We thank James Maeda and Aaron Cooke for assistance with the filming and editing of this video. Funding was provided by the National Science Foundation (OCE-0550468, 1029515).

참고 :이 프로토콜의 3 단계는 척추 동물의 과목을 포함한다. 절차는 동물이 가능한 가장 인도적인 치료를받을 수 있도록 설계되었으며, 노스 캐롤라이나 윌 밍턴 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 1) 조직 추출 <ol><li> 이 이차 대사 산물의 체적 농도를 변경하므로 수화가 아닌 압착, 건조 아웃 또는 지나치게 젖은의 자연 상태에있는 조직을 사용합니다. 리거나 50 ㎖ 원심 분리 튜브에 삽입 할 수있는 부분 또는 슬라이스로 조직 들어온다. 참고 : 신선한 조직은 경우에 따라 사용될 수 있지만,자를 때 압착 적용​​되지 않는 고정 된 조직을 잘게 자르거나하는 것이 낫다. </li><li> (1) 30 ㎖에 조직편을 추가 40 ㎖의 최종 부피까지 졸업 원심 분리 튜브에서 한 혼합물을 디클로로 메탄 (DCM)과 메탄올 (MeOH 중하는)에 도달한다. 의 전송을 포함하는 모든 단계를 수행해야합니다적절한 환기와 흄 후드에서 용매. </li><li> 튜브를 씌우고 그것을 여러 번 반전 한 후 4 시간 추출 기간 동안 반복적으로 선동. 주 :이 기간 동안 물을 MeOH와 결합하고, 생성 된 메탄올 : 물상은 DCM 상으로부터 분리한다. 조직 교대 DCM 및 메탄올에 노출되어 물을 에멀젼으로서 튜브 교반 된 바와 같다. </li><li> 둥근 바닥 플라스크에 DCM 추출물을 전송 및 낮은 열을 이용 회전 증발기에서 증발 건고 (&lt;40 ° C). 최소한의 용매를 사용하여, 20 ㎖의 섬광 바이알에 건조 된 추출물을 전송. 회전 증발기 어댑터 바이알 맞고 다시 낮은 열을 이용 회전 증발기에서 증발 건고 (&lt;40 ° C). 주 : (1) 너트 (2) 와셔, 및 (3) 도토리 너트 : 다음 단계는 나사산로드의 단부 상에 순차적으로 다음 항목을 나사 결합에 의해 조립 될 수있는 제 압축 장비의 사용을 요구한다. 세탁기 중 하나를 천공해야 또는그것은 50 ㎖ 원심 분리 튜브의 내부 지름보다 작도록 끼워. </li><li> 조직 및 메탄올이 포함 졸업 원심 관으로 되돌아 : 물 추출물을 통해 압축 조직에서 추출 매질을 짠다. 메탄올로 이동 : 같은 둥근 바닥 플라스크에 물 추출물을 냉장 보관 (&lt;10 ° C). </li><li> 물 추출물 : 다음 새 메탄올은 메탄올이 들어있는 냉장 둥근 바닥 플라스크에 압축 전송, 지금은 탈수 조직이 2 ~ 6 시​​간 지속 시간의 두 번째 추출에 빠져들 때까지 졸업 원심 분리기 튜브에 메탄올을 추가합니다. 조직이 충분히 추출되지 않았다고 임의 우려가있는 경우, 2 시간 내지 6의 MeOH 추출을 반복한다. </li><li> 낮은 열을 사용하여 회전식 증발기에서 메탄올을 건조시키고 (&lt;40 ° C). 둥근 바닥 플라스크를 헹구어의 MeOH를 사용하여 최소한의 볼륨, 건조 비극성 추출물을 함유하는 섬광 바이알로 둥근 바닥 플라스크에서 남은 수용성 추출물을 전송. </li><li> E진공 농축기에 낮은 열 (&lt;40 ° C)를 사용하여 건조시켜 수용성 추출물을 vaporate. 섬광 유리 병은 이제 조직 10 ㎖의 총 건조 원유 유기 추출물이 포함되어 있습니다. , 산화를 방지하기 위해 N 2 가스로 유리 병의 헤드 공간을 대피 단단히 밀봉 및 저장 냉동 (-20 ° C). </li></ol> 2) 음식 준비 <ol><li> 동결 건조 오징어 맨틀 분말을 준비합니다. 