ADRIAMED

SCIENTIFIC COOPERATION TO SUPPORT RESPONSIBLE FISHERIES IN THE ADRIATIC SEA

GCP/RER/010/ITA GCP/RER/021/EC

Cephalopods Age Determination by Statolith Reading: a Technical Manual

AdriaMed Technical Documents No. 22 GCP/RER/010/ITA/TD-22

Rome (Italy), November 2007

The conclusions and recommendations given in this and in other documents in the Scientific Cooperation to Support Responsible Fisheries in the Adriatic Sea Project series are those considered appropriate at the time of preparation. They may be modified in the light of further knowledge gained in subsequent stages of the Project. The designations employed and the presentation of material in this publication do not imply the expression of any opinion on the part of FAO or MiPAAF or EC concerning the legal status of any country, territory, city or area, or concerning the determination of its frontiers or boundaries.

ii

Preface
The Regional Project Scientific Cooperation to Support Responsible Fisheries in the Adriatic Sea (AdriaMed) is executed by the Food and Agriculture Organization of the United Nations (FAO) and funded by the Italian Ministry of Agriculture Food and Forestry Policies (MiPAAF) and since 2007 from the Directorate General for Fisheries and Maritime Affairs of the European Commission. AdriaMed was conceived to contribute to the promotion of cooperative fishery management between the participating countries (Republics of Albania, Croatia, Italy, Montenegro and Slovenia), in line with the Code of Conduct for Responsible Fisheries adopted by the UNFAO. Particular attention is given to encouraging and sustaining a smooth process of international collaboration between the Adriatic Sea coastal countries in fishery management, planning and implementation. Consideration is also given to strengthening technical coordination between the national fishery research institutes and administrations, the fishery organizations and the other relevant stakeholders of the Adriatic countries.

FAO-AdriaMed Project HQ FAO FIFM Viale delle Terme di Caracalla 00153 Rome, Italy Tel: ++39 06 570 55467 Fax: ++39 06 570 55188 e-mail: faoadriamed@faoadriamed.org URL: http://www.faoadriamed.org

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GCP/RER/010/ITA Publications

The AdriaMed Project publications are issued as a series of Technical Documents (GCP/RER/010/ITA/TD-00) and Occasional Papers (GCP/RER/010/ITA/OP-00) related to meetings and research organized by or conducted within the framework of the Project. Occasionally, relevant documents may be translated into national languages as AdriaMed Translations (GCP/RER/010/ITA/AT-00). Comments on this document would be welcomed and should be sent to the Project headquarters: AdriaMed Project FAO FIFM Viale delle Terme di Caracalla 00153 Roma Italy faoadriamed@faoadriamed.org

For bibliographic purposes this document should be cited as follows: Ceriola, L. and Milone, N. 2007. Cephalopods Age Determination by Statolith Reading: a Technical Manual. Scientific Cooperation to Support Responsible Fisheries in the Adriatic Sea. GCP/RER/010/ITA/TD-22. AdriaMed Technical Documents, 22: 78pp.

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Preparation of this document

This document is the final version of the material used for the preparation of two training courses on age determination of cephalopods by statolith readings organized in 2006 and in 2007 by the FAO Projects AdriaMed and MedSudMed. These notes aim at providing an introductory treatment of some of the basic topics of age determination using statoliths. The notes serve as a reference guide to the methods used during the training courses. These notes are primarily for the scientists involved in the AdriaMed and MedSudMed Project research activities related to the appraisal of fisheries resources, they can also be of interest for students and professionals of fisheries research. However, a comprehensive introduction to the methodologies to be applied is outside the scope of this publication. This manual should be considered a further integration to the information reported by Jereb et al. (1991) in view of the research carried out and the progress made thereafter. It provides an updated and easily accessible tool to facilitate the expansion of statolith analysis in the Mediterranean where, in spite of the great importance of cephalopods in fisheries, research on this mollusc class still has to be developed entirely. Interested readers may find the literature given in Chapter 9, useful for in-depth treatment of the topic and the Glossary annexed to the document.

Acknowledgements
Special thanks are due to Ms Patrizia Jereb for her valuable scientific advice which has led to the completion and the improvement of this document. Many thanks are also due to Dr George Jackson for his support and the technical information provided. The assistance of Ms Caroline Bennett in the preparation of this document is gratefully acknowledged. Ceriola, L. and Milone, N. Cephalopods Age Determination by Statoliths Reading: a Technical Manual. AdriaMed Technical Documents. No.22. GCP/RER/010/ITA/TD-22, Rome, 2007: 78 pp. ABSTRACT The main objective of this document is to provide a useful guideline to the age determination methodologies applied to cephalopods using statolith reading. The document is the result of the training activities on this issue organized by the FAO regional Projects AdriaMed and MedSudMed in 2006 and 2007. A selection of methods to extract, prepare and examine statoliths is described in detail. A brief introduction describing the importance of growth studies in cephalopods, as well as the function, internal structure and shape of statoliths is also included. Chapter 1 contains the background information. Chapter 2 describes the terminology used and illustrates the position, morphology and function of statoliths in cephalopods. In Chapter 3 the equipment, materials and the procedure used to extract, clean and prepare statoliths for reading, and the methods for growth increment counting (statoliths reading) are described. Finally in Chapter 4 the potential results of the age determination by using statolith analysis are illustrated. A Glossary is also included in the manual.

Table of Contents
Acknowledgements............................................................................................................. iv Table of Contents ................................................................................................................ v 1. Background information................................................................................................. 1 1.1 AdriaMed and MedSudMed training activities........................................................ 1 1.2 Cephalopods in the marine ecosystem ...................................................................... 1 1.3 Cephalopod growth estimates ................................................................................... 4 1.4 Aim of this document ................................................................................................ 8 2. Cephalopod statoliths...................................................................................................... 9 2.1 Introduction............................................................................................................... 9 2.2 Shape and current terminology ................................................................................ 9 2.3 Structure and microstructure ................................................................................. 12 3. Statolith analysis ........................................................................................................... 21 3.1 Equipment and materials ........................................................................................ 21 3.1.2.Collecting data sheet......................................................................................... 22 3.1.3 Measuring and dissecting kit............................................................................ 23 3.1.4 Cleaning and storing......................................................................................... 24 3.1.5 Mounting and grinding..................................................................................... 24 3.1.6 Counting (statolith reading) ............................................................................. 27 3.2 Procedure and techniques ....................................................................................... 28 3.2.1 Extraction.......................................................................................................... 28 3.2.1.1 Chemical method ....................................................................................... 29 3.2.1.2 Surgical method ......................................................................................... 29 3.2.2 Cleaning and storage ........................................................................................ 32 3.2.2.1 Cleaning...................................................................................................... 33 3.2.2.2 Storage........................................................................................................ 33 3.2.3 Mounting and grinding..................................................................................... 35 3.2.3.1 Mounting .................................................................................................... 36 3.2.3.2 Grinding ..................................................................................................... 38 3.2.4 Counting............................................................................................................ 42 3.2.4.1 Direct counting........................................................................................... 45 3.2.4.2 Image analysis system ................................................................................ 47 4. Some possible outcomes ................................................................................................ 49 5. References...................................................................................................................... 50 Glossary............................................................................................................................. 61 Annex A: Data sheet.......................................................................................................... 65 Annex B: Statoliths extraction from fresh animals (Sepioidea, Teuthoidea and Octopoda) .......................................................................................................................... 66 Annex C: Ground statoliths.............................................................................................. 74

1. Background information 1.1 AdriaMed and MedSudMed training activities The training component has been central to the AdriaMed and MedSudMed Projects since their outset. During the AdriaMed and MedSudMed Coordination Committee meetings, the necessity to support and develop the Project component on training as a key part of the work programme was emphasised. Other regional experts meetings organised by the Projects over the years, also highlighted this requirement. Several training activities were therefore planned and organized in agreement with the needs expressed by the research institutions involved in the Projects activities. For AdriaMed this was mainly achieved through the Project Working Group on Demersal Fisheries Resources; in particular several training courses were organized by AdriaMed to enhance the standardization of the methodologies used for the appraisal of demersal resources at Adriatic level. An annual, international bottom trawl survey was organized among the eastern countries of the Adriatic Sea, supported by the AdriaMed Project, for the joint appraisal of demersal resources in the region. However, it became increasingly clear from the Adriatic experts that national expertise needed to be developed and improved within the region, and particularly in cephalopod appraisal methods. The same request was expressed by the research institutions participating in the FAO MedSudMed Project (that focuses on the Central Mediterranean). Consequently, a first training course was organized in the framework of MedSudMed activities in July 2006, a second training course was then organized by AdriaMed in May 2007 in Split (Croatia) in order to provide basic knowledge on age reading techniques, in particular concerning the reading of cephalopod statoliths. The didactic material prepared and used during these training exercises has therefore been drawn on to prepare this manual. These notes aim to provide an introductory treatment of some of the basic topics of age determination using statoliths. The notes serve as a reference guide to the methods used during the training courses and are primarily for the junior scientists involved in the AdriaMed and MedSudMed Project research activities related to the appraisal of the fisheries resources. They can also be of interest for students and professionals of fisheries research.

1.2 Cephalopods in the marine ecosystem Cephalopods play an important role in the marine ecosystems, both as predators and prey. They are voracious, opportunistic and highly versatile predators of fish and invertebrates, and also represent the most important prey category of several fish species and top predators such as marine birds, whales and other marine mammals (for extensive reviews see Rodhouse and Nigmatullin, 1996; Piatkowski et al., 2001; Boyle and Rodhouse, 2005). In terms of biomass, on the basis of fisheries data, cephalopod production for the 1988-1991 period was estimated to be 1.88 percent of the total production necessary to sustain total 6 global fishery (i.e. 2.476 x 10 t, mean annual net weight) (Pauly and Christensen, 1995, in Boyle and Rodhouse, 2005). Human fishery for cephalopods has continued to rise since then
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and some information on their biomass has been added due to improved knowledge on the consumption of cephalopods by natural predators. In the Southern Ocean, based on an estimate of the consumption of cephalopods by 6 vertebrate predators (30 x 10 t, Rodhouse et al., 1994a), a standing stock of squid biomass as high as 100 million tonnes was calculated. Further estimates of the whole cephalopod standing stock biomass reached values between 193 and 375 million tonnes (Rodhouse and Nigmatullin, 1996), and the average annual cephalopod consumption by sperm whale alone 6 was estimated to reach 267 x 10 t (Clarke, 1996). According to these estimates, the application of Pauly and Christensens approach (1995) to more recent data would lead to considerably higher values. Although cephalopod incidence in predators diet may be overestimated (e.g. Santos et al., 2001) and fishery data may be not entirely accurate, if considering that top predators harvest squids and fishes generally not available to human fishery (e.g. Trites et al., 1997), values of total cephalopod biomass in the worlds oceans up 6 to 500 x 10 t (Voss, 1973) may not be so unlikely. Being subdominant predators that tend to increase in biomass when other species become depleted, and having been estimated to 9 consume between 2 and 4 x 10 t of food annually (Rodhouse and Nigmatullin, 1996), it is now clear that cephalopods are a dominant component within the marine ecosystem and that ultimately fluctuation in their abundance may seriously affect the abundance of their predators and prey populations (e.g. Jereb et al., 2005). Originally not included among the economically important fishery resources, cephalopods have gained increasing attention in the past decades as an alternative to more traditional marine resources, and their importance as a source of protein for human consumption is likely to continue to increase in the future (Caddy and Rodhouse, 1998; Piatkowsky et al., 2001; Rosa et al., 2002; 2005). Due to a steady increase in cephalopod capture production during the last 30 years, from about 1 to around 3.5 million Mt (FAO, 2006) (Figure 1), the fishery they support became one of the top invertebrate fisheries in the world.

