Genomic DNA samples isolated from geographical purple Washington clam (Saxidomus purpuratus) were obtained from two different regions in Korean Peninsula: Gunsan (Gunsan population; GSP), and Haeju (Haeju population; HJP), a collection area in the vicinity of the West Sea. The seven arbitrarily primers, OPA-07, OPA-09, OPA-18, OPA-20, OPC-03, OPC-06 and OPC-09 were shown to generate the total loci, loci observed per primer, shared loci by each population, specific, and polymorphic loci which could be clearly scored. We also generated the unique shared loci to each population and shared loci by the two populations in purple Washington clam. The size of the DNA fragments also varied wildly, from 50 to 2,400 bp. Here, 304 total loci were identified in the GSP purple Washington clam population, and 282 in the HJP: 91 polymorphic loci (29.9%) in the GSP and 47 (16.7) in the HJP. 198 shared loci, with an average of 28.3 per primer, were observed in the GSP population. The decamer primer OPA-07 generated the shared loci by the two populations, approximately 1,000 bp, between the two Saxidomus populations. The oligonucleotide primer OPC-03 also generated the shared loci by the two populations, approximately 500 bp and 1,000 bp, in GSP population from Gunsan and HJP population from Haeju. The other primer, OPC-06 generated the shared loci by two Gomphina populations (approximately 400 bp). The dendrogram, generated by seven reliable primers, indicates three genetic clusters. The dendrogram obtained by the seven primers indicates three genetic clusters: cluster 1 (GUNSAN 01-GUNSAN 02), cluster 2 (GUNSAN 03-GUNSAN 11), and cluster 3 (HAEJU 12-HAEJU 22). The genetic distance between the two geographical populations ranged from 0.043 to 0.506. Especially, the longest genetic distance displaying significant molecular differences, 0.506, was found to exist between individuals GUNSAN no. 11 of Gunsan and HAEJU no. 17 of Haeju.
연체동물 유래 아미노산 서열 22,138 개에서 NLS가 예측되는 아미노산 서열은 266 개였으며 이는 연체동물 전체 아미노산 중 1.2% 정도였다. 또한 현재 등재되어 있는 연체동물 8,314 종 중 NLS를 포함한 아미노산이 밝혀진 생물은 60여 종에 불과 하였다. 현재 알려진 연체동물 서열 중에는 두족 강의 경우가 NLS를 포함한 아미노산이 많을 것으로 예측되었다. ACKNOWLEDGEMENT이 연구과제는 1999년도 인제대학교 학술연구조성연구사업의 일환으로 수행되었습니다.
localization of a protein containing nuclear localization signals (NLS) has been well studied in many organisms ranging from invertebrates to vertebrates. However, no systematic analysis of NLS-containing proteins available from Mollusks has been reported. Here, we describe in silico screening of NLS-containing proteins using the mollusks database that contains 22,138 amino acids. To screen putative proteins with NLS-motif, we used both predict NLS and perl script. As a result, we have found 266 proteins containing NLS sequences which are about 1.2% out of the entire proteins. On the basis of KOG (The eukaryotic orthologous groups) analysis, we can't predict the precise functions of the NLS-containing proteins. However, we found out that these proteins belong to several types of proteins such as chromatin structure and dynamics, translation, ribosomal structure, biogenesis, and signal transduction mechanism. In addition, we have analysed these sequences based on the classes of mollusks. We could not find many from the species that are the main subjects of phylogenetic studies. In contrast, we noticed that cephalopods has the highest number of NLS-containing proteins. Thus, we have constructed mollusks NLS database and added these information and data to the mollusks database by constructing web interface. Taken together, these information will be very useful for those who are or will be studying NLS-containing proteins from mollusks.
Gonad index, spermatogenesis and the reproductive cycle of Meretrix petechialis were investigated by cytological, histological observations. Monthly changes in the gonad index coincides the gonadal development. The morphology of the spermatozoon had a primitive type and is similar to that of other bivalves having a short mid-piece with five to six mitochondria surrounding the centrioles. The morphology of the sperm nucleus type and the acrosome shape of this species were cylindrical type and cap-like shape, respectively. The spermatozoon was approximately 40-45 <TEX>${\mu}m$</TEX> in length including the sperm nucleus length (about 1.50 <TEX>${\mu}m$</TEX>), acrosome length (0.60 <TEX>${\mu}m$</TEX>) and tail flagellum. The axoneme of the tail flagellum consisted of nine pairs of microtubules at the periphery and a pair at the center. The axoneme of the sperm tail showed 9 + 2 microtubular arrangement. The spawning period was from June to September and the main spawning occurred from July to August when seawater temperatures were higher than <TEX>$20^{\circ}C$</TEX>. The reproductive cycle of this species could be categorized into five successive stages: early active stage (February to March), late active stage (February to May), ripe stage (April to July), partially spawned stage (June to September), and spent/inactive stage (September to February).
