Research article Received: 13 August 2013 Revised: 24 February 2014 Accepted: 6 March 2014 Published online in Wiley Online Library: 1 April 2014 (wileyonlinelibrary.com) DOI 10.1002/jrs.4470 An analysis of bivalve larval shell pigments using micro-Raman spectroscopy Christine M. Thompson,a* Elizabeth W. North,a Sheri N. Whiteb and Scott M. Gallagerb Micro-Raman spectroscopy has been used on adult bivalve shells to investigate organic and inorganic shell components but has not yet been applied to bivalve larvae. It is known that the organic matrix of larval shells contains pigments, but less is known about the presence or source of these molecules in larvae. We investigated Raman spectra of seven species of bivalve larvae to assess the types of pigments present in shells of each species and how the ratio of inorganic : organic material changes in a dorso-ventral direction. In laboratory experiments, we reared larvae of three clam species in waters containing different organic signatures to determine if larvae incorporated compounds from source waters into their shells. We found differences in spectra and pigments between most species but found less intraspecific differences. A neural network classifier for Raman spectra classified five out of seven species with greater than 85% accuracy. There were slight differences between the amount and type of pigment present along the shell, with the prodissoconch I and shell margin areas being the most variable. Raman spectra of 1-day-old larvae were found to be differentiable when larvae were reared in waters with different organic signatures. With micro-Raman spectroscopy, it may be possible to identify some unknown species in the wild and trace their natal origins, which could enhance identification accuracy of bivalve larvae and ultimately aid management and restoration efforts. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: Raman spectroscopy; polyenes; bivalve larvae; chemotaxonomy; classification Introduction each genera being the result of both biological and environmental forces.[6] Recent work has suggested that the organization of the Most research on bivalve shell formation has investigated adult organic matrix of larval shells may differ between species because shells, leaving a gap in research involving bivalve larval shell birefringence patterns that reflect the orientation of aragonite microstructure. Pigments, often polyenes similar to carotenoids crystals in the organic matrix appear to be species-specific.[9–11] or porphyrins, have been identified in adult bivalve shells and Studies of mineralization of larval bivalves have traditionally – in pearls mineralized from bivalves.[1 3] The types of pigments focused on the calcium-carbonate portions of the PDI and PDII which exist in larval shells, how they are distributed, and their using electron microscopy and infrared spectroscopy. This re- origins remain unknown. Knowledge of the role of pigments in search focuses on a novel method for studying the content of the larval shell could enhance our current understanding of larval the shells of bivalve larvae: Raman spectroscopy. Raman spec- shell formation as well as provide applications in larval ecology troscopy is a nondestructive method that provides qualitative research, particularly aiding in identification of larvae in the field. structural information on a mixture of organic molecules in a Much of our current understanding of the larval shell structure sample. By radiating a sample with focused laser light, a spectrum comes from comparisons with adult shells. Unlike adult shells, is produced on the basis of the light scattering from the excited which can be either calcite or aragonite or a mixture of the two, functional groups in organic molecules. Raman spectroscopy has all bivalve larval shells consist of aragonite, a calcium-carbonate been employed to study pigments in calcium-carbonate material polymorph, embedded in an organic matrix.[4] Bivalve larvae start in various mollusk shells,[1,12,13] pearls from bivalves,[2,3] and out by secreting a shell made of amorphous calcium-carbonate, corals.[14,15] Pigments that have been identified in mollusk shells which then becomes a crystalline aragonite after a few days.[5] include substituted polyenes such as carotenoids, unsubstituted Whereas adults can have different shell ultrastructure based on polyenes, and porphyrins.[12] Most commonly, Raman spectra indi- species groups, larval shell ultrastructure observed using electron cate the presence of all-trans-polygenic pigments containing microscopy is similar between species indicating that larval shell formation is a highly conserved evolutionary process.