간행물

한국응용곤충학회 학술대회논문집

권호리스트/논문검색
이 간행물 논문 검색

권호

2010 한국응용곤충학회 정기총회 및 춘계학술발표회 (2010년 5월) 225

21.
2010.05 구독 인증기관·개인회원 무료
Sodium-potassium-chloride co-transporter (NKCC) is a membrane bound channel protein that plays a prominent role in a variety of epithelial absorptive, secretory processes and a direct role in cell volume regulation, in which NKCC transports sodium, potassium, and chloride ions across the cell membrane. It has been known that prostaglandin E2 (PGE2) induces an acute cell lysis of specific hemocyte type, oenocytoid, to release prophenoloxidase into the plasma and ouabain (a specific sodium pump inhibitor) inhibits the oenocytoid cell lysis resulting in preventing phenoloxidase activation. However, it is not clear how the intracellular signaling pathway leads to oenocytoid cell lysis in response to PGE2. This study was designed to analyze functional role of NKCC in the cell lysis to release prophenoloxidase. A gene structure of NKCC was derived from cDNA library of Spodoptera exigua hemocyte, NKCC was expressed in all developmental stages and tissues. A real time quantitative RT-PCR showed that bacterial challenge significantly induced its expression. Specific inhibitors of NKCC, bumetanide and chlorothiazide, clearly prevented the cell lysis in a dose dependent manner. When RNA interference using double stranded RNA (dsRNA) specific to NKCC suppressed its expression, the oenocytoid lysis and PO activation was significantly inhibited in response to PGE2. It also reduced nodule formation to bacterial challenge. These results indicate that NKCC is associated with oenocytoid cell lysis probably by increasing cell volume through inward transport of ions in response to PGE2.
22.
2010.05 구독 인증기관·개인회원 무료
A polydnavirus, Cotesia plutella bracovirus (CpBV), encodes host translation inhibitory factors (HTIFs). Two HTIFs have been identified and named as CpBV15α and CpBV15β. HTIFs specifically inhibit host gene expression at post transcriptional level. This suggests that HTIFs can discriminate host mRNAs for their inhibition. To test this hypothesis, we chose a storage protein 1 (SP1) for a target and CpBV15β for non-target genes. Both target and non-target genes were constructed in a cloning vector by cloning their 5'UTR+ORF and in vitro transcribed. The capped partial mRNAs were in vitro translated using rabbit reticulocyte lysate in the presence or absence of HTIFs. Translation of SP1 mRNA was significantly inhibited by the HTIF, but those of CpBV15α and CpBV15β were not. We prepared chimeric mRNAs by exchanging 5'UTR and ORF of both target and non-target genes. Inhibitory activity of HTIFs was dependent on the 5'UTR structure. Target 5'UTR had more complicated secondary structure than non-target 5'UTR in terms of free energy required for unwinding. To identify the target molecule of HTIF to discriminate 5'UTR, an immunoprecipitation using HTIF antiserum was conducted. HTIF was co-precipitated with eIF4A that would be required for unwinding the secondary structure of mRNA. These results suggest that HTIF of CpBV can sequester host eIF4A, any mRNAs possessing complicated secondary structures in their 5'UTRs would be difficult to recruit initiation complex for efficient translation.
23.
