본 연구는 호랑나비의 사육을 위한 기초적인 생육특성을 구명하고자 수행되었으며, 월동형 번데기의 저장기간에 따른 우화율과 산란실의 크기에 따른 산란특성에 대한 연구와 호랑나비 애벌레이 일반적인 생육특성에 대한 조사를 실시하였다. 조사결과 월동형 번데기의 저장기간에 따른 우화율은 4±1℃의 저온저장고에서 약 10개월 저장후에도 85%의 우화율을 보였으며, 저장기간중 주기별 우화실험결과 평균우화율이 89.6%였고, 기간별 우화율에 차이가 없는 것으로 나타났다. 산란실의 크기에 따른 짝짓기 성공률을 조사한 결과 대형 산란실(6,000×6,000×3,500㎜)에서는 86.7±5.8%로 소형 산란실(2,500×3,000×2,000㎜)의 63.3±15.3%비해 짝짓기가 더 잘 이루어지는 것으로 보였다. 반면에 산란실의 크기에 따른 산란수량은 대형 산란실이 137.0±16.5개, 소형 산란실이 141.7±20.4개로 큰 차이가 없었다. 먹이식물에 대한 산란선호도는 황벽나무가 141.7±27.8개로 67.7±20.6개를 산란한 산초나무나 77±21.8개를 산란한 귤나무에 비해 산란선호도가 우수한 것으로 나타났다. 호랑나비 애벌레의 부화율은 92.2%로 나타났으며, 알기간은 4.4±0.8일로 나타났다. 애벌레기간은 총 19.9±2.1일로 나타났으며, 5령기간이 가장 길어 6.9±1.8일이였다. 호랑나비 애벌레의 령별 두폭은 1령 애벌레가 0.72mm±0.02였으며, 2령 1.19±0.02mm, 3령 1.65±0.05mm, 4령 2.43±0.07mm, 5령 3.21±0.12mm로 각각 나타났다. 용화율은 92%로 나타났으며, 번데기기간은 8.8±0.9일이었고, 우화율은 92.8%였다.

The effect of an artificial diet on the developmental rate, a life history parameter, was examined for the swallowtail butterfly, Papilio xuthus. Artificial insect diets are an essential component of many insect rearing systems that produce insects for research purposes. Complex agar-gelled diets are generally prepared in large batches and used shortly after preparation because the degradation of perishable diet ingredients, such as vitamins and fatty acids, can adversely affect insect quality (Brewer F D 1984). However, the timing of diet preparation may be inconvenient and large batches wasteful if the unused, excess diet is discarded. This study showed that if the artificial diet was fed during the larval stage, the larval and pupal developmental periods, the percentage of pupation, and the pupal weight generally did not differ significantly among the five artificial diets evaluated. The percentage of pupation varied considerably, with no significant differences among diets except for diet C. A maximum pupation percentage of 83% was observed on diet C. Pellet-type diets were investigated with the aim of developing a more easily prepared diet. The extrusion of the artificial diet under high temperature and pressure may induce desirable chemical and physical changes in the extruded product. The purpose of the present study was to develop an artificial diet for rearing P. xuthus.

Papilio xuthus is known as the Asian swallowtail, Chinese yellow swallowtail, or as the Xuthus swallowtail. It overwinters in pupae stage. The study was conducted to investigate the temperature effect on adult emergence of overwintering P. xuthus pupae at four different constant temperatures (15, 20, 25 and 30°C) with photoperiod 10:14(L:D). The lower developmental thresholds of female, male and both sexes combined were 9.71, 11.03, and 10.49°C, respectively. Developmental period of overwintering pupae to adults decreased with increasing temperature between 15 and 30°C. Degree-days for adult emergence to female, male and both sexes combined were 227.27, 175.44, and 200.80DD, respectively. The relationship between the developmental rate and temperature was fitted by linear and five nonlinear developmental rate models (Logan 6, Lactin 1, 2 and Briere 1, 2). The variation of overwintering pupae development was described by the two-parameter Weibull distribution model. The adult emergence of overwintering P. xuthus pupae in spring can be explained and predicted by using developmental rate models and developmental distribution model. The effect of climate change on phenology and distribution of P. xuthus may be presented by the model.

