Methods for the Preparation of Pacifi c Spiny Dogfi sh, Squalus suckleyi, Fin Spines and Vertebrae and an Overview of Age Determination

CINDY A. TRIBUZIO, MARY ELIZABETH MATTA, CHRISTOPHER GBURSKI, NIKKI ATKINS, and WALTER BUBLEY

Introduction To be viable for an age determina- North Pacifi c Ocean, ranging from the tion study, a hard structure must form Koreas and Japan through Russian and Age determination of elasmobranchs visible, annually-formed growth pat- Alaskan waters to the North Ameri- presents unique challenges compared terns. Typically, these growth patterns can west coast, reaching as far south to that of teleosts. Elasmobranchs do consist of alternating light and dark as Baja California (Ebert et al., 2010). not have calcareous otoliths or scales, bands; each pair of growth bands is Squalus suckleyi was previously structures commonly used to age te- termed an “annulus,” representing 1 thought to be identical to its Atlantic leosts. Various techniques, including year of growth. Note that this differs counterpart, S. acanthias, but genetic, bomb-radiocarbon dating, histologi- from the standard English defi nition meristic, and morphological evalua- cal staining, and X-radiography, have of the word annulus, which is derived tion has proven them to be two distinct been applied to hard structures such from the Latin word “anus” meaning species (Ebert et al., 2010). Further, as fi n spines, vertebrae, and caudal “ring,” not “annus” meaning “year” S. suckleyi differs from S. acanthias thorns to age elasmobranchs (Cail- (Panfi li et al., 2002). in several key life history attributes, liet and Goldman, 2004; Carlson and In most elasmobranch studies, sag- including slower growth, larger maxi- Goldman, 2006). ittally sectioned vertebrae are selected mum size, and later maturity. Much of as the primary age structure (Cailliet the existing scientifi c literature from Cindy A. Tribuzio, corresponding author (cindy. and Goldman, 2004), although clarity the North Pacifi c Ocean refers to spiny [email protected]), is with the Auke Bay Labo- of annuli within vertebrae is largely dogfi sh collected there as S. acanthias, ratory, Alaska Fisheries Science Center, Nation- al Marine Fisheries Service, NOAA, 17109 Pt. species-specifi c. For some species, and it is important to note that the spe- Lena Loop Road, Juneau, AK 99801. Mary Eliz- vertebra growth patterns are not dis- cies name is now considered incorrect abeth Matta and Christopher Gburski are with cernible and thus alternative structures in previous literature and should be the Resource Ecology and Fisheries Manage- ment Division, Alaska Fisheries Science Cen- such as the neural arch (McFarlane et considered S. suckleyi. ter, National Marine Fisheries Service, NOAA, al., 2002) or dorsal fi n spine (Clarke The dorsal fi n spine method has 7600 Sand Point Way NE, Seattle, WA 98115. Nikki Atkins is with the Fishery Resource Anal- and Irvine, 2006) must be used. been used to age S. suckleyi and S. ysis and Monitoring Division, Northwest Fish- Squaliform sharks (dogfi shes) are acanthias since the 1930’s. In this eries Science Center, National Marine Fisheries one of two orders of elasmobranchs method, annuli on the enamel of the Service, NOAA, 2032 SE OSU Drive, Newport, OR 97365. Walter Bubley is with the South Car- possessing dorsal fi n spines (Clark and second dorsal fi n spine are viewed olina Department of Natural Resources, Marine Irvine, 2006). Pacifi c spiny dogfi sh, and counted using refl ected light and Resources Research Institute, 217 Fort Johnson Squalus suckleyi (hereafter termed a dissecting microscope or with im- Road, Charleston, SC 29412. “spiny dogfi sh”), is a species of Squal- age analysis software. However, this doi: dx.doi.org/10.7755/MFR.78.1–2.1 iform shark found throughout the may not be the best method to accu- rately describe age and growth of the species. Because the dorsal fi n spine ABSTRACT— The Pacifi c spiny dogfi sh, ability due to diffi culty in interpreting fi n extends from the body into the envi- Squalus suckleyi, is a small shark spe- spine growth patterns. A new method was ronment, breakage and erosion of the cies commonly found in the North Pacifi c recently developed for S. acanthias, a North fi n spine often occurs over time. Thus, Ocean. Age determination for this species Atlantic Ocean congener of S. suckleyi, us- larger, older spiny dogfi sh tend to have has historically been conducted by exami- ing histologically stained thin sections of fi n spines with more wear than small- nation of the dorsal fi n spine with little vertebrae instead of dorsal fi n spines for change in methodology since the 1930’s. age estimation. Here, we apply this histo- er, younger fi sh. Despite extensive use, there are two major logical method to vertebrae of S. suckleyi Ketchen (1975) developed an algo- caveats associated with fi n spines as age and describe the historic methodology for rithm to estimate the number of miss- structures: 1) fi n spines protrude from the dorsal fi n spines. This document presents ing annuli in the worn portion of the body and are subject to damage, requiring detailed procedures for both methods, in- estimation of annuli contained in missing cluding sample collection, sample prepara- fi n spine using the relationship be- portions of the fi n spine and 2) there is a tion, and age estimation criteria for each tween the enamel base diameter and high degree of inter- and intra-reader vari- structure. the number of annuli counted on un-