2002년 1월부터 12월까지 말백합 (Meretrix petechialis)을 대상으로 조직학적 관찰에 의해 생식주기를 조사하였고, 생화학적 분석방법에 의해 폐각근 조직과 내장낭 조직의 생화학적 성분 변화를 조사하였다. 본 종의 생식주기는 초기활성기 (1-3월), 후기활성기 (2-5월), 완숙기 (4-8월), 부분산란기 (7-8월) 그리고 퇴화 및 비활성기 (9-2월)로 연속적인 5단계로 구분할 수 있었다. 총단백질 함량은 내장낭에서 폐각근보다 약 2 배 정도 높게 나타났다. ANOVA test 결과, 총단백질 함량의 월별 변화는 폐각근에서 유의한 차이가 없었고 (p = 0.071), 내장낭에서는 유의한 차이를 보였다 (p < 0.001). 내장낭 내 단백질 함량은 초기활성기 (1-3월), 후기활성기 (3-5월), 및 완숙기에 높았고, 7월에 가장 낮았다. 글리코겐 함량은 내장낭보다 폐각근에서 더 높았다. 글리코겐 함량의 월변화는 폐각근 (F = 237.2, p < 0.001) 과 내장낭 (F = 64.04, p < 0.001) 모두에서 유의성을 나타내었다. 글리코겐 함량은 폐각근의 경우, 완숙기인 4 월에 최대치에 이른 후, 부분산란기 및 퇴화기인 6-9월까지 서서히 감소하였다. 그리고 내장낭의 경우는 초기활성기까지는 비교적 낮은 값을 보인 후, 후기활성기인 5월에 가장 높았다. RNA 함량은 폐각근보다 내장낭에서 더 높게 나타났다. RNA 함량의 월별 변화는 폐각근 (F = 195.2, p < 0.001) 과 내장낭 (F = 78.85, p < 0.001) 모두에서 유의성을 나타내었다. RNA 함량은 폐각근의 경우, 초기활성기인 1-2월에 높았고, 후기활성기인 3-4월에 극감하였으며, 완숙기인 5월에 약간 증가된 후 6-7월에 다시 감소하였다. 그리고 내장낭의 경우는 완숙기인 5월에 가장 높았고, 부분산란기인 6-7월에 급격히 그 값이 감소하였다. 총단백질 함량의 경우 폐각근과 내장낭 사이에 통계적으로 유의한 양의 상관관계가 나타났다 (r = 0.715, p = 0.020). 글리코겐의 경우에는 양의 상관관계가 있는 것처럼 나타났으나 (r = 0.216), 통계적으로 유의한 수준은 아니었다 (p = 0.550). RNA의 경우 음의 상관관계가 나타났으나(r = -0.233), 이 또한 통계적으로 유의한 수준은 아니었다 (p = 0.518). 전반적으로 볼 때, 가리비류와 같이 생식소, 소화맹낭, 중장선이 부위별로 구분이 된 이매패류와 달리 백합은 위 3가지 부분이 명확하게 구분되지 않아 내장낭으로 분석하여 특정시기(월)에 국한되어 폐각근과 내장낭의 생화학적 함량변화가 역상관관계를 나타내는 현상을 보였다. 특히, 폐각근과 내장낭내 총 RNA 함량 변화 양상은 총단백질 함량 변화 양상과 유사한 경향을 보여 RNA 함량 증가시 단백질 함량의 증가가 일어나고 있음을 알 수 있었다.
We investigated the reproductive cycle of the hard clam, Meretrix petechialis with its gonadal development by histological observations. The seasonal changes in biochemical component of the adductor muscle, visceral mass, foot muscle and mantle of the clam were studied by biochemical analysis, from January to December, 2002. The reproductive cycle of this species can be divided into five successive stages: early stage (January to March), late active stage (February to May), ripe stage (April to August), partially spawned stage (July to August) and spent/inactive stage (September to January). Total protein content in the visceral mass was over two times higher than that in the adductor muscle. Monthly changes of total protein content in the adductor muscle were not statistically significant (ANOVA, p = 0.071), while the changes in the visceral mass were significant (p < 0.001). Total protein content in visceral mass was higher during the early active, late active, and ripe stages (from January to May), while the lowest in July. Glycogen content in the adductor muscle was higher than that in the visceral mass. Monthly changes in glycogen contents were statistically significant in both adductor muscle (F = 237.2, p < 0.001) and the visceral mass (F = 64.04, p < 0.001). Glycogen content in the adductor muscle was the highest in the ripe stage (April). Its content was lower in the partially spawned and the spent/inactive stages (June-September). Glycogen contents in the visceral mass were relatively lower until the early active stage, while the highest in the late active stage. RNA content was higher in visceral mass than that in the adductor muscle. Monthly changes in RNA contents were significant in both adductor muscle (F = 195.2, p < 0.001) and visceral mass (F = 78.85, p < 0.001). RNA content in the adductor muscle was high in the early active stage (January-February), and then it decreased rapidly in the late active stage (March-April), thereafter, slightly increased during the partially spawned stage (June-July). RNA content in the visceral mass reached a maximum during the ripe stage (May), and then it decreased rapidly during the partially-spawned stage (June-July). There was significant positive correlation in total protein contents between adductor muscle and visceral mass (r = 0.715, p = 0.020). However, there was no correlation between adductor muscle and visceral mass in glycogen (p = 0.550), while a negative correlation was found between the adductor muscle and visceral mass in RNA (p = 0.518) contents. Especially, changes in RNA content showed a negative correlation between the adductor muscle tissue and visceral mass. Therefore, these results suggest that the nutrient content of the adductor muscle, visceral muscle and foot muscle changed in response to gonadal energy needs.
남해안 여자만에서 출현하는 꼬막, Tegillarca granosa(Linnaeus) 에 관한 연령과 성장에 관하여 연구하였다. 7-8월에 발생한 꼬막 치패는 당해 10월에 각고 약 5.60 mm <TEX>$({\pm}\;1.07)$</TEX>로 최초로 나타났으며, 이 후 고수온기에는 급속히 성장하다가 10월 이후 저수온기에는 거의 성장이 정지하였고, 이듬해 수온 증가와 함께 급격히 성장하였다. 꼬막은 발생 후 약 11-15 개월이 경과하는 6-9월에 가장 빠른 성장을 보였고, 이론적 최대각고 (크기) 의 약 29% 정도가 이 시기에 성장하였다. von Bertalanffy 성장식에 적용한 꼬막의 연령별 성장식은 다음과 같이 산정되었다. <TEX>$I_t(sh_t)=46.1317\;(1-e^{-0.4997(t-0.5828)})$</TEX>.
This paper describes relative age and growth pattern of the blood cockle, Tegillarca granosa (Linnaeus) in Yeoja Bay, southern coast of Korea. The young shell of blood cockle that spawned from July to August reached 5.60 mm <TEX>$({\pm}\;1.07)$</TEX> in shell height in October. Slow growing season was estimated to be from October to the next May with water temperature under <TEX>$23^{\circ}C$</TEX>. With warm water temperature in July to September, the growth was fast. The young shells reached 29 percentage of asymptotic shell height 11-15 months after spawning. Growth was estimated by von Bertalanffy growth function as follows. <TEX>$I_t(sh_t)=46.1317\;(1-e^{-0.4997(t-0.5828)})$</TEX>.
The gonad index, condition index, reproductive cycle and spawning of the pen shell Atrina (Servatrina) pectinata were investigated using samples from the subtidal zone of Nokdo on the Boryeong coastal waters of Korea. Samples were collected monthly by SCUBA divers for one year from January to December, 2001. A. (Servatrina) pectinata is dioecious and oviparous. The spawning season of this species occurred once a year from June to August, with the main spawning occurring between June and July when the seawater temperature was around <TEX>$20^{\circ}C$</TEX>. Ripe oocytes were about 60-65 <TEX>${\mu}m$</TEX> in diameter. The reproductive cycle of this species could be classified into five successive stages; early active stage (November to March), late active stage (February to May), ripe stage (April to July), partially spawned stage (June to August), and spent/inactive stage (August to October). Monthly changes in the gonad index reached a maximum (4.6) in May (ripe stage), thereafter, the GI values gradually decreased from June to August when spawning occurred continuously. Therefore, monthly changes in the GI values showed a similar pattern to the gonadal phase. The condition index (CI) of the meat part without the posterior adductor muscle reached the maximum in June (ripe and partially spawned stage) and the minimum in September (spent/inactive stage), Accordingly, monthly changes in the condition indice of the meat part without the posterior adductor muscle coincided with the gonadal phases.