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

Mt

1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 Year

Figure 1. World capture production for cephalopod (1970-2005).

In the Mediterranean Sea, total cephalopod capture production increased steadily until the end of the 1980s, when a peak was reached (83 000 tonnes in 1988), followed by a decrease

during the 1990s, after which the total commercial landings settled down to about 50 000 tonnes per year (FAO, 2006) (Figure 2).
90000 80000 70000 60000

Tonnes

50000 40000 30000 20000 10000 0 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003

Year

Figure 2. Mediterranean capture production for cephalopods (1970-2004).

A similar scenario characterizes the Adriatic Sea, where a decreasing trend in total cephalopod landings was recorded starting from the mid 1990s (FAO, 2006) (Figure 3).

20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0

Tonnes

1970

1973

1976 1979 1982

1985

1988 1991

1994

1997 2000

2003

Year

Figure 3. Total capture production for cephalopod in the Adriatic Sea (1970-2004).

This decreasing trend, anomalous if related to the continuously increasing trends observed in the other areas of the distributional range, was described in detail and tentatively commented on (Jereb, 2002; Jereb and Agnesi, in press), but no sound explanation has been given to date. Major characteristics of the cephalopod populations studied so far are the relatively short life span, high growth rates and relatively early maturity, along with a remarkable physiological capacity to respond to environmental changes by quickly adjusting these biological features (e.g. Boyle, 1983, 1987; Boyle and Boletzky, 1966; Boletzky, 1994; Roberts et al., 1998;
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Dawe et al., 2001; Rodhouse, 2001; Rodhouse et al., 2001; ODor et al., 2002; Waluda et al. 2004). Their high plasticity in growth-rate, in particular, provides cephalopods with an advantage in the competition with long-living late-maturing finfish species, as highlighted by capture production trends in highly harvested regions (Caddy and Rodhouse, 1998; Balgueras et al., 2000; Pauly et al., 2001; Payne et al., 2006; Ungaro et al., 2006; Ceriola et al., 2007). These unique physiological strategies make cephalopods key-species in regions characterized by considerable environmental fluctuations or high fishing pressure (Trotsenko and Pinchukov, 1994; Laptikowsky and Remelso, 2001; Zeidberg and Hamner, 2002; Jackson and Domeier, 2003; Ceriola et al., 2006; Chen et al., 2006; Dawe et al., 2006; Jackson et al., 2007) and may ultimately result in dramatic abundance fluctuations, due to change in oceanographic conditions (e.g. ODor and Dawe, 1998; Dawe et al., 2000; Bendik, 2001; Dawe et al., 2001; Jackson and Domeier, 2003). Worldwide concern on the level of exploitation of all marine resources was the driving force to increase knowledge for assessment and management purposes. Attention to cephalopods came late, but have proceeded continuously since then and fishery-related studies are rapidly contributing to knowledge on the biology of many cephalopod species (e.g. Rodhouse et al., 1994b; Payne et al., 1998; Rodhouse et al., 2001; Boyle and Rodhouse, 2005; Jereb and Roper, 2005; McIntyre, 2006). Due to the importance of growth (and growth rates) within cephalopods life cycle, the understanding of this key-process is one of the main targets of many recent comprehensive studies focusing on cephalopods (e.g. Jackson and ODor, 2001; Ragonese et al., 2002; Arkhipkin, 2004; Jackson, 2004).

1.3 Cephalopod growth estimates Cephalopod growth can be estimated by applying both indirect and direct methods. Indirect method The indirect method consists in the analysis of the length frequency distributions (LFD) observed in samples obtained from commercial landings or experimental surveys (e.g. Mangold-Wirz, 1963; Sanchez, 1984; Jereb and Ragonese, 1995), as is the practice for fish. This involves the identification and interpretation of the different modes present in the usually polymodal LFD of the selected species. Often this analysis is subjective and the final outputs can vary considerably according to the readers interpretation. In cephalopods, most of the studies have been carried out on squids and several modes usually resulted in the studied LFD. These have been alternatively interpreted either as the result of the overlapping of different sub-cohorts (because of a protracted spawning season) or as sub-year or year classes, with different resulting life-cycle duration estimates (for reviews see Arkhipkin, 2004; Jackson, 2004). The debate is still open, hoewever to summarize, LFD analysis is considered suitable for discrete and well-identified spawning periods (generally corresponding to a unimodal LFD per time interval) as in the case of many fish species, while it seems less appropriate when applied to species that have a protracted spawning activity, such as many cephalopods.

Direct methods a) Rearing A direct method to investigate growth in cephalopods consists in the observation of the animals in aquarium, where the variation in size (both length and weight) over periodic time intervals can be measured (Figure 4). This provides sound information on individual growth rate at different ontogenetic stages and allows an understanding of the influence of abiotic and biotic factors on the growth process (e.g. temperature and food ration, see for example Jackson and Moltschaniwskyj, 2001a; Villanueva 2000a,b; Forsythe et al., 2001; Villanueva et al., 2003; Forsythe, 2004; Chung and Lu, 2005). However, several problems still limit a wide application of this method. To date, not many cephalopod species have been successfully reared in captivity for the entire life cycle, mostly due to a considerably high mortality rate at the early stages. Thus, information on growth of reared animals may not cover the whole life span, referring instead to a fraction of it (Arkhipkin, 2004). In addition, uncertainty about the correspondence of growth in captivity to that in the wild remains, as it is not possible to perfectly reproduce natural environmental conditions in an aquarium. In spite of these negative constraints, studies performed on reared animals helped to understand growth performance in several species and were used to validate results obtained by applying other ageing methods (e.g. to validate the time interval necessary for the formation of growth increments in statoliths and gladii) (Jackson, 1994; Arkhipkin, 2004; Jackson, 2004) a b c

Figure 4. Cephalopod rearing in captivity: a-b eggs maintenance, c adults rearing. From CephBase (http://www.cephbase.utmb.edu/)

b) Tagging and recapture experiments in the wild Tagging cephalopods in the wild and recapturing them, by knowing the time interval (i.e. the time that passes from the tagging and the recapture), offers another direct way to measure growth of this group of species. This method has been used for a long time, from the very early experiments in the late 1920s (for review see Nagasawa et al., 1993), until the recent multi-species electronic Tagging of Pacific Pelagics (TOPP), a pilot program of the Census of Marine Life (Block et al, 2002). Mainly applied to investigate vertical and horizontal migrations and geographic distribution of commercially important ommastrephid populations, this methodology also allows for the collection of important information on the biology, physiology, ecology and stock identity of the investigated populations (Nagasawa et al., 1993). Several tags can be used, depending mainly on the individual size range of the species investigated, on the research targets and on the amount of funding available. Tags more commonly applied to cephalopods are shown in Figure 5. Also in this case, however,
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unavoidable disadvantages have limited the use of the methodology to investigate growth in cephalopods and in squids in particular. Indeed, many squids are highly migratory animals and their recapture rates are very variable (for reviews see Nagasawa et al., 1993; Murata and Nakamura, 1998; Sauer et al., 2000; Markaida et al., 2005; Stark et al., 2005; Semmens et al., 2007); in addition, the tagging process itself may affect animals mantle integrity and swimming capability and eventually their survival, such that, in general, juveniles and small size specimens cannot be tagged (e.g., Semmens et al., 2007).

(a)

(b)

(c)

Figure 5. Tags generally used to mark small fish and cephalopods: (a) Spaghetti tag; (b) Dart tag; (c) T-bar anchor tag.

c) Hard structures analysis All the hard structures in cephalopods soft bodies, with the exception of the chitinous suckers rings, have the potential to memorise ontogenetic events through the formation of periodical marks, or growth increments (see Arkhipkin, 2005, for a review) (Figures 6,7). These structures include statoliths (Young, 1960; Clarke, 1966), gladii (La Roe, 1971), mandibles (Clarke, 1965) crystalline lens of the eye (Williams, 1909) and cuttlebones (Choe, 1963).
Statolith
Gladius

Cuttlebone

Beak

Crystalline lens Figure 6. Schematic illustrations of a cephalopod, showing the location of hard structures potentially suitable for age and growth studies: Statoliths; Bones: Gladius and Sepion; Beak; Crystalline lens. 6

SR

Figure 7. Sucker rings (SR), probably the only hard structure in cephalopods bodies not bearing growth increments.

