The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

Invertebrate animal studies conducted herein are not regulated in the United States; therefore the Institutional Animal Care and Use Committee has no authority for review of such protocols. In lieu of these not falling under the jurisdiction of regulation in the United States, the authors hereby state that these studies were conducted with sincere effort towards the ethical use, care, and treatment of these animals, the number of individuals was minimized and these efforts are consistent with the Basel Declaration and the International Council for Laboratory Animal Science (ICLAS) ethics guidelines.
1. Doryteuthis pealeii (D. pealeii) Dissection
<ol>
	<li>Purchase decapitated squid D. pealeii from the Marine Resources Department at the Marine Biological Laboratory at Woods Hole, MA or from any fresh-fish market. Do not use specimens that have been filleted or previously altered in order to apply this procedure. Specifically, the chromatophore layer must still be intact on the specimen.
	<ol>
		<li>If the animals are not used immediately, store them at -20 &#176;C until use. To defrost the specimens, place them in room temperature water for 1 hr prior to dissection.</li>
	</ol>
	</li>
	<li>Using stainless steel straight fine-point dissection scissors, cut along the posterior side of the animal. Begin at the base of the ventral mantle, and end at the fin region.</li>
	<li>Remove all of the internal organs (stomach, gill, ink gland, reproductive organs, systemic heart, and brachial heart) by lifting them with dissecting forceps and severing their connective tissue using dissection scissors. Discard organs in a specimen bag.</li>
	<li>Use dissecting T-pins to pin the mantle (fin side up) in a standard 11-1/2&#34; x 7-1/2&#34; x 1-1/2&#34; aluminum dissection pan containing a flexible dissection pad. Submerge the tissue in 250 ml of seawater that has been filtered through a disposable polystyrene vacuum filtration system containing 0.2 &#181;m pore sizes.</li>
	<li>Carefully remove the epidermal skin layer (opaque color) with dissecting forceps, keeping the chromatophore containing skin layer intact. Once the epidermal layer is removed, discard it in a specimen bag.</li>
	<li>Dissect small (1 cm x 1 cm) sections of the chromatophore layer with forceps and dissection scissors. Collect samples in 1.5 ml micro-centrifuge tubes (fill one-half of each empty tube with the dissected tissue).</li>
	<li>Add 500 &#181;l of filtered seawater to the tissue containing tubes. The samples can be stored overnight at 4 &#176;C.</li>
</ol>
2. Isolating Chromatophore Pigment Granules
<ol>
	<li>Tissue Digestion
	<ol>
		<li>Preparation of Papain/collagenase
		<ol>
			<li>Weigh out 30 mg of collagenase and 5 mg of papain using a 4&#34; x 4&#34; low nitrogen weigh paper. Transfer these into a 50 ml polypropylene centrifuge tube with a screw cap.</li>
			<li>Measure 10 ml of deionized water using a graduated pipette. Add directly to vial containing papain/collagenase. Vortex on the highest setting until the solution is clear.</li>
		</ol>
		</li>
		<li>Removing Extracellular Tissue
		<ol>
			<li>Centrifuge the collected chromatophore tissue from step 1.7 for 5 min at 14,000 x g. Discard the top layer of sea water into a plastic waste bucket.</li>
			<li>Add 500 &#181;l of the papain/collagenase solution to the tissue using a P1000 variable volume single channel micropipette. Vortex each sample for 1-2 min on the highest setting.</li>
			<li>Sonicate the samples for 5 min at a frequency of 40 kHz to dissociate cells from the surrounding tissue.</li>
			<li>Centrifuge for 5 min at 14,000 x g. Discard the supernatant into a plastic waste bucket.</li>
			<li>Centrifuge the collected chromatophore tissue for 5 min at 14,000 x g. Discard the top layer of sea water into a plastic waste bucket. Repeat once again.</li>
			<li>Manually remove any remaining large tissue sections that did not digest in this process with forceps before proceeding to the next step.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Isolating the Pigment Granules
	<ol>
		<li>Preparation of Homogenization Buffer
		<ol>
