<I>Spirula Spirula</I>

BULLETIN OF MARINE SCIENCE, 78(2): 389–391, 2006 NOTE DNA from beacH-WasHed SHells of THE Ram’S Horn SQuid, SPIRULA SPIRULA Jan Strugnell, Mark Norman, and Alan Cooper Fresh tissue samples of cryptic, rare, and deep-sea molluscan species are often difficult, if not impossible, to obtain for molecular studies. Although museums can provide preserved tissue samples of many rare species, those collected prior to the 1980s are primarily formalin fixed, making the extraction and amplification of DNA improbable. Improvements in the extraction and amplification of DNA from bone, feathers, hair, and soil have led to studies boasting 120 bp of DNA obtained from samples over 400,000 yrs old (Willerslev et al., 2003). The successful application of such DNA extraction techniques to dried molluscan samples (e.g., shells, cuttlebones, opercula) obtained directly from the environment would greatly increase the suite of species readily available for molecular studies. The Ram’s Horn squid, Spirula spirula (Linnaeus, 1758) is such a species. Spirula spirula is a mesopelagic resident of the open ocean in the tropical Atlantic and Indo- Pacific regions (Norman, 2000). It possesses a planispiral and chambered internal calcareous shell. Beach-washed S. spirula shells are commonly found on tropical shorelines throughout their range. However, complete samples of the animal are rarely stranded and the shells never contain external remains of the dead animal. Spirula spirula is the sole member of the family Spirulidae (Owen, 1836). The phylogenetic position of this unusual species is the subject of debate with molecular studies linking it to the suborder Oegopsida (open-eyed squids; Bonnaud et al., 1997; Carlini et al., 2000; Lindgren et al., 2004) or the family Sepiidae (cuttlefishes; Bon- naud et al., 1997; Carlini et al., 2000; Strugnell et al., 2005). The aim of this study was to attempt to extract DNA from beach-washed shells of S. spirula. Shells of S. spirula were collected from the shoreline at Mission Beach in northern Queensland by the second author (M.N.) in 1999. These shells contained no external remnants of flesh. Spirula spirula shells are chambered structures used for buoyancy control. They are divided internally by transverse septa, which create a series of separate gas compartments within the shell. As in chambered nautili and cuttlefishes, buoyancy is mediated by osmotic transport of fluid in and out of the chambers. Chambers are joined by a common living tissue thread known as the siphuncle. Chambers are also lined by a thin layer of tissue known as the pellicle, which aids in transporting water to the siphuncle by capillary action. This study aimed to extract DNA from residual tissue of siphuncle and pellicle in beach-dried S. spirula shells. Care was taken to select unbroken shells of S. spirula as it was thought that the compartments within these shells may be less susceptible to DNA contamination from other marine organisms compared with broken shells. In June 2001, the shells were washed with 70% ethanol prior to extraction in an attempt to remove any con- taminating DNA present on the external surfaces of these samples. Approximately 0.5 g of S. spirula shells were crushed using a mortar and pestle, placed in 20 mL of 0.5 M EDTA (pH 8.0) (in order to decalcify tissues) and agitated overnight at room temperature. Following centrifugation the resulting pellet was used as a starting “tis- Bulletin of Marine Science 389 © 2006 Rosenstiel School of Marine and Atmospheric Science of the University of Miami 390 BULLETIN OF MARINE SCIENCE, VOL. 78, NO. 2, 2006 sue” sample in an extraction protocol modified from Winnepenninckx et al. (1993) and Carlini and Graves (1999). The pellet was placed in a 2000 µL “O-ring” tube containing 500 µL of isolation buffer (50 mM EDTA, 50 mM Trishydroxymethyl amonomethane [Tris], 150 mM NaCl, pH 8.0), 60 µL of 10% sodium dodecyl sulfate [SDS], 10 µL of 10 mg ml−1 ribonuclease A and 10 µL of 25 mg ml−1 proteinase K and was incubated overnight at 37 °C. Subsequently, 10 µL of hexadecyltrimethylammo- niumbromide [CTAB] buffer (10% w/v CTAB, 0.7 M NaCl) was added to the samples, which were then incubated for 20 min at 65 °C and cooled to room temperature. 350 µL of saturated NaCl was added and samples were vortexed at high speed for 3 min and mixed slowly for an additional 15 min on a rotating mixer. The solution was extracted once with phenol, once with phenol:chloroform (1:1), and once with chloroform. Centricons were used (according to the manufacturer’s instructions) to concentrate and desalt the isolated DNA. 12S rDNA was chosen as the target gene because it is a mitochondrial gene and therefore occurs in a greater copy number in cells than nuclear DNA. Furthermore, 12S rDNA has been shown to be extracted from “ancient DNA” in previous studies (Willerslev et al., 2003). 