2010.05 구독 인증기관·개인회원 무료
국내 사과 및 복숭아의 과실류에 피해를 주고 있는 복숭아순나방(Grapholita molesta)은 과실 내부 가해라는 서식 습성에 따라 화학적 방제에 어려움이 있 다. 따라서 야외 노출 발육 시기인 성충을 대상으로 교미교란 처리 기술이 대체 기술로서 제안되었다. 본 해충의 성페로몬을 기반으로 교미교란제를 처리한 포장에서 뚜렷한 교미교란 효과를 얻을 수 있다. 그러나 국내 과수의 소규모 경 영규모에 따라 인접한 농가에서 교미한 암컷의 유입으로 방제의 실효를 거두 지 못하게 되었다. 이를 해결하기 위해 교미한 암컷을 포획할 수 있는 먹이트랩 과 처리 효과의 사각지대를 줄이기 위한 교미교란제의 테두리 처리가 병행될 필요가 있었다. 먹이트랩과 테두리처리가 투입된 교미교란제 처리는 교미교란 제의 단독 처리에 비해 우수한 방제 효과를 나타냈다. Paste 형태의 교미교란제 를 3월과 7월의 2회 처리로 연중 방제 효과를 거둘 수 있었다. 추후 교미교란제 의 투입량을 줄이는 기술과 교미한 암컷의 포획 효율을 높이는 유인제 개발 기 술이 필요하다.
24.
2010.05 구독 인증기관·개인회원 무료
To clarify the molecular mechanism of metamorphosis, we analyzed the Broad-Complex (BR-C) gene in the beet armyworm, Spodoptera exigua. We obtained a partial BR-C sequence from a pyrosequencing cDNA library. The BR-C of S. exigua was expressed only in the final larval instar, at which its expression was clearly detected in the epidermis. A treatment of a juvenile hormone analog, pyriproxyfen, inhibited larval-pupal metamorphosis and suppressed the BR-C expression. The hormonal treatment also inhibited expression of two storage protein genes that were usually expressed during a final larval instar. RNA interference of the BR-C using its double strand RNA suppressed BR-C expression and inhibited the larval-pupal metamorphosis. These results suggest that the BR-C is critical to induce larval-pupal metamorphosis of S. exigua.
25.
2010.05 구독 인증기관·개인회원 무료
A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp encoding two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28~50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. ORF301 and ORF302 were transiently expressed in hemocyte, fat body, gut, and epidermis of P. xylostella. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to non parasitized larvae of P. xylostella, in which the two genes were expressed at least for four days post-injection. The P. xylostella larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population, suppression of immune associated genes including cecropin, pro-phenoloxidase (PO) and serpin1, and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression by the viral segmenttreated larvae, but not by ORF302 specific dsRNA. The larval injected with CpBV-S3 exhibited an enhanced susceptibility to baculovirus infection. These results indicate that ORF301 of CpBV-S3, which containing BEN domain, suppresses both cellular and humoral immune responses in P. xylostella.
26.
2010.05 구독 인증기관·개인회원 무료
Antimicrobial peptides (AMPs) are a group of immune proteins that protect the host from microial infection. Gallerimycin is one of the AMPs most commonly found in Galleria mellonella and Spodoptera frugiperda. In this paper, we found Gallereimycin in Spodoptera exigua by expressed sequence tag library 0analysis. The gallerimycin of S. exigua gene is 332 bp long and the predicted open reading frame contains 75 amino acids with a signal peptide. After removing signal peptide, S. exigua gallrimycin was estimated to be 5.9 kDa and pI at 8.53. The gallerimycin of S. exigua shared maximum sequence homology with that of S. frugiperda. In naive S. exigua larvae, not much gene expression was detected, but strongly induced in fat body and hemocytes following immune challenge with entomopathogenic bacteria and fungus. A recombinant gallerimycin was prepared using a bacterial expression system and showed significant antibacterial and antifungal activities. RNA interference using double stranded RNA could knock down the expression of gallerimycin and significantly suppressed immune capacity.
27.
2010.05 구독 인증기관·개인회원 무료
Bacillus thuringiensis (Bt) is effective to control the diamondback moth, Plutella xylostella. However, its relative slow and unstable control efficacy limits its wide use by farmers. To facilitate pathogenic rate of Bt, a bacterial mixture technique has been developed in this study. Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata temperata (Ptt), possess high immunosuppressive activity against several lepidopteran insects. The mixture treatments using Bt + Xn or Bt + Ptt significantly enhanced Bt pathogenicity in median lethal concentration and time. Though live Xn and Ptt bacterial cells gave significant effect on the pathogenicity, their 48 h culture broth after removing the bacterialcells still possessed the synergistic effect on the Bt pathogenicity. The larvae fed with the bacterial culture broth suffered significant immunosuppression in response bacterial to infection
28.
