臺灣博碩士論文加值系統

鱟又名馬蹄蟹，其祖先出現於古生代泥盆紀，已存活於地球逾四億多年，以活化石的形象被人們廣為所知；現存三屬四種，除美洲鱟只分布於美洲大西洋沿岸外，其餘的三棘鱟、巨鱟、圓尾鱟均為亞洲地區所特有，多分布於東南亞地區。然而隨著棲地破壞與人為過度捕撈，野生鱟的族群數量日益減少，逐漸瀕危。為了深入瞭解鱟在野外的生態習性以及生態區位，食性研究將是一個很好的切入點。本研究將使用分子技術分析並比較亞洲地區不同種鱟之間的捕食取向及地區間食性差異，做為改良人工飼料的參考依據。在收集了鱟的胃腸內容物以萃取DNA之後進行PCR，再通過添加阻斷鱟DNA擴增的PNA阻斷子，可優先得到食物物種的片段，將得到的序列與公共資料庫進行比對，從而得知其捕食的物種與捕食比例，推斷其偏好的成分。根據實驗結果，四個地區的三棘鱟胃腸內容物均明顯的不同於圓尾鱟與巨鱟，在圓尾鱟的腹中檢測出大量的海葵及二枚貝，巨鱟則以多毛綱生物為主。在台灣金門及香港白泥、水口採集到的三棘鱟其胃腸內容物的組成成分較為相似，以多毛綱和寡毛綱為主；日本地區則略有差別，雖然有部分多毛綱生物但以二枚貝為多數。另外，將收集到較多樣本的三棘鱟和圓尾鱟與其棲息地泥灘中的生物進行比較後，發現各個地區的泥灘生物組成差異不大，其中三棘鱟的胃內容物成分和土壤生物的重疊性高，而圓尾鱟沒有食用土壤中佔多數的物種，顯示其對獵物應是具有選擇性。本論文是首次利用分子技術對鱟的胃內容物進行分析，並首次發現其中含有膠態的刺絲胞生物。

Horseshoe crabs are famous as being a living fossil and their blood is widely harvested for medical application. However, over-harvesting and degradation of habitat lead to the decline in horseshoe crab populations. To investigate the ecological niche and the behavior of horseshoe crab in wild, dietary research is a direction. In the present study, the diet composition of Asian horseshoe crab species were studied using molecular technique. The three Asian horseshoe crab species, Carcinoscorpius rotundicauda, Tachypleus gigas and T. tridentatus were collected from Japan, Taiwan, Hong Kong and Singapore. DNA were extracted from the content of intestine and gizzard. Nuclear 18S rRNA gene was amplified from the DNA extract with addition of peptide nucleic acid (PNA) blockers that inhibit amplification of horseshoe crab DNA so that only DNA of prey items were preferentially amplified. The results showed that the prey composition are different in the three species of horseshoe crabs. There were a large proportion of sea anemone and Bivalve in the diet of C. rotundicauda, and T. gigas mainly feed on Polychaeta. Moreover, the diet of T. tridentatus from Kinmen, Shuikou and Pak Nai were similar, mainly with Polychaeta and Oligochaeta. On the other hand, T. tridentatus from Kyushu has its diet consisting of more Bivalve and few Polychaeta. Diet composition from C. rotundicauda has prey composition very different from that find in habitat soil fauna, showing that this horseshoe crab has food selectivity. This is also the first study using DNA metabarcoding to analysis the dietary of horseshoe crabs and successfully detected gelatin prey (Cnidarian) from gut content and feces.

