Comprehensive notes on alternative fish structures for microchemical chronologies (TEA/SIA)

Endoskeleton (up to 40% calcified)

• Composition and structure
  • Bone is a composite of organic and inorganic components.
  • Organic matrix: ~90% collagen fibrils providing the scaffold; proteins like glycoproteins and osteocalcin are associated with the fibrils.
  • Inorganic matrix: hydroxyapatite (bioapatite, Ca10(PO4)6(OH)2) deposited on collagen, providing stiffness and acting as an ion reservoir.
  • Mineral content also includes carbonate, hydroxide, sulfate, fluoride, and small amounts of Na, K, Mg, Cl.
  • Cells involved in bone turnover: osteoblasts (form collagen and mineralize), osteocytes (regulate resorption when embedded in bone), osteoclasts (resorb bone).
  • In teleosts, there are cellular bones (with osteoblasts/osteocytes) and acellular bone (osteoblast-derived matrix with few/no osteocytes).
  • Collagen organization varies with growth: woven-fiber bone (fast growth), parallel-fiber/pseudo-lamellar bone (intermediate growth), lamellar bone (slow growth).
• Turnover, remodeling, and aging implications
  • Bone acts as an ionic reservoir and participates in homeostasis; turnover rates depend on metabolism and bone type.
  • Remodeling: resorption by osteoclasts followed by deposition of secondary bone; cementing lines can indicate remodeling events.
  • Collagen turnover in fishes appears slow, but turnover can be metabolism-dependent and vary by bone type; some studies suggest slower turnover in collagen but experimental turnover for d15N in juvenile bone collagen can show faster turnover than previously assumed (heart < bone collagen < blood < muscle; juvenile growth context).
  • Turnover rates for Ca, Sr in bone are slow relative to other tissues; turnover of bone collagen d15N can be tissue- and age-dependent; juvenile rapid growth may shorten apparent records.
• TEA (trace-element analysis) and SIA (stable-isotope analysis)
  • TEA on endoskeletons used to classify geographic origin and to distinguish wild vs. cultured fish; Sr isotopes can reflect ambient water or diet contributions to bone.
  • Sr incorporation pathways: diet and ambient water both contribute; temperature and salinity can influence skeletal Sr incorporation.
  • Commonly studied elements: Sr, P, Ca, K, Zn, Ba, Mn, Cu, Pb, Cd; some studies also measure d18O in bone for diadromous migrations and hydrologic history.
  • SIA on bone collagen: stable C and N isotopes (δ13C, δ15N) record trophic level and basal resource use; δ13C and δ15N enrichment in bone collagen compared to other tissues varies by species and environment.
• Advantages and considerations
  • Advantages: bone has high mineral content; suitable for TEA/SIA; robust matrix enables long-term records; can inform geographic origin and migratory histories; non-lethal options exist when sampling non-pathological specimens (though bone sampling can be destructive).
  • Considerations: remodeling and resorption can confound chronology; vascularized bone may show turnover that reduces temporal resolution; need species-specific turnover data and standardized demineralization and lipid-extraction protocols for comparability.
  • Practical notes: management of bone remodeling is essential to correctly interpret age and movement from TEA/SIA; cement lines and secondary bone must be distinguished from primary bone.
• Selected findings and examples
  • Sr-based differentiation of salmon populations by vertebral Sr; diadromous migrations inferred from Sr in vertebrae; diet and water contributions to Sr incorporation documented.
  • Direct relationships observed between water and skeletal concentrations for Mn, Zn, Pb, Ba, Cd in several species; Mg deficiency can alter Ca and Na concentrations, reflecting ion transport coupling.
  • Remodeling events and resorption can re-distribute elements; interpretation must consider whether measured elements reflect the depositional age.
• Key equations and concepts
  • Bioapatite mineral: $\text{Ca}<em>{10}(\text{PO}</em>4)<em>6(\text{OH})</em>2$ is the primary mineral phase in bone; trace elements substitute for Ca in the hydroxyapatite lattice.
  • Isotope ratios in bone collagen follow trophic enrichment; typical enrichment (per trophic step) for nitrogen is in the order of $\Delta\delta^{15}N \approx 2.0\,\text{to}\,4.0\,\text{‰}$; for carbon in collagen, \Delta\delta^{13}C \approx 3.9\,\text{to}\,5.3\,\‰ (context-dependent, species-specific).

