Onların işbirliği çok takdir - Yazarlar olan tekneler biz örnekleme için izin verildi, tüm New England ticari lobstermen teşekkür etmek istiyorum. Buna ek olarak, biz Little Bay Lobster Şirketi (Newington, NH) ve bize kendi tesislerinde örnekleme yapmak izin Champlains Seafood (Point Judith, RI) teşekkür ederim. Aşağıdaki UNH öğrenciler bu çalışma boyunca değerli yardım sağladı: Haley Beyaz, Françoise Morrison, Sarah HAVENER, Audra CHAPUT ve Mayıs grose. Bu proje whw (proje # R/CFR-11) ve JSG bir UNH Deniz Programı Hibe NH SeaGrant hibe tarafından desteklenmiştir.

Kısım A: Alan Örnekleme Tekniği 1.. Kadın Ölçümler <ol><li> Her dişi ıstakoz, karapas uzunluğu (CL) ve kaliperler bir çift kullanarak en yakın 1.0 mm, ikinci karın segmentinin genişliğini ölçmek. NOT: (dişiler yumurta taşıyan) karın genişliği cinsel olgunluk göstergesidir çünkü ikinci karın segmenti ölçülür. Buna ek olarak, (adım 1.2), daha sonra üreme aşaması ile, bu veriler için gerekli dökücü sahne tespit edilmesi avantajlı olabilir. </li><li> Karın pleopods birinde (İstakozlarda standart bir metodolojik protokol) kapalı bir küçük distal bölümünü Klip ve daha sonra bir diseksiyon mikroskobu 9 görüntüleme için temiz deniz suyunda pleopod saklayın. NOT: Bu adım aynı zamanda dişiler yumurtalarını 10 a&#39;ya hazırlanıyor bir göstergesidir pleopods, çimento bezlerinin varlığını ortaya çıkarabilir. </li></ol> 2. İğne Insertion <ol><li> (18, G Tablo 1 e bakınız), şırınga iğnesi kaydedin ~ 45 ° (Şekil 1) ° &#39;de, kuyruk doğru açılı seminal deponun içine 1 cm. Not: önce seminal deponun altındaki sperm en yüksek konsantrasyonu (Şekil 1) ulaşmak için sperm tamponundan itilir gibi dokunulmamış sperm fişli ıstakoz, iğne, sokma esnasında bir direnç karşılayabilir. NOT: fiş varlığı, kendi içinde, ıstakozlar evlendirilen olduğunu güvenilir bir göstergedir. Bazı hayvanlarda hiçbir spermatofor mevcut olabilir, çünkü çok az direnç olabilir, spermatofor henüz bir fiş içine sertleştirilmiş edilmemiştir, ya da daha önce yumurta bir veya daha fazla kavramaları dölleme için kullanılmış olabilir küçüktür. </li></ol><img alt="Şekil 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; Şekil 1. İğne ekleme ve ıstakoz seminal kabın görüntüle. Sperm örneği ayıklamak için ıstakoz seminal yuvasına künt uçlu iğne takma. İğnenin açısı ve derinliği de tutarlı bir numune elde etmek için kritiktir. Sağ: disseke ve dikey olarak bölünmüş olan bir seminal deponun göster. Spermatozoa deponun dibinde (derin noktası) seviyesinde yer alan ise sperm fiş, kabın en üst ya da en dış kısmını kaplar. Bu bir derinlikten numune kabını delinerek kan kirlenmiş numunesi neden olabilir dikkat etmek önemlidir. <a href="https://www.jove.com/files/ftp_upload/50498/50498fig1highres.jpg" target="_blank">resmi büyütmek için buraya tıklayın</a> . 3. Sperm Tak Penetran <ol><li> Başlangıçtaki yerleştirme sonrası, (vücut eksenine dik olarak) yaklaşık dikey iğne açısını yükselmek ve çok yavaş çalışmaya başlaraşağı kabın içine. </li><li> Uç kuyruk doğru hafifçe işaret edilmektedir, böylece belirli bir açıda eğimli iğne tutun. Sonunda matris içine nüfuz kadar bu durumda, iğne ucu, tıpanın üst sıkıştırma edilir. </li><li> Direnç kez daha hissedilir kadar yavaş yavaş aşağıya doğru itmek için iğne devam - bu kabın alt ve numune alınır burada. </li></ol> 4. Sperm Kaldırma <ol><li> Haznenin dibinden bir örnek (çoğunlukla katı fiş madde, sıvı, sperm ve diğer sıvı) çıkarın ve 2.0 ml işaretlenmiş bir plastik tüpe örnek yatırmak. </li><li> Soğuk deniz suyu ~ 0.1-0.5 ml yuvasını yıkayın. </li><li> Aynı tüp içerisinde sıvı ve yeri, yaklaşık 0.3 ml çıkarın. </li><li> Buz üzerinde mağaza sperm numuneleri laboratuvarda incelenir kadar. Bölüm B: Lab Sperm Örneklerinin İncelenmesi Not: Bireysel l seminal kapların kaldırılır Örnekler<html> obsters sperm hücreleri mevcut veya yok olmadığını belirlemek için muayene edilmelidir. </li><li> Her plastik tüp, bir kapak kayma ile bir cam slayt üzerinde bir yerde, ve bir bileşik mikroskop ile 100X at görünümünde ~ sıvının 50 ul çıkarın. </li><li> Gibi örnekler Puan: kıt sayıda, ya da hiç sperm (bu çalışmada gerçek sperm sayıları, Şekil 2 ölçmek vermedi). </li></ol><img alt="Şekil 2," fo:content-width="5in" fo:src="/files/ftp_upload/50498/50498fig2highres.jpg" src="/files/ftp_upload/50498/50498fig2.jpg" width="600px" /> Şekil 2. Istakoz sperm örneği görüntüler. 40X büyütmede bir bileşik mikroskop ile bakıldığında, bir dişinin seminal priz alınan ıstakoz sperm örneklerinin Görüntüler. Soldaki resim, diğer (sağ), seyrek bir örneği (ölçek bar = 10 mikron) göstermektedir ise çok sayıda sperm bulunan bir örnek gösterir.. Jpg &quot;target =&quot; _blank &quot;&gt; büyük resmi görebilmek için buraya tıklayın.