Table of Content

摘要……………………………………………………………………I
Abstract……………………………………………………………II
List of figure……………………………………………………III
List of table………………………………………………………IV
1. Introduction………………………………………………1
1.1 About horseshoe crabs…………………………………1
1.2 Biology of horseshoe crabs……………………………1
1.3 Aquaculture and restocking of horseshoe crabs…2
1.4 Research Objectives……………………………………3
2. Materials and Method……………………………………5
2.1 Sampling……………………………………………………5
2.2 Analysis of soil fauna diversity of horseshoe crab habitat…………………………………………………………5
2.3 DNA extractions from horseshoe crab gut content and soil fauna………………………………………………………5
2.4 Development of PNA probe blocking primer…………7
2.5 Polymerase Chain Reaction (PCR)……………………7
2.6 Agarose Gel Electrophoresis…………………………7
2.7 Gel DNA Purification……………………………………8
2.8 Illumina sequencing……………………………………9
2.9 Data analysis……………………………………………9
3. Results……………………………………………………10
3.1 Successful amplification……………………………10
3.2 Saturation curve………………………………………10
3.3 Overall composition…………………………………10
4. Discussion………………………………………………12
4.1 Metabarcoding……………………………………………12
4.2 Intraspecfic and Interspecfic in diet composition ………………………………………………………………………13
4.3 Geographic variation of soil fauna………………13
References…………………………………………………………16

List of Figure

Figure 1. Sampling locations for horseshoe crab dietary analyses……………………………………………………………21
Figure 2. The environment photos of sampling locations22
Figure 3. The gizzard and intestine dissected from horseshoe crab……………………………………………………23
Figure 4. Sequence alignment of the 18S rRNA……………24
Figure 5. Gel photo of PCR results…………………………25
Figure 6. Saturation curve of observed MOTUs……………26
Figure 7. Mean proportion of diet by locations with standard deviation………………………………………………27
Figure 8. RRA bar chat by individual………………………28
Figure 9. FOO bar chat by locations…………………………29
Figure 10. PCoA by group_all horseshoe crab samples……30
Figure 11. PCoA by group_all horseshoe crabs except Kyushu samples……………………………………………………31
Figure 12. PCoA by group_all horseshoe crabs with soil fauna…………………………………………………………………32
Figure 13. PCoA by MOTU_all horseshoe crab samples……33
Figure 14. PCoA by MOTU_all horseshoe crab except Kyushu samples………………………………………………………………34
Figure 15. PCoA by MOTU_all horseshoe crabs with soil fauna…………………………………………………………………35


 
List of Table

Table 1. Information for locations, number and size of samples………………………………………………………………36
Table 2. List of macro fauna found in soil………………37
Table 3. Basic statistic for Illumina sequencings………38
Table 4. Frequency of MOTU……………………………………39
Table 5. RRA mean process with standard deviation………47
Table 6. FOO calculation………………………………………48

Akbar, J. B., Kamaruzzaman, B. Y., Jalal, K. C. A., & Zaleha, K. (2012). Feeding ecology and food preferences of Carcinoscorpius rotundicauda collected from the Pahang nesting grounds. Sains Malaysiana, 41(7), 855–861.
Alexander, S. A., Hobson, K. A., Gratto-Trevor, C. L., & Diamond, A. W. (1996). Conventional and isotopic determinations of shorebird diets at an inland stopover: the importance of invertebrates and Potamogeton pectinatus tubers. Canadian Journal of Zoology, 74, 1057–1068.
Berkson, J. M., & Shuster, C. N. Jr. (1999). The horseshoe crab: the battle over a true multiple use resource. Fisheries, 24, 6–10.
Bråte, J., Logares, R., Berney, C., Ree, D. K., Klaveness, D., Jakobsen, K. S., & Shalchian-Tabrizi, K. (2010). Freshwater Perkinsea and marine-freshwater colonizations revealed by pyrosequencing and phylogeny of environmental rDNA. ISME Journal, 4, 1144–1153.
Botton, M. L. (1984). Diet and food preferences of the adult horseshoe crab Limulus Polyphemus in Delaware Bay, New Jersey, USA. Marine Biology, 81, 199–207.