[4–6] Larval shells have two main components: the youngest part of the shell, * Correspondence to: Christine M. Thompson, University of Maryland Center for prodissoconch I (PDI), which is secreted <24 h after fertilization, Environmental Science, Horn Point Laboratory, Cambridge, MD 21613, USA. and the prodissoconch II (PDII), which is secreted approximately E-mail: [email protected] 40 h after fertilization and contains regions of mineral between a University of Maryland Center for Environmental Science, Horn Point Labora- darker growth bands.[7] Genera of adult bivalves have different [8] tory, Cambridge, MD, 21613, USA organic matrix organizations reflecting a unidirectional miner- 349 alogical evolution at the superfamily level with divergences in b Woods Hole Oceanographic Institution, Woods Hole, MA, 02543, USA J. Raman Spectrosc. 2014, 45, 349–358 Copyright © 2014 John Wiley & Sons, Ltd. C. M. Thompson et al. chains of conjugated C=C bonds (i.e., polyenes). These pigment We compared the spectra from the PDI region to the later larval bands exhibit strong absorption due to coupling of electronic and shell, and we compared spectra between growth band and vibrational translations and can be detected at low concentrations mineral areas. The third part of the study tested whether PDI in the shells even among other biological materials.[3,16] Although shells of early larvae incorporate pigments from their surround- structurally similar to β-carotene and other natural carotenoids, ing environment. We reared three clam species in the laboratory these polyenes showed different spectral bands when compared in waters with different organic signatures to test if pigment with those of pure β-carotene, indicating that they have different peaks in Raman spectra differed between treatments and chain lengths, lack methyl groups (unsubstituted), and occur in thereby determine if early larvae that are not feeding might multiple combinations.[1–3,12,15,17] In bivalves and pearls, different incorporate pigment from surrounding waters into their shells. pigment peaks were seen in different colored regions of the specimens,[1,2] indicating that they are responsible for coloration. Different chain lengths, combinations, and relative proportions of Methods pigments can be responsible for coloration, but pigments can be detected in low concentrations due to resonance even if they do We carried out micro-Raman analysis on seven species of lab- not play a role in coloration.[3] In mollusks, polyenes likely form spawned or hatchery-spawned larvae and two species which were complexes with organic matrix proteins[3] and may play additional grown in experimental conditions. This analysis targeted the pig- roles stabilizing the aragonite crystals and mediating crystal- ment regions of the spectra to determine (1) whether pigments lization.[2] In corals, C=C bands of polyenes shifted to lower in the larval shell differed between species, (2) how pigment com- wavenumbers after demineralization, indicating interactions with position differed within a shell, and (3) if early PDI larval shells incor- the mineral constituents.[18] A Raman mapping study on an adult porate pigments from their surrounding environment. gastropod shell revealed thin peaks of polyenes associated with growth lines.[13] Similar molecules are present in bivalve larvae Differences between species and may have comparable roles (S.M. Gallager, unpub. data). Raman spectroscopy of bivalve larval shells could have useful We obtained 7-day-old (7 d) Argopecten irradians (bay scallop), applications for identification or tracing origins of bivalve larvae. Mercenaria mercenaria (hard clam), and Crassostrea virginica Although Raman spectroscopy has been applied as a chemotax- (eastern oyster) larvae from the Aquaculture Research Corpora- onomic method to distinguish microbial cells[19,20] and species of tion in Dennis, MA, in 2011 and 6–8 days Mulinia lateralis (coot lichens and other fungi,[21,22] it has not been tested for bivalve clam), Tagelus plebeius (razor clam), Ischadium recurvum (hooked larvae. In addition, it is not known if the pigment material in larval mussel), and Rangia cuneata (common rangia) larvae from shells could be associated with waters of their natal origin. If adults spawned in the laboratory at University of Maryland these pigments are associated with other organic molecules Center for Environmental Science, Horn Point Laboratory, in known to accumulate in growth bands,[23] we might see more 2009. Larval samples had been stored in ethanol or formalde- presence of pigments in this region. Raman spectra of fish oto- hyde prior to analysis. liths have shown higher organic signals in dark bands; however, Before the micro-Raman analysis, it was necessary