2010.05 구독 인증기관·개인회원 무료
As an environment-friendly phytosanitary measure, CATTS (controlled atmosphere temperature treatment system) has been developed to kill several quarantine insect pests infesting subtropical agricultural commodities. This study tested any possibility to apply CATTS to apples to effectively eliminate the peach fruit moth, Carposina sasakii, which has been regarded as a quarantine insect from the imported countries. When the larvae of C. sasakii were directly exposed to 46℃ (an installed lethal temperature of CATTS), they showed a median lethal time at 14.66 min. Addition of high carbon dioxide to the temperature treatment enhanced the thermal limit susceptibility of C. sasakii to 46℃. The larvae internally infesting apples were tested using this CATTS device and showed 100% lethality after 60 min exposure to a treatment of 46℃ under 15% CO2 in the chamber. This study suggests a possibility that CATTS can be applied as a quarantine measure to kill the larvae of C. sasakii locating inside the apples. To understand the CATTS effect, a heat shock protein was cloned. Hsp90 was partially sequenced and showed its expression in response to heat treatment. CATTS was likely to suppress hsp90 expression.
29.
2010.05 구독 인증기관·개인회원 무료
Based on Drosophila model, Toll and Imd signals have regared as central intracellular pathways in insect immune cells in response to various pathogens. Current insect genome studies have identified the corresponding orthologs in other insets. This study reports two immune signaling genes, Se-Toll-1 and Se-Relish-1, and suggests Toll and Imd pathways in the hemocytes of Spodoptera exigua. Partial Se-Toll-1 and Se-Relish-1 share high sequence homologies with known Toll and Relish genes of lepidopteran and dipteran species. Their expressions were detected from all developmental stages. In larval stage, there two genes were expressed in all tested tissues including hemocytes. Real time quantitative RT-PCR indicates that expression of both genes were highly up-regulated by bacterial and fungal infections. Various antimicrobial peptides (AMPs) were expressed in the hemocytes of S.exigua, in which their expressions appeared to be controlled by Se-Toll-1 and Se-Relish-1. However, Se-Toll-1 and Se-Relish-1 were proved to controlled different AMP genes from their RNA interference assays. These results suggest Toll and Imd signals in the hemocytes of S. exigua.
30.
2010.05 구독 인증기관·개인회원 무료
Previous studies indicated that Matsumuraeses phaseoli and M. falcana (Lepidoptera: Tortricidae) are separate species since a few differences were observed in genitalia morphology and female sex pheromone composition. A clear difference was detected in the DNA sequences of cytochrome oxidase I of the two species separately collected in different plants and regions. A hybridization test also showed that a post-zygotic reproductive isolation occurred between the species. In field monitoring, however, both species have been caught simultaneously and together in the separate sex pheromone traps installed for the two species around neighboring soybean and red bean fields. Molecular marker-assisted identification with several adults sampled from the trapped insects showed that only ca. 40% of M. phaseoli adults identified as the species by genitalia morphology was the M. phaseoli, while ca. 97% of M. falcana adults identified as the species was the M. falcana. The result indicated that the observation of genitalia did not make a decisive criterion for classification of the insects. Conclusively, it suggested that the sex pheromones of the two species should be studied more precisely although there is a possibility that the two species are hybridized in fields as in laboratory, and speciation is under process.
31.