			<li>Weigh 2.38 g of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES, 100 mM), 0.095 g of magnesium chloride (10 mM), 0.856 g of potassium-aspartate (50 mM), and 0.015 g of dithiothreitol (1 mM). Transfer into a 150 ml polystyrene container with a screw cap. Add one commercial mini tablet protease inhibitor.</li>
			<li>Measure 100 ml of deionized water using a graduated cylinder. Add directly to the container containing the homogenization salts. Vortex until the solution is clear.</li>
		</ol>
		</li>
		<li>Homogenization of Pigment Granules
		<ol>
			<li>Centrifuge each sample from step 2.1 at 14,000 x g for 5 min. Discard the supernatant into a plastic waste bucket. This sample should be void of extracellular tissue.</li>
			<li>Add 500 &#181;l of homogenization buffer to each tube and vortex for 1-2 min each to thoroughly mix the samples. Sonicate the samples for 30 min (~40 kHz) and follow with 5 min of centrifugation at 14,000 x g.</li>
			<li>Discard the supernatant into a plastic waste bucket, and repeat the previous step 2-3 times to ensure the complete digestion of the chromatophore sacculus and protein tethers. The remaining solution should contain a light yellow supernatant and a red-colored pellet containing the isolated pigment granules.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Pigment Extraction
<ol>
	<li>Preparation of Acidic Methanol (HCl-MeOH)
	<ol>
		<li>Carefully add 25 &#181;l of concentrated (36.5-38 % (w/w)) hydrochloric acid (HCl) to 5 ml of methanol (MeOH). Store the HCl-MeOH solution in a screw cap glass sample vial. Swirl gently to uniformly mix the solution. 
		NOTE: The more acidic the solution, the more pigment will be extracted during the first extraction.</li>
	</ol>
	</li>
	<li>Extraction of Pigment
	<ol>
		<li>Centrifuge the pigment granule suspension (from 2.2.2) for 5 min at 14,000 x g and discard the supernatant into a plastic waste bucket.</li>
		<li>Add 500 &#181;l of the HCl-MeOH solution and vortex each sample for ~3-4 min. Sonicate the samples for 10 min (~40 kHz), then centrifuge for 5 min at 14,000 x g.</li>
		<li>Using a transfer pipette, collect the supernatant in a 1 dram screw-top glass vial. The supernatant should be dark red in color.</li>
		<li>Repeat the extraction step 3.2.2 until no further color is extracted (~4-5 washes). 
		NOTE: Each extraction will yield ~1.5 ml of pigment. The remaining colorless pellet is the pigment extracted granules. NOTE: Both colorless granules and extracted pigments can be stored in water at 4 &#176;C until further use.</li>
	</ol>
	</li>
</ol>
4. Characterization throughout Extraction Process
<ol>
	<li>Scanning Electron Microscopy
	<ol>
		<li>Image the granules (unreacted from step 2.2.1 and pigment-extracted from step 3.2.3) using scanning electron microscopy (SEM) to verify the purity of the extraction process.</li>
		<li>To prepare granule samples for imaging, first centrifuge both unreacted granules and pigment extracted granules for 5 min at 14,000 x g, and discard the supernatant into a plastic waste bucket. Re-suspend these in 1 ml of ethanol, and deposit 2-3 drops of the granule suspension onto aluminum SEM stubs using a transfer pipette.</li>
		<li>After drying the samples overnight, sputter coat with gold-platinum coating for 3 min to achieve a 15 nm coating.</li>
		<li>Image using a SEM with a Schottky emitter and a secondary electron detector. Use a 5-7 kV beam voltage and a working distance between 8 and 9 mm to image these materials.</li>
	</ol>
	</li>
	<li>Ultraviolet-visible (UV-vis) Spectrophotometric Measurements
	<ol>
		<li>Monitor the absorbance profiles for all 3 conditions (unreacted granule from 2.2.2, extracted pigment from 3.2.3, and pigment extracted granules from 3.2.4) using a dual beam spectrophotometer from 200-800 nm.</li>
		<li>Collect a blank measurement using deionized water in a 1 ml cuvette for the unreacted granules and the pigment extracted granules. Collect UV-Vis absorption spectra for both samples.</li>
		<li>Collect a blank measurement using the acidic methanol solution in a 1 ml cuvette for the extracted pigment. Collect UV-Vis absorption spectrum for sample.</li>
		<li>Determine maximum wavelength of each sample by identifying the wavelength at which the absorbance peak reaches its maximum relative height.</li>
	</ol>
	</li>
</ol>