Botton, M. L. (2001). The conservation of horseshoe crabs: what can we learn from the Japanese experience? In: Tanacredi, J. T. (eds.) Limulus in the limelight. Kluwer Academic/Plenum Publishing, New York, pp. 41–51.
Botton, M. L., & Haskin, H. H. (1984). D2istribution and feeding of the horseshoe crab, Limulus polyphemus, on the continental shelf off New Jersey. Fishery Bulletin, 82, 383–389.
Botton, M. L., Loveland, R. E., & Tiwari, A. (2003). Distribution, abundance, and survivorship of young-of-the-year in a commercially exploited population of horseshoe crabs Limulus polyphemus. Marine Ecology Progress Series, 265, 175–184.
Botton, M. L., Shuster, C. N. Jr., & Keinath, J. A. (2003). Horseshoe crabs in a food web: who eats whom. In: Shuster, C. N. Jr., Barlow, R. B., & Brockmann, H. J. (eds) The American horseshoe crab. Harvard University Press, Cambridge, MA, pp 133–153.
Botton, M. L., Loveland, R. E., Tanacredi, J. T., & Itow, T. (2006). Horseshoe crabs (Limulus polyphemus) in an urban estuary (Jamaica Bay, New York), and the potential for ecological restoration. Estuarine Coasts, 29, 820–830.
Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., Fierer, N., Peña, A. G., Goodrich, J. K., Gordon, J. I., Huttley, G. A., Kelley, S. T., Knights, D., Koenig, J. E., Ley, R. E., Lozupone, C. A., McDonald, D., Muegge, B. D., Pirrung, M., Reeder, J., Sevinsky, J. R., Turnbaugh, P. J., Walters, W. A., Widmann, J., Yatsunenko, T., Zaneveld, J., & Knight, R. (2010). QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7, 335–336.
Carmichael, R. H., & Brush, E. (2012). Three decades of horseshoe crab rearing: a review of conditions for captive growth and survival. Reviews in Aquaculture, 4, 32–43.
Carmichael, R. H., Rutecki, D., & Valiela, I. (2003). Abundance and population structure of the Atlantic horseshoe crab Limulus polyphemus in Pleasant Bay, Cape Cod. Marine Ecology Progress Series, 246, 225–239.
Carmichael, R. H., Rutecki, D., Annett, B., Gaines, E., & Valiela, I. (2004). Position of horseshoe crabs in estuarine food webs: N and C stable isotopic study of foraging ranges and diet composition. Journal Experimental Marine Biology Ecology, 299, 231–253.
Carmichael, R.H., Gaines, E., Sheller, Z., Tong, A., Clapp, A., & Valiela, I. (2009) Diet composition of juvenile horseshoe crabs: implications for growth and survival of natural and cultured stocks. In: Tanacredi, J. T., Botton, M. L., & Smith, D. R. (eds) Biology and conservation of horseshoe crabs. Springer, New York, pp 521–534.
Chatterji, A., Mishra, J. K., & Parulekar, A. H. (1992). Feeding behaviour and food selection in the horseshoe crab, Tachypleus gigas (Muller). Hydrobiologia, 246, 41–48.
Chatterji, A., Vijaykumar, R., & Parulekar, A. H. (1988). Growth and morphometric characteristic in the horseshoe crab, Carcinoscorpius rotundicauda (Latreiile) from Canning (West Bengal), India. Pakistan Journal of Scientific and Industrial Research, 33, 352–355.
Chen, C. P., Yeh, H. Y., & Lin, P. F. (2004). Conservation of the horseshoe crab at Kinmen, Taiwan: strategies and practices. Biodiversity Conservation, 13, 1889–1904.
Chen, Y., Lau, C. W., Cheung, S. G., Ke, C. H., & Shin, P. K. S. (2010). Enhanced growth of juvenile Tachypleus tridentatus (Chelicerata: Xiphosura) in the laboratory: a step towards population restocking for conservation of the species. Aquatic Biology, 11, 37–46.