Statoliths, in particular, are considered as true archives of cephalopod life cycles (Rhoads and Lutz, 1980 in: Lipinski, 2001) and can be used to investigate several aspects of their physiology, ecology and life style (e.g. Clarke and Maddock, 1988; Bizikov, 1991; Lipinski, 2001; Arkhipkin, 2005; Villanueva et al., 2007). Most important to the purpose of this manual, all these structures have the necessary characteristics to be used as ageing tools: (i) the presence of interpretable increment microstructures; (ii) the possibility to correlate these microstructures progression/evolution with a regular and determinable time scale; (iii) continuous growth at a measurable rate throughout the whole life span, especially in squids (Beamish and McFarlane, 1983 in: Arkhipkin, 2005). The first to notice periodical marks on statoliths was Young (1960) who studied the statocysts in Octopus vulgaris. However, only in the middle of the 1960s was the role of statoliths as recording structures recognized, when growth increments in the statolith microstructure of several squid species were described (Clarke, 1966). Since then, several studies have been carried out and outstanding progresses have been made in understanding statoliths structure, function and their possible use to investigate cephalopod growth (e.g. Rodhouse and Hatfield, 1990; Jereb et al., 1991; Arkhipkin and Perez, 1998; Arkhipkin and Bizikov, 2000; Jackson and ODor, 2001; Lipinski, 2001; Bettencourt and Guerra, 2000; Arkhipkin, 2004; Jackson, 2004; Arkhipkin, 2005; Ceriola, 2007; Jackson et al., 2007, Zumholtz et al., 2007a,b). Other than for age and growth estimates, these small calcareous structures proved to be useful for species identification (e.g. Clarke, 1978; Lipinski et al., 1993; Clarke, 1998; Lombarte et al., 2006) and to investigate several aspects of cephalopod life history, including age, hatching date and hatching period, growth rate, ontogenetic shifts, accidental events, number of mating events, variations in environmental conditions, population structure,
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systematic, population dynamic and life style (e.g. Jackson, 1994; Jackson and Moltschaniwskyj, 2001b; Arkhipkin, 2003; Jackson and Domeier, 2003; Steer et al., 2003; Villanueva et al., 2003; Forsythe, 2004; Chung and Lu, 2005; Semmens et al., 2007; Villanueva et al., 2007; Zumholz et al., 2006, 2007c). Indeed, their potential as actual life recorders was eventually acknowledged (Arkhipkin, 2005). As for age determination, several problems still wait to be solved, calling for further research and studies: the pillar hypothesis of the daily periodicity of growth increments (i.e. 1 ring = 1 day), was only partially validated and only in one case did the validation occur in the natural environment (Lipinski et al., 1998); many species have not been studied yet; statolith analysis is laborious and very time consuming; the number of samples is often limited, making age and growth estimates challenging; changes in statolith structure and growth increment deposition rate do occur along with the animals growth and maturity, as changes in the increment width do, and biases related to statolith preparation and differences in the methods of interpreting and enumerating increments have been widely acknowledged (e.g. Lipinski and Durholtz, 1994; Gonzalez et al., 2000; Bettencourt and Guerra, 2001). In spite of all these problems, to date statoliths microstructure analysis is the most widely used method to investigate age and growth in cephalopods species, particularly squids (for reviews see Arkhipkin, 2004; Jackson, 2004). Therefore, broad scale and collaborative studies on their use are highly welcomed, to contribute to evaluating precision and to increase consistency among investigators.

1.4 Aim of this document To promote statolith analysis and gather further information on cephalopods and especially on squid growth, in areas where statoliths are not yet currently used for age and growth studies (e.g. many regions in the Mediterranean basin), practical guidelines are necessary. A first manual on age determination by statolith analysis was published by Jereb et al. (1991) and collects the proceedings of an international workshop held at the Istituto di Tecnologia della Pesca e del Pescato of the Consiglio Nazionale delle Ricerche in Mazara del Vallo (Sicily, Italy) in 1989, it includes a laboratory guide in which basic methods and techniques used for statolith analysis are described. This manual does not intend to replace the precious contribution provided by Jereb et al. (1991), but rather to integrate the information therein reported, in view of the research carried out and the progress made in the almost 20 years that have passed. The aim of this work is to provide an updated and easily accessible tool to help spread the use of statolith analysis in the Mediterranean areas and the Adriatic Sea in particular, where research and studies on cephalopods are currently developing.

2. Cephalopod statoliths 2.1 Introduction Cephalopod statoliths are paired calcareous structures associated with the sensory epithelia (i.e. the macula statica princeps) located on the wall of the anterior chambers of the two adjacent sac-like equilibrium organs called statocysts (Figure 8). First described by Ovsjannikov and Kowalevsky (1867) and intensively studied thereafter (for brief reviews see Budelmann, 1990; Arkhipkin and Bizikov, 2000), statocysts are cavities of irregular shape, located in the posterior/ventral part of the cranial cartilage and consist of two chambers (anterior and posterior), partially separated by finger-like projections and filled with liquid. They can be considered analogous to the vertebrate semicircular system (Stephens and Young, 1978; Williamson and Budelmann, 1985) and have two receptor systems (i.e. the gravity and the angular acceleration receptor system) which give the animal proper information about its position and movement in the water and enable it to compensate its eye movements (Budelmann, 1977). The complex macula-statolith is responsible for the detection of gravity and a possible role of statoliths in the detection of angular accelerations was recently hypothesized (Arkhipkin and Bizikov, 1998). The potential of the statocists to detect vibration stimuli was also investigated and the capability to detect low-frequencies sounds was recorded and commented (e.g. Hanlon and Budelmann, 1987, Hanlon and Messenger, 1996).

STATOLITHS

STATOCYSTS
Figure 8. Statocyst structure (anterior part) and position of the statoliths (from Arkhipkin and Bizikov, 2000 modified).

2.2 Shape and current terminology Statolith shape is species-specific and varies greatly both in the same species, during the ontogenesis from a simple droplet in paralarvae (Figure 9) to the differentiated statoliths in

all adult specimens (Arkhipkin, 2003), and among the different cephalopod groups, octopods, squids and cuttlefishes (Figure 10).

(A)

(B)

20 m
Figure 9. Illex illecebrosus statoliths: A) from a newly hatched paralarval (0 day old); B) from a 7 days old specimen (from Sakai et al., 2004).

0.5 mm

(A)

(B)

(C)

Figure 9. Statoliths of (A) Loligo vulgaris, (B) Sepia officinalis, (C) Eledone moscata at the transmitting light microscope.

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Clarke and Maddock (1988) suggested that statolith shape depends more on the evolutionary relationships of cephalopods rather than on the statoliths function. Arkhipkin and Bizikov (2000) confirmed this hypothesis for squids and sepioids and distinguished two main statolith morphologies: the demersal, typical of near-bottom decapods, and the pelagic, typical of all pelagic squids. Teuthoid and sepioid adult statoliths are considerably different from octopod statoliths and their external morphology is characterised by four main regions: the dorsal dome, the lateral dome, the rostrum and the wing (Clarke, 1978) (Figure11). The wing, which is the area of attachment to the statocyst wall (Dilly, 1976), has a dorsal and a ventral indentation, separated by a structure called the spur. Statoliths are predominantly hard and translucent except for the wing, which is softer and opaque white. For convenience and clarity in the present document, to describe statolith orientation (anterior, posterior, dorsal, ventral, medial, lateral, marginal) and sections (sagittal, transversal, frontal), the nomenclature reported by Lipinski et al. (1991) is adopted (Figure 12).

DD (a) DS LD S W (b)

Figure 11. Adopted terminology to describe the external morphology of the statolith: (a) lateral view, (b) anterior views; LD = lateral dome; DD = dorsal dome; R = rostrum; W = wing; S = spur; LS = lateral spur. From Arkhipkin, 2005 modified.

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(1)

(2)

(3)
Figure 12. Adopted terminology to indicate statolith orientation and section planes. Left: D = dorsal; V = ventral; L = lateral; M = medial; A = anterior; s = sagittal; t = transversal; f = frontal; m = marginal. (From Lipinski et al., 1991 modified). Right: (1) = lateral view/section; (2) = frontal view/section; (3) transversal view/section. The shape and proportions in the figures are only indicative.

2.3 Structure and microstructure Statoliths are composed of calcium carbonate (CaCO3) crystallised as aragonite, which lie within a matrix of organic material that has been ascertained to be mucoprotein (Radtke, 1983). The aragonite crystals are about the 95% in weight of the whole structure, with the mucoprotein matrix accounting for the remaining 5%. However, an inverse relationship was observed between this proportion and the individual size/age: the quantity of organic matter declines with age, i.e. statoliths become more calcified as the animal grows up (Arkhipkin and Perez, 1998; Bettencourt and Guerra, 2000). Statolith formation results from a process called biomineralization (i.e. the deposition of a mineral structure in a living creature) that was recently reviewed and described in detail for Loligo vulgaris and Sepia officinalis by Bettencourt and Guerra (2000). These authors suggested that the different biochemical composition of the statocyst endolymph found in the two species is responsible for different crystallization processes, which results in a different microstructure and, ultimately, in a different definition of the growth increments. In addition, they formulated the working hypothesis that dark rings (rich in organic matter) are deposited during daylight, while light rings (rich in CaCO3) during darkness. The crystals of carbonate and the protein matrix are deposited around a starting point named focus (Natsukari et al., 1988) (Figure 13), which usually consists of one single concretion (Arkhipkin and Perez, 1998). Around the focus statolith development proceeds periodically
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with the deposition of new strata (or growth increments) outside the pre-existing external surface. This process continues during the individuals entire life.

F F

10 m

5 m

Figure 13. Detail of the statolith microstructure in ommastrephids (left) and loliginids (right); the arrows point to F, the focus, the initial point of the statolith.

The width of each growth increment (0.5-10 m) varies depending on the deposition rates and, indirectly, on the environmental variations or the ontogenetic shifts experienced by the animal during the lifespan; for example, hatching and the transition between ontogenetic phases have direct consequences on the deposition process inducing a marked anomaly in the formation of the growth increments (Arkhipkin, 2005). The understanding and interpretation of the growth increment periodicity, that is the period of time necessary for the development of a complete increment, is one of the main problems concerning the use of statoliths for age and growth studies (for reviews see Arkhipkin, 1991; Lipinski et al., 1991; Jackson, 1994; Arkhipkin and Perez, 1998; Arkhipkin, 2004; Jackson, 2004). Increasing evidence suggested that increments are formed with a daily periodicity and that their total number represents the individuals age in days (e.g. Lipinski, 1978; Hurley and Beck, 1979; Hurley et al., 1979; Jackson and Choat, 1992; Jackson, 1994; Arkhipkin, 2004). The hypothesis one increment = one day was then validated for several species by means of different experimental methods (for reviews see e.g. Arkhipkin, 1991; Arkhipkin, 2004; Jackson, 2004). Even though this assumption is now widely accepted, it is dutiful to point out that results of statolith analysis should be taken with caution and the derived growth estimates should be considered putative in species for which this validation has not been carried out yet (Lipinski and Durholtz, 1994; Lipinski et al., 1998). In general, statolith microstructure reflects its periodical formation: when observed in a proper section a number of concentric rings (for comments on the terminology see Lipinski et al., 1991; and Lipinski, 1993) become visible with the centre in the focus (Figure 14). These rings (hereunder also referred to as growth rings or growth increments) correspond to the statoliths growth increments. They are composed of the paired dark and light growth layers produced over a 24h period (Figure 15) (e.g., Arkhipkin, 1991; Lipinski
13

et al., 1991; Jackson, 1994; Bettencourt and Guerra, 2000; Sakai et al., 2004; Arkhipkin, 2005; Jackson et al., 2005).

