Os autores gostariam de agradecer a todos os pescadores de lagosta comerciais da Nova Inglaterra, cujos barcos foram autorizados a amostra a partir de - a sua cooperação é muito apreciada. Além disso, agradecemos a Little Bay Lobster Company (Newington, NH) e de Champlin frutos do mar (Point Judith, RI), que permitiu a realização de amostragem em suas instalações. Os seguintes estudantes UNH prestou assistência valiosa ao longo deste estudo: Haley Branco, Françoise Morrison, Sarah Havener, Audra Chaput, e maio Grose. Este projecto foi apoiado por subsídios da NH SeaGrant a WHW (projeto # R/CFR-11) e um Programa Bolsa UNH Marine para JSG.

Parte A: Técnica Campo Amostragem 1. Feminino Medidas <ol><li> Para cada lagosta fêmea, medir o comprimento da carapaça (CL) e a largura do segundo segmento abdominal com uma aproximação de 1,0 milímetros usando um par de pinças. NOTA: O segundo segmento abdominal é medida porque a largura do abdômen (onde as fêmeas carregam seus ovos) é um indicador da maturidade sexual. Além disso, pode ser vantajoso para determinar a fase muda depois para combinar estes dados com a fase reprodutiva (ver passo 1.2). </li><li> Clipe uma pequena porção distal de um dos pleópodos abdominais (um protocolo metodológico padrão em lagostas) e armazenar o pleópodo na água do mar limpa para posterior visualização com um microscópio de dissecação 9. NOTA: Esta etapa também pode revelar a presença de glândulas de cimento nos pleópodos, que são uma indicação de que as mulheres estão se preparando para expulsar seus ovos 10. </li></ol> 2. Agulha Insertion <ol><li> Insira a agulha da seringa (18 G, ver Tabela 1) ~ 1 cm no receptáculo seminal, em ângulo em direção à cauda em ~ 45 ° (Figura 1). NOTA: Em lagostas com bujões de esperma intactas, a agulha pode encontrar alguma resistência durante a inserção, como é empurrado através do tampão de esperma antes de alcançar a maior concentração de esperma no fundo do receptáculo seminal (Figura 1). NOTA: A presença do plugue, em si, é um indicador confiável que as lagostas acasalaram. Em alguns animais pode haver pouca resistência, possivelmente porque não spermatophore está presente, o spermatophore é pequeno, ele não tenha endurecido num tampão, no entanto, ou pode já ter sido utilizado para fertilizar uma ou mais garras de ovos. </li></ol><img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50498/50498fig1highres.jpg" src="/files/ftp_upload/50498/50498fig1.jpg" width = &quot;600px&quot; /&gt; Figura 1. Inserção da agulha e visão de lagosta receptáculo seminal. Inserção da agulha de ponta romba no receptáculo seminal da lagosta para extrair uma amostra de esperma. O ângulo e profundidade da agulha são ambos críticos para a obtenção de amostras consistentes. Direita: Vista de um receptáculo seminal que foi dissecado e dividida verticalmente. A ficha de esperma ocupa a porção de topo ou exterior do recipiente, enquanto os espermatozóides são localizados na parte inferior (ponto mais fundo) do receptáculo. É importante notar que a amostragem abaixo desta profundidade pode perfurar o recipiente e resultar em uma amostra contaminada com sangue. <a href="https://www.jove.com/files/ftp_upload/50498/50498fig1highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a> . 3. Penetrando o esperma plug <ol><li> Após a inserção inicial, steepen o ângulo da agulha, a quase vertical (perpendicular ao eixo do corpo), e muito lentamente começa a trabalharpara baixo, para dentro do receptáculo. </li><li> Manter a agulha inclinada a um ângulo de modo que a ponta está apontado ligeiramente para a cauda. Neste caso, a ponta da agulha está a comprimir a parte superior do obturador, até que, eventualmente, penetra na matriz. </li><li> Continue a empurrar lentamente a agulha para baixo até sentir resistência, mais uma vez - este é o fundo do recipiente e onde a amostra é colhida. </li></ol> 4. Remoção de esperma <ol><li> Retirar uma amostra (muitas vezes o material tampão sólido, esperma líquido e outro fluido) a partir do fundo do recipiente, e depositar a amostra para um tubo de plástico de 2,0 ml marcado. </li><li> Lave o recipiente com ~ 0,1-0,5 ml de água do mar fria. </li><li> Retire cerca de 0,3 ml de líquido e coloque no mesmo tubo. </li><li> Amostras de esperma loja no gelo até que possam ser examinadas em laboratório. Parte B: exame de amostras de esperma no laboratório NOTA: As amostras retiradas dos receptáculos seminais do indivíduo l<html> obsters deve ser examinado para determinar se as células do esperma estavam presentes ou ausentes. </li><li> Remover ~ 50 mL de fluido de cada tubo de plástico, coloque em uma lâmina de vidro com uma lamela e vista no 100X com um microscópio composto. </li><li> Pontuação amostras como: esperma escassos, numerosos, ou ausente (este estudo não quantificar o número de esperma reais, figura 2). </li></ol><img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/50498/50498fig2highres.jpg" src="/files/ftp_upload/50498/50498fig2.jpg" width="600px" /> Figura 2. Imagens de amostra de esperma Lobster. Imagens de amostras de lagosta esperma retiradas de receptáculo seminal da fêmea, como visto com um microscópio composto em 40X de ampliação. Imagem à esquerda mostra uma amostra que continha numerosos espermatozóides, enquanto o outro (direita) ilustra uma amostra escassa (barra de escala = 10 mm).&quot;Target =&quot;. Jpg _blank &quot;&gt; Clique aqui para ver imagem ampliada.