78(1–2) 1 worn fi n spines. An alternative analyti- vertebrae do not wear or break over southwestern edge of its range, in par- cal method for estimating lost annuli time as fi n spines do, therefore reduc- ticular, S. japonicus, S. blainville, and was proposed by Cheng (2012); how- ing one source of variability and error S. brevirostris. However, S. suckleyi is ever, few laboratories have adopted in the age determination process (i.e., generally easy to distinguish from the this method to date. the need to use a modeled estimate for other Squalus species because of the Taylor et al. (2013) conducted a de- the number of missing annuli in worn white spots along the sides and be- tailed examination of the Ketchen and fi n spines). Furthermore, age estimates cause the origin of the fi rst dorsal fi n Cheng analytical approaches and de- derived from vertebrae are far more spine is posterior to the rear free tips termined that both methods produced precise than those from spines (Bubley of the pectoral fi ns, whereas in most questionable age estimates for larger, et al., 2012). species of Squalus occurring in the older fi sh, and they further recom- Results are not presented in this western North Pacifi c Ocean the fi rst mended an examination of new meth- paper as it is intended solely as a dorsal fi n and spine are located above ods. Attempts to improve Ketchen’s technical guide for the collection, lab- the pectoral fi ns (Compagno, 1984). algorithm (McFarlane and King, 2009) oratory processing, and interpretation The fi rst steps in spiny dogfi sh col- have not addressed problems of error of age structures. This paper is part of lection are to sex and measure each from other sources (e.g., natural vari- a larger spiny dogfi sh age determina- fi sh. Sex can be determined externally; ability, reader error); therefore, the tion project. While both methods are males have paired claspers attached to historical method continues to be used presented here to improve precision the pelvic fi ns. Special care may be (Tribuzio et al., 2010). and document ageing criteria for use needed when identifying sex of very Fin spine-based age estimates of by other laboratories, it is important small (immature) animals, as male both S. suckleyi and S. acanthias have to note that at this time, the vertebra claspers can be small. There are four been validated using bomb-derived method does not appear to be appro- commonly used length measurements radiocarbon (Campana et al., 2006). priate for older spiny dogfi sh (Tibuzio for spiny dogfi sh (Tribuzio et al., Furthermore, annual periodicity of et al., in press). Documenting age de- 2009), measured from the tip of the the fi n spine banding pattern has been termination methods is imperative, as snout to 1) the dorsal pre-caudal pit verifi ed by oxytetracycline (OTC) in- criteria used for identifying annuli can (pre-caudal length, PCL); 2) the deep- jections and tag/recapture methods on drift over time and among agencies. est indentation of the tail fork (fork S. suckleyi that were at liberty up to This paper provides a central reference length, FL); 3) the dorsal tip of the 21 yr (McFarlane and Beamish, 1987; for all laboratories involved in spiny tail in its natural position (natural to- McFarlane and King, 2009). However, dogfi sh age determination and will tal length, TLnat); and 4) the dorsal tip the low precision of fi n spine-based promote consistency between the two of the tail with the upper lobe of the age estimates (CV = 19%; Rice et al., methods and among spiny dogfi sh age caudal fi n depressed to align with the 2009; Tribuzio et al., 2010) is prob- readers, in the hopes of improving in- horizontal axis of the body (extended lematic, and systematic bias among ter- and intra-laboratory precision. total length, TLext) (Fig. 1). However, age determination laboratories oc- PCL has the least amount of mea- curs despite age validation (Rice et Sample Collection surement error and is often the easi- al., 2009). Measurement errors among Squalus suckleyi is a small shark est to measure. This is the preferred readers, potential systematic errors with medium-brown to gray coloration length measurement for the species, among laboratories, and process errors dorsally and white ventrally; white but equations exist to convert among associated with estimating the number spots are often present along the up- length measurements (Tribuzio et al., of worn annuli all combine to produce per body. A prominent spine is present 2009; Tribuzio and Kruse, 2012). the low precision in age estimates and along the anterior edge of each dorsal Two age structures are collected: 1) therefore growth parameters (Tribuzio fi n. A complete description of identify- the posterior dorsal fi n spine (hereaf- et al., 2010). This error has ramifi ca- ing characteristics is available in Ebert ter termed “spine” and 2) a section of tions for population modeling and et al. (2010). While there are at least the vertebral column. The posterior biological reference points for fi sh- 25 species of the genus Squalus occur- spine is preferred to the anterior spine ery management (Tribuzio and Kruse, ring throughout the world, S. suckleyi because it is larger and generally sub- 2011). is the only one occurring in the east- ject to less wear. Vertebra samples can To address these issues, a new meth- ern North Pacifi c Ocean. However, the be collected at various positions along od of age determination using vertebrae species can be confused with members the vertebral column. The authors have has been developed for S. acanthias in of the Triakidae family (e.g., brown found that vertebrae collected more the northwest Atlantic Ocean (Bubley smoothhound, Mustelus henlei), which anteriorly are generally larger, making et al., 2012). This method employs his- possess an anal fi n and do not have them easier than posterior vertebrae tologically stained vertebra thin sec- dorsal fi n spines. In the western North to prepare and read. If time allows, it tions and presents two advantages over Pacifi c Ocean, S. suckleyi may overlap is preferable to collect vertebra sam- the old dorsal fi n spine method. First, with other Squalus species near the far ples ventral to the anterior dorsal fi n;

2 Marine Fisheries Review Figure 1.—External measurements for spiny dogfi sh. All measurements start at the snout and extend to either the pre-caudal length (PCL), fork length (FL) or total length (TL, both natural and extended). The dashed line shows the horizontal axis of the body for measuring TL extended). For this sampling plan only the PCL is used. The arrow points to the pre-caudal notch. From Tribuzio et al. (2009). however, it is quickest to collect the Heating will create a strong odor and spine can be initially cleaned by care- vertebrae and posterior spine simulta- it is recommended to use a well-ven- fully scraping away the tissue with a neously (Fig. 2). tilated area or to process under a fume fi ngernail or other soft tool. Remain- For collection, a vertical incision hood. Spines may be heated either us- ing tissue on the exterior of the spine is made behind the posterior spine, ing a microwave or hotplate. To micro- can be carefully removed with forceps slicing down (ventrally) through the wave, spines should be placed, either or another fi ne-tipped tool. The car- vertebral column (Fig. 2A). Then an individually or as batches if each is tilage plug on the inside of the spine incision about 4 inches long is made in an individually labeled microwave- will generally slide out as one piece horizontally towards the head, keeping safe bag, in a microwave-safe con- and can be pulled out with forceps or a the blade ventral to the vertebrae (Fig. tainer fi lled with water. If spines are dental pick. Once the plug is removed, 2B). The section is removed by cut- in individual bags, the bags should any tissue remaining inside the spine ting vertically to the dorsal side (Fig. be punctured to allow water to fl ow can be scraped away without concern 2C). Excess muscle tissue should be through. Depending on the power of of damage to the interior of the spine. trimmed to make storage easier (Fig. the microwave, the size of the contain- Spines can be stored long term in 2D, 2E). Samples should be stored in er, and the number of spines, it should individual paper coin envelopes. After individually labeled, single-specimen only require 2–6 min to adequately cleaning, each spine should be allowed reclosable bags and frozen. Vertebra heat the spines. Alternatively, spines to air dry for at least 24 h to eliminate and spine samples from the same ani- may be heated in large, water-fi lled all moisture and potential sources of mal can be stored in the same bag. The beakers or in a divided basket within decay and to prevent the spine from same cutting and sample storage/pres- a large tray placed on a hotplate for sticking to the paper storage enve- ervation method can be used regard- several minutes. Tissue should easily lope. Spines may become brittle after less of whether the vertebra samples scrape off the surface of the spine with many years of storage, so care is war- are removed from the anterior or pos- gentle rubbing. ranted when handling archived enve- terior portions of the vertebral column. Tools that are helpful for cleaning lopes. Some laboratories use a barcode Specimen Preparation the inner surfaces of spines include system to track samples; if this is the forceps, microprobes (i.e., dissecting case, the barcode sticker can be placed Dorsal Fin Spines needles), and dental picks. For clean- inside the envelope just below the fold Various techniques and tools can ing the outer enamel, the best tool is a of the envelope’s fl ap. Barcodes can be used to prepare spines for age de- fi ngernail; however, a soft toothbrush still be scanned from this location, and termination. The frozen spines and (used gently) or a cloth can be useful. if the adhesive weakens, the barcode vertebrae need to be separated and Metal tools should be used with cau- label will not be lost. tion on the outside of the spine be- the vertebrae retained for further pro- Vertebrae cessing as described in the next sec- cause they may damage the enamel tion; it is advisable to do this with the surface used for age determination. To prepare spiny dogfi sh verte- structures frozen or partially frozen to All soft tissue needs to be removed brae for age determination, they must prevent tissue degradation of the ver- from the exterior and interior of the be individually dissected, thin-sec- tebrae prior to processing. spine. While working on a spine, it tioned, stained, and mounted on glass Spines need to be heated to aid in is helpful to keep the spine wet, oth- slides. Histological staining methods tissue removal, taking care not to dam- erwise the tissue becomes sticky and have been adapted from Bubley et al. age the spine enamel or base structure. diffi cult to remove. The outside of the (2012). Supplies needed to prepare