Fin spines (30–35% calcified)

• Structure and physiology
  • Fin spines are anterior, unsegmented, rigid, and more calcified than soft fin rays; composed of dermal bone with hydroxyapatite as the main mineral phase.
  • Can incorporate 1–3 fin-ray substructures into the spine; metabolic pathways for element incorporation resemble those of vertebrae.
  • Growth via vascularization and inward-outward resorption; annuli can be obscured by resorption, particularly in highly active species (epipelagic taxa like billfishes and tunas).
  • Fin spine resorption and vascularization can limit chronological microchemical analyses by destroying early-life annuli.
• TEA (trace-element analysis)
  • Used to study stock structure, movement, environmental histories, and pollutant exposure.
  • Techniques include LA-ICP-MS and X-ray-based methods; development of matrix-matched standards for spine analysis (e.g., hydroxyapatite pellets) for LA-ICP-MS.
  • Examples: Sr concentrations in spines linked to marine-derived water sources in sturgeon; TEA used to classify catfish origins and to differentiate wild vs. farmed individuals; temporal variation observed in analyte concentrations along spines of tuna species.
• SIA (stable-isotope analysis)
  • TEA and SIA have been applied to fin spines; SIA of organic matrix in spines remains less explored; d13C and d18O used in some studies (classification of capture locations in catfish and other species).
• Advantages and considerations
  • Advantages: non-lethal sampling; suitable when otoliths cannot be collected (e.g., endangered species or management concerns); strong potential for stock-structure and environmental-history assessments.
  • Considerations: vascularization and resorption can erase early annuli; potential for external contamination during storage/handling; careful cleaning (e.g., 5% nitric acid wash) can remove external contaminants.
  • Temporal persistence of water chemistry differences is required for robust environmental-history inferences.
• Key points and examples
  • Fin spines provide a pathway to non-lethal TEA/SIA and have shown correlation with vertebral chemistry in juveniles and adults; TEA used to track migratory histories in sturgeon and to classify catfish origins; some work demonstrates a relationship between spine and muscle metal content.

Fin rays (23–29% calcified)

• Structure and physiology
  • Fin rays consist of both mineral and organic components; organic matrix rich in type-I collagen, ~40% by weight; inorganic phase similar to endoskeletal bone.
  • Growth is additive: new layers are added over old ones; annuli form from birth to adulthood with oldest layers at the core.
  • Ray regeneration after damage is possible; regeneration involves new osteoblasts and restoration of function, enabling non-lethal sampling in some contexts.
  • Vascularization and resorption occur primarily at the core; older, metabolically active fish show more resorption in rays.
• TEA and SIA
  • TEA: used to infer migratory histories (e.g., freshwater vs. marine environments) by tracking elements like Ba and Sr over time in rays; “elemental tagging” experiments show persistence of trace elements through regrowth; supporting use in stock identification and environmental history.
  • SIA: both TEA and SIA evidence suggest rays can archive life-history information; isotopes (δ13C, δ15N) are enriched in fin rays relative to muscle due to inclusions in inorganic matrix; some studies show ray-based SIA can reconstruct geographic and trophic histories.
• Advantages and considerations
  • Advantages: non-lethal sampling; suitable for endangered or management-sensitive species; rays provide a long-term chemical archive across the lifespan; aging alignment with otoliths has been demonstrated in reef fishes and some others.
  • Considerations: resorption can still affect early annuli in fast-metabolizing species; edge crowding of annuli in older individuals; small species may pose preparation challenges; careful interpretation needed when comparing to otoliths.
• Key examples
  • TEA used to document diadromous patterns and river-origin signals in sturgeon and esocids; elemental tagging and migrations have been tested in several taxa; ray chemistry linked to freshwater vs. marine transitions;
  • SIA-based approaches have been used for life-history, diet, and geographical origin inference, with some studies showing ray tissues provide robust isotope histories.

Scales (<20% calcified)

• Structure and physiology
  • Elasmoid scales with osseous outer layer and basal organic layer (elasmodin) composed of partially mineralized collagen.
  • Growth occurs via circuli (concentric rings) and annuli; scales can record growth history akin to otolith rings.
  • Elements can be deposited at growing edges or integrated into the hydroxyapatite (Ca-phosphate) of the scale; scales can reflect ambient water chemistry and diet signals.
• TEA (trace-element analysis)
  • Scale microchemistry pioneered in the 1970s showing Sr uptake from ambient water and intra-structure element distributions; LA-ICP-MS enabled spatially resolved analyses of scales.
  • Key elements tracked include Sr, Ba, Mn, Mg, and heavy metals (Cd, Pb, Zn, etc.).
  • Scale-element patterns often reflect environmental histories and migrations in diadromous species (e.g., salmonids) and can distinguish habitats along estuarine-to-seawater gradients.
  • Some comparisons show scale Sr:Ca tracks water chemistry and otolith chemistry in some cases, but relationships can be inconsistent across species and life stages due to differences in deposition and remobilization.
• SIA (stable-isotope analysis)
  • d13C and d15N measured in whole scales, or at specific circuli to resolve life-history events; d34S used in some freshwater contexts.
  • Scales can be used for non-lethal isotope sampling and have been applied to estuarine and freshwater ecology, long-term trophic histories, and ecosystem-level changes.
  • Scale isotopes have proven complementary to trace-element data in ecological storytelling and can help in isoscape development when background baselines are established.
• Advantages and considerations
  • Advantages: non-lethal sampling; scales are widely available in many species; archival value in preserved collections and sediments; allows replication across scales from a single fish; useful to monitor environmental metals and isoscape patterns.
  • Considerations: scale chemistry can be affected by mucus and organic debris; inorganic carbonate can bias d13C measurements unless scales are acid-washed (common practice is 1.2 M HCl for ~2 minutes); acid washing effects on isotopes can be variable and species dependent; scale collagen vs muscle d13C differences require correction factors; scales can regenerate, so early-life histories may be incomplete; post-depositional reworking and lipid residues can bias results.
  • Lipids can depress d13C values; lipid extraction or normalization may be necessary to compare across tissues or studies; scale-to-muscle offsets for d13C can range roughly from about -10.2‰ to -4.0‰ depending on species and methods.
• Key issues and adjustments
  • Underplating: juvenile collagen may be overprinted by later-deposited collagen, dampening expected d15N differences between life stages; corrections may be possible but require species- and age-specific calibrations.
  • Whole-scale versus annulus-specific analyses: whole-scale analyses can bias interpretation; targeted circuli or edge analyses may provide more temporally-resolved information.
  • Scales are regenerable; non-regenerated scales are preferred for long-term chronologies; regenerated scales may still be informative for recent periods if aging is known.
• Examples and applications
  • Scale microchemistry used to track estuarine and freshwater migrations, to differentiate farmed vs wild salmon using Sr and Mn signals, and to track water-quality histories (e.g., dissolved nitrogen, heavy metals).
  • Archival scales have been used to link climate oscillations and planktonic community changes to salmonid mortality and trophic shifts.

