The red-spotted apollo butterfly, Parnassius bremeri Bremer, 1864 (Lepidoptera: Papilionidae), has been listed as an endangered species in Korea. We developed microsatellite markers by nest-generation sequencing (NGS), selected 12 markers, and applied the markers to available South Korean populations to understand population genetic characteristics. The genotyping of 40 P. bremeri individuals from three localities in South Korea showed that at each locus, the observed number of alleles ranged from 17 to 43, the observed and expected heterozygosities were 0.84722-0.90556 and 0.76045-0.79208, respectively, and FIS was –0.155 to –0.121. STRUCTURE analysis supported the presence of two genetic pools in all three populations, although an immediate reason for this subdivision is not known. The population based FST, RST, and front wing length collectively suggest that at least Samchuk population in Gangwon Province has a significant distance. Further scrutinized analysis is undergoing.

This study was conducted to investigate exact life cycle and Parnassius bremeri’s response to heat shock stress from 2012 to 2016 in Holoce Ecosystem Conservation Research Institution through captive breeding. About 16 days(15.7±0.3days) later embryo in eggs were developed as pharate first instar and the eggs of Parnassius bremeri possessed the ability to undergo an extended aestivation hosting a fully developed pharate 1st instar larvae within the chorion. Pharate 1st instar in egg hatched from late in November to early in December about 194 days(194.3±1.1days) later. All pharate 1st instar in egg and out of egg survived at 25℃, 35℃. In case of 45℃ survival rate of pharate 1st instar in an egg (93.8±6.3%) was significantly higher than those (22.2±10%) of pharate 1st instar out of an egg. All was dead at 47.5, 50, 52.5 and 55℃.

Egg SCP ( –47.2 ±1.0℃) of Red-spotted Apollo butterfly, Parnassius bremeri, was lower than larva(–35.0 ±0.9℃). The difference (12℃) between egg and larva was revealed by chorion through scanning electron microscope (COXEM EM-30, Korea). But considering of about 6 months’ pharate 1st instar in egg from June to November, function of egg seems not to be overwintering mechanism but rather estivation. In order to identify estivation function of chorion heat stress was carried out. Experiment was conducted on the 4 conditions such as high temperatures of 25, 35, 45 and 55 °C for 2 hour, then allowed them to recover at 25 °C for 1 hour. Total 65 samples 0f 31 eggs and 34 pharate 1st instar out of an egg were used and replicated 3 times. All pharate 1st instar in egg and pharate 1st instar out of egg survived at 25℃ and 35℃. In case of 45℃ survival rate of pharate 1st instar in an egg (91.6±3.5%) was significantly higher than those (37.8±9.4%) of pharate 1st instar out of an egg. All was dead at 55℃.

SCPs on larvae and eggs of red-spotted apollo butterfly, P. brimeri were measured according to the method of Kim and Kim (1977) with a thermocouple, BTM-4208SD (LT Lutron, Taipei, Taiwan), to detect the release of the latent heat of fusion as body water freezed. SCP of larvae during March goes below –27.4±1.7℃ and egg scp during November goes –47.2±1.0℃. In order to identify the reason of the difference(-17℃) between egg and larva we took photographed egg through scanning electron microscope (COXEM EM-30, Korea). Chorion of P. bremeri were 100.1㎛, Papilio machaon and Sericinus montela in same family was 10.8㎛, 5.5㎛ respectively. P. bremeri was 10 times, 20times thicker than another species within the same family .

The red-spotted apollo butterfly, Parnassius bremeri, immatures grow during winter and spring. Supercooling point of larvae during January goes much below -20℃. Morphologically, the larvae appear to be adapted to cold temperatures. Dark-colored body surface is useful to absorb solar energy and spiny integument may prevent any external ice formation on the body surface. Biochemically, P. bremeri larvae elevate glycerol as a cryoprotectant. This study reports two genes associated with glycerol biosynthesis in P. bremeri. Larval transcripts were analyzed using RNA-Seq technique. A total of 14 Gb transcripts were read by Illumina HiSeq and assembled to be 127,279 contigs. To specify the the genes associated with glycerol biosynthesis, a biosynthetic pathway to synthesize glycerol from dihydroxyacetone-3-phosphate was predicted with two genes of glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK). Both genes were annotated in the transcriptome of P. bremeri. Pb-GPDH encodes 166 amino acid residues containing NAD+-binding region, catalytic site, and calcium binding region. The predicted amino acid sequence was clustered with other lepidpopteran GPDH genes. Three Pb-GK genes were annotated from the transcriptome. Pb-GK1 encodes a full open reading frame of 514 amino acid residues. A ohylogenetic analysis showed that these three GKs were separately clustered. Interestingly, Pb-GK1 was clustered with other GKs that were known to be associated with rapid cold hardiness.

The 15,389-bp long complete mitogenome of the endangered red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, which have the gene order of tRNAMet, tRNAIle, and tRNAGln at the beginning. Due to the uncertainty the start codon for COI gene in insect has been discussed extensively. We propose the CGA sequence as the start codon for COI gene in lepidopteran insects, based on complete mitogenome sequences of lepidopteran insects including our P. bremerii and additional sequences of the COI start region from a diverse taxonomic range of lepidopteran species (a total of 51 species belonging to 15 families). As has been suggested in other sequenced lepidopteran insects the 18 bp-long poly-T stretch and the downstream conserved motif ATAGA that were previously suggested to serve as a structural signal for minor-strand mtDNA replication also was found at the 3’-end region of the P. bremerii A+T-rich region. In an extensive search to find out tRNA-like structure in the A+T-rich region, each one tRNATrp-like sequence and tRNALeu (UUR)-like sequence were found in the P. bremeri A+T-rich region, and most of other sequenced lepidopteran insects were shown to have tRNA-like structure within the A+T-rich region, thereby indicating that such feature is frequent in the lepidopteran A+T-rich region. Phylogenetic analysis using the concatenated 13 amino acid sequences and nucleotide sequences of PCGs of the four macrolepidopteran suferfamilies together with Tortricoidea and Pyraloidea well recovered a monophyly of Papilionoidea and a monophyly of Bombycoidea. However, Geometroidea and Noctuoidea were unexpectedly clustered as one group and placed this group to the sister group to Bombycoidea, instead of Papilionoidea in most analyses.

The 15,389-bp long complete mitochondrial genome (mitogenome) of the redspotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, but differs from the most common type, as the result of the movement of tRNAMet to a position 5’-upstream of tRNAIle. No typical start codon of the P. bremeri COI gene is available. Instead, a tetranucleotide, TTAG, which is found at the beginning context of all sequenced lepidopteran insects was tentatively designated as the start codon for P. bremeri COI gene. All protein-coding genes (PCGs), but COII (T) have complete termination codon TAA or TAG. One of the most unusual feature of the P. bremeri mitochondrial genome is the presence of two tRNA-like structures, such as the tRNATrp-like sequence and tRNALeu (UUR)-like sequence with proper anticodon and clover-leaf structures in the 514-bp long A+T-rich region. Furthermore, the A+T-rich region possesses three sequences that have the potential to form stem-and-loop structures, flanked by the conserved sequences, "TA(A)TA" at the 5’ end and "G(A)nT’ at the 3’ end. After more genomic and phylogenetic analyses are performed, further detailed information will be available.