주 : 오징어 맨틀 다른 저생 무척추 동물에 필적하고, 2.2의 하위 단계에 성분으로 사용될 영양의 공급원을 제공한다. <ol><li> 따뜻한 탈 (DI) 물 오징어 맨틀의 해동 냉동 반지, 다음 고속 믹서기에서 그들을 퓌 레. </li><li> 얕은 쿠키 시트 및 동결 (-20 ° C)에 퓌레 오징어 맨틀의 얇은 층을 붓고, 그 다음 작은 조각으로 동결 건조 할을 냉동 오징어 퓌레의 시트를 휴식. </li><li> FR의 운영 절차에 따라 냉동 오징어 맨틀 퓌레를 동결 건조eeze 건조기. </li><li> 분말을 형성 고속 블렌더 오징어 맨틀 퓌레의 동결 건조 된 조각을 분쇄. </li><li> 흄 후드에서 회전 밀가루를 체로 치는으로 분말 오징어 맨틀을 부어 미세 분말로 조직의 큰 덩어리를 분리하는 선별. </li><li> 밀봉 용기에 미세 분말 오징어 맨틀을 전송합니다. 산화를 방지하고 냉동 저장하는 N 2 가스로 용기 헤드 공간을 대피 (-20 ° C). </li></ol></li><li> 식품 혼합물을 준비합니다. 참고 : 필요한 경우 연속 된 여러 분석을 실행할 때, 그것은 ~ 음식을 혼합 100 ㎖를 준비하는 것이 실용적이다, 그러나이 조리법은 작은 볼륨을 확장 할 수 있습니다. <ol><li> 150 ㎖ 비이커에 3g 알긴산 DI 물 100㎖로 5g을 동결 건조 오징어 맨틀 분말 혼합물을 결합. 분말이 완전히 수화하고, 혼합물이 균질해질 때까지 몇 분 동안 격렬히 교반하여 microspatula. 원하는 경우, 식용 색소는 본 보안 목표 명세서에 추가 할 수 있습니다 참고EP : 오히려 첨가하여 추출 처리 혼합물의 색상을 일치하려고하기보다는 (추출물 처리 혼합물 추출물의 천연 색소 마​​스킹) 모두 처리 및 제어의 혼합물을 생성 식품 혼합물에 염료를 추가하는 것이 더 쉽다 제어 염료 혼합물. 녹색을 띤 갈색 또는 식품 색상 조 추출물에서 어떠한 안료를 마스크하는 것이 바람직하다. </li><li> 졸업 주사기로 음식을 혼합 10 mL를 정확하게 넣습니다. 이 과정에서 기포의 혼입을 방지하기 위해주의한다. </li><li> 냉동 건조 원유 유기 추출물 20 ㎖ 섬광 유리 병을 제거합니다. 드롭을 추가 또는 메탄올의 두 사람은 다음 microspatula와 균일 한 혼합물에 추출물을 저어. </li><li> 20 ㎖ 섬광 유리 병에 음식 행렬의로드 10 ML의 주사기를 꺼내 추출물 처리 된 음식 혼합물을 균질화 microspatula 저어. R 다음, 그것은 작은 단위 (예에서 주사기를 배출하는 데 도움이 될 수 2 ml의 배출 및 균질화 :. 참고모든 10 ml의 때까지 EPEAT)의 균질화되고있다. </li></ol></li><li> 분석 알약을 준비합니다. <ol><li> 주사기로 추출 혼합물 (~ 1 ㎖)의 매우 작은 체적을로드하고, 0.25 M의 CaCl2를 용액에 주사기 팁 잠수함. 긴 형상 스파게티 스트랜드를 형성하도록 주사기의 내용물을 꺼내기. </li><li> 몇 분 후, 다음, 면도날 유리 커팅 보드에 4mm 긴 펠릿으로 들어온다, 경화 가닥을 제거 해수를 세정한다. </li><li> 반복 제어 알약을 만들기 위해 조직 추출물을 포함하지 않고 2.3.1와 2.3.2 단계를 반복합니다. 용매의 동등한 볼륨 제어 알약을 치료해야 용매 추가 할 제어 (단계 2.2.3에서 처리 혼합물에 메탄올을 첨가 참조). 음성 대조군, 즉 분석 물고기가 먹이 단념 할 수 확인이 MG ml의 농도에서 denatonium 벤조 에이트를 추가 -1 원시 음식 혼합물 (27)로하는 것이 바람직합니다. </li></ol></li></ol> 3) 기호성생물 검정 <ol><li> 자연산 노란색 상 bluehead 놀래기과의 바닷물 고기, Thalassoma의 bifasciatum와 공급 분석을 수행, 실험실 수조의 불투명 양면 구획의 세 그룹으로 유지했다. </li><li> 고무 전구 유리 피펫을 사용하여 해수의 비이커에서 식품 알약을 제공합니다. 