Chiu, H. M. C., & Morton, B. (1999). The distribution of horseshoe crabs (Tachypleus tridentatus and Carcinoscorpius rotundicauda) in Hong Kong. Asian Marine Biology, 16, 185–194.
Debnath, R., Nag, S. K., Choudhury, A., Dasgupta, R., & Sur, R. K. (1989). Feeding habit and digestive physiology of the Indian horseshoe crab, Tachypleus gigas (Muller). Indian Journal of Physiology and Allied Sciences, 43, 44–49.
Edgar, R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics, 26, 2460–2461.
Eldredge, N. (1991). Fossils, The Evolution and Extinction of Species, New York.
Fincham, J. (2016). Norfolk's starlet anemone: a brief popuation update. In Biodiversity News, 73, 28-30.
Gaines, E. F., Carmichael, R. H., Grady, S. P., & Valiela, I. (2002). Stable isotopic evidence for changing nutritional sources of juvenile horseshoe crabs. Biological Bulletin, 203, 228–230.
Garie´py, T. D., Kuhlmann, U., Gillott, C., & Erlandson, M. (2007). Parasitoids, predators and PCR: the use of diagnostic molecular markers in biological control of arthropods. Journal of Applied Entomology, 131, 225–240.
Gauvry, G. (2009). Community building : an integrated approach to horseshoe crab conservation. In: Tanacredi, J. T., Botton, M. L. & Smith, D. R. (eds) Biology and Conservation of Horseshoe Crabs, Springer, New York, pp 417-438.
Hand, C., & Uhlinger, K. R. (1994). The unique, widely distributed, estuarine sea anemone, Nematostella vectensis, Stephenson: A review, new facts and questions. Estuaries, 17, 501-508.
Holechek, J. L., Vavra, M., & Pieper, R. D. (1982). Botanical composition determination of range diets: a review. Journal of Range Management, 35, 309-315.
Hsieh, H. L. (1995). Spatial and temporal patterns of polychaete communities in a subtropical mangrove swamp: influences of sediment and microhabitat. Marine Ecology Progress Series, 127, 157–167.
Hsieh, H. L., & Chen, C. P. (2009). Conservation program for the Asian horseshoe crab Tachypleus tridentatus in Taiwan: characterizing the microhabitat of nursery grounds and restoring spawning grounds. In: Tanacredi, J. T., Botton, M. L., & Smith, D. R. (eds.) Biology and Conservation of Horseshoe Crabs, Springer, New York, pp 417–438.
Ingerson-Mahar, J. (2002). Relating diet and morphology in adult carabid beetles. In: Holland, J. (eds.) The Agroecology of Carabid Beetles, Intercept, Andover, UK, pp 111–136.
Itow, T. (1993). Crisis in the Seto Inland Sea: the decimation of the horseshoe crab. EMECS Newsletters, 3, 10–11.
Itow, T., Sugita, H., & Sekiguchi, K. (1991). A phenomenal decrease of the horseshoe crab in the Inland Sea of Japan and its causes. Bull Dept Manage Information Sciences Jobu University, 4, 29–46 (in Japanese).
Kartzinel, T. R., Chen, P. A., Coverdale, T. C., Erickson, D. L., Kress, W. J., Kuzmina, M. L., Rubenstein, D. I., Wang, W., & Pringle, R. M. (2015). DNA metabarcoding illuminates dietary niche partitioning by African large herbivores. Proceedings of the National Academy of Sciences of the United States of America, 112, 8019–8024.
King, R. A., Tibble, A. L., & Symondson, W. O. C. (2008). Opening a can of worms: unprecedented sympatric cryptic diversity within British.
King, R. A., Read, D. S., Traugott, M., & Symondson, W. O. C. (2008). Molecular analysis of predation: a review of best practice for DNA-based approaches. Molecular Ecology, 17, 947–963.