50 m

Figure 14. Statolith microstructure; narrow and large growth increments are highlighted.

Four main growth zones or regions, each characterised by rings of different width, are generally observed within the statolith microstructure of adult squids: the nucleus and the postnuclear, dark and peripheral zones (Figure 16). Each zone is formed during different ontogenetic phases and the shift between adjacent zones is usually related to ontogenetic shifts (Arkhipkin, 2005).

5 m

Figure 15. Dark and light layers alternate in the statolith growth increments.

14

PZ

DZ

PNZ

Figure 16. The four main zones within the statolith microstructure: N= nucleus; PNZ = post nuclear zone; DZ = dark zone; PF = peripheral zone. The arrow points to the focus (F).

The nucleus is the area surrounding the focus and its outer border appears as an unmistakable marked ring when observing the statolith in a prepared section (Figure 17). Considered an indirect effect of the stress associated with hatching (e.g. Villanueva et al. 2003), it was named hatch-check or natal-ring (Hureley and Beck, 1979; Lipinski, 1986; Natsukari et al., 1988; Jackson, 1989), and can be considered the appropriate location/ring from which to begin post-hatching age estimation.

15

(A)

NR

NR

(B)

Figure 17. Detail of the statolith microstructure in (A) ommastrephids and (B) loliginids; NR, the natal ring, is highlighted.

The natal ring corresponds to the first growth increment in ommastrephid squids (e.g. Radtke, 1983; Balch et al., 1988; Arkhipkin and Perez, 1998; Villanueva et al., 2003; Steer et al., 2003) and probably in most oegopsid squids, as growth increments in this group are only deposited after hatching (Arkhipkin, 2004, 2005). On the contrary, in myopsid squids growth increment formation begins during the embryogenesis and the newly hatched statoliths already have several embryonic increments inside the natal ring (e.g., Morris, 1991; Villanueva et al., 2003; Jackson and Moltschaniwsky, 2001a; Jackson, 2004) (Figure 18).

16

NR

IRs

50

NR

IRs

50

NR IRs

25

Figure 18. Natal ring and some internal rings in the statolith microstructure; NR = natal rings; . IRs = internal rings.

17

The postnuclear zone (Figure 19) is generally formed within the immediate post-hatching or paralarval phase. This zone is well defined in Ommastrephidae which are characterised by having a special paralarval phase, named rhynchoteuthion (Chun, 1903), in which both tentacles are fused into a proboscis.

PNZ

PNZ

Figure 19. Details of the statolith microstructure: the arrows indicate the inner and the outer boundaries of the postnuclear zone (PNZ).

This constitutes a very delicate phase in the squids life cycle (Dawe and Brodziak, 1998), since the animals seem unable to attack prey (Boletzky and Hanlon, 1983) and they probably feed on suspension material (ODdor et al., 1985). The transformation to predatory juveniles may be critical, by causing temporary starvation (e.g. Froeman, 1984) which results in a
18

decreasing growth phase, recorded as prominent check in the statoliths microstructure (e.g. Laptikhowsky et al., 1993; Arkhipkin, 2005). Other Oegopsid squids do not have a distinct paralarval phase; thus, such a distinct boundary between the postnuclear and the following zone is not evident in their statoliths (e.g. Arkhipkin and Perez, 1998; Arkhipkin, 2005). Both the subsequent dark and peripheral zones are characterised by regularly spaced growth increments (Figure 20). The influence of several factors on the formation of these zones was studied, and the transition from the dark to the peripheral region was alternatively attributed to changes in the food regime, as well as to habitat shifts (e.g. Jackson, 1993; Arkhipkin, 1997; Arkhipkin and Perez, 1998; Arkhipkin, 2005).

PZ DZ F

PNZ

Figure 20. Statolith microstructure main four regions: (F) focus, (PNZ) postnuclear zone, (DZ) dark zone, (PZ) peripheral zone.

Conspicuously marked rings or checks (Lipinski et al., 1991) are often noticeable within the statoliths microstructure (Figure 21). These prominent rings are assumed to reflect the occurrence of specific events in the cephalopods life, and were associated to various stressful episodes such as behavioural changes, migrations, starvations, thermal shocks, mating etc. (e.g., Spratt, 1978; Kristensen, 1980; Lipinski, 1981; Arkhipkin and Murzov, 1986; Arkhipkin, 1988, Arkhipkin and Perez, 1998, Arkhipkin, 2005).

19

check

check

Figure 21. Checks in the statolith microstructure of adult females, in response of mating event.

20

3. Statolith analysis 3.1 Equipment and materials Statolith analysis is a multi-phase procedure, which includes statolith extraction, cleaning, mounting (on an appropriate support), grinding and, eventually, reading (i.e. growth increments counting). The following commonly used laboratory tools and specific materials and equipments are required to proceed (Figure 22): cephalopods samples data collecting sheets dissecting and measuring kits cleaning and storing kits mounting and grinding equipment ring counting (statolith reading) equipment

A detailed description of the necessary equipment is hereby provided and the most important characteristics of specific materials (e.g. glue paper for grinding) are described and commented on.

(a)

(d)

(b)

(e)

(c)

(f)

Figure 22. Equipment and materials necessary to collect and prepare the statoliths: cephalopod samples (a) cephalopod samples; (b) dissecting kit; (c) lapping films; (d) microscope slide, (e) mounting medium; (f) transmitting light microscope. 21

3.1.1.Samples Fresh, frozen or alcohol-preserved cephalopods can be used for statolith extraction and studies (Figure 23). Statoliths will be considerably easy to find in the cephalic cartilage of fresh animals and also in defrosted specimens, whilst it can be difficult to extract them from specimens preserved for a long time (more than two years) in ethyl alcohol (Lipinski, 1980; Dawe and Natsukari, 1991; Jackson and Choat, 1992). Specimens preserved in formalin are not suitable for statolith studies because formalin deeply damages calcareous structures.

Figure 23. Fresh samples: Sepia officinalis (left); Illex coindetii (right)

3.1.2.Collecting data sheet The preparation of the data sheets for data recording should be carefully planned. A single data sheet that permits the recording of a wide range of information (e.g. sampling source and collecting date, name of the recorder, morpho-biometric characteristics of the specimen considered, as well as all the necessary information to identify the individual to which the statoliths belong) should be prepared and used for each specimen (Figure 24, Annex A)

Figure 24. Example of a data collecting sheet for biological studies.

22

3.1.3 Measuring and dissecting kit A complete kit to measure and dissect cephalopods is necessary (Figure 25). This includes a ruler (1 mm) and a scale (at least to 0.1 g, but to 0.01 g is recommended) for body measurements (length and weight), scissors, a surgical blade or a scalpel, surgical tweezers and a dissecting needle, to dissect cephalopods and extract the statoliths. When handling very small individuals (i.e. embryos or paralarval individuals; mantle length < 1 mm), a dissecting microscope (or a stereomicroscope) with 40X - 50X total magnification and very fine tweezers and dissecting needles are necessary. Larger specimens require progressively less magnification (10 X 20 X for specimens of approximately 210 mm DML), whereas individuals with mantle length over 100 mm generally do not require a microscope for dissection (e.g. Dawe and Natsukari, 1991;Villanueva, 2000a,b; Villanueva et al., 2003). (a)

(b)

(c) (d)

Figure 25. Kit for specimens dissection and measurements: (a) capsulae, blazers and scissor; (b) surgical blade and pins; (c) ichtyometer; (d) ruler (0.1 cm). 23

3.1.4 Cleaning and storing Distilled water, absolute ethanol, or other chemical solutions such as sodium hypochlorite (1% - 5%) and NaOH (10%), can be used to clean the statoliths. Ultrasonic cleaners can also be used (e.g. Dawe and Natsukari, 1991; Chung and Lu, 2005). To store the statoliths any plastic box or capsule, as well as oil-paper envelopes are potentially useful, as statoliths can be stored dry; however ethanol (70% or 96%) gelatine or glycerol can also be used as storing media (Rodhouse and Hatefield, 1990; Dawe and Natsukari, 1991; Jackson and Yeatman, 1996). The use of a plastic 96-well immunoassay microplates as illustrated in Figure 26 is recommended. A label recording the statoliths basic references is also necessary, i.e. a link to associate the statoliths to the specimens from which they were extracted. Generally a paper label will do.

Figure 26. Plastic 96-well immunoassay microplates

3.1.5 Mounting and grinding Before grinding, statoliths have to be mounted on an appropriate support, which is generally a microscope slide. Accordingly, to mount a statolith on a microscope slide, a dissecting microscope (20 X 40 X magnitude) a surgical needle and a proper mounting medium (i.e. a glue or paste) are necessary (Figures 27, 28). The choice of the mounting medium is very important, as it should guarantee strong adhesion to glass surfaces, good viscosity, fast drying and transparency. Several glues are used as mounting media, all of them presenting both advantages and disadvantages: the Canada balsam, which needs a long time to dry, but has a good viscosity (Lipinski, 1978); plastic substances as the Lakeside 70 cement (Arkhipkin, 1991); some transparent resins like the Protexx syntetic mountant (Dawe et al., 1985), which also require some time to complete hardening, or the Polarbel 812 resin (Morris and Aldrich, 1985); and thermoplastic cements, like the Crystal Bond (Jackson, 1990) and the Buehler (Zumholz et al., 2006). The DPX (dibutyl-phtalate-polystirene-xylene), a mounting medium used for histological studies, has also been used for statolith analysis with double function, glue and
24

clearing agent, especially for small species or sub-adult individuals statoliths, which generally do not require grinding (e.g. Jackson, 1989; Dawe and Natsukari, 1991; Jackson and Choat, 1992; Jackson, 1994).

(a)

(b)

(c)

Figure 27. (a) Fragment of Crystal Bond 509, the mounting medium suggested; (b) hot plate; (c) microscope slide.

Currently, one of the most frequently used media is the Crystal Bond, which proved to be an excellent choice as it is completely translucent, it is not fluorescent under UV irradiation, melts at low temperatures and hardens relatively rapidly after removal from heat (Jackson, 1990; Jackson, 1994; Jackson et al., 2005). Several types of Crystal Bond are commercialised, with different melting temperatures and properties; among these, the Crystal Bond 509 (softening temperature 71 C, flow point 135 C, viscosity at flow point 6000 cps) can be considered an ideal medium for statolith mounting (e.g. Jackson, 1990; Bettencourt and Guerra, 2001). Therefore, when using the Crystal Bond or any type of similar thermoplastic cement for mounting statoliths, a hot plate capable to achieve the melting temperature (110-130 C for Crystal Bond) will also be necessary.

25

Figure 28. Dissecting microscope (stereomicroscope 10-40 X total magnification) and microscope slide. The arrow points to a statolith placed on the microscope slide.

To grind statoliths, the use of several different types of sandpapers, each with a proper grit, is recommended. Among recent available commercial sandpapers, lapping films gave good results (e.g. Zumholz et al., 2006) and in this manual the use of a complete set of lapping films with 30, 12, 5, 3 or 0.05 grades is recommended to achieve the best results from grinding1 (Figure 29). For the grinding process, the coarsest lapping film will be used first, the others will follow in sequence following a decrease in coarseness. A flat and clean surface is also required as a basis for the grinding process. A glass slide large enough to contain the lapping films can be considered an optimal solution, but any other material (e.g. metal or plastic) with the same characteristics will do as well (Figure 30).

The 3M

lapping film currently is one of the most used sandpapers. 26

(00.5 m)

(5 m)

(12 m)

(30 m)

Figure 29. Lapping films for grinding, with different grit grades (in parenthesis).

(00.5 m)

(5 m)

(12 m)

(30 m)

Figure 30. To grind the statolith, a glass slide or any other flat and clean surface is needed as a support for the lapping films.

3.1.6 Counting (statolith reading) For the analysis of statolith microstructure (e.g. to count growth increments, or to measure hatch rings) a transmitting light microscope with a total magnification between 100 X 400 X (generally ocular 10 X with micrometer, objectives 10 X, 20 X, 40 X) is generally required, although a magnitude of 600 X will be needed for observation of embryonic or paralarvae statoliths. In some cases, the potential need for even higher magnification (i.e. 600-1000X) has been highlighted (Arkhipkin, 2005), while the use of polarized light can improve ring identification and counting (e.g. Dawe and Natsukari, 1991; Jackson and Moltschaniwskyj, 1999; Jackson et al., 2003) (Figure 31). The use of a system capable of displaying statoliths microstructure on a monitor is highly recommended. Standard systems
27

generally consist of a camera mounted on a microscope and connected to a computer monitor. In some cases, on the basis of the experience carried out in studies on fish otoliths (see Campana, 1992 and Jearld, 1995 for reviews), a computer-based image analysis system (Campana, 1987) was used for statolith investigation (Arkhipkin, 1996; Gonzalez et al., 1996). An automatic, or semi-automatic ring counting, through an image analysis system, can also be carried out. However, due to the variation of the growth increment width even in the same statolith, the automatic ring counting has to be considered with care and it will not be described in this manual.

Figure 31. Transmitting light microscope with 200X-600X total magnification minimum (top left) and an image analysis system, with a camera (oval) mounted on a microscope (right) connected to a monitor (down left).

3.2 Procedure and techniques 3.2.1 Extraction Two different methods can be used for statolith extraction: surgical and chemical. The surgical methods are highly recommended, however the chemical method has also been used (e.g. Hurley and Beck, 1979), thus a brief description of the latter method is given as well.

28

3.2.1.1 Chemical method The method involves the extraction of the posterior-ventral portion of the cartilaginous skulls and its dissolving in a proteolyitic enzyme solution (e.g. pepsin and sodium hypochlorite, Hurley and Beck, 1979; trypsine and boric acid, Amaratunga and Budden, 1982) within an appropriate vial. After the cartilage has dissolved, the two statoliths can be collected from the bottom of the vial. In order to extract the skull, set the individual with the ventral side up and anterior side toward, and remove the funnel and the skin beneath it. The ventral part of the cephalic cartilage will be exposed and can be removed by severing it from the head. The use of chemicals may apparently simplify the extraction, but is not suitable when the statolith sampling occurs on board vessels, as secure conditions to store and handle potentially dangerous chemical components are required. Furthermore the use of chemicals is not appropriate to process large samples. In addition, chemicals may eventually damage the statoliths (Dawe and Natsukari, 1991).

3.2.1.2 Surgical method Techniques for surgical statolith extraction have been described in many of the publications dealing with cephalopod age and growth, the most useful being reviewed by Rodhouse and Hatfield (1990), Arkhipkin, 1991 and Dawe and Natsukari (1991). In this manual two surgical techniques for statolith extraction are described. 1) Place the animal ventral side up and anterior side towards you, and cut the fusion between funnel and body from the anterior part with a surgical blade or a scalpel. After cutting the fusion, the statocysts will be visible through the skin in fresh and defrosted squids, even in large specimens. In octopods and sepioids the skin and muscles beneath the funnel generally prevent the view of the statocysts, thus this tissue has to be removed by means of horizontal/longitudinal cuts (along the sagittal plane) performed holding the animal with one hand and flexing the head dorsally. Once the cephalic cartilage is exposed, carefully remove thin vertical/transversal cartilage slices to cut off the anterior side of the statocysts. While doing this, care should be taken not to force the surgical blade too deeply or too far forward, to avoid damaging other parts of the head. This could cause internal white fluids to flow over, which could cover the statocysts and prevent location of the statoliths. Stop the cutting when the two statoliths become visible in the anterior side of the statocysts (two distinct cavities will appear and the two statoliths will be visible as clear particles in the middle of them). Once the statoliths are exposed, pick them up with the surgical tweezers or with the aid of the scalpel/blade tip. Generally speaking, care must be taken to handle the specimens gently and avoid crashing or moving their heads too roughly, since statoliths may detach from the anterior wall of the statocysts and drop into the bottom of the cavity. Some phases of statolith extraction following this technique are shown in sequence below (Figure 32a-e) and in Annex B.

29

a)

b)

Figure 32. Some steps of a surgical technique for the statoliths extraction in Sepioidea: a) Set the individual ventral side up and anterior side towards you; b) Remove the fusion between the funnel and the body from the anterior side; c) After removing the tissue covering the statocysts, make one or more transversal cut in order to expose the statoliths; 4) Stop the cutting when the two statoliths become visible in the anterior side of the statocysts (two distinct cavities will appear and the two statoliths will be visible as clear particles in the middle of them); e) Pick the two statoliths up with thin tweezers or with the tip of the surgical blade. 30

c)

d)

Figure 32. Continued

31

e)

Figure 32. Continued

2) Place the animal ventral side up and sever the head in correspondence with the anterior end of the mantle. As in 1), the statocysts will generally be visible in fresh squids and the statoliths will appear as opaque white structures within them. Remove the anterior cartilage wall by gently cutting away thin cartilaginous slices, until the two maculae with their statoliths are visible and the statoliths exposed. Collect them gently with the surgical tweezers or with the aid of the scalpel/blade tip.

Both procedures are also suitable to process very small specimens as embryos or paralarvae, and generally animals with dorsal mantle length < 10 mm, under a dissecting microscope; in this a case very thin sharpened blade or scalpel and tweezers are needed.

3.2.2 Cleaning and storage After extraction, statoliths should be prepared for analysis or stored. As the grinding process removes surface material, cleaning the statoliths will not be necessary at all if they have to be ground on both sides. On the contrary, thin and translucent statoliths (mainly from paralarvae and embryos) need either to be ground on one side only (Dawe and Natsukari 1991; Arkhipkin and Roa-Ureta, 2005; Villanueva 2000 a,b; Villanueva et al., 2003) or not to be
32

ground at all (e.g. Jackson and Yeatman, 1996; Jackson and Moltschaniwskyj, 2001b; Jackson et al., 1997): in these cases cleaning is required. However, it is always preferable at least to rinse the statoliths in distilled water and let them dry at room temperature before storing them or proceeding with the analysis. As statoliths from very small specimens are difficult to locate and handle, it is advisable to not remove them and store the entire specimen until the actual analysis (e.g. Dawe and Natsukari, 1991).

3.2.2.1 Cleaning Several methods are suitable to clean statoliths, including rinsing them either in distilled water, ethanol (absolute or 75%), sodium hypochlorite, or NaOH solution (e.g. Rodhouse and Hatfield, 1990; Dawe and Natsukari 1991; Jackson and Wadley, 1998; Steer et al., 2003), as well as ultrasonic cleaning (Dawe and Natsukari, 1991; Natsukari, 1998; Chung and Lu, 2005) as summarized below. a) Place the statoliths in a vial (or in the plastic 96-well immunoassay microplates previously described) with distilled water or ethanol (75% - 100%) and let them dry at room temperature (e.g. Jackson and Moltschaniwskyj, 1999; Arkhipkin, 1997; Sakai et al., 2004; Triantafillos, 2004; Jackson et al., 2007). b) Set the statoliths on a glass surface (e.g. a microscope slide), rinse them in distilled water and dehydrate them by rinsing in absolute ethanol (e.g. Jackson, 1989; Rodhouse et al., 1994b; Jackson and Wadley, 1998; Bettencourt and Guerra, 2000). c) Place the statoliths in a solution of sodium hypochlorite (from 1 % to 5 %) for different time-ranges, depending on the strength of the solution (the stronger the solution, the shorter the time interval) and the size of the statoliths. Usually a few minutes will be necessary for thick statoliths (e.g. Jackson, 1990; Jackson and Forsythe, 2002), less than 1 minute for small and thin statoliths (e.g. Ikeda et al., 1999). After cleaning, rinse the statoliths in distilled water and dehydrate them with 100% ethanol. d) Place the statoliths in an ultrasonic cleaner for 1-3 minutes, then soak the statoliths in a strong alkaline solution (e.g. 10 % NaOH) overnight (Natsukari, in Dawe and Natsukari 1991; Chung and Lu, 2005).

3.2.2.2 Storage Statoliths are acellular mineralized structures, that will not decompose under relatively dry conditions (Rodhouse and Hatfield, 1990). Accordingly, they can be stored dry in small glass or plastic vials (e.g. Triantafillos, 2004; Jackson et al., 2007) (Figure 33). Storage in a wet/liquid medium such as oil-paper envelopes (Arkhipkin, 2003) 70 % or 96 % ethanol is also possible (e.g. Arkhipkin, 1997; Challier et al., 2002; Miyahara et al., 2006). However, when storing statoliths in ethanol within closed capsules, these should be kept in cool conditions. Indeed, high room temperature (e.g. > 30C) may cause an increase in the partial
33

pressure of the ethanol determining the opening of the capsules and the loss of the statoliths. The use of glycerol and gel capsules is also appropriate, but more expensive and less easy to apply. Several researchers store statoliths dry or in ethanol (70% to 96%) (Dawe and Natsukari, 1991) and in general no differences in growth increment visibility were found examining statoliths kept dry and those preserved in alcohol for one year (Nastsukari, 1998). Therefore, ultimately, the storage method will be selected based on personal preferences/choices. However, to store statoliths dry in the plastic microplate already mentioned sheltering with paraffin eventually closing with a proper cover is suggested (Figure 34). As already stated, when having to store statoliths of embryos or paralarvae and/or animals smaller than 30-50 mm ML, it is advisable to store the whole specimens instead of the statoliths extracted. These animals should be preserved in 95 % ethanol, after a 15-20 immersion in 60 % ethanol (Arkhipkin, 1991). For long-term storage, ethanol should be changed periodically (every 6 8 months). Whatever system is used to store the statoliths, each pair has to be labelled separately, in order to link the results of the analysis to the specimen to which the statolith belongs. It is useful to underline that plastic microplates can be used both for cleaning and storing by filling the holes with distilled water or 96% ethanol, setting the paired statoliths in the bottom of one single hole and letting them dry at room temperature. Once the holes are completely dry, cover them with paraffin and/or sticky-tape to prevent the statoliths from moving and close the box with its cover.

Figure 33. Statoliths can be stored in plastic vials (dry or in ethyl alcohol) or in any other box.

34

Figure 34. The use of a plastic 96-well immunoassay microplate to clean and store the statoliths (both dry or in ethyl alcohol), is recommended.

3.2.3 Mounting and grinding While in paralarvae or small juveniles of some species growth increments are visible without grinding, large juveniles and adult statoliths require preparation before grinding, i.e. mounted on an appropriate support (generally a microscope slide) and ground to be read.

Several procedures were described in Jereb et al. (1991) and used thereafter, either as they were originally described, or slightly modified (e.g. Bettencourt et al., 1996; Arkhipkin, 1997; Lipinski et al., 1998; Raya et al., 1999; Gonzlez et al., 2000; Bettencourt and Guerra, 2001; Challier et al., 2002; Villanueva et al., 2003). In this volume a comparatively easy procedure is reported and to this extent the use of the thermoplastic cement Crystal Bond (or equivalent) as a mounting medium is assumed, as its characteristics gives several advantages during the statoliths preparation (e.g. it is possible to melt a thermoplastic cement at any time during the procedure allowing for changes in the statoliths position/orientation when necessary).

35

3.2.3.1 Mounting Mounting the statoliths on a microscope slide (or on any other substrate) in order to prepare them for grinding is a relatively easy but extremely important process. It is mostly during this phase that the plane and the area to be ground are decided, as well as the section of the statolith that will be observed. Accordingly, the statolith should be mounted with the proper orientation to enable the operator to grind it following a well-defined plane; this should allow a section of a predefined area to be obtained and create the best conditions for the observation of the microstructure (Figure 35a,b) (e.g. Dawe and Natsukari, 1991; Dawe and Beck, 1997; Linpinski et al., 1998; Hendrickson and Hart, 2006; Jackson et al., 2007).

a
MM F

b
MM

Figure 35. Different statolith orientation on the microscope slide: a = concave side up; b = concave side down. MM = mounting medium; F = focus

Several regions of the statolith can be used for ring observation, differing in increment width as well as in increment sequence, which can be complete (i.e. without interruption or white areas from the natal ring to the statolith margin) or not. Generally, a transversal section of the domes (dorsal and lateral domes) is used for ring examination and counting (e.g. Jackson, 1989; Arkhipkin, 1991; Jackson and Choat, 1992; Dawe and Beck, 1997; Bettencourt and Guerra, 2001; Quetglas and Morales-Nin, 2004; Challier et al., 2005; Hendrickson and Hart, 2006; Ceriola, 2007; Jackson et al., 2007), even though in some cases (i.e. for several loliginids and some oegopsids) a transversal section of the rostrum is preferred (e.g. Natsukari and Komine, 1992; Bettencourt et al., 1996; Arkhipkin, 1997; Raya et al., 1999; Arkhipkin, 2005).

36

In order obtain a transversal section of the statoliths, under a binocular dissecting microscope (10-40X magnification), set a fragment2 of the thermoplastic cement in the middle of a microscope slide and place the statolith close to this fragment with the anterior or the posterior side up (the cement fragment size should be equivalent to that of the statolith; larger quantities also can be used, but this may increase the time of grinding and compromise the final clearness of the ground surface). Place the microscope slide on the hot-plate (previously warmed to around 120 C) to melt the thermoplastic resin and wait for a few seconds. After the resin has melted, move the slide back under the microscope and set the statolith in the middle of the melted cement (Figure 36).

Figure 36. Placing the statolith on the melted mounting medium

While placing the statolith in the melted cement, it is important to not turn it upside-down in order to keep the predefined grounding plane. In addition, the statolith orientation should be accurately checked under the binocular microscope, so as to have the possibility to modify it, if necessary, while the cement is hardening. If the statolith orientation is correct, let the cement harden; on the contrary, melt the thermoplastic cement again by replacing the glass slide on the hot-plate and modify the statoliths position or orientation. The Crystal Bond hardening and the microscope slide cooling down will require 1 or 2 minutes at room temperature, after which the statolith will be ready for grinding. The first orientation of the statolith varies depending on the species and on the side/s to be ground (whether only one or both). When both the anterior and the posterior side of the statolith have to be ground, beginning with the concave side down rather than with the concave side up is generally a choice based on the personal experience of the author and in some extent to the characteristics of the statolith/species. Likewise, when only one side of the statolith needs to be ground the choice is related to the species to analyse. For example,
2

Small fragments of resin are obtained from larger pieces by using a strong needle. 37

grinding the anterior side of the statolith results in a better ring identification in Illex coindetii, whereas the opposite is true for Loligo vulgaris (Ceriola pers. observ.). Help in taking this kind of decision may come from literature, as well as from personal experience.

3.2.3.2 Grinding Grinding is the most delicate phase of the whole procedure of statolith preparation, since the rings identification depends on proper grinding and, ultimately, the results counting process too. Statoliths from embryos, paralarvae and small loliginids and sepioids do not need grinding, as they are thin and translucent enough to allow a direct ring counting3 (e.g. Jackson, 1989; Jackson et al., 1997; Jackson and Moltschaniwskyj 2001b). The grinding procedure consists of two phases: the first of main grinding and the second of polishing. Generally a coarse waterproof sandpaper is used for the main grinding, i.e. to remove the external side of the statolith, while a finer sandpapers are used to polish the ground surface. It was noted that statolith reading improves when statoliths are ground with sandpaper moistened with cold water (Arkhipkin, 1991), however also grinding with dry sandpaper gives excellent results. To grind the statolith, set four small squares/rectangles of lapping film (each of 12 x 8 cm approximately) with 30, 12, 5 and 3 or 0.05 grades respectively on a glass surface (or any other flat surface). Then, holding the microscope slide with the mounted statolith upside down, start grinding the statolith on the first lapping film (30 ) with a very light pressure and circular movements (Figure 37). The extent and intensity of grinding, as well as the grinding plane should be monitored continuously by observing the ground surface using a dissecting microscope (40 X magnification) and/or a transmitting light microscope (up to 100 X magnification). Continue grinding until the focus becomes visible beneath the ground surface, when focusing at the microscope or when the ground surface almost reaches the edge of the statolith. Stop using the coarsest lapping film before the edge of the statolith is reached and continue the process using the second lapping film (12 grades). Changing the sandpaper ensures that: 1) grinding decelerates and does not proceed so far as to obliterate the focus or to damage the external edge; 2) some of the scratches are removed from the ground area (Figures 38, 39).

For such small statoliths, the use of clearing agents like Eukitt, Euparal xylene and DPX (dibutyl-phthalatepolystyrene-xylene) for mounting (Jackson, 1989; Dawe and Natsukari, 1991) may improve the visibility of growth increments. The same effect was obtained by Villanueva et al. (2003) by using tetracycline, a marking agent, when incubating Loligo vulgaris eggs. 38

main grinding
(30 ) (12 ) (5 )

(30 )

(12 )

(5 )

(0.05 )

polishing
(5 ) (0.05 )

(12 )

(5 )

(0.05 )

Figure 37. Sequence of grinding. In parenthesis the suggested lapping films grades

Figure 38. Subsequent phases in the grinding process; the clear area is the ground surface.

39

(A)

Ground area

(B)

Ground area

Figure 39. Subsequent phases in the grinding process of a real statolith. (A) Only the dorsal and lateral domes were ground; (B) Almost the whole surface was ground.

Continue the grinding process with the 12 grades lapping film until the ground surface, slightly above the focus, reaches the most external point of the dorsal dome (the edge of the statolith). After that, keep on grinding with the two finer lapping films (5 and 3 or 0.05 grades), in order to polish the ground area. When the surface of the statolith is polished from all the scratches, examine the statolith at the transmitting light microscope (10 X ocular and objectives 10 X, 20 X and 40 X or more) in order to decide if the growth rings are adequately visible (i.e. properly identifiable) or if grinding the opposite surface is necessary.
40

If the statolith needs grinding on the second surface, it has to be turned upside down (that is with the ground surface on the glass) and further ground on the second side. To turn the statolith upside down, place the microscope slide on the hot plate (set at about 120 C) until the Crystal Bond melts, then place the microscope slide under the dissecting microscope and change the statolith orientation with a needle. By gently touching the statolith margin, this will remain attached to the tip of the needle and it will be easy to raise the statolith and turn it upside down, with the flat surface on the glass. Once the cement and the glass support have cooled down and the whole structure has hardened (generally within 1 or 2 minutes), start with the grinding procedure again, exactly as done for the first surface. As a general rule, and especially for inexperienced researchers, when grinding the second surface (but also for the first one) it is advisable to stop before reaching the statolith edge, to avoid exceeding in the operation (overgrinding), as in over-ground statoliths is very difficult to properly identify rings and to estimate the individuals age. A scheme of the grinding process (set to obtain a transversal section) and the different levels of grinding are reported in Figure 40 (convex side up) and Figure 41 (convex side down).

Figure 40. Longitudinal section of a mounted statolith (convex side up) to illustrate the grinding process as it proceeds on both sides of the statolith. The dashed line indicates the grinding plane. 41

Figure 41. Longitudinal section of a mounted statolith (convex side down) to illustrate the grinding process as it proceeds on both sides of the statolith. The dashed line indicates the grinding plane.