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FAO and Agriculture Organization of the United Nations). Fisheries and Aquaculture Department. , (2012).</li><li>MacDiarmid, A., Butler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+economy+and+limitation+in+spiny+lobsters.">Sperm economy and limitation in spiny lobsters.</a> Behav. Ecol. Sociobiol. 46, 14-24 (1999).</li><li>Kendall, M. S., Wolcott, D. L., Wolcott, T. G., Hines, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+male+size+and+mating+history+on+sperm+content+of+ejaculates+of+the+blue+crab+Callinectes+sapidus.">Influence of male size and mating history on sperm content of ejaculates of the blue crab Callinectes sapidus.</a> 230, 235-240 (2002).</li><li>Sato, T., Ashidate, M., Goshima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+male+mating+frequency+and+male+size+on+ejaculate+size+and+reproductive+success+of+female+spiny+king+crab,+Parolithodes+brevipes.">Effects of male mating frequency and male size on ejaculate size and reproductive success of female spiny king crab, Parolithodes brevipes.</a> Mar. Ecol. Prog. Ser. 296, 251-262 (2005).</li><li>Sato, T., Goshima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impacts+of+male-only+fishing+and+sperm+limitation+in+manipulated+populations+of+an+unfished+crab,+Hapalogaster.">Impacts of male-only fishing and sperm limitation in manipulated populations of an unfished crab, Hapalogaster.</a> 313, 193-204 (2006).</li><li>Rondeau, A., Sainte-Marie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+mate-guarding+time+and+sperm+allocation+by+male+snow+crabs+(Chionoecetes+opilio)+in+response+to+sexual+competition,+and+their+impact+on+the+mating+success+of+females.">Variable mate-guarding time and sperm allocation by male snow crabs (Chionoecetes opilio) in response to sexual competition, and their impact on the mating success of females.</a> Biol. Bull. 201, 204-217 (2001).</li><li>Waddy, S. L., Aiken, D. E., Bauer, R. T., Martin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mating and insemination in the Ameri can lobster,Homarus americanus. Crustacean sexual behavior. , 126-144 (1990).</li><li>Johnson, K. J., Goldstein, J. S., Watson, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+methods+for+determining+the+fertility+status+in+early-stage+American+lobster,+Homarus+americanus,+eggs.">Two methods for determining the fertility status in early-stage American lobster, Homarus americanus, eggs.</a> J. Crust. Biol. 31, 693-700 (2011).</li><li>Aiken, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proecdysis,+setal+development,+and+molt+prediction+in+the+American+lobster+(Homarus).">Proecdysis, setal development, and molt prediction in the American lobster (Homarus).</a> 30, 1337-1344 (1973).</li><li>Aiken, D. E., Waddy, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cement+gland+development,+ovary+maturation,+and+reproductive+cycles+in+the+American+lobster+Homarus+americanus.">Cement gland development, ovary maturation, and reproductive cycles in the American lobster Homarus americanus.</a> J. Crust. Biol. 2, 315-327 (1982).</li><li>Pugh, T. L., Goldstein, J. S., Lavalli, K. L., Clancy, M., Watson, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=At-sea+determination+of+female+American+lobsters+(Homarus+americanus)+mating+activity:+Patterns+vs.+expectations.">At-sea determination of female American lobsters (Homarus americanus) mating activity: Patterns vs. expectations.</a> Fish. Res. 147, 327-337 (2013).</li><li>Bauer, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+trends+in+sperm+transfer+and+storage+complexity+in+decapod+crustaceans.">Phylogenetic trends in sperm transfer and storage complexity in decapod crustaceans.</a> J. Crust. Biol. 6, 313-325 (1986).</li><li>Talbot, P., Helluy, S., Factor, 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=Reproduction+and+embryonic+development.">Reproduction and embryonic development.</a> Biology of the Lobster: Homarus americanus. , 177-216 (1995).</li><li>Cooper, R. A., Uzmann, J. R., Cobb, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ecology of juvenile and adult Homarus. The biology and management of lobsters. 2, 97-142 (1980).</li><li>Estrella, B. T., McKiernan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch-per-unit-effort+and+biological+parameters+from+the+Massachusetts+coastal+lobster+(Homarus+americanus)+resource:+description+and+trends.">Catch-per-unit-effort and biological parameters from the Massachusetts coastal lobster (Homarus americanus) resource: description and trends.</a> NOAA Tech. Rep. NMFS. 81, (1989).</li><li>Waddy, S. L., Aiken, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+fertilization+and+consecutive+spawning+in+large+American+lobster,+Homarus+americanus.">Multiple fertilization and consecutive spawning in large American lobster, Homarus americanus.</a> Can. J. Fish. Aquat. Sci. 43, 2291-2294 (1986).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlantic+States+Marine+Fisheries+Commission+(ASMFC).+American+lobster+stock+assessment+report+for+peer+review.">Atlantic States Marine Fisheries Commission (ASMFC). American lobster stock assessment report for peer review.</a> ASMFC Stock Assessment. , (2009).</li></ol>

Os autores declaram não haver conflitos de interesse.

Este vídeo e papel esboço e demonstrar um método para determinar se as lagostas fêmeas acasalaram com sucesso. Esta abordagem é simples o suficiente para que ele possa ser utilizado para a amostragem em larga escala de lagostas no mar por uma variedade de utilizadores. O método baseia-se na detecção da presença de um spermatophore no receptáculo seminal que é transmitido a partir do sexo masculino para lagostas fêmea durante o acasalamento. Estes spermatophores parcialmente endurecer e formar um tampão de e...

<html> Lagosta americana (Homarus americanus) é uma das pescas mais produtivas do Atlântico Norte (~ 56 mil toneladas em 2011, avaliado em mais de $ 390000000) 1. No entanto, há uma falta geral de compreensão sobre a dinâmica reprodutiva da espécie em populações selvagens. Gerando estimativas mais precisas de produção reprodutiva, incluindo o número e tamanho de indivíduos participando ativamente na reprodução, pode melhorar o processo de avaliação de estoque. Por exemplo, as mulheres que são impedidos de alcançar seu pleno potencial reprodutivo, devido à limitação do esperma foram identificados como uma preocupação para vários crustáceos marinhos explorados comercialmente incluindo: lagostas, caranguejos azuis 2 3, rei caranguejos 4, caranguejos de pedra 5 e caranguejos da neve 6. O objetivo geral é determinar se a limitação de esperma pode ser um fator em certas regiões da pesca da lagosta americana. nt &quot;&gt; Os primeiros sinais de um potencial problema de limitação de esperma em lagostas foram observadas durante a realização de um estudo de marcação relacionado com lagostas fêmeas ovígeras (com visivelmente intactos, ovos recentemente extrudados). recapturar relatos de pescadores indicaram que aproximadamente 15% desses animais caiu suas desovas após apenas 1-2 meses. A hipótese de trabalho foi que algumas lagostas estavam carregando os ovos que não foram fertilizados e, como resultado, &#39;caiu&#39; 7. Dados preliminares de um estudo posterior confirmou o fato de que as lagostas vontade expulsar os ovos, mesmo que eles não tenham acasalado com sucesso e esses ovos não fertilizados só são realizadas para ~ 1 mês 8. Portanto, dada a observação de mulheres que levam os ovos não fertilizados em populações naturais, buscou-se determinar até que ponto a limitação de espermatozóides pode estar contribuindo para um nível submáximo de reprodução por lagostas sexualmente maduros. Para atingir este objetivo, a técnica foi desenvolvida para detectar a spermatophore depositado pelos machos durante o acasalamento. Este artigo e vídeo descrever um método não invasivo, simples desenvolvido para determinar o sucesso de acasalamento de lagostas do sexo feminino. A técnica pode ser utilizada de forma rápida e confiável no mar, ou a bordo de investigação ou de embarcações de pesca comercial. Os pormenores deste método de amostragem, bem como alguns resultados representativos são apresentado para ilustrar a aplicação da técnica.