2010.05 구독 인증기관·개인회원 무료
Three acetylcholinesterases (AChEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus. Sequence comparison with known AChEs in conjunction with three-dimensional structure analysis suggested that all BxAChEs share typical characteristics of AChE at the major catalytic structures. BgAChE3 was most predominantly transcribed and then followed by AChE1 and AChE2. Immunohistochemistry using anti-BxAChEs antibodies revealed that BxAChE1 is most widely distributed whereas BxAChE2 exhibits more localized distribution in neuronal tissues. BxAChE3 was detected from entire body together with some limited tissues, including mouth parts and alimentary lining, and determined to be the only soluble AChE, suggesting its localization in hemolymph or/and extracellular space. Kinetic analysis of in vitro expressed BxAChEs revealed that BxAChE1 has the highest substrate specificity whereas BxAChE2 has the highest catalytic efficiency with BxAChE3 having the lowest catalytic efficiency. Interestingly, presence of BxAChE3 in the pool of BxAChEs significantly reduced the inhibition of BxAChE1 and BxAChE2 by inhibitors. Knockout of BxAChE3 by RNAi significantly increased the toxicity of nematicides, suggesting the protective role of BxAChE3 against these toxicants. Based on several features, including tissue distribution, expression level, substrate kinetics and inhibition property, it appeared that BxAChE1 is the major AChE with the function of postsynaptic transmission whereas BxAChE3 has been evolved to acquire the function of chemical defense, perhaps intrinsically against secondary toxic compounds from host pine trees, such as α-pinene and limonene. BxAChE2 appears to play a role in post-synaptic transmission in specialized neurons but its detailed physiological function still remains to be elucidated.
32.
2010.05 구독 인증기관·개인회원 무료
tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for Bursaphelenchus xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2,304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions or bands via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two non-identical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.
33.
2010.05 구독 인증기관·개인회원 무료
경남지역 진주, 하동, 고성 자운영 재배지에서 알팔파바구미의 발생을 포충 망조사, 육안조사, 식물체 수거 후 밀도조사를 통해 년중 발생생태를 조사하였 다. 자운영 포장에서 효과적인 알팔파바구미의 초기 유충의 조사방법으로 초 기 발생하는 유충은 포충망 조사로 확인이 어려우므로 10×10cm 넓이 면적의 식물체를 자른 후 용기에 담아 흔들어서 떨어진 유충 및 식물체에 남은 유충을 조사하는 것이 효과적이었다. 초기에 발생하는 유충은 식물체의 아래부분에 발생하고 크기가 작아서 포충망 조사로는 4월중순까지 발생확인이 안되었으 나 식물체 수거후 용기에 흔들어서 조사하는 방법으로는 3월중순부터 확인이 되었다. 산란을 위해 자운영답으로 이동한 성충을 조사하는데는 Pitfall trap을 이용 하는 것이 좋은 결과를 얻었다. 육안조사와 포충망 조사로는 확인할 수 없었으 나 Pitfall trap에서는 1월부터 발생을 확인할 수 있었다. 알파파바구미의 발생생태는 이동성충이 전년도 10월 하순경 자운영답으로 이동하여 산란을 시작하였고 유충은 3월상중순, 번데기는 4월하순, 1세대 성충 은 5월중순부터 발생하며 벼의 정식이 시작되면 주변 새로운 기주로 옮겨가서 서식하다가 다시 10월하순경 자운영이 올라오면 이동하여 산란을 개시하였다
34.
2010.05 구독 인증기관·개인회원 무료
2008년과 2009년 제주도 녹차 재배지(도순, 한남, 서광)에서 발생하는 풍뎅이류의 피해와 밀도 및 발생 생태를 조사하였다. 세 지역 모두 홈줄풍뎅이 (Bifurcanomala aulax)가 우점하였고, 청동풍뎅이(Anomala albopilosa)와 꽃무지(Eucetonia pilifera)가 발생하였다. 세 종 모두 유충은 차나무의 뿌리를 가해하였고, 청동풍뎅이는 차 잎을 가해하였다. 홈줄풍뎅이는 2령 또는 3령으로 월동하였고, 5월 하순에 번데기가 된 후 6월 하순에 성충이 출현하였다. 부화 된 1령충은 8월 하순에 발견되었다. 녹차 재배지에서 청동풍뎅이의 우화는 6월 25일 최초로 이루어졌고, 7월 14일까지 계속 되었으나 이후 8월 중순까지는 우화되는 개체가 없었다. 유아등에서는 7월 5일 유인 된 후 8월 초순부터 2차 유인이 된 후 9월 초순까지 계속 되었고, 유인 최성기는 9월 초순이었다.