Proportion of Tegillarca granosa surviving after 2-6 hrs air exposure with 12 hrs interval at 20℃ and 28℃ for 20 days showed 85-100%, 80-100%, respectively. The survival rate was somewhat lower at high temperature but not significant (p < 0.05). Subsequent exposures for 7-9 days showed survival rate of 8.0-24.1% at 20℃ and 28℃. Oxygen consumption rates and filtration rates were significantly higher for 4 to 6 hrs exposures, compared with the preceding exposures. On the other hand, at 28℃, oxygen consumption rates in adult granular ark for 6 hrs exposure during 20 days had significantly decreased. Filtration rates in study groups increased a little over extended period of exposure, compared with those in control groups, and were similar irrelevant to the time of exposure and size of experimental animals. It is concluded, in view of their viability and oxygen consumption rate during air exposure, that ark shells have quite a high resistance to air exposure with their limited range of responses.
바지락의 중금속의 농축 방식 및 해독기작을 연구하기 위해 소화선, 소화관 및 아가미등 세 기관을 선택하여 면역조직화학적 방법, 미세구조적 연구 그리고 SEM-EDS를 사용한 MT와 중금속의 분포 양상을 지역별로 채집한 샘플과 Cd-Cu에 시간별로 노출시킨 샘플에서 각각 살펴보았다. 중금속에 의해 유도된 MT 단백질이 면역조직화학적인 방법에 의해 붉은 색으로 표지되었다. 노출시간이 길어질수록 간췌장에서는 diverticula의 외곽으로 강한 반응을 보였으며, 아가미에서는 중금속의 노출시간이 길어질수록 노출되지 않은 조직에 비해 전반적으로 더 넓은 지역에서 강하게 반응을 보였다. 간췌장과 아가미를 투과전자현미경으로 관찰한 결과 중금속(Cd) 에 더 오래 노출될수록 세포질에서 inclusion body와 중금속으로 추정되는 과립들을 다수 관찰할 수 있었으며, 핵막이 swelling 되는 현상을 관찰할 수 있었다. 또한 발달된 조면 소포체와 미토콘드리아가 관찰되었다. SEM-EDS 결과 중금속의 분포 양상이 지역별로 상당히 상이한 결과를 나타났는데, 이는 지역별로 중금속의 오염정도와 오염원이 되는 중금속의 종류가 다르기 때문으로 추측된다. 그리고 지역별로 채집한 바지락을 이용한 실험과 S와 Zn의 수치가 높게 나타나는 것으로 보아 MT가 관여하고 있음을 간접적으로 확인할 수 있었다.
The present study was conducted to confirm that a bivalve Ruditapes philippinarum can be used as a biomarker for the monitoring of the heavy metal pollution in the silt of the marine environment. The clams were collected from the silt of Cheonsu-bay, Buheung-ri, and Tan-island of the West Sea, Korea. To observe the normal structures of the target organs (hepatopancreas and gill), they were dissected out for the immunohistochemical study and the electron microscopy with TEM, SEM, and SEM-EDS. The immunohistochemical study showed that the interdiverticular connective tissues of the hepatopancreas, and the outer epithelium of the gill lamellae was strongly reacted to anti-metallothionein (MT), indicating the presence of MT, a metal-binding protein, involved in metal detoxifying process. According to the examinations under the TEM, the epithelial cells of the hepatopancreas of the clams collected from polluted area (Tan-island) showed certain changes such as swollen rER, swollen nuclear envelope and inclusion bodies in the nulcei. In the SEM-EDS analysis, tissue of the hepatopancreas showed relatively higher concentration of S, Zn, and Cd. These elements are supposed to be concerning with the MT-reaction in the hepatopancreas. Considering that the coastal bivalve R. philippinarum showed immediate subcellular responses to heavy metal pollution in the overall experiments conducted, this species might act as one of efficient biomarkers for the heavy metal contamination in the marine environment.