<ol>
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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. 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Yazarlar çıkar çatışması beyan ederim.

Bu video ve kağıt anahat ve dişi ıstakoz başarıyla evlendirilen varsa belirlemek için bir yöntem ortaya koymaktadır. Bu yaklaşım kullanıcıların çeşitli denizde ıstakoz büyük ölçekli örnekleme için kullanılabilir olması yeterince basittir. Bu yöntem çiftleşme sırasında erkek ikinci dişi ıstakoz geçirilen seminal hazne içinde bir spermatofor varlığını tespit dayanmaktadır. Bu spermatophores kısmen da hazne içinde yatırılır kısa bir süre sonra, bir sperm fiş halinde sertleşme...

<html> Amerikan ıstakoz (Homarus americanus) Kuzey Atlantik (~ $ 390,000,000 üzerinde değerli 2011 yılında 56.000 mt) 1 en verimli balıkçılık biridir. Ancak, vahşi popülasyonlar bu türün üreme dinamikleri konusunda anlayış genel bir eksikliği vardır. Aktif üreme katılan bireylerin sayısı ve boyutları da dahil olmak üzere üreme çıkış daha doğru tahminler, üreten bir stok değerlendirme sürecini iyileştirmek olabilir. Dikenli ıstakoz 2, mavi yengeç 3, kral yengeç 4, taş yengeçler 5 ve kar yengeç: Örneğin, nedeniyle sperm sınırlaması tam üreme potansiyelini elde engellenir kadın dahil olmak üzere birçok ticari istismar deniz kabuklular için bir endişe olarak tespit edilmiştir 6. Genel amaç sperm sınırlama yanı sıra Amerikan ıstakoz balıkçılığın belirli bölgelerinde bir faktör olabilir eğer belirlemektir. nt &quot;&gt; (gözle görülür sağlam, son zamanlarda sıkılır yumurta) ovigerous kadın ıstakoz ile alakasız bir etiketleme çalışması yapılırken İstakozlarda potansiyel bir sperm sınırlama sorununun ilk belirtileri gözlendi. ~ bu hayvanların% 15 düşmüş olduğu belirtilen balıkçıların raporları Recapture onların yumurta kavramaları sadece 1-2 ay sonra. çalışma hipotezi bazı ıstakoz döllenmiş olmasaydı yumurta taşıma ve, sonuç olarak, &#39;düştü&#39; 7., bir sonraki çalışmasının ön veriler gerçeği doğruladı olduğunu olduğunu ıstakoz olacak başarıyla evlendirilen değil ve bu döllenmemiş yumurta, sadece ~ 1 ay 8 uygulansa bile yumurta a&#39;ya. Dolayısıyla, doğal popülasyonlarının döllenmemiş yumurta taşıyan dişilerin gözlem verilen, biz sperm sınırlama katkıda olabilir için kapsamını belirlemek için çalıştı cinsel olgun ıstakozlarına üreme bir submaksimal düzeyde. Bu hedefe ulaşmak için, teknik spermatositlerde algılamak için geliştirilmiştirPhore çiftleşme sırasında erkek tarafından yatırılır. Bu kağıt ve video kadın ıstakoz çiftleşme başarısını tespit etmek için geliştirilen bir invaziv olmayan, basit bir yöntem. Teknik denizde, ya araştırma ya da ticari balıkçı teknelerinde hızlı ve güvenilir şekilde kullanılabilir. Bu numune alma yöntemin ayrıntıları yanı sıra bazı temsili bulgular tekniğinin uygulanmasını göstermek için sunulmuştur.