<ol><li>Bell, G. R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatophore+radial+muscle+fibers+anchor+in+flexible+squid+skin%5BTitle%5D)+AND+%22Invertebr+Biol%22%5BJournal%5D)">Chromatophore radial muscle fibers anchor in flexible squid skin.</a> Invertebr Biol. 132 (2), 120-132 (2013).</li>
<li>Crookes, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reflectins%3A+The+unusual+proteins+of+squid+reflective+tissues%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Reflectins: The unusual proteins of squid reflective tissues.</a> Science. 303 (5655), 235-238 (2004).</li>
<li>Deravi, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure-function+relationships+of+a+natural+nanoscale+photonic+device+in+cuttlefish+chromatophores%5BTitle%5D)+AND+%22J.+R.+Soc.+Interface%22%5BJournal%5D)">The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores.</a> J. R. Soc. Interface. 11 (93), 1-9 (2014).</li>
<li>Mäthger, L. M., Denton, E. J., Marshall, N. J., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+and+behavioural+functions+of+structural+coloration+in+cephalopods%5BTitle%5D)+AND+%22J.+R.+Soc.+Interface%22%5BJournal%5D)">Mechanisms and behavioural functions of structural coloration in cephalopods.</a> J. R. Soc. Interface. 6 (2), 149-163 (2009).</li>
<li>Mäthger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomical+basis+for+camouflaged+polarized+light+communication+in+squid%5BTitle%5D)+AND+%22Biol.+Lett%22%5BJournal%5D)">Anatomical basis for camouflaged polarized light communication in squid.</a> Biol. Lett. 2 (4), 494-496 (2006).</li>
<li>Mäthger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Malleable+skin+coloration+in+cephalopods%3A+selective+reflectance%2C+transmission+and+absorbance+of+light+by+chromatophores+and+iridophores%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Malleable skin coloration in cephalopods: selective reflectance, transmission and absorbance of light by chromatophores and iridophores.</a> Cell Tissue Res. 329 (1), 179-186 (2007).</li>
<li>Cloney, R. A., Brocco, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatophore+Organs%2C+Reflector+Cells%2C+Iridocytes+and+Leucophores+in+Cephalopods%5BTitle%5D)+AND+%22Amer.+Zool%22%5BJournal%5D)">Chromatophore Organs, Reflector Cells, Iridocytes and Leucophores in Cephalopods.</a> Amer. Zool. 23 (3), 581-592 (1983).</li>
<li>Hanlon, R. T., Messenger, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adaptive+coloration+in+young+cutttlefish+%28Sepia+officinalis+L%29-+The+morphology+and+development+of+body+patterns+and+their+relation+to+behaviour%5BTitle%5D)+AND+%22Philos+Trans+R+Soc+Lond+B+Biol+Sci%22%5BJournal%5D)">Adaptive coloration in young cutttlefish (Sepia officinalis L)- The morphology and development of body patterns and their relation to behaviour.</a> Philos Trans R Soc Lond B Biol Sci. 320 (1200), 437-487 (1988).</li>
<li>Sutherland, R. L., Mäthger, L. M., Hanlon, R. T., Urbas, A. M., Stone, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cephalopod+coloration+model.+II.+Multiple+layer+skin+effects%5BTitle%5D)+AND+%22J.+Opt.+Soc.+Am%22%5BJournal%5D)">Cephalopod coloration model. II. Multiple layer skin effects.</a> J. Opt. Soc. Am. 25 (8), 2044-2054 (2008).</li>
<li>Florey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructure+and+function+of+cephalopod+chromatophores%5BTitle%5D)+AND+%22Amer.+Zool%22%5BJournal%5D)">Ultrastructure and function of cephalopod chromatophores.</a> Amer. Zool. 9 (2), 429-442 (1969).</li>
<li>Florey, E., Kriebel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+and+mechanical+responses+of+chromatophore+muscle+fibers+of+squid%2C+Loligo+opalescens%2C+to+nerve+stimulation+and+drugs%5BTitle%5D)+AND+%22Z.+Vergl.+Physiol%22%5BJournal%5D)">Electrical and mechanical responses of chromatophore muscle fibers of squid, Loligo opalescens, to nerve stimulation and drugs.</a> Z. Vergl. Physiol. 65 (1), 98-130 (1969).</li>
<li>Williams, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+of+Phenoxazone-Based+Pigments+to+the+Structure+and+Function+of+Nanostructured+Granules+in+Squid+Chromatophores%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Contributions of Phenoxazone-Based Pigments to the Structure and Function of Nanostructured Granules in Squid Chromatophores.</a> Langmuir. , (2016).</li>
<li>Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ommochrome+pigmentation+of+the+linea+and+rosa+seasonal+forms+of+Precis+coenia+%28Lepidoptera%3A+Nymphalidae%29%5BTitle%5D)+AND+%22Arch.+Insect.+Biochem.+Physiol%22%5BJournal%5D)">Ommochrome pigmentation of the linea and rosa seasonal forms of Precis coenia (Lepidoptera: Nymphalidae).</a> Arch. Insect. Biochem. Physiol. 36 (3), 215-222 (1997).</li>
<li>Van Den Branden, C., Decleir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Study+of+the+Chromatophore+Pigments+in+the+Skin+of+the+Cephalopod+Sepia+Officinalis%5BTitle%5D)+AND+%22L.+Biol.+Jb.+Dodonaea%22%5BJournal%5D)">A Study of the Chromatophore Pigments in the Skin of the Cephalopod Sepia Officinalis.</a> L. Biol. Jb. Dodonaea. 44 (2), 345-352 (1976).</li>
<li>Aubourg, S., Torres-Arreola, W., Trigo, M., Ezquerra-Brauer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+Jumbo+Squid+Skin+Pigment+Extract+and+its+Antioxidant+Potential+ina+Marine+Oil+System%5BTitle%5D)+AND+%22Eur.+J.+Lipid+Sci.+Technol%22%5BJournal%5D)">Characterization of Jumbo Squid Skin Pigment Extract and its Antioxidant Potential ina Marine Oil System.</a> Eur. J. Lipid Sci. Technol. 118 (0), (2016).</li>
</ol>