참고 :이 방법으로 음식을받을 물고기를 양성하는 몇 일이 걸릴 수 있습니다. 음식의 전달을 앞에 조절 자극 (수족관 유리 피펫의 예를 몇 가지 탭은) 음식 펠릿의 추가를 기대하는 물고기를 훈련하는 것이 도움이 될 수있다. </li><li> 알약을 채점. 쉽게 물고기에 의해 소비되는 경우 펠릿 허용 고려하십시오. 자신의 구강에 걸릴 하나 이상의 물고기에 의해 세 번 최소 후에 먹게하지 않을 경우 펠릿이 거부 고려, 펠릿 접근하고 하나의 시도 후 무시 된 경우에는. </li><li> 점수 샘플. 참고 : 분석 절차는 그림 1에서 플로우 차트로 묘사된다 식사를 거부 물고기의 그룹.프로토콜의 모든 단계에서 제어 펠렛을 추가로 간주되지 않습니다. 분석의 단일 실행의 두 가지 잠재적 인 결과가 있습니다 샘플 허용 또는 거부 중 하나. <ol><li> 물고기의 그룹이 협력 여부를 확인하기 위해서 제어 펠렛과 함께 시작합니다. 처리 된 펠렛을 제공합니다. 물고기가 처리 된 펠렛을 수락하면 수락으로, 샘플 점수. 물고기가 처리 된 펠렛을 거부 할 경우, 물고기가 먹이를 중단 여부를 결정하기 위해 후속 제어 펠렛을 제공합니다. 물고기가 후속 제어 펠렛을 적용하는 경우 거부로, 샘플 점수. </li></ol></li><li> 복제. 각 추출물에 대한 물고기의 열 독립적 인 그룹과 분석 절차를 반복합니다. </li></ol> 4) 평가 의의 <ol><li> 피셔의 정확한 시험 (26)의 수정 된 버전으로 처리 펠릿 VS 제어 소비의 차이의 중요성을 평가한다. 컨트롤에 대한 한계 합계 및 치료 펠릿 고정되도록 테스트를 수정그들에게 모두 무작위 샘플을 치료. 참고 :이 페이지 제공 = 0.057 7 펠렛을 먹을 때; 7 이상의 펠릿을 먹을 경우 따라서, 어떤 추출물을 6 개 이하의 펠릿을 먹을 경우 억지력으로 간주하고 맛이있다. </li><li> 추출물의 집단 간의 상대적 기호성을 비교하기 위해, 각 그룹 내에서 먹을 펠릿의 평균 수를 계산합니다. 펠릿의 평균 수는 + 먹을 경우 너무 복제 추출물의 그룹이 억지력으로 간주됩니다 6 펠릿 임계 값을 유지 표준 오차 (SE) ≤6. 주 : 대표적인 결과, 그룹 할당은 종이므로 복제 추출물을 구별 개체로부터 와서 상대 기호성 종간 비교 될 수있다. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphitoxin,+a+new+high+molecular+weight+antifeedant+pyridinium+salt+from+the+Caribbean+sponge+Amphimedon+compressa.">Amphitoxin, a new high molecular weight antifeedant pyridinium salt from the Caribbean sponge Amphimedon compressa.</a> Journal of Natural Products. 58 (5), 647-652 (1995).</li><li>Assmann, M., Lichte, E., Pawlik, J. R., K&#246;ck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical defenses of the Caribbean sponges Agelas wiedenmayeri and Agelas conifera. Marine Ecology Progress Series. 207, 255-262 (2000).