78(1–2) 3 Figure 2.—Dorsal fi n spine and vertebrae dissection. A) A downward incision is made posterior to the dorsal fi n spine, followed by B) a horizontal incision ventral to the vertebral column, and fi nally C) an upward incision is made to simultaneously remove the spine and a portion of vertebral column. D) Excess tissue is removed from the spine and vertebrae to produce E) a single sample ready to be frozen until further processing in the laboratory is possible. vertebrae are listed in Table 1. Per- thawed, a scalpel or sharp knife is used trum; fi ne forceps can be used to re- sonal protective equipment such as to cut excess tissue from around the move stubborn tissue. It is often not eye goggles, latex or nitrile gloves, and vertebral column section. The axial possible to remove all the tissue; how- laboratory coats should be worn when processes (neural and hemal arches) ever, this will not impact age deter- handling chemicals. A chemical fume may be left attached to the vertebrae to mination. Several vertebra centra per hood should be used where indicated, simplify cleaning, and any remaining animal can then be stored in individual and refer to material safety data sheets soft tissue can then be scraped off with vials containing 70% ethanol to await (MSDS) for proper handling, storage, the back of a knife. sectioning and staining. If desired, and disposal of all chemicals. Individual centra are separated from the remainder of the vertebral column the vertebral column using a scalpel. may be stored frozen. Dissection The axial processes and remaining soft Vertebral column sections must be tissue can then be trimmed from each Thin-sectioning at least partially thawed before excess centrum. Care should be taken when Silicone molds (suggested size: 64 soft tissue can be scraped away. Once removing excess tissue from each cen- × 70 × 12 mm) are used to encase

4 Marine Fisheries Review rows of vertebra centra in resin for Table 1.—Supplies used for sectioning, staining, and mounting vertebra centra. sectioning multiple specimens simul- Sectioning supplies Staining supplies taneously. A brand of resin that works Wax cups marked in 2 oz increments Histology tissue cassettes well for this purpose is Polytranspar Tongue depressors Large beakers 1 Strips of paper ~ 1 cm wide (to label blocks) Graduated cylinders Artifi cial Water , a clear casting res- Dried, cleaned vertebrae Containers to store reagents Silicone molds Funnel in that requires a catalyst to harden Polyester resin Glass slides (available at taxidermy supply busi- Forceps Cover slips Probe Slide boxes nesses). A new batch of resin must be Paper towels Hot plate mixed for each use because it starts to Scalpel Orbital shaker or stir plate with bars Scissors Stopwatch harden immediately once the catalyst Precision saw with diamond blade Small and large forceps has been added. Wax cups pre-marked Dissecting microscope Scalpels Ruler Flammable chemical disposal container in 2 oz increments are quite helpful Calipers Dissecting microscope Plastic tub for mixing the resin as they can be Trays disposed of once the unused catalyzed Paper towels Distilled water resin hardens. Modifi ed Harris hematoxylin First, working in a fume hood, a Gelatin Glycerol batch of resin is mixed and a thin layer Listerine is poured into the mold such that it just Hydrochloric acid (12 M) Ethanol (100%) barely covers the bottom of the mold. RDO Rapid Decalcifi er The mold should be left undisturbed for about 40 min to allow the resin to harden until tacky. A line should be drawn in the resin, using a probe and acceptable as long as stain penetration to mold formation and must therefore a ruler as a guide, for aligning multi- and annulus clarity are not affected. be changed more frequently. ple vertebra centra. Either the anterior We recommend checking the thick- Kaiser glycerin jelly is used to ad- or the posterior end of each centrum ness of the distal ends of each section here stained specimens to glass slides is then pressed into the resin with the with calipers to verify that the saw is and affi x coverslips. It is made using focus on the guideline, to result in a cutting accurately. Thin sections can 40 ml distilled water, 7 g Knox gela- longitudinal section. Space should be be stored in 70% ethanol indefi nitely. tin, 50 ml glycerol, and 10 ml Lister- left at the top of each block to place Excess resin should be trimmed away ine antiseptic mouthwash (any fl avor a label with the specimen numbers for from the vertebra sections using a which has thymol as an active ingredi- each row. Each block can accommo- sharp scalpel prior to staining. ent). The water and glycerol are mixed date 2–3 rows of vertebrae, with 4–7 together, then sprinkled with the gela- Solution Preparation vertebrae per row. tin, and allowed to sit for 5 min. The Once aligned, the vertebrae need to Several of the solutions used to stain solution is then melted at low heat us- dry in place (about 30 min) so they are and mount specimens can be prepared ing a hot plate or double boiler (gela- held fi rm in the bottom layer of resin. in advance: acid-alcohol, a series of tin’s melting point is 40°C). After the This prevents the vertebrae from shift- concentrated glycerin solutions, and gelatin has dissolved, the solution is ing as the top layer of resin is poured. Kaiser glycerin jelly. Acid-alcohol, removed from the heat and Listerine A new batch of resin is then poured used to destain specimens, is prepared added, stirring slowly to prevent for- over the centra so that they are fully using 65% distilled water, 35% etha- mation of bubbles. The Kaiser glycerin covered while striving to keep block nol, and 12 drops of 12M hydrochloric jelly solution is then allowed to cool thickness to a minimum. Excess resin acid per 300 ml water/ethanol mixture. until it is completely solid prior to use. can cause diffi culties when sectioning. Acid alcohol should not be reused and The resin should be allowed to dry a should be disposed of according to lo- Staining minimum of 2 days in the fume hood. cal and federal regulations. Vertebra thin sections are placed in After the resin is fully cured, verte- A series of increasingly concentrat- histology tissue cassettes labeled with brae are sectioned with a high-speed ed glycerin solutions (25, 50, 75, and specimen numbers. Multi-chamber saw such as an IsoMet 5000 (Buehler- 100%) is used to hydrate stained thin cassettes may be used to prepare many ITW, Lake Bluff, IL). Optimal section sections to prepare them for mount- specimens simultaneously if desired. thickness for spiny dogfi sh vertebrae ing. Distilled water is added to 100% Cassettes are placed in a large bea- is 0.4 mm, although some variation is glycerin to make the glycerin solutions ker on an orbital shaker and fully im- (25, 50, and 75%). Each glycerin so- mersed in RDO Rapid Decalcifi er 1Mention of trade names or commercial fi rms is lution can be stored in a separate con- (Apex Engineering Products Corp.). for identifi cation purposes only and does not im- ply endorsement by the National Marine Fisher- tainer and reused, although the weaker A partially full bottle of water or Er- ies Service, NOAA. solutions (25 and 50%) are more prone lenmeyer fl ask may be nested within