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			<td height="40" style="height:40px;width:163px;">Calipers: Mitutoyo Digimatic</td>
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			<td style="width:231px;">http://www.globalindustrial.com</td>
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			<td style="width:231px;"></td>
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			<td height="40" style="height:40px;width:163px;">3 mL Luer-Lok&#8482; syringe</td>
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			<td style="width:135px;">309585</td>
			<td style="width:231px;">www.bd.com</td>
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			<td height="60" style="height:60px;width:163px;">Monoject aluminum hub blunt needles, 18-G x 1</td>
			<td style="width:129px;">Webster Veterinary</td>
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			<td height="40" style="height:40px;width:163px;">2.0 mL plastic storage tubes</td>
			<td style="width:129px;">Eppendorf&#160;</td>
			<td style="width:135px;">wu-06333-72</td>
			<td style="width:231px;">http://www.coleparmer.com</td>
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			<td height="40" style="height:40px;width:163px;">Styrofoam cooler with ice</td>
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			<td height="40" style="height:40px;width:163px;">Compound light microscope</td>
			<td style="width:129px;">Olympus&#160;</td>
			<td style="width:135px;">BH System</td>
			<td style="width:231px;">http://www.olympusamerica.com</td>
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			<td height="10" style="height:10px;width:163px;"></td>
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			<td height="40" style="height:40px;width:163px;">Glass sides with cover slip</td>
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			<td style="width:231px;">75 mm x 25 mm, ~ 1 mm thickness</td>
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			<td height="41" style="height:41px;width:163px;">Pipettor with disposable tips</td>
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a noninvasive method for in situ determination of mating success in female american lobsters (homarus americanus)

Apesar de ser uma das mais produtivas da pesca no Noroeste do Atlântico, muito permanece desconhecido sobre a dinâmica reprodutiva naturais de lagostas americanas. Trabalhos recentes em populações de crustáceos exploradas (caranguejos e lagostas) sugere que há circunstâncias em que fêmeas maduras não são capazes de alcançar o seu pleno potencial reprodutivo, devido à limitação do esperma. Para examinar esta possibilidade em diferentes regiões da pesca de lagosta americano, um método confiável e não invasivo foi desenvolvido para provar um grande número de lagostas fêmeas no mar. Este método envolve a inserção de uma agulha de ponta romba no receptáculo seminal da fêmea para determinar a presença ou ausência de um tampão de espermatozóides e para retirar uma amostra que pode ser examinada para a presença de esperma. Uma série de estudos de controle foram realizados no cais e no laboratório para testar a confiabilidade desta técnica. Estes esforços de amostragem envolveu 294 lagostas fêmeas para confirmar que a presença oplugue esperma fa era um indicador confiável de esperma dentro do recipiente e assim, o acasalamento. Este documento detalha a metodologia e os resultados obtidos a partir de um subconjunto da totalidade da amostra fêmeas. Dos 230 lagostas fêmeas amostradas de Banco de George e Cape Ann, MA (intervalo size = 71-145 mm de comprimento da carapaça), 90,3% foram positivas para o esperma. Explicações potenciais para a ausência de espermatozóides em algumas fêmeas incluem: imaturidade (falta de maturidade fisiológica), avaria da tomada de esperma depois de ser utilizado para fertilizar uma ninhada de ovos, e falta de atividade de acasalamento. As pesquisas indicam que esta técnica para analisar o sucesso de acasalamento de lagostas do sexo feminino é um proxy confiável que pode ser usado no campo para documentar atividade reprodutiva em populações naturais.

