 균형에 기여하는 것으로 알려져 있다. 그러나 무분별한 농약 사용과 서식지 파괴로 인해 개체수가 감소되고 있어 2017년 환경부에서 애기뿔소똥 구리를 멸종위기 야생생물 Ⅱ급으로 지정하였다. 애기뿔소똥구리의 미토콘드리아 유전자를 활용한 선행연구가 발표되었지만 유전자원 확보는 여전히 미비한 실정이다. 이번 연구는 애기뿔소똥구리의 성장, 면역 및 생식과 관련된 유전 정보를 확보하기 위해 Illumina HiSeq 4000 platform을 활용하여 전사체 분석을 실시하였다. Illumina HiSeq 4000을 통해 확보된 전사체 데이터를 Trinity 프로 그램을 통해 de novo assembly를 진행하여 contigs를 생성하였다. 생성된 contigs를 TGICL 프로그램을 통해 clustering 하여 unigenes을 확보하였다. 이후 확보된 unigenes는 PANM DB 및 Swiss-Prot, KOG, InterProScan, GO, KEGG를 기반으로 한 BLASTx를 사용하여 annotation을 진행하였다. 25,106개의 unigene 중에서 23,289개가 PANM DB에 annotation 되었으며, Swiss-Prot, InterProScan에서는 각각 19,660개, 13,545개의 unigene이 annotation 되었다. KOG 분석에서는 ‘general function prediction only’ 범주에서 높은 비율로 나타났으며, GO 분석에서는 ‘Molecular Function’ 카테고리에서 가장 많이 annotation 되었다. KEGG 분석을 통해서는 ‘Environmental information processing’ 항목이 높은 발현을 보였다. 이번 연구를 통해 확보된 기능적 데이터는 야생에서의 보존 계획을 수립하는 데 있어 기초 자료로써 활용될 수 있을 것으로 사료된다.

The dung beetle, Copris tripartitus, has been listed as an endangered insect in Korea. In contrast to typical genomic characteristics, the 15,457-bp long, the complete mitochondrial genome (mitogenome) of C. tripartitus has a few unusual characteristics. The genome has the trnP-trnT arrangement at the ND4L-ND6 junction, which is found only in two species of Scarabaeoidea, including C. tripartitus, in 53 available species. Twelve protein coding genes (PCGs) have typical ATT and ATG start codons, whereas COII had the start codon GTA, which is unprecedented in Scarabaeidae. The 297-bp A + T-rich region, which is the shortest available in Scarabaeidae, harbors three repeat units (26, 29, and 21 bp), each consisting of duplicated identical sequences interspersed in the A + T-rich region. Phylogenetic analyses were performed using the concatenated sequences of the 13 PCGs and two rRNA genes using both the Bayesian inference (BI) and maximum likelihood (ML) methods for the families Scarabaeidae and Lucanidae that are available for their mitogenomes in Scarabaeoidea. Our consensus phylogeny yielded monophylies of each family and the subfamily Scarabaeinae in Scarabaeidae with the high nodal supports (0.99-1 by BI and 79-100 by ML). Among the three tribes, represented by multiple genera in Scarabaeinae each Oniticellini and Onitini was supported as monophyletic groups with the highest nodal supports (1 by BI and 100 by ML), whereas Onthophagini was non-monophyletic group, presenting the tribe relationships: ((((Oniticellini + Onthophagini [Caccobius, Milichus, Onthophagus]) + Onthophagini [Digitonthophagus, Phalops]) + Onitini) + (Phanaeini + Eurysternini) + Coprini).

Previously, a partial mitochondrial COI and CytB gene sequences have been used to infer genetic diversity and gene flow of the species. In this study, we additionally collected C. tripartitus from one previous and two new localities, sequenced the COI and CytB genes. Sequence divergence of current samples showed slightly lower values, but nearly equivalent to previous study. Haplotype diversity was still high and nucleotide diversity indicating that Seogwi-dong showed the highest estimates in both gene sequences. Gene flow among populations is high, but a significance difference was detected between Gulupdo and Anmado; and between Gulupdo and Seogwi-dong only in COI sequences (P < 0.05). Considering the high genetic diversity and gene flow in C. tripartitus populations, one of the major issues regarding conservation seems not to be the recovery of genetic diversity as has been suggested before.