We have nothing to disclose.

We have demonstrated a method to extract pigments from squid chromatophore granules. By specifically targeting the granules, our goal is to determine their role in mediating adaptive coloration. This method differs from previous reports designed to characterize cephalopod pigments using bulk tissue samples14 or freeze-dried skin15.
While this protocol is effective at extracting chromatophore pigments, it is limited to small reaction scales. Dissecting one squid yields ~11...

Cephalopods such as squid, cuttlefish, and octopus have the ability to dynamically alter their appearance for camouflaging and signaling.1-6 This capability is supported in part by the selective areal expansion of pigmented organs known as chromatophores.4,7-9 Chromatophores are soft actuators that contain a network of nanostructured pigment granules confined within a cytoelastic sacculus that is anchored radially by muscle fibers.1,3 As they are actuated, chromatophores expand by 500% in presented surface area distributing the granules throughout the organ.3,7,10,11 When this action is concerted over a number of chromatophores, the overall coloration of the animal is changed. While it is known that the pigment granules contribute to this color change, their composition remains unknown. We describe a procedure to isolate and purify chromatophore pigments which may be adapted for future compositional studies.
The isolation of pigment granules involves a multistep extraction, homogenization, and purification procedure.3,12 Chromatophore containing tissue is harvested through careful extirpation from the cephalopod. A digestion and homogenization process is then used to dissociate the surrounding tissue and separate the chromatophore cells. The nanostructured granules are then isolated and purified from the remaining chromatophores using repeated sonication and centrifugation. Upon purification, pigments are extracted from the granules in a process that is adapted from the extraction of visible color from butterfly wings using acidic methanol solutions.13 Scanning electron microscopy (SEM) and spectrophotometry are used to confirm that the chromatophore pigments are successfully extracted using this process.
This method describes the isolation of chromatophore granules which is used to explore the molecular contributions to coloration in cephalopods.12 Small molecule extractions from whole animals can often be a long and tedious process. The aim here is to inform future researchers of an effective and facile protocol for the acquisition of pigments from the nanostructured granules in cephalopods.

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

Chromatophores are dissected from the D. pealeii dorsal mantle (Figure 1A, 1B). Once they are removed, chromatophores are lysed and purified using centrifugation and washing cycles to isolate out the pigmented granules (Figures 2A, 2B). Acidic methanol solutions (HCl-MeOH) are used to extract the pigment from the granules (Figure 2C), yielding a soluble pigment extract and insoluble, colorless pi...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

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Biology

9.4K Views.  University of New Hampshire. A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

Video: A Method for Extracting Pigments from Squid Doryteuthis pealeii

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.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

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

Environment

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Genetics

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Chemistry

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

Biochemistry

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 &micro;m mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 &deg;C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Immunology and Infection

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Engineering

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

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Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish