Conus Marmoreus Research Articles

Characterization of the Native Disulfide Isomers of the Novel χ-Conotoxin PnID: Implications for Further Increasing Conotoxin Diversity.

χ-Conotoxins are known for their ability to selectively inhibit norepinephrine transporters, an ability that makes them potential leads for treating various neurological disorders, including neuropathic pain. PnID, a peptide isolated from the venom of Conus pennaceus, shares high sequence homology with previously characterized χ-conotoxins. Whereas previously reported χ-conotoxins seem to only have a single native disulfide bonding pattern, PnID has three native isomers due to the formation of different disulfide bond patterns during its maturation in the venom duct. In this study, the disulfide connectivity and three-dimensional structure of these disulfide isomers were explored using regioselective synthesis, chromatographic coelution, and solution-state nuclear magnetic resonance spectroscopy. Of the native isomers, only the isomer with a ribbon disulfide configuration showed pharmacological activity similar to other χ-conotoxins. This isomer inhibited the rat norepinephrine transporter (IC50 = 10 ± 2 µM) and has the most structural similarity to previously characterized χ-conotoxins. In contrast, the globular isoform of PnID showed more than ten times less activity against this transporter and the beaded isoform did not display any measurable biological activity. This study is the first report of the pharmacological and structural characterization of an χ-conotoxin from a species other than Conus marmoreus and is the first report of the existence of natively-formed conotoxin isomers.

Anti-Ovarian Cancer Conotoxins Identified from Conus Venom.

Conotoxins constitute a treasury of drug resources and have attracted widespread attention. In order to explore biological candidates from the marine cone snail, we isolated and identified three novel conopeptides named as Vi14b, Vi002, Vi003, three conotoxin variants named as Mr3d.1, Mr3e.1, Tx3a.1, and three known conotoxins (Vi15a, Mr3.8 and TCP) from crude venoms of Conus virgo, Conus marmoreus and Conus texile. Mr3.8 (I-V, II-VI, III-IV) and Tx3a.1 (I-III, II-VI, IV-V) both showed a novel pattern of disulfide connectivity, different from that previously established for the µ- and ψ-conotoxins. Concerning the effect on voltage-gated sodium channels, Mr3e.1, Mr3.8, Tx3a.1, TCP inhibited Nav1.4 or Nav1.8 by 21.51~24.32% of currents at semi-activated state (TP2) at 10 μmol/L. Certain anti-ovarian cancer effects on ID-8 cells were exhibited by Tx3a.1, Mr3e.1 and Vi14b with IC50 values of 24.29 µM, 54.97 µM and 111.6 µM, respectively. This work highlights the role of conotoxin libraries in subsequent drug discovery for ovarian cancer treatment.

Isolation and characterization of five novel disulfide-poor conopeptides from Conus marmoreus venom.

Background: Conopeptides from cone snail venom have aroused great interest related to the discovery of novel bioactive candidates, due to their excellent prospects for the treatment of various health problems such as pain, addiction, psychosis and epilepsy. In order to explore novel biopeptides, we investigated the structure and function of five novel conopeptides isolated from the venom of Conus marmoreus from South China Sea.Methods: C. marmoreus crude venom was prepared, fractionated and purified by HPLC system. The primary sequences of the five novel disulfide-poor conopeptides Mr-1 to Mr-5 were identified by comprehensive analysis of de novo MALDI-TOF tandem mass spectrometry and Edman degradation data. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their biological effects at several heterologous rat nicotinic acetylcholine receptor (nAChR) subtypes (α1β1δε, α3β2, α3β4, α4β2) were determined by electrophysiological technique.Results: Five novel disulfide-poor conopeptides were identified and named as follows: Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPP NTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. The sequences of conopeptides Mr-2 to Mr-5 do not belong to any category of the known disulfide-poor conopeptides. No significant activity against the above nAChR subtypes were observed for the five conopeptides at 100 µM. Conclusion: We purified and structurally characterized five novel disulfide-poor conopeptides from C. marmoreus crude venom and first investigated their nAChR inhibitory effects. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from this cone snail venom.

'Messy' Processing of χ-conotoxin MrIA Generates Homologues with Reduced hNET Potency.

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus, the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that “messy” processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.

The Marbled Cone, Conus marmoreus, stranded on Nama beach, Yonaguni-jima, Yaeyama Islands, southwestern Japan

The Marbled Cone, Conus marmoreus, stranded on Nama beach, Yonaguni-jima, Yaeyama Islands, southwestern Japan

In silico Characterization and Docking of ?-Conotoxin Mr1.7a from Conus marmoreus Targeting Neuronal nAChR ?3?2

In silico Characterization and Docking of ?-Conotoxin Mr1.7a from Conus marmoreus Targeting Neuronal nAChR ?3?2

Crystallography and Textural Aspects of Crossed Lamellar Layers in Arcidae (Bivalvia, Mollusca) Shells

Bivalve shell microstructures are important traits that can be used for evolutionary and phylogenetic studies. Here we examine the crossed lamellar layers forming the shells of the arcoids, Arca noae, Glycymeris glycymeris and Glycymeris nummaria in order to better understand the crystallography of this complex biomaterial. Textural aspects and crystallography of the outer crossed lamellar layer of these species have been clarified using high-resolution electron microscopy and X-ray diffraction (XRD) techniques. These shells are made of aragonite crystals in a crossed lamellar arrangement with a high preferred crystal orientation (texture). The distribution of maxima in the pole figures implies that there is not a single crystallographic pattern within the measured area, but a continuous variation between two classes of distinct patterns. In the first of these, there is a set of four crystal orientations (referred to as upper set). These four crystal orientations are distributed in two pairs (which are coplanar), with the crystals of each pair being twinned on {110}. The pairs are tilted with respect to each other by approximately 20-40º around an axis perpendicular to the {110} common twinning plane. In the second pattern (referred to as lower set), the crystal orientations of each pair are rotated around a <–110> direction until the a-axes of the diametrically opposing crystal orientations of two different twinned pairs become parallel. As a result of this rotation, in the lower set the crystal orientations no longer form pairs twinned on {110}.These crystallographic relationships are unknown in inorganic aragonite. Our results are similar to those reported for the neogastropod Conus marmoreus. Thus, the common crossed lamellar crystallography in the arcoid bivalves and in C. marmoreus is a striking example of convergence in the development of crossed lamellar microstructures.

Will jumping snails prevail? Influence of near-future CO2, temperature and hypoxia on respiratory performance in the tropical conchGibberulus gibberulus gibbosus

Tropical coral reef organisms are predicted to be especially sensitive to ocean warming because many already live close to their upper thermal limit, and the expected rise in ocean CO2 is proposed to further reduce thermal tolerance. Little, however, is known about the thermal sensitivity of a diverse and abundant group of reef animals, the gastropods. The humpbacked conch (Gibberulus gibberulus gibbosus), inhabiting subtidal zones of the Great Barrier Reef, was chosen as a model because vigorous jumping, causing increased oxygen uptake (ṀO2 ), can be induced by exposure to odour from a predatory cone snail (Conus marmoreus). We investigated the effect of present-day ambient (417-454 µatm) and projected-future (955-987 µatm) PCO2 on resting (ṀO2 , rest) and maximum (ṀO2 , max) ṀO2 , as well as ṀO2 during hypoxia and critical oxygen tension (PO2 , crit), in snails kept at present-day ambient (28°C) or projected-future temperature (33°C). ṀO2 , rest and ṀO2 , max were measured both at the acclimation temperature and during an acute 5°C increase. Jumping caused a 4- to 6-fold increase in ṀO2 , and ṀO2 , max increased with temperature so that absolute aerobic scope was maintained even at 38°C, although factorial scope was reduced. The humpbacked conch has a high hypoxia tolerance with a PO2 , crit of 2.5 kPa at 28°C and 3.5 kPa at 33°C. There was no effect of elevated CO2 on respiratory performance at any temperature. Long-term temperature records and our field measurements suggest that habitat temperature rarely exceeds 32.6°C during the summer, indicating that these snails have aerobic capacity in excess of current and future needs.

An efficient transcriptome analysis pipeline to accelerate venom peptide discovery and characterisation

Transcriptome sequencing is now widely adopted as an efficient means to study the chemical diversity of venoms. To improve the efficiency of analysis of these large datasets, we have optimised an analysis pipeline for cone snail venom gland transcriptomes. The pipeline combines ConoSorter with sequence architecture-based elimination and similarity searching using BLAST to improve the accuracy of sequence identification and classification, while reducing requirements for manual intervention. As a proof-of-concept, we used this approach reanalysed three previously published cone snail transcriptomes from diverse dietary groups. Our pipeline method generated similar results to the published studies with significantly less manual intervention. We additionally found undiscovered sequences in the piscovorous Conus geographus and vermivorous Conus miles and identified sequences in incorrect superfamilies in the molluscivorus Conus marmoreus and C. geographus transcriptomes. Our results indicate that this method can improve toxin detection without extending analysis time. While this method was evaluated on cone snail transcriptomes it can be easily optimised to retrieve toxins from other venomous animals.

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes.

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I β-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

Isolation, purification and functional characterization of alpha-BnIA from Conus bandanus venom

We report the isolation and characterization by proteomic approach of a native conopeptide, named BnIA, from the crude venom of Conus bandanus, a molluscivorous cone snail species, collected in the South central coast of Vietnam. Its primary sequence was determined by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman's degradation of the pure native fraction. BnIA was present in high amounts in the crude venom and the complete sequence of the 16 amino acid peptide was the following GCCSHPACSVNNPDIC*, with C-terminal amidation deduced from Edman's degradation and theoretical monoisotopic mass calculation. Sequence alignment revealed that its –C1C2X4C3X7C4– pattern belongs to the A-superfamily of conopeptides. The cysteine connectivity of BnIA was 1–3/2–4 as determined by partial-reduction technique, like other α4/7-conotoxins, reported previously on other Conus species. Additionally, we found that native α-BnIA shared the same sequence alignment as Mr1.1, from the closely related molluscivorous Conus marmoreus venom, in specimens collected in the same coastal region of Vietnam. Functional studies revealed that native α-BnIA inhibited acetylcholine-evoked currents reversibly in oocytes expressing the human α7 nicotinic acetylcholine receptors, and blocked nerve-evoked skeletal muscle contractions in isolated mouse neuromuscular preparations, but with ∼200-times less potency.

MrIC, a Novel α-Conotoxin Agonist in the Presence of PNU at Endogenous α7 Nicotinic Acetylcholine Receptors

α-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Their high selectivity and affinity for the various subtypes of nAChRs have led to significant advances in our understanding of the structure and function of these key ion channels. Here we report the discovery of a novel 4/7 α-conotoxin, MrIC from the venom duct of Conus marmoreus, which acts as an agonist at the endogenous human α7 nAChR in SH-SY5Y cells pretreated with PNU120596 (PNU). This unique agonist activity of MrIC at α7 nAChRs may guide the development of novel α7 nAChR modulators.

Systematic interrogation of the Conus marmoreus venom duct transcriptome with ConoSorter reveals 158 novel conotoxins and 13 new gene superfamilies

BackgroundConopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining.ResultsHere we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies.ConclusionsTaken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.

Expression and secretion of functional recombinant μO-conotoxin MrVIB-His-tag in Escherichia coli

μO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.

High accuracy mass spectrometry comparison of Conus bandanus and Conus marmoreus venoms from the South Central Coast of Vietnam

Cone snail (genus Conus) venoms provide a rich source of small bioactive peptides known as conopeptides or conotoxins, which are highly interesting in pharmacological studies for new drug discovery. Conus species have evolved expressing a variety of conopeptides, adapted to the biological targets of their own specific preys at their living environments. Therefore, the potential proteomic evaluation of Conus venom components, poorly studied, is of great interest. Early studies supposed about 5% overlap in venom peptides from different Conus species. In this study, we compare using nano-liquid chromatography coupled with electrospray ionisation-mass spectrometry and bioinformatics, the molluscivorous Conus bandanus venom to that of its close-relative Conus marmoreus of the South Central Coast of Vietnam. With this approach, we demonstrate with high precision that 92 common conopeptides are present in the venom of the two mollusc-hunting cone snails, representing 24.4% (out of 376 peptides) and 18.4% (out of 499 peptides) of C. bandanus and C. marmoreus components, respectively. The proteomic comparison of the two close-relative interspecies suggests both common and different strategies for mature conopeptide production in the two species. The overall estimation of putative conopeptide disulphide bridges reveals 75% and 61% of “disulphide-rich” peptides in C. bandanus and C. marmoreus venom components, respectively. The same amino acid sequence for Bn1.1 and Mr1.1, determined at the genomic level, was also found in the two venoms, besides other common conopeptides. Confidently, the broader distribution of C. bandanus compared to C. marmoreus guarantee new opportunities for discovering conopeptides with original pharmacological properties.

Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom

Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.