<html> Os dados aqui apresentados foram para um subconjunto de lagostas fêmeas que se originou a partir de ambos nearshore e offshore. O grupo nearshore consistiu de 44 lagostas que foram capturados nas águas costeiras perto de Cape Ann, Massachusetts e transportados em caixas térmicas para o laboratório para exame. Um total de 186 lagostas no mar foram capturados em Banco de George e amostrado a uma instalação de detenção comercial em New Hampshire. As amostras de esperma foram obtid...

Watch this Scientific Journal Video about A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus at JoVE.com

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Mudanças induzidas pela pesca para a pesca de crustáceos explorados, tais como a pesca de lagosta americano, poderia influenciar a sua dinâmica reprodutiva, levando a uma redução no sucesso de acasalamento. O objetivo deste estudo foi desenvolver um método não invasivo para determinar o sucesso de acasalamento de lagostas fêmeas que podem ser fisiologicamente ou funcionalmente maduro.

Um método não invasivo para<em&gt; Em situ</em&gt; Determinação do sucesso de acasalamento em fêmeas lagostas americanas (<em&gt; Homarus americanus</em&gt;)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

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A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

10.9K visualizações.  University of New Hampshire.  Mudanças induzidas pela pesca para a pesca de crustáceos explorados, tais como a pesca de lagosta americano, poderia influenciar a sua dinâmica reprodutiva, levando a uma redução no sucesso de acasalamento. O objetivo deste estudo foi desenvolver um método não invasivo para determinar o sucesso de acasalamento de lagostas fêmeas que podem ser fisiologicamente ou funcionalmente maduro. 

Vídeo: A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Uma técnica para Screen americana Beech para Resistência ao Beech Scale Insect (<em&gt; Cryptococcus fagisuga</em&gt; Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

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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, um método melhorado para Inex<em&gt; In Situ</em&gt; Medição de partículas e dissolvidos compostos Processado por alimentadores de suspensão ativa

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

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

Procedimento otimizado para determinar a adsorção de fosfonatos para Granular hidróxido férrico, usando um método de determinação de fósforo miniaturizado

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

In Situ Técnicas de hibridização para amostras de Coral adultas parafina

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

Um ensaio de amplificação isotérmica (lâmpada) mediada por Loop para identificação rápida de Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

Implantação de uma nova marca acústica Micro no juvenil Pacífico lampreia e enguia americana

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&amp;#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Uma plataforma telemétrica e gravimétrica para fenotipagem fisiológica em tempo real de interações vegetais-ambientais

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Mistletoe Eradicator - A Novel Tool for Simultaneous Mechanical and Chemical Control of Mistletoe

Some species of mistletoes, an aerial plant hemiparasites, play a Janus-like role in ecosystems by being rich biodiversity hotspots due to their trophic and topic associations with various organisms and at the same time acting as biological pirates, exhausting the host trees by stealing water and nutrients via the haustorium. Many crop and timber trees are attacked by mistletoes, for example, dwarf mistletoes parasitize conifers, representatives of genus Tapinanthus target guava, and Phoradendron mistletoe target pecans. Recently, an increasing infestation of the European mistletoe (Viscum album subsp. album L.) in monoculture stands of common walnuts (Juglans regia L.)&#160;was recorded for the first time during 2018 in different regions of Kashmir Valley, India. The common chemical approach based on the spraying of mistletoe exophytes with the growth regulators, ethephon and glyphosate, was not efficient since V. album resprouted in the next vegetation season. Hand pruning of mistletoe was time-consuming, and a polyethylene covering resulted in the localized rotting of host tissues. The most efficient approach to eradicate mistletoe was the use of a novel tool called Mistletoe Eradicator, which allows to use mechanical and chemical means of mistletoe control simultaneously. It consists of a manipulating pole with the terminal pruning saw having a container filled with the growth regulator, from which the liquid slowly drops onto the saw blade through the nozzle. It allows working both from the ground and in the tree canopy. After the targeted application of 5%-10% ethephon or glyphosate to the base of the just pruned V. album stems (stubs), exophyte resprouting was significantly reduced 9 months after the pruning as compared to the only pruned or sprayed exophytes. An alternative variant of Mistletoe Eradicator was constructed in Ukraine filled just with a food color additive to track the liquid dropping and was successfully tested.