3.2.4 Counting Growth increment counting, i.e. the statolith reading, is the final phase of the whole procedure and, under the assumption that one growth increment is deposited during one day, it allows estimation of the individuals age in days. The statolith reading consists of the enumeration of the growth increments visible in the statolith microstructure. Starting from the first growth increment around the focus, either the dark or the light layers have to be counted, i.e. only the dark (suggested) or the light rings (Figure 42). However, the natal ring has to be identified first in order to begin the counting process. In addition, since the increment width and visibility generally vary between the different zones within the statolith microstructure, the best areas for increments identification and counting also have to be identified.
42

5 4 3 2 1

Figure 42. Ring identification for counting: either the dark or the light layers have to be counted. The arrows point to the dark layers.

The natal ring is identifiable as a marked and complete check in the region immediately around the focus. Its maximum diameter varies among species (from less then 100 to 250300 microns, Villanueva, 2000a,b; Steer et al., 2003; Sakai et al., 2004) and the check itself can be more or less evident depending on the extent of the grinding process as well as on the characteristics of the statolith (Figure 43). Sometimes in the region immediately around the focus several checks are visible, making it difficult to identify the proper natal ring. As general rule, the innermost complete marked check should be considered as the proper natal ring4 (Arkhipkin, 1991; Jackson, 1994; Arkhipkin and Perez, 1998; Villanueva, 2000a,b; Steer et al., 2003; Arkhipkin, 2004; Ceriola, 2007). Once the natal ring has been identified, the best areas (or best paths) to be followed for ring counting have to be searched for in the statoliths microstructure. Statolith shape and microstructure are species-specific so that growth increment identification can be easier in some regions of the statolith rather than in others depending on the species. Moreover, different regions of the statolith can be suitable for reading depending on the extent of grinding. However, as a general feature, the narrowest increments are observed close to the nucleus (in the postnuclear zone), while the broadest and more regular rings are observed in the dark and peripheral zones. The dorsal and lateral domes (in transversal or frontal section) have been shown to be the best regions for increment counting (e.g. Jackson, 1989; Arkhipkin, 1991; Jackson and Choat, 1992; Dawe and Beck, 1997; Bettencourt and Guerra, 2001; Quetglas and MoralesNin, 2004; Challier et al., 2005; Hendrickson and Hart, 2006; Ceriola, 2007; Jackson et al., 2007), but in some cases (mainly in loliginids) the rostrum in transversal section has been preferred (e.g. Natsukari and Komine, 1992; Bettencourt et al., 1996; Arkhipkin, 1997; Raya et al., 1999; Arkhipkin, 2005). Accordingly, it is within these regions that the best path for counting is to be sought (Figure 44).
4 As already mentioned (see page 22), generally in the ommastrephids the natal ring is the first ring after hatching and can be identified in the microstructure as the innermost ring, while in many loliginids several rings are deposited during the embryonic development but usually not as marked/evident as the natal ring, so for these species the innermost marked ring should be considered the natal ring. Ultimately, only individual experience will help identify the proper natal ring both in species with embryonic increments and in individuals with several checks in the postnuclear zone.

43

Natal ring

Natal ring

Natal ring

Figure 43. Natal rings in statolith microstructure of adult specimens. 44

WA 3 AA

Figure 44. The best path for ring counting is indicated by progressive numbers. WA = White Area; AA = Adjacent Area where the number of rings of the WA can be estimated from.

3.2.4.1 Direct counting Place the microscope slide with the ground statolith under the microscope and identify the best magnification for ring identification; find the natal ring and the region/s where the
45

increments are most visible; try to define a path to follow from the natal ring to the statolith edge and begin counting, with the natal ring as increment number one. Due to the plane of grinding, ring definition may change from the innermost to the external regions. In this case it is necessary to slightly change the focus (by gently focusing) to achieve the best resolution; in this phase, the use of polarized light may help. When counting in different regions (e.g. along the lateral dome for the postnuclear zone, off the dorsal dome in the dark zone and in the lateral dome again for the peripheral zone), it is possible to move from one region to the other by following the rings border (Figure 45).

...
30 29 28 27 26 25

19 18 17 16 15 14 14 13 12 11 10

19

25 24 23 22 21 20

Figure 45. Ring enumeration following a well defined path: begins from the right, continues on the left, goes back to the right. Numbers are an exemplification.

If areas where rings are not visible (i.e. white areas) are present in the observed section, the rings number in these areas can be approximated as the number of increments in the adjacent (just above or below) region of the same width, since in adjacent regions ring width does not change dramatically. However, this procedure has to be used with caution when white areas occur in proximity of the boundary between two different regions (i.e. postnuclear/dark zone or dark/peripheral zones), because in this case shifting from one region to another may result
46

in different ring widths. When the white areas cover more than the 15% of the whole statolith surface, the statolith should be rejected (Gonzlez et al., 1996).

3.2.4.2 Image analysis system In its simplest form, an image analysis system (IAS) can store pictures in memory and reproduce them, unaltered, whenever required. In practice, however, images can be enhanced before being redisplayed, providing the main advantage over visual examination. To visualize an enlarged image of the statolith microstructure on a monitor considerably facilitates the process of reading and it is recommended with both photographs (on acquired images) and live images (directly observing the statoliths under the microscope). Working on live images, the best regions and focus for the rings counting will be defined by looking at the monitor, as if looking directly at the microscope. This makes it easier to follow the rings progression from the natal ring to the statolith edge and significantly shortens the reading procedure. Working with photographs, the best available pictures of the ground statolith regions (from the natal ring to the external edge) have to be chosen and read in sequence; alternatively, an assemblage can be done to carry out the counting process on a single image.

47

A statolith section as it generally appear after grinding on two sides

48

4. Some possible outcomes Individual age data, coupled with data on size (individual length and weight) and maturity can be used to investigate several aspects of cephalopod life history. - Age and life span: individual age and species life span are the first and basic information deriving from the analysis of the statoliths. - Hatch data and time range: based on the individual age in days and on the date of capture, the individual hatch data can be back-calculated (hatch data = date of capture age in days); consequently, the population hatching period can be defined. This approach is used to identify possible monthly or seasonal cohorts within a population. - Growth shape: size-at-age data (mantle length or total weight)5 can be used to define growth shape and to compute growth curves; growth curves obtained for different monthly (or seasonal) cohorts can then be compared, and the potential effect of monthly (or seasonal) parameters highlighted. For example, faster growth in spring-summer hatched animals, with respect to autumn-winter hatched animals was highlighted by Arkhipkin et al. (2000) in I. coindetii.; - Growth rate: growth rate can also be estimated, per age class or time interval (e.g. Arkhipkin and Bizikov 1991; Gonzalez et al. 1996; Jackson et al. 1997; Ceriola, 2007) as follows:

Instantaneous growth rate: G =

(ln R2 lnR1) t2 - t1

* 100

Absolute growth rate: AGR =

R2 R1 t2 t1 ln (R1) ln (R0) * 100 T

Individual growth rate: IGR =

Where: Rx = length or weight at the time x t1 and t2 = extreme (begin and end respectively)of the age class considered T = total age. Ontogenetic shifts: by defining the boundary between two or more adjacent regions (when present) in the statolith microstructure, it is possible to define the duration of each ontogenetic phase.

The use of length-at-age rather than weight-at-age data has been widely debated (e.g. Bello, 1991) and the analysis of both data is suggested here, when possible. 49

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Glossary
Adult Mature individual, a female that has mature eggs (generally stored in the oviducts), or a male that has produced spermatophores (generally stored in the Needams sac). Angular acceleration receptor system Receptor system in the statocyst deputed to monitor angular acceleration. It is subdivided into four sensory epithelia or cristae. Anterior Toward the head end. Aragonite crystal (or statoconia) Subunits of calcium carbonate (CaCO3) which forms the mineral fraction of statolith. Beak (or mandible) Chitinous jaws of cephalopods, bound in powerful muscles. It is formed by a dorsal (or upper) beak, which is inserted within a ventral (or lower) beak, to tear tissue with a scissor-like cutting action. Biomineralization A sequence of events in which the minerals secreted by the cells crystallise and form a structure following a defined plan of construction. Canada balsam A viscous, yellowish, transparent resin obtained from the balsam fir (Abies balsamea) used as cement to mount the statolith on a glass surface. Cephalic cartilage Cartilage-like tissue that envelops the posterior part of the brain of cephalopods and that encompasses the statocysts. The cartilage has a large central foramen through which the oesophagus passes and minor foramina for nerves and blood vessels. Cephalopoda The class within the Mollusca characterised by bilateral symmetry, anterior head appendages and funnel, posterior mantle, mantle cavity with organs, and shell and fins when present. Check (or Mark) A highly evident (prominent) growth increment. They can be complete (developed along the entire outline of the statolith) or incomplete (developed only in one region of the statolith). Complete check See check Complete mark See check Crystal Bond Thermoplastic cement used to mount statolith on a glass surface. Crystalline lens Anatomic component of the cephalopod eye. It consists of idrosolubil proteins organized to form two planoconvex components. It can be potentially used for ageing studies. Cuttlebone (or sepion) The calcareous (chalky) oblong, supporting protective and buoyancy shield in the dorsal part of the mantle of cuttlefishes. It can be used to study age and growth in sepioids. Dark zone The region of the statolith external to the postnuclear zone. It is characterised by regularly spaced growth increments. It appears brownish in transmitted light, white opaque in reflected light. Direct rings counting The counting of growth increments in the statoliths microstructure without grinding procedure. Dissecting microscope A microscope used to collect statoliths from small individuals (mantle length generally < 10 mm). It is also used to check the proper orientation of statolith on the glass support before grinding. It is generally a reflected light microscope with 40-50X total magnification. Dome, dorsal and lateral Regions of the statolith translucent in transmitted light. The microstructure of these regions is generally characterised by well defined growth increments. Dorsal The uppermost, or back, surface of a cephalopod, opposite the ventral surface where the funnel is located. Dorsal dome Region of the statolith. See also Dome. Dorsal mantle length The standard measure of length in coleoid cephalopods. In sepioids and teuthoids it is measured along the dorsal midline from the mantle margin to the posterior tip of the body. In octopods it is
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measured from a line joining the mid-point of the eyes to the posteriormost area of the mantle. Dorsal spur Region of the statolith. Embryos Pre-hatching individuals. Individuals during the development within the eggs. Focus The initiation point of the statolith. It usually consists of one single aragonite concretion. Focusing The process of slightly focus changing at the microscope. Funnel The ventral, sub-conical tube through which water is expelled from the mantle cavity during locomotion and respiration (reproductive and waste products, as well as the ink, pass through the funnel). Gladius (pen) The feather or rod-shaped chitinous supporting structure in the dorsal midline of squids; the homologue of the shell of ancestral forms. Gravity accelerator system Receptor system in the statocyst deputed to monitor gravity and to control compensatory eye and head movements. It is subdivided into three sensory epithelia or maculae. Ground surface Area of the statolith which has been ground. It is used for ring identification and counting. Growth increment counting (statolith reading) The process to enumerate the growth increments within the statolith microstructure. Growth ring See growth increment Hot plate A laboratory electric tool used to heat microscope slide in order to melt resin for statolith mounting. Incomplete check See check Incomplete mark See check Increment See growth increment Lapping film (sandpaper) The paper used to grind and polish the surface of statolith. It can be of different grit: coarse paper (smaller grit) grinds statolith, fine paper (grater grit) polishes ground surface.

