A Method for Extracting Pigments from Squid Doryteuthis pealeii

Summary

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

Abstract

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.

Introduction

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 chromatopho....

Protocol

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

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Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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