The embryos of marine bivalves have been commonly used in bioassays for quality assessments of marine environments. Although several standard protocols for the developmental bioassay of bivalves have been proposed, only a few trials for application of these protocols in environmental assessments or for the development of a new protocol with Korean species have been conducted. As such, there is a strong need to establish standard bioassay protocols with bivalves commonly found in Korean waters. To determine the sensitivity of Mytilus galloprovincialis to establish a standard bioassay, their fertilized eggs were exposed to six metals (Ag, Cd, Cr, Cu, Ni, and Zn). The order of biological impact was Ag > Cu > Ni > Zn > Cr > Cd and their lowest observed effective concentration were 5, 16.4, 25.4, 142, 187 and 1,500 μ g/l, respectively. The proportion of normal larvae appeared to decrease linearly with the logarithm of each toxicant concentration within the tested range. The average values of median effective concentrations (EC50) from the triplicate experiments for Ag, Cd, Cr, Cu, Ni, and Zn were 6.8, 1,797, 786, 16.6, 68.1, and 139.2 μg/l, respectively. There was a more than 100-fold difference in EC50 values of Cu and Cd. The value of EC50 or median lethal concentration of Cu was within the range observed for other bivalve developmental bioassays. The overall sensitivity of M. galloprovincialis in the present developmental bioassay was also similar to that of other marine organisms commonly used in aquatic bioassays (e.g. sea urchins, oysters). Hence, the bioassay using the embryo of M. galloprovincialis is considered to be a useful tool to monitor and evaluate the quality of marine aquatic environments.
본 연구는 2004년 3월 10일 남해 강진 만에서 채취한 피조개 어미 1,200 마리를 확보하여 수온조절에 의한 인위적인 성성숙을 유도하였으며, 성숙 유도된 어미로부터 잔존율, 비만도, 생식소 조직검사, 적산수온 및 성숙소요일수를 조사하였다. 성 성숙유도된 어미의 잔존율은 <TEX>$20^{\circ}C$</TEX>가 94.8％로 가장 높았고, <TEX>$23^{\circ}C$</TEX>가 90.3％, 대조구(자연수온구)가 84.8％이었고, <TEX>$26^{\circ}C$</TEX>가 18.1％로 온도가 높을수록 잔존율이 낮은 경향이 있었다. 42일 째 생식소 조직의 암컷의 완숙기 출현율은 <TEX>$23^{\circ}C$</TEX> 실험 구에서 67%로 가장 높았고, <TEX>$20^{\circ}C$</TEX> 실험구에서 56%, <TEX>$26^{\circ}C$</TEX>에서 22%였으며, 자연수온 실험구에서는 성숙이 매우 늦어 완숙기에 이르지 못하였다. 수컷의 경우, <TEX>$23^{\circ}C$</TEX> 실험구에서 완숙기 출현율이 100%로 가장 높았고, <TEX>$20^{\circ}C$</TEX> 실험구에서 87%, <TEX>$26^{\circ}C$</TEX>에서 73%였으며, 자연수온 실험구에서는 50% 순으로 나타났다. 성숙소요일수는 <TEX>$23^{\circ}C$</TEX>와 <TEX>$20^{\circ}C$</TEX>에서 각각 42일 및 49일, 대조구인 자연수온 <TEX>$(9.5-14.8^{\circ}C)$</TEX>에서는 79일이였으며, 성 성숙 유도율은 <TEX>$20^{\circ}C$</TEX>와 <TEX>$23^{\circ}C$</TEX>에서 각각 55.5% 및 60.5%로 타 실험구와는 유의한 차이를 나타내었다 (p < 0.05). 따라서 <TEX>$20^{\circ}C$</TEX> 실험구 (적산수온: <TEX>$1,295^{\circ}C$</TEX>) 가 피조개 어미를 인위적으로 성 성숙을 유도하는데 가장 좋은 것으로 판단된다.
Artificial induction of maturation by heating of the ark shell, Scapharca broughtonii (Schrenck) broodstock was investigated from 10th March to 21th May in 2004. Maturity at the fixed water temperature group of <TEX>$23^{\circ}C$</TEX> was 60.5％, and it was the highest in maturity among experimental groups cultured for seven weeks. The proportions of organism surviving to start of S. broughtonii in the raised water temperature experimental groups (20 and <TEX>$23^{\circ}C$</TEX>) were over 90.5％, as similar to the control group (natural water temperature, 84.8％). But, the proportions of organism surviving to start of S. broughtonii in the fixed water temperature experimental groups (20, 23, and <TEX>$26^{\circ}C$</TEX>) were decreased with the increasing of the water temperature. In the fixed water temperature experimental groups of <TEX>$26^{\circ}C$</TEX>, the survival was 18.1％. Base on these results, the fixed water temperature of <TEX>$20^{\circ}C$</TEX> (accumulated water temperature; <TEX>$1,295^{\circ}C$</TEX>) was the best condition for artificial induction of sexual maturation.