12S rDNA sequences from six species: Loligo forbesi Steen- strup, 1856 [AY545099]; Sepia officinalis Linnaeus, 1758 [AY545098]; Sepia phara- onis Ehrenberg, 1831 [AY616875], Lolliguncula brevis (Blainville, 1823) [AY616867], Sepioteuthis lessoniana Férussac, 1831 in Lesson, 1830–1831 [AY616869], and Octo- pus bimaculoides Pickford and McConnaughey, 1949 [AY545086]) were aligned by eye and the primer, 12S′Spirulabck (5′AAC TTA AAA ARY TTG GCG GT 3′), was designed in a conserved region. This primer was used in conjunction with the 12Sa forward primer (5′AAG AGC GAC GGG CGA TAT GTA 3′) for DNA amplification and sequencing (Simon, 1990). DNA amplification (hotstart 94° C for 2 min, then 35 cycles of denaturation 94 °C for 40 s, annealing 50 °C for 40 s, extension 72 °C for 1.5 min) produced a single band of 310 bp. The amplified product was purified using the QiaGen PCR purification kit Q( iagen Ltd., U.K.). The sequencing reaction was performed on both forward and reverse strands using a PRISM BigDye terminator v3 cycle sequencing ready reaction kit (Applied Biosystems, U.K.). A clean sequence of 310 bp was sequenced on an ABI 3700 according to the manufacturer’s instructions (DNA Sequencing Facility, Zoology Department, Oxford University; AY935647). Over a year after this sequence was obtained, a fresh tissue sample of S. spirula was kindly donated by K. Warnke (Institut für Geologische Wissenschaften Paläontolo- gie, Berlin). The sample was collected from Fuerteventura on 17 September 2002. A 0.1 g sample of this tissue was used as a starting tissue sample for DNA extraction and was extracted using the methods outlined above. The primers 12Sa and 12Sb (Si- mon, 1990) were used to amplify and sequence a 399 bp fragment of 12S rDNA. The resulting sequence (AY545097) was identical to that sequenced a year earlier from the S. spirula shells, thereby validating this sequence and extraction procedure. This finding has significant implications for future molluscan phylogenetic studies. It is likely that DNA from other mitochondrial and also nuclear genes can be extracted from S. spirula shells, thereby removing the necessity of expensive trawling expeditions in order to obtain fresh tissue samples. Furthermore, it is possible that similar DNA extraction techniques can also be applied to other cephalopod shells (i.e., cuttlebones and Nautilus shells.) NOTES 391 Acknowledgments We are especially grateful to K. Warnke for donating Spirula spirula tissue used in this study. This project was funded by the Rhodes Trust and the BBSRC (43/G16942). Literature Cited Bonnaud, L., R. Boucher-Rodoni, and M. Monnerot. 1997. Phylogeny of cephalopods inferred from mitochondrial DNA sequences. Mol. Phy. Evol. 7: 44–54. Carlini, D. B. and J. E. Graves. 1999. Phylogenetic analysis of cytochrome oxidase I sequences to determine higher-level relationships within the coleoid cephalopods. Bull. Mar. Sci. 64: 57–76. ___________, K. S. Reece, and J. E. Graves. 2000. Actin gene family evolution and the phylogeny of coleoid cephalopods (Mollusca: Cephalopoda) Mol. Biol. Evol. 17: 1353–1370. Lindgren, A. R., G. Giribet, and M. K. Nishiguchi. 2004. A combined approach to the phylogeny of Cephalopoda (Mollusca). Cladistics 20: 454–486. Norman, M. 2000. Cephalopods. A world guide. Conch Books, Hackenheim. 318 p. Simon, C. 1990. Evolution of mitochondrial ribosomal RNA in insects as shown by the poly- merase chain reaction. Mol. Evol. 235–244. Strugnell, J., M. Norman, J. Jackson, A. J. Drummond, and A. Cooper. 2005. Molecular phylogeny of coleoid cephalopods (Mollusca: Cephalopoda) using a multigene approach; the effect of data partitioning on resolving phylogeneies in a Bayesian framework. Mol. Phy. Evol. 37: 426–441. Willerslev, E., A. J. Hansen, J. Binladen, T. B. Brand, M. T. P. Gilbert, B. Shapiro, M. Bunce, C. Wiuf, D. A. Gilichinsky, and A. Cooper. 2003. Diverse plant and animal DNA from Holo- cene and Pleistocene sedimentary records. Science. 300: 791–795. Winnepenninckx, B., T. Backeljau, and R. De Watcher 1993. Extraction of high molecular weight DNA from molluscs. Trends in Genet. 9: 407. Date Submitted: 3 December, 2004. Date Accepted: 6 April, 2005. Addresses: (J.S., A.C.) Molecular Evolution, Department of Zoology, South Parks Road, Ox- ford OX1 3PS, U.K. (M.N.) Museum Victoria, Melbourne, Victoria, 3001, Australia. Corre- sponding Author: (J.S.) Marine Systems Research Group, School of Biology and Biochem- istry, Queen’s University, Belfast, BT9 7BL, U.K. Current Address: (J.S.) British Antarctic Survey, High Cross, Madingley Rd., Cambridge CB4 0ET, U.K. E-mail: <[email protected]> or <[email protected]>..

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