78(1–2) 5 the beaker to keep the cassettes sub- can be repeated until the desired moved using a razor blade so the slides merged. Sectioned vertebrae should level of staining is achieved. won’t stick together during storage. be soaked in RDO until softened (fl ex- 3) De-stain the specimens with acid ible) and yellowed. It is recommended alcohol solution for about 4 min Age Determination to check sections after 5–10 min and to make the banding pattern more re-immerse in RDO if not adequately visible and improve contrast be- Dorsal Fin Spines decalcifi ed. In general, larger vertebra tween light and dark bands. As described, accurate age estima- sections require longer soak times, but 4) Place the cassettes in a tub fi lled tion of spiny dogfi sh spines is diffi cult a total soak time of roughly 15 min is with water in the sink and rinse because the spines are external to the adequate for most specimens. Soak- using agitation for about 1 min. body and are often broken or worn, re- ing too long will cause the sections Again, check sections under the sulting in “missing” annuli, the num- to decalcify to the point where annuli microscope to ensure de-staining ber of which must be estimated using are not visible after staining. Once the is adequate. A pattern of alternat- regression methods based on morpho- specimens are suffi ciently decalcifi ed, ing light and dark bands, purple metric relationships. Several differ- cassettes are removed from the RDO or red in color, should be evident. ent measurements taken on each spine and placed in a beaker under running If necessary, Steps 3 and 4 can be (Fig. 3) are used to calculate the num- water for 1 h. RDO can be reused until repeated, although the time spent ber of potentially missing annuli. Mea- precipitates form. Following decalcifi - in acid alcohol may be adjusted surements can be taken using either cation, cassettes need to be soaked in based on staining strength. calipers or image analysis software, 100% distilled water for at least fi ve 5) Rinse the cassettes in the sink un- but the method should be consistent minutes, although the process can be der running water for 10 min. for all specimens in a given study. stopped here overnight. 6) Soak the cassettes in distilled wa- In the Ketchen (1975) approach, The following seven steps need to ter for 2 min. the two key measurements used in be conducted sequentially, i.e., no 7) Soak the cassettes in gradual- estimating lost annuli are the enamel overnight breaks. Solutions are placed ly increasing concentrations of base diameter (EBD) and the diameter in a beaker on an orbital shaker to en- glycerin: 10 min each in 25, 50, at the last readable point (LRP, also sure constant movement. 75, and 100% glycerin. Verte- called the “no-wear point” and the di- 1) Soak the cassettes in modifi ed brae can be left in 100% glycerin ameter at this point is LRD) (Fig. 3). Harris hematoxylin (Richard-Al- overnight if there is insuffi cient For the Cheng (2012) approach, the len or Thermo Fisher Scientifi c) time for slide-mounting. mid-point measurement (MID) is used for about 8 min. Total soak time in addition to the EBD and LRD (Fig. Specimen Mounting may vary depending on size and 3). The MID is somewhat subjective, thickness of vertebra sections, To mount specimens, a stained ver- based on the reader’s interpretation and it is recommended to do tebra thin section and a small (pea- of the spine, and represents the loca- some test samples to determine sized) amount of Kaiser glycerin jelly tion along the spine where the reader the best soak time to achieve is placed next to each other on a la- feels that the readability of the spine optimal staining. Fresh hema- beled glass slide. The slide is then degrades and confi dence in the inter- toxylin should be used because placed on a hot plate set to the lowest pretation of annuli decreases. Three this chemical loses potency af- temperature that will melt the jelly in a additional measurements, total length ter expiration. Precipitates may period of 30–60 sec. The temperature (TL), stem length (SL), and spine base sometimes form within open should be set as low as possible to pre- diameter (SBD) (Fig. 3) can be used containers of hematoxylin and vent formation of air bubbles, which for other applications such as deter- can be fi ltered out using conical can occlude specimens. mining relationships with animal size; paper coffee fi lters. Once the jelly is melted, a coverslip however, they are not necessary for es- 2) Rinse the cassettes in a labora- is placed over the vertebra. It is best timating the number of missing annuli. tory sink by soaking in multiple to hold the cover slip at roughly a 30° Further descriptions of the spine ref- water baths until the water runs angle to the slide, touching the jelly, erence its orientation in situ (Fig. 4). clear. The staining level should and then slightly drag it towards the The enamel on the tip (dorsal edge) is be checked for adequacy by ex- specimen, which will cause the jelly to typically the most worn portion of the amining sections under a dissect- melt around and underneath the speci- spine. Wear progresses ventrally from ing microscope. Staining should men when the cover slip is placed on the anterior to the posterior edge of be uniform and dark, and an- top (i.e., there is no need to move the the spine. It is important to accurate- nuli should be visible although vertebra on top of the melted jelly). ly identify the LRP, which is the most contrast between light and dark The slide is then allowed to cool dorsal point of enamel on the anterior bands may be low. If additional completely. Excess jelly that leaks edge (Fig. 5A). The LRD is measured staining is required, Steps 1 and 2 from under the cover slip should be re- on the enamel between the anterior

6 Marine Fisheries Review Figure 3.—Measurements taken for each fi n spine: LRD=last readable point diameter; MID=mid-point diameter; EBD=enamel base diameter; SBD=spine base diameter; SL=stem length; TL=total length.

To estimate spine age, the dark bands are counted. For some spines, this is a relatively easy task (e.g., Fig. 5), but for others it can be much more challenging. In addition to be- ing stacked upon each other (Fig. 6), annuli can appear compressed and al- most indistinguishable in portions of the spine (Fig. 7). Annuli can also be very faint or diffuse, especially closer to the tip (Fig. 8A). For smaller ani- mals, it appears that there are mul- tiple “false annuli,” or “checks,” at the base of the spine. These fade with age and are less noticeable in older animals. This is likely caused by feed- ing pulses occurring in years of faster growth. While spiny dogfi sh are gener- Figure 4.—Fin spine orientation. alist feeders, they are also opportunis- tic and have been documented gorging when seasonal food sources are abun- and posterior edge (Fig. 5B). In gen- location would result in an underes- dant (Tribuzio, 2010). For larger ani- eral, a relatively larger LRD indicates timation of the true diameter at that mals, the dark bands near the base are a greater degree of wear. point and a potential underestimation more distinct and are more likely to be If the LRP is improperly identi- of the number of worn annuli. true annuli. It is also possible to fi nd fi ed, over- or underestimation of the Annuli are laid down as the spine spines with a bold white band at the number of worn annuli can occur. For grows in length. The oldest annuli are enamel base (Fig. 8). This is the area example, if the location of the annu- at the dorsal tip and those most re- where new annuli form but the enamel lus just ventral to the worn enamel is cently formed are at the ventral edge has not been laid down yet. If a dark assumed to be the LRP, then the in- of the enamel. The enamel gland de- area appears ventral to this white band, formation in the area between that posits enamel as the spine grows from it is not counted in the age estimate. annulus and the worn edge of the the base. In periods of slower growth There are a number of methods that enamel is lost, resulting in a potential annuli can be close together and some- have been used to make identifying overestimate of the number of worn times appear to be stacked upon one annuli easier. Newer ventral portions annuli. Conversely, if annuli are evi- another (Fig. 6). Growth patterns on of the enamel tend to have ridges as- dent dorsal to the worn edge of the the enamel do not appear to match sociated with the dark bands (the old- enamel, as in both spines in Figure those on dentine or the base cones er ridges, located more dorsally, are 5, it is inappropriate to identify the (white structure) of the spine and the generally more worn), and dynami- LRP dorsal to the worn edge of the deposition mechanisms are differ- cally adjusting the light source at dif- enamel. This is because it is not pos- ent (Irvine et al., 2006). Thus, it is ferent angles can be helpful in seeing sible to accurately measure the spine not possible to use the base cones or the ridges. Only the most prominent diameter at locations lacking enamel. dentine to verify when annuli appear ridges should be counted, as the small- Thus, measuring the diameter at this stacked on top of each other. er ridges between them are likely false

78(1–2) 7 annuli. A thin layer of mineral oil is also helpful to increase light refl ec- tion. When viewing the dorsal por- tion of the spine, where the enamel is thinner, shining refl ected light on the spine from directly above can make it easier to see the color differences between dark bands while providing more contrast. Additionally, spines can be viewed either dorsally or laterally to assist with identifying annuli and aid in fol- lowing annuli around the spine. True annuli typically encircle the spine, whereas false annuli are often incom- plete. Displaying spines on a monitor and either zooming out or standing back from the screen may aid in the identifi cation of prominent dark bands. Applying color fi lters to high-reso- lution images of spines using image software such as Adobe Photoshop can also be helpful (Fig. 9). There are unlimited combinations of potential image transformations, and differ- ent transformations may be used for examining different portions of the spine. Transmitted light or polarizing fi lters have also been found to be use- ful when examining small or embry- onic spines. Interpretation of annuli can vary substantially between readers. To ad- dress this imprecision, a reference set of photographs of known-age speci- mens has been assembled, the ages of which were validated using bomb- radiocarbon dating (Campana et al., 2006). These specimens range in dif- fi culty of interpretation from “easy” (Fig. 8A), where annuli are consistent- ly spaced and relatively easy to iden- tify, to “hard” (Fig. 8B), where annuli are more closely spaced and diffi cult Figure 5.—A) Identifi cation of the Last Readable Point (LRP) on worn fi n spines to discern from one another on cer- from two different spiny dogfi sh. White arrows point to the LRP on the anterior tain locations of the spine. These high edge of the enamel. The three small green arrows on the top fi n spine highlight dark bands that should not be counted by the age reader because they are dorsal resolution images and a set of calibra- to the LRP. B) Side view, with white lines showing measurements of the fi n spine tion spines, for which multiple readers diameter corresponding to the LRP. have come to an agreed age, are stored as part of the permanent collection of the NMFS Alaska Fisheries Science Center’s Age and Growth Program and are available for future calibrations be- tween readers and laboratories. Such reference sets are invaluable for eval- uating accuracy of age estimates and