35.
2010.05 구독 인증기관·개인회원 무료
and evaluated mainly physico-chemical properties. In the present study, several biological indices were applied to determine whether biological indices could provide a more comprehensive understanding in terms of soil quality assessment in urban forests. Microbe and invertebrate biological indices (i.e. biodiversity of both microbe and invertebrate, enzyme activity of microbe, feeding activity of invertebrate) were examined at 6 urban forests with different levels of disturbance in Seoul, Korea. The results showed that feeding activity and biodiversity were significantly and positively intercorrelated, but not with the enzyme activity. We also examined whether these biological indices could be modeled as functions of soil physico-chemical characteristics. To develop a predictive model, we applied principal component regression. The results showed that first principal component represented more than 33% of the total variance of biological indices and gave a good relationship with soil physico-chemical characteristics (R2=0.71). The predictive model developed in this study can be used for qualitative but not for quantitative assessment of soil quality.
36.
2010.05 구독 인증기관·개인회원 무료
Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) causes losses in several crops in Korea. Release of non-viable refrigerated eggs of R. pedestris is known to enhance natural parasitism by Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) and Gryon japonicum (Ashmead) (Hymenoptera: Scelionidae) in soybean fields. In this study, we conducted an experiment of cage-exclusion design to verify the former results in more manipulative approach in a soybean field. Agakong field (45×26 m2) located at Songcheon, Andong was divided into 15 plots (10×6 m2) with each experimental arena of 3×2 m2 in the center. There were three treatments: (1) release of refrigerated eggs of R. pedestris, (2) release of refrigerated eggs with one time spray of thiamethoxam, and (3) untreated control. A fine mesh cloth with iron poles was used to encircle the arenas. Refrigerated eggs of R. pedestris were released (100/arena) twice before sampling. One-day old eggs of R. pedestris were released (60/arena) in all the experimental arenas at an interval of 6 days, and 30 eggs from each were collected to record parasitism. We found no significant difference in the eggs and nymphs population of R. pedestris among the treatments. However, adult density was significantly reduced in the treated plots during final two sampling days compared to the control. We found significantly higher parasitism by G. japonicum on the eggs collected from treated plots (9-25%) compared to the control plots (1-9%). It is verified that releasing non-viable eggs of R. pedestris help to enhance natural parasitism in soybean field.
37.
2010.05 구독 인증기관·개인회원 무료
Chrysanthemum flower model trap developed by modifying an artificial yellow chrysanthemum flower was reported to be more attractive to flower thrips than a commercial yellow sticky trap. The installation of the traps (20 traps per 50 m2 plot), especially, reduced the seasonal populations of Frankliniella intonsa Trybom (Thysanoptera: Thripidae) on strawberry flowers in greenhouse by 82% compared to the untreated control. In this study, we tested if the installation of the flower model traps can reduce thrips population on a red pepper field located in Seokdong, Andong. The pepper field was treated two times with pesticides during the period of experiment. The traps were installed in plant canopy at different densities (0, 5, 10, 20 traps) in 20 plots (3×5 m2 each) using a completely randomized design. Population of thrips was examined on the collected pepper flowers from 1 July to 29 July in 2009. Thrips found on the flowers were all F. intonsa. Significance effect of treatment and sampling date was found from repeated-measure analysis of variance. The highest density of traps significantly reduced female and male F. intonsa population by 60% and 46% compared to the control, respectively. However, no difference in immature population was found among the treatments. These results indicate flower model trap can be an additional tool for the management of flower thrips on field red pepper.