Comparative analysis of proteases in the injected and dissected venom of cone snail species

The venom of cone snails has been the subject of intense studies because it contains small neuroactive peptides of therapeutic value. However, much less is known about their larger proteins counterparts and their role in prey envenomation. Here, we analyzed the proteolytic enzymes in the injected venom of Conus purpurascens and Conus ermineus (piscivorous), and the dissected venom of C. purpurascens, Conus marmoreus (molluscivorous) and Conus virgo (vermivorous). Zymograms show that all venom samples displayed proteolytic activity on gelatin. However, the electrophoresis patterns and sizes of the proteases varied considerably among these four species. The protease distribution also varied dramatically between the injected and dissected venom of C. purpurascens. Protease inhibitors demonstrated that serine and metalloproteases are responsible for the gelatinolytic activity. We found fibrinogenolytic activity in the injected venom of C. ermineus suggesting that this venom might have effects on the hemostatic system of the prey. Remarkable differences in protein and protease expression were found in different sections of the venom duct, indicating that these components are related to the storage granules and that they participate in venom biosynthesis. Consequently, different conoproteases play major roles in venom processing and prey envenomation.

Biophysical properties of Nav1.8/Nav1.2 chimeras and inhibition by µO‐conotoxin MrVIB

Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming α-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na(v) 1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na(v) α-subtypes in neurons, Na(v) 1.8 is most strongly inhibited by the µO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na(v) 1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na(v) 1.8 and the TTX-S Na(v) 1.2 using a domain exchange strategy. Wild-type and chimeric Na(v) channels were expressed in Xenopus oocytes, and depolarization-activated Na⁺ currents were recorded using the two-electrode voltage clamp technique. MrVIB (1 µM) reduced Na(v) 1.2 current amplitude to 69 ± 12%, whereas Na(v) 1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na(v) 1.8. A similar reduction in Na⁺ current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na(v) 1.8. In contrast, MrVIB had only a small effect on Na⁺ current for chimeras containing the corresponding region of Na(v) 1.2. Taken together, these results suggest that domain II of Na(v) 1.8 is an important determinant of MrVIB affinity, highlighting a region of the α-subunit that may allow further nociceptor-specific ligand targeting.

Diversity and evolution of conotoxins in Conus virgo, Conus eburneus, Conus imperialis and Conus marmoreus from the South China Sea

The venom peptides of cone snails are encoded by a large gene family, and can selectively bind to voltage-gated ion channels (Na+, K+ and Ca2+ channels) and to membrane receptors (nAChR, 5-HT3R, NMDAR). To identify novel conotoxin genes and analyze the evolution of typical conotoxin superfamily genes from different Conus species, we have constructed cDNA libraries derived from the venom ducts of Conus virgo, Conus eburneus, Conus imperialis and Conus marmoreus, which were collected from the South China Sea. 1312 transcripts from four Conus venom duct cDNA libraries were analyzed and 38.7–49.6% of the transcripts encoded conotoxin sequences. In addition to known conotoxins, 34 novel conotoxins have been identified and can be classified into eleven superfamilies, some of which showed unique patterns of cysteines or different signal peptide sequences. The evolutionary trees of T- and A-superfamily conotoxins were analyzed. Likelihood approaches revealed that T-superfamily conotoxins from the four Conus species undergo positive selection, mostly located in the mature toxin region. These findings contribute to a better understanding of the diversity and evolution of conotoxins from the South China Sea, and some novel conotoxins are valuable for further functional investigations.

Crystallographic relationships in the crossed lamellar microstructure of the shell of the gastropod Conus marmoreus

The crossed lamellar microstructure of mollusk shells shows a very complex hierarchical architecture constituted of long rod-shaped aragonite crystals stacked parallel to each other inside each first order lamella, which are almost perpendicular to the ones contained in parallel neighboring lamellae. To better understand the construction and properties of the crossed lamellar microstructure we have performed a detailed study to determine the crystallographic characteristics and their evolution during shell growth using scanning electron microscopy, transmission electron microscopy and X-ray diffraction texture analysis. The arrangement of crystals is rationalized by a set of twin law relationships between aragonite crystals. Specifically, the aragonite rods, or third order lamellae within each first order lamella, internally consist of polysynthetic twins bounded by {1 1 0} mirror planes. In turn, the polysynthetically twinned aragonite crystals also show a constant crystallographic orientation with respect to aragonite crystals in adjacent first order lamellae. It can be seen as another twin law in which crystals from adjacent lamellae are bounded by (1 1 0) planes but with their c-axes rotated within this plane by 30°. Thus there are two sets of twin laws that relate crystal units at lower (third order lamellae) and higher (first order lamellae) length scales. These hierarchical relationships play a crucial role in the construction, organization and properties of this complex microstructure. The later orientational relationships have never been described in geological aragonite and are only found in biogenic materials with a crossed lamellar microstructure. Their occurrence is probably determined by the presence of shell organic components which regulate crystal growth and may favor unusual crystallographic relationships.