</li><li>Kubanek, J., Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+antifeedant+triterpene+glycosides+from+the+Caribbean+sponge+Erylus+Formosus.">New antifeedant triterpene glycosides from the Caribbean sponge Erylus Formosus.</a> Natural Product Letters. 15 (4), 275-285 (2001).</li><li>Pawlik, J. R., McFall, G., Zea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+odor+from+sponges+of+the+genus+Ircinia+protect+them+from+fish+predators.">Does the odor from sponges of the genus Ircinia protect them from fish predators.</a> Journal of Chemical Ecology. 28 (6), 1103-1115 (2002).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defenses+of+Caribbean+sponges+against+invertebrate+predators.+I.+Assays+with+hermit+crabs.">Defenses of Caribbean sponges against invertebrate predators. I. Assays with hermit crabs.</a> Marine Ecology Progress Series. 195, 125-132 (2000).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense+of+Caribbean+sponges+against+invertebrate+predators.+II.+Assays+with+sea+stars.">Defense of Caribbean sponges against invertebrate predators. II. Assays with sea stars.</a> Marine Ecology Progress Series. 195, 133-144 (2000).</li><li>Burns, E., Ifrach, I., Carmeli, S., Pawlik, J. R., Ilan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+anti-predatory+defenses+of+Red+Sea+and+Caribbean+sponges.+I.+Chemical+defense.">Comparison of anti-predatory defenses of Red Sea and Caribbean sponges. I. Chemical defense.</a> Marine Ecology Progress Series. 252, 105-114 (2003).</li><li>Jones, A. C., Blum, J. E., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+for+defensive+synergy+in+Caribbean+sponges:+Bad+taste+or+glass+spicules.">Testing for defensive synergy in Caribbean sponges: Bad taste or glass spicules.</a> Journal of Experimental Marine Biology and Ecology. 322 (1), 67 (2005).</li></ol>