Phoretic mites were collected from Copris ochus and C. tripartitus (Coleoptera: Scarabaeidae). These beetles were collected from Holoce Ecosystem Conservation Research Institution, Gangwon-do. Five unrecorded species of Eviphis hastatellus Berlese, 1910 (Eviphididae), Holostaspella scatophila Takaku, 1994 (Macrochelidae), Macrocheles japonicus Evans & Hyatt, 1963 (Macrochelidae), Pachylaelaps siculus Berlese, 1921 (Pachylaelapidae) and Parasitus consanguineus Oudemans & Voigts, 1904 (Parasitidae) were recorded from Korea for the first time. However, Parasitus consanguineus Oudemans & Voigts (Parasitidae) was already recorded by Oudemans & Voigts in 1904 but since then no one has recorded. We have rerecorded this species in Korea.

The dung beetle, Copris tripartitus, (Coleoptera: Scarabaeidae), is one of the insect species listed as endangered wildlife in Korea. In order to establish conservation strategy an investigation on nation-wide genetic magnitude and nature of genetic diversity would be required. In this study, we sequenced each partial sequences of mitochondrial COI (658 bp) and CytB (433 bop) genes and complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (411~420 bp) from 70 individuals of C. tripartitus collected from five Korean localities. The sequence divergence of mitochondrial genes was unexpectedly substantial as 5.0% (33 bp) for COI and 4.6% (20 bp) for CytB, whereas that of ITS2 was only 1.9% (6 bp), revealing much higher variability in mitochondrial DNA. In phylogenetic analysis, each 57 and 47 haplotypes obtained from COI and CytB gene sequences was subdivided into two groups (groups A and B), but the node supports for each group was not strong enough to consider each group as independent monophyletic groups in both genes. This interpretation was further supported by the same analysis with ITS2, showing no subdivision at all. These findings reinforce importance for the inference of phylogenetic result using both mitochondrial and nuclear encoded sequences. The C. tripartitus occurring in Korean peninsula was genetically well connected to each other and no obvious population genetic structure was detectable.

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3'rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

To find some antibacterial peptides responsible for bacterial resistance, we performed differential hybridization with total cDNA probes which synthesized from normal and immunized larvae. Thirteen individual cDNA transcripts were expressed differentially in a total 1,862 random cDNA clones. One of upregulated genes is a novel member of the insect defensin-like peptide(Coprisin), a family of antibacterial peptide. Northern blot analysis showed that Coprisin was up-regulated at 4h and reached the highest point level at 16h after injection of E.coli. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with predicted molecular weight of 8.6 kDa and PI of 8.72. Comparison of the deduced amino acid mature portion of Coprisin with defensin-like peptide of other insect indicated that it has 79.1% and 67.4% identity with Anomala cuprea and Allomyrina dichotoma, respectively. To find antibacterial active region of Coprisin, we synthesized four peptides corresponding to amino acid residues 1V-43N-NH2(CopN1), 5-16(CopN2), 19-30(CopN3) and 31-43(CopN4) of coprisin having amidated amino acid residues at their Cterminal. A 12-mer amidated at its C-terminus, ACALHCIALRKK-NH2 (Ala19-Lys30-NH2) was synthesized based on the deduced amino acid sequence, assumed to be an active site sequence. This peptides showed antibacterial activity against E.coli, Staphylococcus aureus, MRSA, Psedomonas syringae, and Pectobacterium carotovorium. Modified 9-mer peptide, LRCIALRKK-NH2, showed strong antibacterial activity than mellitin peptide used as a positive control against gram-negative and gram-positive bacteria. This peptide showed no haemolytic activity and quite stable at 100℃ for several hours of incubation and in a wide pH range(pH2-12). Therefore, this peptide may be a good candidate for the development of new drug with potent antibacterial activity without cytotoxicity.

Copris tripartitus Waterhouse is a coprophagus insect which enhances the soil fertility as conveying dung into the soil. We compared preimaginal development, mortality, and size of C. tripartitus at various temperature condition (15, 17.5, 20, 25, 27.5, 30). Total preimaginal periods ranged from 118.0d at 17.5℃ to 49.3d at 27.5℃. Development threshold temperature (DT) and total effective temperature for the development of egg, larva, pupa and for complete development (egg to emergence) were 12.1, 11.2, 12.1 and 9.2℃ and 82.7, 462.0, 225.7 and 947.2 degree days, respectively. The mortality of egg and larva was the lowest at 25℃. The optimum temperature for hatchability and on the development of C. tripartitus was estimated to the 25℃.