38.
2010.05 구독 인증기관·개인회원 무료
Modeling the complex system often provide better understanding of the behavior of the system given that parameters for the modeling are appropriate. The honeybee mite, Varroa destructor Anderson and Trueman, is one of the most serious pests of honeybees in Korea. Even with vast amount of ecological information of this parasite and its host, Apis mellifera, in the world, limited information is available in Korean environment. This paper provides some basic framework of the varroa mite population dynamics modified from published works in western world to adapt to Korean beekeeping environment. Overall population growth rate was in agreement to those previous published. However the detail behavior was quite different mainly because of splitting honeybee colony during the summer season. Although this framework provide reasonable, logical theoretical linkage to varroa mite population growth, implementation of the results provided from this model is not appreciated mainly because this paper is intended to show the basic framework, not the simulated results. Thus, further incorporation of realistic parameters from honey bee and its parasites in Korean environment would provide better insight of the population behavior and management options. Also, research gaps which need to be filled are further discussed.
39.
2010.05 구독 인증기관·개인회원 무료
관행관리와 친환경관리를 하는 경북 상주시 공검면 일대 10개의 배과원에서 2005년부터 5년간 복숭아순나방과 복숭아심식나방의 발생을 페로몬트랩을 이 용하여 모니터링하였다. 복숭아심식나방은 5월말 1세대 성충이 발생이 시작되 어 7월 중순 최성기를 보였으며 9월 중순 이후 성충은 급감하였다. 년간 누적 발생량의 변이계수는 20% 이내로 과원별로 발생량의 차이가 매우 적은 것으 로 나타났다. 반면 년차별 발생량의 차이는 누적온일도와 정의 상관을 보였다. 또한 관행관리과원에 비하여 약제가 적게 처리되는 친환경관리 과원에서 발생 량이 많았다. 반면 복숭아순나방은 월동 세대 성충이 5월말에 발생 최성기를 이루고, 누적온일도 모델에 적용시킨 결과 4세대의 성충이 발생하는 것으로 파 악되었다. 복숭아순나방은 매년 발생이 증가하는 패턴을 보였으며 이는 누적 온일도와는 상관이 없었다. 과원별 연간 누적 발생량의 변이계수는 5-46%로 과원별로 발생량의 차이가 매우 다. 상주지역 배 과원에서 복숭아순나방과 복 숭아심식나방의 발생량 간에는 상관관계가 나타나지 않았다.
40.
2010.05 구독 인증기관·개인회원 무료
In an effort to gain a better understanding of the nature of the population genetic structure of the pest insect, Spodoptera litura (Lepidoptera: Noctuidae), tobacco cutworms were collected from six Korean and five Chinese localities and their mitochondrial A+T-rich region and nuclear internal transcribed spacer 2 (ITS2) regions were cloned and sequenced. A total of 106 A+T-rich region haplotypes and 92 ITS2 sequence types were obtained from 158 individuals. Compared to the preliminary mitochondrial COI gene sequence data that provided all identical sequence types in all samples, the A+T-rich region and nuclear ITS2 are highly variable. Overall, a low level of genetic fixation in the A+T-rich region (FST = 0 ~ 0.02965) and nuclear ITS2 (FST = 0 ~ 0.34491), and no discernable isolated population was noted to exist among most S. litura populations. The presence of a majority of within-population variations, rather than variations among populations or between China and Korea, indicates that the S. litura populations are composed of heterogeneous individuals. The fixation index at hierarchical level of among regions shows no significance, suggesting that the S. litura populations in both Korea and China are profoundly interrelated with one another. This finding is in accord with the current knowledge that S. litura has sufficient flight capacity for dispersal.
1 2 3 4 5