8 Marine Fisheries Review periodicity (i.e., marginal increment analysis), confi rming location of the birthmark, and correlations between growth and annulus deposition. Vertebrae accrete material around the focus as the shark grows. The an- nuli located closest to the focus are the earliest deposited, while those located furthest are the most recently depos- ited (Fig. 12). The growth pattern of an individual changes as it gets older, with the annuli closer to the focus be- ing broader and more diffuse than the more distal (outer) annuli, which are thinner with sharper edges. The dis- tance between annuli also decreases with increasing distance from the fo- cus, such that annuli can become quite compressed in appearance near the distal edge of the vertebra (Fig. 12). To determine age, fi rst the loca- tion of the birthmark (age-0) must be Figure 6.—Magnifi ed image of spine enamel showing annuli stacked on top of one identifi ed. The birthmark usually cor- another. White dots identify ridges corresponding to annuli. responds with an angle change at the interface of the corpus calcareum and intermedialia (Goldman, 2004) (Fig. periodically testing inter-reader and nuli are most easily seen on the cor- 12). The birthmark is excluded from inter-laboratory precision. pus calcareum and radiate outwards the annulus count, and the following from the focus. In many shark spe- Vertebrae dark band is the fi rst one counted in cies, annuli can be followed through the age estimate. The birthmark’s loca- Stained spiny dogfi sh vertebra thin the intermedialia to the other edge of tion may be confi rmed by measuring sections are typically viewed with the corpus calcareum, but the interme- vertebra radii of full-term embryos ob- transmitted light, and fi sh age is es- dialia are absent or inconsistent in the tained from gravid females and com- timated by counting the dark purple Squalus species examined to date. paring the average to measurements bands (Fig. 10). As with viewing Vertebra measurements, if desired, from the focus to the presumed birth- spines, a number of fi lters either at- should be taken along a single tran- mark on adult specimens. tached to the microscope or applied in sect. The vertebra radius is taken from Interpreting annuli on vertebrae, as imaging software can enhance contrast the focus (center) to the distal (outer) with spines, is essentially counting the between light and dark bands. The edge of the vertebra along the cor- number of dark bands deposited. In morphological features of sectioned pus calcareum (Fig. 11). Measure- some cases, determining what consti- vertebrae are shown in Figure 11. An- ments are important for determining tutes an annulus can be challenging,

Figure 7.—Spine showing area with annuli very closely spaced or compressed. White dots identify selected individual annuli.

78(1–2) 9 Figure 8.—Fin spines that are A) easy and B) diffi cult to interpret, with ages validated by bomb radiocarbon dating. Dots rep- resent annuli. Images courtesy of the Canadian Shark Research Laboratory, Bedford Institute of Oceanography, Nova Scotia, Canada.

Figure 9.—Image analysis. Examples of four possible image transformations (performed using Adobe Photoshop Elements). From the top: inverted color, inverted then copper gradient, copper gradient only, rainbow gradient. Backgrounds are 1 mm x 1 mm grids.

10 Marine Fisheries Review Figure 10.—Identifying bands and annuli on Figure 11.—Vertebra anatomy, with orientation and vertebra radius mea- vertebrae. surement shown.

Figure 12.—Sectioned and stained vertebra. Each dark band is marked with a white Figure 13.—Annulus width on ver- dot. The birthmarks are noted with red arrows. Note that annuli become more com- tebra (indicated by colored lines). pressed with increased distance from the focus. Note the change in distance be- tween annuli from the edge of the corpus calcareum nearest (red ar- row) and furthest from the interme- dialia (blue arrow).

78(1–2) 11 Acknowledgments The authors would like to give special thanks to Cal Blood, Sandy Rosenfeld, and Patrick McDonald, who helped with the development of this paper and participated in method development and inter-laboratory stud- ies. This project was funded by the North Pacifi c Research Board (www. nprb.org), project number 1106 and is publication number 463. The fi ndings and conclusions in the paper are those of the authors and do not necessar- ily represent the views of the National Marine Fisheries Service, NOAA. Literature Cited Bubley, W. J., J. Kneebone, J. A. Sulikowski, and P. C. W. Tsang. 2012. Reassessment of spiny dogfi sh Squalus acanthias age and growth using vertebrae and dorsal-fi n spines. J. Fish Biol. 80(5):1300–1319. (doi: https://doi. Figure 14.—Two different vertebrae with dark bands and marginal increment ratio org/10.1111/j.1095-8649.2011.03171.x). (MIR) measurements identifi ed. The blue line represents the edge width (EW) and Cailliet, G. M., and K. J. Goldman. 2004. Age the red line represents the last annulus width (LAW). The yellow arrow and dot determination and validation in chondrichthy- mark the edge of the centrum. an fi shes. In J. C. Carrier, J. A. Musick, and M. R. Heithaus (Editors), Biology of sharks and their relatives, p. 399–447. CRC Press, Boca Raton, Fla. Campana, S. E., C. Jones, G. A. McFarlane, and S. Myklevoll. 2006. Bomb dating and while others are relatively easy to dif- look somewhat granulated under high- age validation using the spines of spiny ferentiate. By examining vertebrae us- er magnifi cation. It is sometimes easi- dogfi sh (Squalus acanthias). Environ. Biol. ing some of the following techniques, er to distinguish annuli near the focus Fish. 77(3/4):327–336. (doi: https://doi. org/10.1007/s10641-006-9107-3). the reader can have more reproducible by reducing magnifi cation when view- Carlson, J. K., and K. J. Goldman (Edi- age estimates. 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