Eye lenses (0% calcified)

• Structure and physiology
  • Eye lenses form during gastrulation from surface ectoderm; composed of lens epithelial cells at the anterior surface and lens fiber cells that accumulate laminae.
  • Lens fibers are hexagonal in cross-section, elongate as they mature, and are subjected to a process called attenuated apoptosis, leaving a membrane but no organelles in mature lens fiber cells.
  • Crystallin proteins (alpha, beta, gamma crystallins) dominate lens composition and are produced by lens epithelial cells; crystallin composition affects optical properties.
  • Laminae form as layers of accumulated lens material; newer laminae reside in the outer cortex, older laminae near the nucleus.
• Trace-element analysis
  • Metals can be measured in lens tissue; Measured elements include Ba, Co, Cu, Fe, Hg, Mn, Pb, Rb, Sr; metals may bind to crystallin sulfhydryl groups rather than substitute for Ca in a hydroxyapatite lattice.
  • Outer cortex is continuously perfused by aqueous humor, which can alter trace-element signatures after protein synthesis ceases, potentially reducing temporal resolution in some contexts.
• Stable-isotope analysis (SIA)
  • Eye lenses contain high protein content (crystallin; up to ~50% of lens mass) and thus can be used for long-term δ13C and δ15N analysis, providing spatially broader isotopic baselines.
  • Wallace et al. (2014) demonstrated eye lenses as long-term recorders of trophic and geographic histories in fishes; isotopic variation in lenses reflects isotopic baselines and isoscapes and can align with known life-history patterns.
• Advantages and considerations
  • Advantages: potential long-term isotopic record with seasonal or longer resolution; tissue without turnover in mature lens fiber cells provides a stable archive; sampling can be done without micromilling; one lens can be used for a screening, another for high-resolution later analysis.
  • Considerations: lethal sampling required to access lenses (in most cases); temporal resolution depends on laminae formation rates; interpretation requires accurate aging linkage to otoliths or other known-age markers; metals may diffuse or be redistributed via aqueous humor, challenging TEA.
• Practical implications and future potential
  • Eye lenses offer a unique complement to bone/scale-based records, particularly because they can provide d15N data (nitrogen-rich protein matrix) with potentially seasonal to lifetime resolution depending on laminae formation.
  • Delamination-based sampling is a practical method for non-destructive or minimally destructive sampling when compared to micro-milling otoliths; however, aging eye-lens laminae requires establishing age links to known-life-history markers.

General considerations across alternative structures

• Temporal conservation and turnover
  • Structures without turnover (e.g., eye lenses) can provide longer, more stable records than vascularized tissues that turnover and remodel.
  • Non-vascularized tissues (endoskeleton, fin spines, fin rays, scales, eye lenses) are more likely to preserve historic chemical signatures, though metabolic and remodeling processes can still modulate signals.
• Methods and instrumentation
  • TEA: mainly done with mass spectrometric techniques (LA-ICP-MS, laser ablation ICP-MS; X-ray fluorescence; AAS/AES); calibration relies on matrix-matched standards when possible.
  • SIA: common isotopes include $\delta^{13}C$, $\delta^{15}N$, $\delta^{34}S$; compound-specific isotope analysis is possible but expensive; lipid extraction can affect isotopic measurements in scales and bones.
• Sampling ethics and management implications
  • Fin spines, fin rays, scales offer non-lethal sampling options, advantageous for endangered or protected species or brood stock where otoliths are not feasible.
  • Eye lenses and some calcified tissues may require lethal sampling; ethics and management policies must be considered when designing studies.
• Cross-structure integration and interpretation
  • Using multiple structures in tandem increases inference robustness and offsets the limitations of any single structure.
  • Differences among structures in elemental incorporation pathways, turnover rates, and baseline isotopic values require cross-calibration and careful interpretation.
• Practical cautions and calibration needs
  • Cleaning procedures (e.g., acid washes) can alter isotopic signatures; corrections or species-specific calibrations are essential.
  • Remobilization and post-depositional alteration (especially in scales and spines) must be accounted for when interpreting chronological microchemistry.
  • Standardization of sampling, preparation, and data interpretation is crucial for cross-study comparability.