The authors declare that they have no competing financial interests.

본원에 기술 된 절차에 antipredatory 해양 생물 화학적 방어를 평가하기위한 상대적으로 간단하고, 생태 학적으로 중요한 실험 프로토콜을 제공한다. 여기에서 우리는 방법이 세트에 만족하는 중요한 기준을 검토 : (1) 적절한 육식 동물.이 공급 분석은 bluehead 놀래기과의 바닷물 고기, Thalassoma의 bifasciatum, 카리브해 전역의 산호초에 가장 풍부한 물고기 중 하나를 ?...

화학 생태학 화학자 및 생태 학자의 협력을 통해 개발했다. 지상파 화학 생태학의 subdiscipline 몇 시간 동안 주변되었습니다 있지만, 해양 화학 생태학의는 몇 십 년 오래지만 해양 생물 1-8의 진화 생태학과 사회 구조에 중요한 통찰력을 제공하고 있습니다. 스쿠버 다이빙과 NMR 분광법의 응급 기술을 활용, 유기 화학자들은 빠르게 1970 년대와 1980 년대 9 저서 무척추 동물과 조류의 소설 대사를 설명하는 출판물의 큰 숫자를 생성합니다. 이차 대사 산물 경험적 증거도없이 새로운 화합물에 생태 학적으로 중요한 특성을 관찰 작용이 책의 많은 어떤 목적을 봉사해야한다는 것을 가정. 약 동시에, 생태도 분포 이전에 이리저리 알려진 저서 동물과 식물의 풍부함을 스쿠버 다이빙의 출현을 활용하고 설명했다준설 상대적으로 비효율적 인 샘플링 방법을 해요. 이러한 연구자의 ​​가정은 고착 아무것도 부드러운 바디 화학적 육식 동물 (10)에 의해 소비를 방지하기 위해 방어해야이었다. 종 중 농도에 달리 설명 작업 사용했던 경험주의를 소개하는 노력의 일환으로, 일부 생태 독성 분석 (11)에서 화학 방어를 외삽하기 시작했다. 대부분의 독성 분석은 절반 분석 유기체를 죽이는에 대한 책임 추출물의 건조 질량 농도의 후속 결정으로, 전체 물고기 나 무척추 동물 조직의 원유 유기 추출물의 수성 현탁액에 다른 생물의 노출을하고있었습니다. 그러나 독성 분석은 잠재적 인 포식자가 자연 상태에서 먹이를 인식하는 방식을 모방하지 않으며, 후속 연구는 독성과 기호성 12 ~ 13 사이의 관계를 발견했다. 그것은 권위있는 저널에 출판이 거의 또는 전혀 ecologica을 가진 기술을 사용하는 것은 놀라운 일이다L 관련성 14-15 것을이 연구가 아직 널리 인용된다 오늘. 그것은 독성 데이터를 기반으로 연구 16-18을 게시 할 계속주의하는 것이 더욱 놀랍다. 여기에 설명 된 생물 검정 방법은 antipredatory 화학 방어를 평가하기 위해 해양 화학 생태에 대한 생태 학적으로 중요한 접근 방법을 제공하기 위해 1980 년대 후반에 개발되었다. 방법은 생태 학적으로 의미있는 독성 데이터보다 기호성 데이터를 제공, 식품 영양 학적으로 유사한 매트릭스 천연 농도 대상 유기체로부터 조 유기 추출물을 샘플링 포식자 모델을 필요로한다. (1) 적절한 제너럴 육식 동물이 먹이 분석에 사용되어야한다, (2) 모든 극성 유기 대사가 철저의 조직에서 추출해야합니다 해양 생물의 조직의 antipredatory 활동을 평가하는 일반적인 방법은 네 가지 중요한 기준을 포함 생물체를 대상으로, (3) 대사 ㄴ해야같은 체적 농도 영양 적절한 실험 음식에 혼합 전자는 그들이 추출하고, (4) 실험 설계 및 통계 방법은 의미있는 메트릭이 상대 distastefulness을 표시하기 위해 제공해야하는 유기체에서 발견. 아래에 설명 된 절차는 카리브해 해양 무척추 동물에 antipredatory 화학 방어를 평가하기 위해 설계되었습니다. 이 종은 카리브해 산호초에 공통이며, 저서 무척추 동물 (19)의 넓은 구색을 샘플링 알려져 있기 때문에 우리는 모델 육식 물고기 bluehead 놀래기과의 바닷물 고기, Thalassoma의 bifasciatum을 사용합니다. 표적 조직은 유기체로부터 먼저 추출 식품 혼합물과 결합하고, 마지막 T. 그룹에 제공되며 bifasciatum은 추출물 처리 된 음식을 거부 여부를 관찰합니다. 이 방법을 사용하여 분석 데이터는 해양 생물 12,20-21, L의 수비 화학에 중요한 통찰력을 제공하고 있습니다IFE의 역사 절충 22-24, 지역 사회 생태 25-26.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Scientific</td>
			<td>D37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A41220</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C614-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryocool Heat Transfer Fluid</td>
			<td>Fisher Scientific</td>
			<td>20-548-146</td>
			<td>For vacuum concentrator</td>
		</tr>
		<tr>
			<td>Alginic Acid Sodium Salt High Viscosity</td>
			<td>MP Biomedicals</td>
			<td>154723</td>
			<td></td>
		</tr>
		<tr>
			<td>Squid mantle rings</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Can be purchased at grocery store</td>
		</tr>
		<tr>
			<td>Denatonium benzoate</td>
			<td>Aldrich</td>
			<td>D5765</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml graduated centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>20 ml scintillation vial</td>
			<td>Fisher Scientific</td>
			<td>03-337-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bulbs for Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>03-448-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Red bulbs for pellet delivery</td>
			<td>Fisher Scientific</td>
			<td>03-448-27</td>
			<td></td>
		</tr>
		<tr>
			<td>250 ml round-bottom flask</td>
			<td>Fisher Scientific</td>
			<td>10-067E</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation vial adapter for rotavap</td>
			<td>Fisher Scientific</td>
			<td>K747130-1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Weightboats</td>
			<td>Fisher Scientific</td>
			<td>02-202B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-10</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml graduated syringe</td>
			<td>Fisher Scientific</td>
			<td>14-817-53</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml graduated syringe</td>
			<td>Fisher Scientific</td>
			<td>14-817-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

여기에 우리가 일반적으로 카리브해 스폰지의 여섯 종 (그림 2)이 생물 검정의 결과를보고합니다. 이러한 데이터는 처음 Pawlik 등. (12)에 의해 1995 년에 출판 및 공동 발생하는 분류군 중 화학 방어 전략의 차이를 조사하기 위해이 방식의 힘을 보여되었다. 결과는 표준 오차 (SE) 각 종 + 먹은 음식 펠릿의 평균 숫자로보고되었다. 거의 펠릿 Agelas의 clathrodes, Amphimedon compre...

Watch this Scientific Journal Video about A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms at JoVE.com

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

이러한 생물학적 검정은 식품 영양 학적으로 유사한 매트릭스를 이용 농도 천연 해양 생물의 조직 유기 추출물로부터 병입 억지 대사의 존재를 평가하기위한 모델 육 물고기를 이용한다.

물고기 먹이 실험 생물 검정은 해양 생물의 조직에서 이차 대사 산물의 Antipredatory 활동을 평가하기 위해

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

a-fish-feeding-laboratory-bioassay-to-assess-antipredatory-activity

Research

JoVE Journal

Environment

9.4K 조회수.  University of North Carolina Wilmington.  이러한 생물학적 검정은 식품 영양 학적으로 유사한 매트릭스를 이용 농도 천연 해양 생물의 조직 유기 추출물로부터 병입 억지 대사의 존재를 평가하기위한 모델 육 물고기를 이용한다. 

비디오: A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

물 화학 및 해양 미생물에 필드 가이드 - 바다에서 보이지 않는 선수를 해명

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

연구

환경과학

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

작은 볼륨 생물 검정은 물 - 펄스 진폭 변조 (WATER-PAM) 형광 측정을 사용하여 세균 / 식물 플랑크톤의 공동 문화를 평가하는

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

자동 젤 사이즈 선택은 환경 물 샘플에서 준비 차세대 시퀀싱 라이브러리의 질을 향상시키기 위해

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP, 개선 INEX 방법에 대한<em&gt; 현장에서</em&gt; 미립자의 측정 및 용존 화합물은 액티브 서스펜션 피더에 의해 처리

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

선캄브리아 해양 용승 시스템 광합성 박테리아의 성장을 탐구하는이 풍부한 철 (II)의 실험실 시뮬레이션

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

해양 박테리아와 생태 독성 학적 방법<em&gt; 비브리오 앵 귈라 룸</em&gt; 환경 오염 물질의 급성 독성 평가

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

플라스틱 펠렛 및 플라스틱 유형 분석에서 유기 염소 농약의 추출

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

공장 실험실에서 측정 하는 표면에 박테리아의 부착

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

수 중 스테레오 비디오 도구를 사용 하 여 물고기 밀도 측정을 위한 새로운 방법의 개발

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

해양 모델-따개 비 Balanus improvisus 경작 및 유전자 발현

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...