The protocol describes an easy-to-handle tool called Mistletoe Eradicator which combines mechanical and chemical approaches and is efficient in the control of mistletoe infestation on the host trees. The manuscript uses the European mistletoe (Viscum album subsp. album L.) as an example.

Erradicador de Visco - Uma nova ferramenta para controle mecânico e químico simultâneo do Visco

Some species of mistletoes, an aerial plant hemiparasites, play a Janus-like role in ecosystems by being rich biodiversity hotspots due to their trophic ...

A Complete Method for Evaluating the Performance of Photocatalysts for the Degradation of Antibiotics in Environmental Remediation

Various antibiotics such as tetracycline, aureomycin, amoxicillin, and levofloxacin are found in large quantities in groundwater and soil systems, potentially leading to the development of resistant and multi-drug resistant bacteria, posing a threat to humans, animals, and environmental systems. Photocatalytic technology has attracted keen interest due to its rapid and stable treatment and direct use of solar energy. However, most studies evaluating the performance of semiconductor catalysts for the photocatalytic degradation of organic pollutants in water are currently incomplete. In this paper, a complete experimental protocol is designed to comprehensively evaluate the photocatalytic performance of semiconductor catalysts. Herein, rhombic dodecahedral silver phosphate was prepared by a simple solvent phase synthesis method at room temperature and atmospheric pressure. BrSubphthalocyanine/Ag3PO4 heterojunction materials were prepared by the solvothermal method. The catalytic performance of as-prepared materials for the degradation of tetracycline was evaluated by studying different influencing factors such as catalyst dosage, temperature, pH, and anions at atmospheric pressure using a 300 W xenon lamp as a simulated solar light source and a light intensity of 350 mW/cm2. Compared with the first cycle, the constructed BrSubphthalocyanine/Ag3PO4 maintained 82.0% of the original photocatalytic activity after five photocatalytic cycles, while the pristine Ag3PO4 maintained only 28.6%. The stability of silver phosphate samples was further tested by a five-cycle experiment. This paper provides a complete process for evaluating the catalytic performance of semiconductor catalysts in the laboratory for the development of semiconductor catalysts with potential for practical applications.

Presented here is a protocol to explore a universal set of experimental procedures for comprehensive laboratory evaluation of photocatalysts in the field of environmental purification, using the example of photocatalytic removal of antibiotic organic pollutant molecules from water by phthalocyanine sensitized silver phosphate composites.

Um método completo para avaliar o desempenho de fotocatalisadores na degradação de antibióticos na remediação ambiental

Various antibiotics such as tetracycline, aureomycin, amoxicillin, and levofloxacin are found in large quantities in groundwater and soil systems, ...

In Situ Soil Moisture Sensors in Undisturbed Soils

Soil moisture directly affects operational hydrology, food security, ecosystem services, and the climate system. However, the adoption of soil moisture data has been slow due to inconsistent data collection, poor standardization, and typically short record duration. Soil moisture, or quantitatively volumetric soil water content (SWC), is measured using buried, in situ sensors that infer SWC from an electromagnetic response. This signal can vary considerably with local site conditions such as clay content and mineralogy, soil salinity or bulk electrical conductivity, and soil temperature; each of these can have varying impacts depending on the sensor technology.
Furthermore, poor soil contact and sensor degradation can affect the quality of these readings over time. Unlike more traditional environmental sensors, there are no accepted standards, maintenance practices, or quality controls for SWC data. As such, SWC is a challenging measurement for many environmental monitoring networks to implement. Here, we attempt to establish a community-based standard of practice for in situ SWC sensors so that future research and applications have consistent guidance on site selection, sensor installation, data interpretation, and long-term maintenance of monitoring stations.
The videography focuses on a multi-agency consensus of best-practices and recommendations for the installation of in situ SWC sensors. This paper presents an overview of this protocol along with the various steps essential for high-quality and long-term SWC data collection. This protocol will be of use to scientists and engineers hoping to deploy a single station or an entire network.

In Situ Soil Moisture Sensors in Undisturbed Soils

The determination of soil water content is a critical mission requirement for many state and federal agencies. This protocol synthesizes multi-agency efforts to measure soil water content using buried in situ sensors.

In situ Sensores de umidade do solo em solos não perturbados

Soil moisture directly affects operational hydrology, food security, ecosystem services, and the climate system. However, the adoption of soil moisture ...