Comparative highlights and practical implications

• What to choose when otoliths are unsuitable
  • Endoskeleton (bone) provides a long record for TEA/SIA but requires understanding remodeling; good for geographic origin and diadromous migrations.
  • Fin spines offer a non-lethal alternative with TEA/SIA utility, though early-life annuli may be erased by vascularization in fast-growers.
  • Fin rays provide a durable, additive-record structure with TEA/SIA potential; suitable when otoliths are not available, but aging and resorption must be considered.
  • Scales provide a non-lethal archive with broad historical utility and preserved material for decades; careful handling and corrections for scale mineral content are needed.
  • Eye lenses provide long-term isotopic records with potential annual-resolution capacity; sampling is typically lethal, limiting their use in protected species.
• Synthesis and guidance
  • For robust life-history reconstruction, combine multiple structures (e.g., scales and fin rays, or bone and scales) to cross-validate age, migration, and diet inferences.
  • Explicitly account for structure-specific turnover and remodeling rates; implement species-specific turnover studies where possible.
  • Consider using both TEA and SIA in complement: TEA to track movement/habitat shifts and SIA to infer trophic regime and resource use, while accounting for potential confounding factors (lipids, adsorption/desorption, and isoscape baselines).
• Ethical, philosophical, and practical implications
  • Non-lethal approaches align with conservation ethics and fishery management goals; where feasible, prefer non-lethal tissues (scales, fin spines, fin rays).
  • When lethal sampling is unavoidable (eye lenses), ensure strict justifications, permits, and minimal-impact protocols; the insights gained must justify the costs to populations.
  • The ability to reconstruct long-term ecological and environmental histories from historical archives (scales, preserved bones) provides valuable baselines for understanding climate change, pollution, and fisheries dynamics.

Seven recommended directions for advancing the field

1) Combine multiple structures for TEA and SIA and develop methods to evaluate concordance and discrepancies among structures; use structure-specific turnover rates to interpret cross-structure patterns.
2) Compare different fish groups (freshwater vs marine, diadromous vs non-diadromous, habitats, trophic levels, life stages) to understand how turnover and deposition influence microchemical chronologies.
3) Identify turnover rates for each structure to determine applicability for specific questions (e.g., short-term vs long-term histories).
4) Search for new analytes that may document movement, diet, physiological events, or environmental conditions; consider anions that substitute into hydroxyl groups (OH−) or other lattice sites (e.g., Cl−, F−).
5) Document how dynamic environmental conditions affect incorporation (stress, temperature, salinity, feeding regime) and how these factors modify TEA and SIA signals.
6) Develop element maps and isoscapes for comparison with analytes of interest; track background value changes over time and space to create marine isoscape baselines.
7) Continue to search for new alternative structures with conserved analyte records and evaluate their utility in concert with established structures.

Glossary (selected terms)

• Acellular – Lacking cells; in bone, lack of osteocytes/osteoblasts.
• Annuli – Ring-like growth features on calcified structures used for aging.
• Aqueous humor – Fluid in the eye between cornea and lens; nourishes the lens and maintains pressure.
• Bioapatite – Alternative term for hydroxyapatite in bone.
• Circuli – Growth rings in scales, similar to annuli.
• Crystallin – Protein in the eye lens; crystallin composition affects lens transparency.
• Epithelium, lens – Tissue layers in the lens with distinct cellular organization.
• Elasmodin – Layer in scales formed by collagen bundles (plywood-like structure).
• Lamellar – A bone tissue type with layered structure; associated with slow growth.
• Osteoblast/osteocyte/osteoclast – The three major bone cell types involved in formation, maintenance, and resorption of bone.
• Pseudo-lamellar – Another term for parallel-fiber bone.
• Woven fiber – Loosely organized bone fibers typical of rapidly formed bone.

These notes summarize the major and minor points from the transcript, emphasizing the roles of five alternative fish structures (endoskeleton, fin spines, fin rays, scales, and eye lenses) as chronological recorders for microchemical analyses (TEA and SIA). They highlight structure-specific physiology, methods, advantages, limitations, empirical examples, and future directions, with attention to ethical considerations and practical applications in fisheries science, ecology, and conservation.