Age determination in squid using statolith growth increments

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  • Fisheries Research, 8 (1990) 323-334 323 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

    Age Determination in Squid using Statolith Growth Increments


    Marine Life Sciences Division, British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET (Gt. Britain)

    (Accepted for publication 19 April 1988 )


    Rodhouse, P.G. and Hatfield, E.M.C., 1990. Age determination in squid using statolith growth increments. Fish. Res., 8: 323-334.

    Growth increments have been reported to occur in the squid beak, radula, gladius and statolith. Of these, the statolith, which is part of the organ responsible for detection of linear and angular acceleration, has proved most promising for age determination. Growth increments in the stato- lith are formed from aragonite crystals in an organic matrix. They are best viewed after sectioning the statolith or after decalcification in weak acid. The statolith grows in concert with the rest of the squid. Experiments with squid in which chemical markers have been incorporated at a known time in the statolith, and experiments with cultured squid of known age, appear to confirm the hypothesis that growth increments in the statolith are laid down daily. Increments are produced in the laboratory in the absence of tidal, feeding or temperature cycles, which suggests that there is a firmly entrained endogenous circadian rhythm associated with their formation. However, the possibility that increment formation can be disrupted by environmental factors, or that rings in the statolith are produced coincidentally at the rate of approximately one per day, should not be fully discounted without further experimental corroboration. Data on squid age, derived from growth increments in the statolith, clearly have value in fisheries investigations, but they should be treated with caution until they have been validated.


    Increasing recognition of the considerable commercial potential of cephal- opod stocks worldwide {Worms, 1983; Rathjen and Voss, 1987) has highlighted the necessity for effective management of the fishieries for these molluscs. The requirement for information on growth rate and life span, to support studies of population biology, has recently led cephalopod biologists to focus attention on the problem of assessing age in squid.

    Apparent growth increments have been found in the squid beak (Tinbergen and Verwey, 1945; Clarke, 1965), the radula (Wirz, 1963 ), the gladius (Spratt, 1978) and were first described in the statolith by Clarke (1966). Increments

    0165-7836/90/$03.50 1990 Elsevier Science Publishers B.V.

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    in the beak and radula have subsequently received little attention but there have been a number of studies on growth increments in the statolith. In this paper, we review data on the structure of the squid statolith and the techniques used to prepare them for age studies. We consider the published evidence in support of the hypothesis that growth increments in the statolith are formed daily.


    Squid statoliths are paired calcareous concretions that lie loosely attached at the anterior end of the statocysts. These saccular organs, in the ventro- posterior region of the cartilaginous skull behind the brain, are the sense or- gans responsible for the detection of linear and angular acceleration in squid (Budelmann, 1975, 1977; Stephens and Young, 1978, 1982; Young, 1984; Mad- dock and Young, 1984), and the statolith is functionally analogous to fish oto- liths. It should be noted that in squid the functional body axis is almost per- pendicular to the longitudinal embryonic axis. For convenience and clarity, orientation is referred to, in this review and generally elsewhere, in terms of the functional axis.

    A description of the external morphology of the statolith and an account of interspecific differences in squid, cuttlefish and octopods is given by Clarke (1978) and Clarke and Maddock (1988a,b). The statolith consists of 4 parts: the dorsal dome, lateral dome, rostrum and wing (Fig. 1 ). It is mostly a hard,

    ~/~ / ~ Dorsal dome

    ~ Lateral dome

    ~.. N~ Area o f attprCihnm:;: ~~ to macula w ing ~/ ~ Rostrum

    Fig. 1. Major structural features of a squid statolith: right-hand statolith, anterior view.

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    Fig. 2. Ground, anterio-lateral sections of the statoliths of (a) Illex argentinus (male: mantle length, 245 mm; statolith length, 1.14 mm) and (b) Loligo gahi (male: mantle length, 110 mm: statolith length, 1.73 mm) showing growth increments. Scale bars: 250 #m.

    translucent structure, but the anterior side of the wing, which is the area of attachment to the macula princeps (Dilly, 1976), is softer, opaque white and composed of loosely-packed crystals.

    Statoliths are composed of calcium carbonate in the aragonite crystal form, with an organic matrix (Dilly, 1976; Radtke, 1983). The crystaline subunits, the statoconia, vary in size but are usually elongated and hexagonal with pointed ends. In Illex illecebrosus from Newfoundland, the organic matrix comprises 4.5-5.6% of the total weight of the statolith and over this range the organic component shows a significant negative linear relationship with squid size (Radke, 1983 ). The organic matter is proteinaceous and largely comprised of acidic amino acids, notably aspartic acid, glutamic acid and glycine, which are known to be implicated in biological calcification (Hare, 1963; Weiner and Hood, 1975; Miterer, 1978; Weiner, 1979).

    During development, the statolith of/. illecebrosus, and other ommastrephid species, passes through 5 recognisable stages before attaining the final com- plexity of the adult stage (Morris and Aldrich, 1984; Arkhipkin and Murzov, 1986). The primordial stage has a lachrymiform shape and it develops after the formation of the nucleus. The definitive stage follows, in which all the

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    features illustrated in Fig. 1 become visible, and then there is continuous de- velopment through the pre-juvenile and juvenile stages to the adult stage. This is followed by an advanced stage.

    Although growth increments can sometimes be seen in the untreated stato- lith, they are best viewed after sectioning by grinding on the concave anterio- lateral plane (Hurley and Beck, 1979). Scanning electron microscope exami- nation shows that increments are formed by aragonite crystals in a protein matrix. The crystals radiate from the kernel and disruptions and/or thicken- ings define the boundaries of the increments (Radtke, 1983). In section, the increment-bearing portion of the statolith of adult/, illecebrosus is divided into 3 regions according to variations in the widths of the increments. These regions reflect different stages in the growth and development of the squid (Morris and Aldrich, 1985).

    A growth increment consists of 2 lamellae, one light and one dark (Fig. 2). Pannella (1980) makes the distinction between growth rings and increments in fish otoliths. Each dark lamella is termed a growth ring and an increment consists of one light lamella and the immediately following dark lamella. This terminology has been adopted for the analogous features in the squid statolith (Morris, 1983; Morris and Aldrich, 1985). For the purposes of counting, how- ever, the ring or increment count is effectively identical.


    Examination of statoliths for growth studies requires dissection and removal from the skull, mounting, sectioning and observation.

    Dissection and removal

    Three techniques exist for dissecting statoliths from the squid head. The first is by removing the funnel, flexing the head dorsally and making a series of horizontal incisions through the cephalic cartilage to expose the statocysts (Clarke, 1978; Arkhipkin and Murzov, 1985 ). Alternatively, the funnel can be removed, the head placed on its dorsal surface and vertical incisions made through the cartilage exposing the statocysts; a horizontal incision through the ventral wall of the statocysts then exposes the statoliths (Morris and Aldrich, 1984). The head may also be placed on its ventral surface and a series of ver- tical incisions made through the cephalic cartilage, starting at the posterior end; slicing until the statocysts are exposed.

    Statoliths have also been extracted by dissolving the cartilaginous skull in sodium hypochlorite (Hurley and Beck, 1979).

    As with fish otoliths, statoliths are acellular, mineralized structures, so de- composition should not occur under relatively dry conditions (Campana and Neilson, 1985). However, Dawe et al. (1985) found that drying sometimes

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    causes the statolith to become opaque. This is overcome by storage in absolute alcohol, 70% ethanol, gelatin capsules or glycerol.

    Tissue fragments attached to the statolith after dissection are removed be- fore mounting. Morris and Aldrich (1985) achieved this by soaking in 5% hy- pochlorite solution in water. Careful removal of debris using a mounting needle and fine forceps, followed by immersion in 90% industrial methylated spirit (IMS) for 5 min, produces a specimen suitable for mounting.

    Mounting Whole statoliths may be mounted in liquid paraffin (Lipinski, 1986) but for

    preparing sections, hard-setting resins are used. Suitable mountants for light microscopy include: "Protexx" (Rosenberg et al., 1980; Dawe et al., 1985; Hur- ley et al., 1985), "Lakeside 70" (Kristensen, 1980), "Epon 812" (Morris and Aldrich, 1985 ) and "Polarbed 812".

    Statoliths must be mounted at the correct angle with the concave, anterior surface facing upwards (Morris, 1983 ). Occulting crystals on the anterior sur- face are then ground away to expose the increments below. Increments are obliterated from the periphery of the statolith if the angle of orientation is incorrect. Dawe et al. (1985) mounted statoliths with the convex dorsal surface facing upwards. However, the nucleus is easily obliterated when the statolith is mounted in this way.

    Preparation Grinding is done with silica carbide (carborundum) paper or powder. Wet-

    grinding with water or glycerine using 1000-grit carborundum paper, glued to a glass slide, gives good results. Media used to enhance increments and coun- teract scratches produced during grinding include "Lakeside 70" (Kristensen, 1980), immersion oil (Rosenberg et al., 1980) and glycerol (Morris, 1983).

    Observation A drawing arm mounted on a microscope allows a permanent record of growth

    increments to be made and enhances counting accuracy. High-resolution pho- tomicrographs are difficult to produce, because the growth increments do not all occur in the same focal plane under high power (Dawe et al., 1985 ). Morris and Aldrich (1985) used a microprojector to produce an image of the incre- ments onto paper via a mirror. The resulting magnification (690 X ) gave good results and facilitated accurate counting.

    Two studies have used whole statoliths to view increments. Hixon and Vil- loch (1983) and Yang et al. (1986) decalcified statoliths in a 1:1 mixture of 4% EDTA in distilled water and 0.2 M cacodylate buffer (pH 7.4). Using this method it was not possible to count increments in squid older than 65 days because decalcification distorted larger statoliths. Lipinski (1986) dissolved the inorganic part of the statolith in 0.1 N hydrochloric acid and 10% trichlor

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    acetic acid and stained the resultant 'ghost' with Coomassie Brilliant Blue G- 250. This technique revealed the increments very clearly, except near the edge of the statolith.

    Scanning electron microscopy (SEM) has been used to examine statolith microstructure (Dilly, 1976; Radtke, 1983; Dawe et al., 1985; Lipinski, 1986), but light microscopy is more suitable for routine examination in growth studies.

    Storage Statoliths readily dissolve in weak acid and so are rarely found in specimens

    of squid fixed in unbuffered formalin (Kristensen, 1980). The effect of long- term storage on clarity of growth increments following removal of the statolith is unknown. However, when stored for up to 7 months in glycerol, they are easy to read after processing. Statoliths from squid frozen for 18 months can be read as easily as those from fresh specimens. After sectioning, growth increments fade on prolonged exposure to air. Increments in statoliths of I. argentinus and Loligo gahi, have faded after 6 months (unpublished observation, 1987) and in statoliths from/, illecebrosus they have faded after 12 months (C.C. Morris, personal communication, 1987).


    Micro-growth increments are laid down in the shells of bivalve molluscs (cockles) at intervals corresponding to a semi-diurnal period of tidal immer- sion (Richardson et al., 1980b). Increment formation in cockles is subject to an endogenous rhythm which is entrained and reinforced by regular tidal im- mersions (Richardson et al., 1980a). In fish, micro-growth increments are laid down daily in otoliths and scales (Pannella, 1971, 1974; Brothers et al., 1976; Ralston, 1976; Struhsaker and Uchiyama, 1976; Taubert and Coble, 1977; Ottaway, 1978). Sub-daily increments have also been noted in some species (Campana and Neilson, 1985). There is apparently an endogenous circadian rhythm of increment formation in fish, which is thought to be entrained by photoperiod (Campana and Neilson, 1985 ). Daily growth markings, related to temperature, salinity and feeding, are also present in the cuttlefish shell (Choe, 1963).

    By analogy with the fish otolith, and with regard to the fact that cephalopod physiology is influenced by a diurnal rhythm, it is proposed that increments in the squid statolith are produced on a daily basis (Spratt, 1978; Kristensen, 1980; and see review by Dawe, 1981 ). In addition, Kristensen (1980) has also suggested that second-order rings are laid down at fortnightly and monthly intervals.

    In a number of squid species there is a strong positive relationship between statolith size and measurements of squid body size, such as mantle length, and strong relationships also exist between the number of growth increments in

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    the statolith, size of the statolith and mantle length (Hurley and Beck, 1979; Kristensen, 1980; Radtke, 1983, Morris and Aldrich, 1985). These observa- tions support the view that the statolith grows in concert with the rest of the animal and that increments are laid down regularly during growth.

    If growth increments are formed daily in the squid statolith, it should be possible to follow the modal length of a wild squid population by regular sam- pling, and relate the increase in number of increments, in the statoliths from squid at the mode, to the number of days elapsed between samples. Sampling and counting increments in this way with populations of Loligo opalescens (Spratt, 1978) and L illecebrosus (Hurley and Beck, 1979) indicated that the number of rings produced underestimated the number of days elapsed. This may have...


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