Liao, Y. Y., Hong, S. G., & Li, X. M. (2001). A survey on the horseshoe crabs in the north of South China Sea. Acta Zoology Sinica, 47, 108–111 (Chinese with English Abstract).
Loveland, R. E., Botton, M. L., & Shuster, C. N. Jr. 1997. Life history of the American horseshoe crab (Limulus polyphemus) in Delaware Bay and it importance in a commercial resource. In: Farrell, J., & Martin, C. (eds.) Proceedings of the Horseshoe Crab Forum: Status of the Resource. University of Delaware Sea Grant Program, 15–22.
McInnes, J. C., Alderman, R., Lea, M. A., Raymond, B., Deagle, B. E., Phillips, R. A., Stanworth, A., Thompson, D. R., Catry, P., Weimerskirch, H., Suazo, C. G., Gras, M., & Jarman, S. N. (2017). High occurrence of jellyfish predation by black-browed and Campbell albatross identified by DNA metabarcoding. Molecular Ecology, 26, 4831–4845.
Mishra, A., & Choudhury, A. K. (1985). Polychaetous annelids from the mangrove swamps of Sunderbans, India. In: Leela, J. B. (eds.) Professional National Symposium Biology Utility Construction Mangroves, 448-452.
Mishra, J. K., & Mishra, A. (2011). Anthropogenic effect on the horseshoe crab breeding ground and impact on their population along the North-East Coast of India. International workshop on scientific conservation of Asian horseshoe crabs, Hong Kong.
Moreby, S. J. (1988). An aid to the identification of arthropod fragments in the feces of gamebird chicks (galliformes). Ibis, 130, 519–526.
Novitsky, T. J. 1984. Discovery to commercialization: the blood of the horseshoe crab. Oceanus, 27, 13–18.
Ostrom, P. H., Colunga-Garcia, M., & Gage, S. H. (1997). Establishing pathways of energy flow for insect predators using stable isotope ratios: field and laboratory evidence. Oecologia, 109, 108–113.
Pompano, F., Geagle, B. E., Symondson, W. O. C., Brown, D. S., Jarman, S. N., & Taberlet, P. (2012). Who is eating what: diet assessment using next generation sequencing. Molecular Ecology, 21, 1931-1950.
Rudkin, D. M., & Young, G. A. (2009). Horseshoe crabs – an ancient ancestry revealed. In: Tanacredi, J. T., Botton, M., & Smith, D. R. (eds.) Biology and conservation of horseshoe crabs. Springer, New York, pp 25–44.
Rudloe, A. (1982). Man’s influence as an environmental threat to Limulus. In: Bonaventura, J., Bonaventura, C., & Tesh, S. (eds.) Physiology and biology of horseshoe crabs: studies on normal and environmentally stressed animals, vol. 81, Inc., New York, pp 297–300.
Schreibman, M. P., & Zarnoch, C. B. (2009). Aquaculture methods and early growth of juvenile horseshoe crabs (Limulus polyphemus). In: Tanacredi, J. T., Botton, M. L., & Smith, D. R. (eds.) Biology and Conservation of Horseshoe Crabs, Springer, New York, pp 417–438.
Sekiguchi, K. (1988). Biology of Horseshoe Crabs. Science House, Tokyo, pp 1–428.
Sekiguchi, K., & Nakamura, K. (1979). Ecology of the extant horseshoe crabs. In: Cohen, E. (eds.) Biomedical Applications of the Horseshoe Crab (Limulidae), New York, pp 37–45.
Sheader, M., Suwailem, A. M. & Rowe, G. A. (1997). The anemone, Nematostella vectensis, in Britain: considerations for conservation management. Aquatic Conservation: Marine and Freshwater Ecosystems, 7, 13-25.
Sheppard, S. K., & Harwood, J. D. (2005). Advances in molecular ecology: tracking trophic links through predator-prey food webs. Functional Ecology, 19, 751–762.