Lateral dome Region of the statolith. See also Dome. Macula Sensory epithelium plates placed in the anterior chamber of the statocyst. Macula statica princeps Sensory epithelia of the gravity receptor system in the statocyst where the statolith is attached. Mandible Element of cephalopod beak or jaw. See also beak. Mantle The fleshy (muscular) tubular or sac-like body of cephalopods; provides propulsion through jet-like expulsion of water; contains the viscera Mark See check. Mature Terms referred to sexual maturity which is determined for females by the presence of ova (mature eggs) free in the coelom or in the oviducs, and for males by the presence of spermatophores in Needams sac (see adult). Mollusca One of the major invertebrate phyla. Some of the common molluscs are snails and clams. The Cephalopoda is a class within the Mollusca. Myopsin squids A high-level taxon (order) within the Decapodiformes. In recent classification, the Myopsida (contains the family Loliginidae) have been considered the sister group of the Oegopsida and the two groups together compose the Teuthoidea (squids). At present the phylogenetic relationships within the Decapodiformes are unresolved and the correct classification of myopsids is in limbo. Natal ring The external border of the nucleus in the statolith microstructure of adult individuals. It is the external surface of the statolith in newly hatched individuals and appears as a marked ring when the whole statolith is observed in section. Nucleus The innermost region of the statolith in adult individual. It corresponds to the statolith of newly hatched individual. It is also named as pre-hatching statolith. When observed in section it is surrounded by the natal ring.
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Octopodiformes Higher-level taxon that includes all 8-limbed cephalopods. Because of the long history of referring to these cephalopods by the common name octopods, the latter is used as the common name for the Octopodiformes. Octopods Common name for Octopodiformes. Oegopsin squid A high-level taxon (order) within the Decapodiformes. In recent classification, the Oegopsida (oceanic squids) has been considered the sister group of the Myopsida and the two groups together composed the Teuthoidea (squids). At present the monophyletic nature of the Oegopsida and the relationship between this taxon and the Myopsida are unresolved. Paralarva Terms indicating the first freeliving life history stage (typically planktonic) for those cephalopods that differs in both morphology and vertical distribution from older juveniles. Pen See gladius. Peripheral zone The most external region of the statolith (external to the dark zone). It is characterised by regularly spaced growth increments. It appears transparent in transmitted light. Polarized light Light characterised by an electromagnetic field vibrating along to a well defined plane. Postnuclear zone The region of statolith which is immediately outside to the nucleus. It corresponds to the statolith of paralarval. It is characterised from very narrow growth increments. Pre-hatching statolith Statolith of newly hatched individual. Ring See growth increment Rostrum Region of the statolith that is translucent in transmitted light. The microstructure of this region in some species is characterised by growth increments not easily identifiable. Rynchoteuthion Paralarval stage in the Ommastrephidae. It is characterised by the

fusion of the tentacles into a trunk-like proboscis. Sandpaper Substrate used to grind and polish the surface of statolith. See also lapping film. Sepioidea A high-level taxon within the Decapodiformes. Typically, this taxon includes the Sepiidae, Idiosepiidae, Sepiolidae, Sepiadariidae and Spirulidae, but the monophyletic nature of the group has been questioned. At present the phylogenetic relationships and classification within the Decapodiformes are unresolved. It can also be referred as sepioids. Sepioids Common name for Sepioidea Sepion (= cuttlebone) The calcareous, laminate, dorsal supportive buoyancy structure in the mantle of cuttlefishes Spur Region of the statolith. Squid Common name given to members of the Teuthoidea and some members of the Sepiolidae. Statoconia The crystalline subunits in the statolith structure. See also Aragonite crystal Statocyst A sense-organ that detects gravity, angular accelerations and low frequency sound. The statocyst is embedded within the cephalic cartilage and contains the statoliths. Statolith A calcareous stone (particle) in the statocyst that detect linear acceleration, angular acceleration and orientation. They can be used to estimate age in cephalopods. Statolith cleaning Process to remove stain and tissue residuals from the external surface of the statolith. It is mostly necessary either when the statoliths do not need grinding or need to be ground from one side only. Statolith extraction Process to remove the statoliths from the cephalic cartilage. Statolith grinding The process to remove the external surface of statolith in order to produce a thins section for the growth increment identification. Statolith growth Increasing in size of the statolith.

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Statolith growth increment A new surface deposited around the pre-existing external surface in the statolith. When the statolith microstructure is observed in section a growth increment is visible as pair of dark and light layers. Statolith microstructure Internal structure of the statolith when observed at the microscope. Statolith mounting Process to set statolith on a glass support, generally a microscope slide. Statolith reading Process of growth ring identification and enumeration. See growth ring counting. Subadult Stage at which all of the characters that typically define the species are present, but the reproductive system is not mature and functional. It follows the juvenile stage and precedes the adult stage. A subadult stage is defined in cephalopods as the adult phase is frequently abbreviated. Sucker Muscular, suction-cup- structure/s on the arms tentacles (occasionally on the buccal membrane) of cephalopods. It consists of a cup-shaped portion (acetabulum) and a flat, distal ring (infundibulum), which contacts the substrate.

Sucker ring Chitinous, often serrated or toothed, ring that encircles the opening of suckers of squids and cuttlefishes. Teuthoidea The higher taxon that includes all squid-like decapods; now archaic. The monophyly of this taxon is questionable. It can also be referred as teuthoids. Teuthoids common name for Teuthoidea. Ventral The lowermost or belly surface of cephalopod, the surface where the funnel is located. Opposite to the dorsal surface. White area Region of the statolith microstructure where the ring are not proper identifiable (when this region exceed the 20% of the whole ground surface, the statolith should be rejected). Wing The region of the statolith which is attached to the macula statica princes. The structure of this region differs from that of the rest of the statolith, displaying a very disordered crystal arrangement, with no evidence of growth increments. grade Unit of measure to describe the lapping film grit. It is inversely proportioned to the grit scale, the greater is the number of grades, the finest is the lapping film.

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Annex A: Data sheet

Based on a model provided by George Jackson, Institute of Antarctic and Southern Ocean Studies (IASOS), University of Tasmania.
Taxa: _______________________________________ ID No.: _____________________________ Operator:_________________________ Institution:___________________ Date: _____________ Collection Data: __________________________________________________________________ Condition: Fresh; Frozen; 70% ethanol; other ________________________

Comments:_______________________________________________________________________ ________________________________________________________________________________ Observed damage: _________________________________________________________________ Fixed/Preserved: 10% formalin*; 70% ethanol; other _________________________

Mantle length: ____________________________ mm Total weight: _____________________________ g Sex: Indeterminate; Male; Female

Maturity: 1 + 2 + 3 + 4 + 5 + 6 Comments: ______________________________________________________________________ FEMALE Mated: yes; no No. sperm.______ MALE Testis weight: __________________ g All other repro weight:____________ g

Nidamental gland length: ____________ mm Nidamental gland weight: _____________ g Oviducal gland weight: _______________ g Oviduct weight: _____________________ g Ovary weight: ______________________ g Mantle weight: ______________________ g Fin weight: _________________________ g

Stomach fullness: 0 1 2 3 4 5 Stomach weight: ___________________ g Stomach collected: yes; no

Comments: _____________________________________________________________________ Number of statoliths collected: 0 1 2

Tray ID: _________________________ Row: A B C D E F G H Column: 1 2 3 4 5 6 7 8 9 10 11 12

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Annex B: Statoliths extraction from fresh animals (Sepioidea, Teuthoidea and Octopoda)
The main steps of statolith extraction from fresh or defrosted animals are shown below on sequence: 1) positioning of the specimen; 2) removing of the fusion between the funnel and the body, and the tissue covering the statocysts; 3) exposure of the statoliths (finding of the statoliths in the anterior side of the statocysts); 4-5) picking up of the statoliths. Sepioidea

1)

1) Set the individual ventral side up and anterior side towards you.

2)

2) Remove the fusion between the funnel and the body from the anterior side.
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3)

3) After removing the tissue covering the statocysts, make one or more transversal cut in order to expose the statoliths.

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4a)

4b)

4c)

4) Stop the cutting when the two statoliths become visible in the anterior side of the statocysts (two distinct cavities will appear and the two statolith will be visible as clear particles in the middle of them).

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Teuthoidea

1)

1) Set the individual ventral side up and anterior side toward 2)

2) Remove the fusion between the funnel and the body from the anterior side.

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3)

3) After removing the tissue covering the statocysts, make one or more transversal cut in order to expose the statoliths.

4)

4) Stop the cutting when the two statoliths become visible in the anterior side of the statocysts (two distinct cavities will appear and the two statolith will be visible as clear particles in the middle of them).
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Octopoda

1)

1) Set the individual ventral side up and anterior side toward.

2)

2) Remove the fusion between the funnel and the body from the anterior side.

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3a)

3b)

3) After removing the tissue covering the statocysts, make one or more transversal cut in order to expose the statoliths.

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4)

3a)

4) Stop the cutting when the two statoliths become visible in the anterior side of the statocysts (two distinct cavities will appear and the two statolith will be visible as clear particles in the middle of them).

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Annex C: Ground statoliths

Not-cleaned statoliths that have been ground on one side only: stains and tissue residuals (preventing from growth increments identifications) are clearly visible.

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Some scratches and glue residuals can appear on the statoliths surface after grinding with the coarsest lapping films (e.g. 30 and 12 grades); they can be removed using the finest lapping films (e.g. 3 and 0.05 grades).

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Ground statoliths

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Sometimes growth increment identification and counting is still possible in broken statoliths.

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