Name: a fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms
Uploaded: 2015-01-11T14:00:00.000+00:00
Duration: 16 min 3 s
Description: 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

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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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><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 ...

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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

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

호수 송어에 PCB의 동족체의 순 영양 전송 효율의 실험실 평가 (<em&gt; Salvelinus namaycush</em&gt;) 먹이에서

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

연구

환경과학

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

에서 약탈 먹이 행동을 분석<em&gt; Pristionchus</em&gt; 및 기타 선충

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

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 ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

생물 검정 기반 분류를 사용 하 여 바다 심고 페로몬의 식별

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

생화학

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

애벌레에서 행동 응답 프로필을 조사 하기 위한 실험 프로토콜: 신경 흥분 제 카페인에 응용

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

간단한 생물 분석법에 의한 바다 아네몬의 조추출물에서 용혈성 및 포스포리파아제 활성의 확인

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

아르테미아 살리나 L을 사용한 치사율 생물학적 분석법

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

화학

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

갑각류와 장기 치명적인 독성 시험 Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

생물학

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (AVEXIS)

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

성인 제브라피쉬의 신경 독성 효과를 평가하기 위한 행동 테스트 배터리

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

저자 스포트라이트: 성체 제브라피시의 환경 및 약물 유발 행동 변화 평가 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

호수 송어에 PCB의 동족체의 순 영양 전송 효율의 실험실 평가 (

에서 약탈 먹이 행동을 분석

해양 박테리아와 생태 독성 학적 방법

생물 검정 기반 분류를 사용 하 여 바다 심고 페로몬의 식별

애벌레에서 행동 응답 프로필을 조사 하기 위한 실험 프로토콜: 신경 흥분 제 카페인에 응용

간단한 생물 분석법에 의한 바다 아네몬의 조추출물에서 용혈성 및 포스포리파아제 활성의 확인

아르테미아 살리나 L을 사용한 치사율 생물학적 분석법

갑각류와 장기 치명적인 독성 시험 Artemia franciscana

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (AVEXIS)

단일 탱크에서 행동 테스트 배터리를 사용한 성인 Danio rerio 의 신경 독성 평가