Name: a method for extracting pigments from squid doryteuthis pealeii
Uploaded: 2016-11-09T15:00:00
Description: Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

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 Counc...

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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.

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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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