<table><tbody><tr><td>Calipers</td><td>Mitutoyo Digimatic</td><td>500-196-20</td><td>http://www.globalindustrial.com</td></tr><tr><td>3 ml Luer-Lok syringe</td><td>Beckton-Dickinson</td><td>309585</td><td>www.bd.com</td></tr><tr><td>Monoject aluminum hub blunt needles, 18 G x 1</td><td>Webster Veterinary</td><td>8881202348</td><td>www.mywebstervet.com</td></tr><tr><td>2.0 ml Plastic storage tubes</td><td>Eppendorf</td><td>wu-06333-72</td><td>http://www.coleparmer.com</td></tr><tr><td>Styrofoam cooler with ice</td><td></td><td></td><td></td></tr><tr><td>Compound light microscope</td><td>Olympus</td><td>BH System</td><td>http://www.olympusamerica.com</td></tr><tr><td>Glass sides with cover slip</td><td></td><td></td><td>75 mm x 25 mm, ~1 mm thickness</td></tr><tr><td>Pipettor with disposable tips</td><td></td><td></td><td></td></tr></tbody></table>

a noninvasive method for in situ determination of mating success in female american lobsters (homarus americanus)

Kuzeybatı Atlantik&#39;te en verimli balıkçılık biri olmasına rağmen, çok Amerikan ıstakoz doğal üreme dinamikleri hakkında bilinmemektedir. Sömürülen kabuklu nüfus (yengeç ve ıstakoz) son çalışmalar olgun kadın nedeniyle sperm sınırlaması tam üreme potansiyeline ulaşmak mümkün durumlar vardır olduğunu göstermektedir. Amerikan ıstakoz balıkçılık farklı bölgelerinde bu olasılığı incelemek için, güvenilir ve ağrısız bir yöntem denizde kadın ıstakoz çok sayıda örnekleme için geliştirilmiştir. Bu yöntem, bir sperm fiş varlığını veya yokluğunu belirlemek için ve sperm varlığı açısından incelenebilir bir örnek geri alma dişinin seminal deponun içine bir küt uçlu bir iğne ekleme içerir. Kontrol çalışmalarında bir dizi bu tekniğin güvenilirliğini test etmek için dock ve laboratuarda yapılmıştır. Bu çabalar 294 kadın ıstakoz onaylamak için örnekleme getirdiğini varlığı ofa sperm fiş güvenilir bir kap içinde sperm göstergesidir ve dolayısıyla, çiftleşme oldu. Bu kağıt metodoloji ve örneklenen toplam kadın bir alt elde edilen sonuçları göstermektedir. George Bankası ve Cape Ann, MA örneklemeyi 230 kadın ıstakoz (boyut aralığı = Kabuk uzunluğu olarak 71-145 mm),% 90.3 sperm için pozitiftir. Bazı kadınlarda sperm yokluğu için potansiyel açıklamalar şunlardır: gelişmemişlik (fizyolojik olgunluk eksikliği), yumurtası döllemek için kullanılan sonra sperm fiş dökümünü ve çiftleşme aktivite eksikliği. Anketler kadın ıstakoz çiftleşme başarısını incelemek için bu teknik doğal popülasyonlarının üreme aktivitesini belgelemek için alanda kullanılabilecek güvenilir bir vekil olduğunu göstermektedir.

<html> Burada bildirilen veriler nearshore ve offshore yerlerde hem kökenli kadın ıstakoz bir alt kümesi için vardı. Nearshore grup Cape Ann, Massachusetts yakın kıyı sularında yakalanan ve inceleme için laboratuvara soğutucularda taşınan 44 ıstakoz oluşuyordu. 186 deniz ıstakoz toplam George Bank yakalanan ve New Hampshire&#39;da bir ticari tutma tesiste örneklenmiştir. Sperm numuneleri yukarıda tarif edilen yöntemlere göre tüm hayvanların elde edilmiş ve hemen sperm...

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

Böyle Amerikan ıstakoz balıkçılık gibi sömürülen kabuklu balıkçılık için balıkçılık kaynaklı değişimler, potansiyel çiftleşme başarısında düşüşe neden üreme dinamiklerini etkileyebilir. Bu çalışmanın amacı, fizyolojik veya işlevsel olgun olabilir dişi ıstakoz çiftleşme başarısını tespit etmek için non-invaziv bir yöntem geliştirmek için oldu.

A Noninvaziv Yöntemi<em&gt; In situ</em&gt; Kadın American Istakoz&#39;ların çiftleşme Başarı (Belirlenmesi<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 Görüntüleme.  University of New Hampshire.  Böyle Amerikan ıstakoz balıkçılık gibi sömürülen kabuklu balıkçılık için balıkçılık kaynaklı değişimler, potansiyel çiftleşme başarısında düşüşe neden üreme dinamiklerini etkileyebilir. Bu çalışmanın amacı, fizyolojik veya işlevsel olgun olabilir dişi ıstakoz çiftleşme başarısını tespit etmek için non-invaziv bir yöntem geliştirmek için oldu. 

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

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Genç tatlı su midye içinde in Situ maruz kalma yöntemlerle inci için Bioindication akış ortamı uygunluğu test

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Çevre

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Bireysel Tigriopus kopepodların ve kantitatif analiz dostum koruyan davranışlarının kültür

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

Cohoused suda yaşayan hayvanların uzun süreli Video izleme: Norveç ıstakoz (Nephrops norvegicus) ve günlük Lokomotor aktivite bir vaka çalışması

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Impedance Pneumography for Minimally Invasive Measurement of Heart Rate in Late Stage Invertebrates

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by environmental temperature, this has the potential to alter thermal physiological performance in a variety of marine organisms. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography to understand how cardiac performance in late stage invertebrates changes under acute thermal stress. The protocol presents a minimally invasive technique that allows for real-time collection of heart rate during a temperature ramping experiment. Data are easily manipulated to generate an Arrhenius plot that is used to calculate Arrhenius break temperature (ABT), the temperature at which heart rate begins to decline with increasing temperatures. This technique can be used in a variety of late stage invertebrates (i.e., crabs, mussels, or shrimps). Although the protocol focuses solely on the impact of temperature on cardiac performance, it can be modified to understand the potential for additional stressors (e.g., hypoxia or hypercapnia) to interact with temperature to influence physiological performance. Thus, the method has potential for wide-ranging applications to further understand how marine invertebrates respond to acute changes in the environment.

Measuring heart rate during a thermal challenge provides insight into physiological responses of organisms as a consequence of acute environmental change. Using the American lobster (Homarus americanus) as a model organism, this protocol describes the use of impedance pneumography as a relatively noninvasive and nonlethal approach to measure heart rate in late stage invertebrates.

Geç Evre Omurgasızlarda Kalp Hızının Minimal İnvaziv Ölçümü İçin Empedans Pnömografisi

Temperatures in oceans are increasing rapidly as a consequence of widespread changes in world climates. As organismal physiology is heavily influenced by ...

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Tortricidae Zararlı Böceklerinde Kütle Yetiştirme ve Moleküler Çalışmalar

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Ölçme sperm rehberlik ve motilite içinde Caenorhabditis elegans hermafrodit üreme yolu

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Gelişim Biyolojisi

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

Gastropod Molluskların Yumurta döflenmesi erkek Aksesuar Bezi Ürünlerinin Etkisi

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

Biyoloji

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

A Whole Dağı<em&gt; In Situ</emGastropoda yumuşakça için&gt; Hibridizasyon Yöntemi<em&gt; Lymnaea stagnalis</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Anbürlerde Ovaryalı Izleme ve kontrol için üreme teknikleri

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, the species has undergone a drastic decline since the mid-1960s. The cause of decline is unknown, and research is actively underway to address this knowledge gap. These efforts are hindered by an inability to sex the species in hand, which to date was only possible using molecular markers. Here, we present a protocol to sex loggerhead shrikes by visually analyzing the coloration and pattern in the sixth primary feather. The application of the method will facilitate our ability to identify threats on a finer scale than has been possible to date and to address various ecological and evolutionary hypotheses. The methodology is simple and results reliable&#8211;we encourage including this method for research of both in situ and ex situ populations.

Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern

We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather.

Görsel Sexing Loggerhead Shrike (Lanius Ludovicianus) Plumage Renklendirme ve Desen kullanarak

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, ...

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