Papilio macilentus is the longest tail butterfly of Papilionidae found in Japan, China and Korea and has distinctive morphological differences between male and female. P .macilentus feeds on Zanthoxylum schinifolium and has three generations per year. The lower developmental threshold and thermal constant for adult emergence of P. macilentus overwintering pupae were estimated at four different constant temperatures (15, 20, 25 and 30°C) with photoperiod 10:14 (L:D). The lower developmental threshold of both sexes combined was 7.95°C. The development of overwintering pupae decreased with increasing temperature between 15 and 30°C. Thermal constant of both sexes combined was 251.26DD. The relationship between the developmental rate and temperature was fitted by linear and five nonlinear developmental rate models (Logan 6, Lactin 1, 2 and Briere 1, 2). The development of overwintering P. macilentus pupae was described by the two-parameter Weibull distribution model. The temperature-dependent adult emergence model of P. macilentus developed in this study could be used to predict the full-cycle phenology model of P. macilentus and applied to understand the effects of climate change on the emergence distribution of overwintering pupae.

Attacin is a well-studied glycine-rich antibacterial protein in insect immune response, which has limitary antibacterial effect to some Gram-negative bacteria. A cDNA encoding the attacin gene was screened and isolated from the immunized larvae of the swallowtail butterfly, Papilio xuthus. The complete P. xuthus attacin cDNA is 949 nucleotides encoding a 250 amino acid precursor that contains a putative 18-residue signal peptide, a common 42-residue propeptide sequence and a presumed 190-residue mature protein with a theoretical mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48%~52% and 24%~30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. The attacin transcript was induced at significant level after injection with bacterial lipopolysaccharide (LPS). Recombinant attacin was highly expressed in E. coli BL21 (DE3) cells by fusing with an N-terminal S-tag/thrombin cleavage site configuration protein to avoid the cell death during induction. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC). After desalting and cleavage with thrombin, the recombinant attacin was released and showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35. Our results proved that this protein family with a potent antibacterial activity may play a role in the immune response of butterflies.

Cecropin is an antimicrobial peptide that is synthesized in fat body cells and hemocytes of insect in response to a hypodermic injury or bacterial infection. A 503 bp cDNA encoding a cecropin-like antimicrobial peptide was isolated by employing annealing control primer (ACP)-based differential display PCR and 5'-RACE from immunized Papilio xuthus larvae. The open reading frame (ORF) of isolated cDNA encoded a 63 amino acid prepropeptide with a putative 22-residue signal peptide, a 3-residue propeptide and a 38-residue mature peptide with a theoretical mass of 4060.89 Da. The deduced amino acid sequence of peptide showed significant identities with other Lepidopteran cecropins. This peptide was named as papiliocin. RT-PCR revealed that the papiliocin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). Based on the deduced amino acid sequence of papiliocin, a 38-mer mature peptide was chemically synthesized by Fmoc method, and analyzed antimicrobial activity. The synthetic papiliocin peptide had a broad spectrum of activity against fungi, Gram-positive and negative bacteria, and also showed no hemolytic activity against human red blood cell.

Attacin is an antibacterial protein that is secreted by fat body cells of insect larva in response to bacterial infection. A 949 bp cDNA encoding the antibacterial protein attacin was isolated by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from immunized Papilio xuthus larvae. The attacin cDNAs encoded 250 amino acid residues open reading frame with 60 residues prepropeptide. The deduced amino acid sequence of P. xuthus attacin showed significant identities with other Lepidopteran attacins. The attacin transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The predicted mature attacin was expressed as soluble fusion protein efficiently in bacterial expression system. To increase productivity and solubility, attacin was translationally fused with thioredoxin (Trx) protein and expressed in E. coli cells that are highly sensitive to the mature attacin. The recombinant attacin exhibited antibacterial activity against Gram-negative bacteria.

We have previously shown that the larvae of swallowtail butterfly, Papilio xuthus, exhibit substantial antibacterial activity in the hemolymph, upon challenging with bacterial lipopolysaccharide (LPS). Here we report the isolation and molecular characterization of several immune inducible genes that are specifically expressed by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from P. xuthus the larva. Using 120 arbitrary ACPs, we identified 24 differentially expressed genes (DEGs) that are up-regulated in response to injected LPS. Sequence analysis showed that 18 DEGs revelaed a high sequence similarity to the previously characterized genes of other insects, although 6 DEGs showed no significant similarity to any known genes. Among these inducible transcripts we found 8 putative immune-related genes including cecropin and attacin. Finally, we analysed the expression profiles of potential immune-related genes by RT-PCR and found all of them were considerably increased in the mRNA levels by LPS injection.