Shin, P. K. S., Li, H., & Cheung, S. G. (2009). Horseshoe crabs in Hong Kong: current population status and human exploitation. In: Tanacredi, J. T., Botton, M. L., & Smith, D. R. (eds.) Biology and conservation of horseshoe crabs. Springer, New York, pp 347–359.
Shuster, C. N. Jr. (1948). On the gross anatomy and histology of the alimentary tract in early developmental stages of Xiphosura (= Limulus) polyphemus Linn, M.S. thesis, Rutgers University, New Brunswick, New Jersey.
Shuster, C. N. Jr. (1979). Distribution of the American horseshoe ‘‘crab,’’ Limulus polyphemus (L.). In: Cohen, E. (eds.) Biomedical Applications of the Horseshoe Crab (Limulidae). Alan R. Liss, New York, pp 3–26.
Shuster, C. N. Jr. (1982). A pictorial review of the natural history and ecology of the horseshoe crab Limulus polyphemus, with reference to other Limulidae. In: Bonaventura, J., Bonaventura, C., & Tesh, S. (eds.) Physiology and biology of horseshoe crabs: studies on normal and environmentally stressed animals, vol. 81. Inc. New York, pp 1–52.
Shuster, C. N. Jr. (2001). Two perspectives: horseshoe crabs during 420 million years, worldwide, and the past 150 years in the Delaware Bay area. In: Tanacredi, J. T. (eds.) Limulus in the Limelight. Kluwer Academic/ Plenum, New York, pp 17–40.
Shuster, C. N. Jr. (2003). King crab fertilizer: a once-thriving Delaware Bay industry. In: Shuster, C. N. Jr., Barlow, R. B., Brockmann, H. J. (eds.) The American horseshoe crab. Harvard University Press, Cambridge, MA, pp 341–357.
Shuster, C. N. Jr., & Anderson, L. I. (2003). A history of skeletal structure: clues to relationships among species. In: Shuster, C. N. Jr., Barlow, R. B., & Brockman, H. J. (eds.) The American Horseshoe Crab. Harvard University Press, Cambridge, pp 154–188.
Smith, O. R., & Chin, E. (1951). The effects of predation on soft clams, Mya arenaria. National Shellfish Association, 1951, 37–44.
Sonnenberg, R., Arne, W., Nolte, A. W., & Tautz, D. (2007). An evaluation of LSU rDNA D1-D2 sequences for their use in species identification. Frontiers in Zoology, 4, 6.
Sunderland, K. D., Powell, W., & Symondson, W. O. C. (2005). Populations and communities. In: Jervis, M. A. (eds.) Insects as Natural Enemies: A Practical Perspective, pp 299–434.
Sutela, T., & Huusko, A. (2000). Varying resistance of zooplankton prey to digestion: implications for quantifying larval fish diets. Transactions of the American Fish Society, 129, 545–551.
Symondson, W. O. C. (2002). Molecular identification of prey in predator diets. Molecular Ecology, 11, 627–641.
Traugott, M., Pazmandi, C., Kaufmann, R., & Juen, A. (2007). Evaluating 15N ⁄ 14N and 13C ⁄ 12C isotope ratio analysis to investigate trophic relationships of elaterid larvae (Coleoptera: Elateridae). Soil Biology Biochemistry, 39, 1023–1030.
Valentini, A., Miquel, C., Nawaz, M. A., Bellemain, E., Coissac, E., Pompanon, F., Gielly, L., Cruaud, C., Nascetti, G., Wincker, P., Swenson, J. E., & Taberlet, P. (2009). New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach. Molecular Ecology Resources, 9, 51–60.
Widener, J. W., & Barlow, R. (1999). Decline of a horseshoe crab population on Cape Cod. Biology Bulletin, 197, 300–302.
Zhou, H., & Morton, B. (2004). The diets of juvenile horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda (Xiphosura), from nursery beaches proposed for conservation in Hong Kong. Journal of National History, 38, 1915–1925.