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Ontogeny of the brain in oval squid Sepioteuthis lessoniana (Cephalopoda: Loliginidae) during the post-hatching phase

Published online by Cambridge University Press:  26 March 2013

Shiori Kobayashi
Affiliation:
Department of Marine and Environmental Sciences, Graduate School of Engineering and Science, University of the Ryukyus, Okinawa 903-0213, Japan
Chitoshi Takayama
Affiliation:
Department of Molecular Anatomy, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0213, Japan
Yuzuru Ikeda*
Affiliation:
Department of Marine and Environmental Sciences, Graduate School of Engineering and Science, University of the Ryukyus, Okinawa 903-0213, Japan
*
Correspondence should be addressed to: Y. Ikeda, Department of Marine and Environmental Sciences, Graduate School of Engineering and Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan email: ikeda@sci.u-ryukyu.ac.jp
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Abstract

Among invertebrates, cephalopods have one of the most well-organized nervous systems. However, with respect to the ontogeny of the nervous system, the post-embryonic development of the cephalopod brain has only been documented for a few species. Here, we investigated the development of the brain of captive oval squid Sepioteuthis lessoniana during the post-hatching phase. The central part of the brain of the oval squid is divided into four main regions, namely, the supraoesophageal, anterior suboesophageal, middle suboesophageal, and posterior suboesophageal masses, each consisting of several lobes. At various ages in juvenile squid, the total volume of the central part of the brain (except the optic lobe) is significantly correlated with its body size, indicated by mantle length and wet body weight. The vertical lobe, superior frontal lobe, and anterior subesophageal mass drastically increase in relative volume as the squid grows. In contrast, the middle suboesophageal mass and posterior suboesophageal mass do not increase in volume with increasing squid age and body size. The effects of these results have been discussed in relation to the onset of squid behaviours during post-hatching.

Type
Research Article
Copyright
Copyright © Marine Biological Association of the United Kingdom 2013 

INTRODUCTION

Coleoid cephalopods (squid, cuttlefish and octopus) have the most complex and largest brain or central nervous system (CNS) among all invertebrates (Budelmann, Reference Budelmann, Breidbach and Kutsch1995). The relative volume of a coleoid cephalopod brain falls between that of fishes and reptiles and that of birds and mammals (Packard, Reference Packard1972). Cephalopods live in various marine environments, are highly mobile during active predation and/or migration, and have relatively short life spans, typically one year. In spite of their short lifespan, cephalopods are equipped with all the major sense organs, including a human-like lens eye (Hanlon & Messanger, 1996). These advanced features of well-developed mobility and senses can be explained as a result of coevolution with their rival marine vertebrate-like fishes (Packard, Reference Packard1972).

The cephalopod CNS has three main functional parts, which are classified according to their functional levels. One major part of the CNS, the optic lobes, lies outside the brain capsule, whereas the other major part, the central part, comprises the supraoesophageal and suboesophageal masses (SPM and SBM, respectively). Structurally, the SPM is the most complex region of the cephalopod CNS. In Coleoidea, the SBM can be divided into anterior, middle, and posterior areas (MSA, MSM and MSP, respectively) (Nixon & Young, Reference Nixon and Young2003). Lying inside the cartilaginous brain capsule and surrounding the oesophagus, the brain comprises approximately 30 lobes. According to a number of stimulation and lesion experiments, these lobes have been suggested to attribute to different functions that respond to various behaviours (Boycott, Reference Boycott1961; Young, Reference Young1977; Boyle, Reference Boyle and Willows1986; Maddock & Young, Reference Maddock and Young1987; Nixon & Mangold, Reference Nixon and Mangold1996).

The available documented data on the anatomy and neurophysiology of the cephalopod brain were obtained after the analysis of adult cephalopods (Abbott et al., Reference Abbott, Williamson and Maddock1995). These data have provided a basis for biomedical models, such as that of the giant nerve systems, reflexes driven by sense organs, and short- and long-term memory systems (Gilbert et al., Reference Gilbert, Adelman and Arnold1990; Abbott et al., Reference Abbott, Williamson and Maddock1995). Conversely, limited data from only three species are available on the development of the cephalopod brain (octopods: Octopus vulgaris, Amphioctopus fangsiao; sepiids: Sepia officinalis). This is partly because only a few cephalopod species can be cultured for the complete duration of their life cycle (Gilbert et al., Reference Gilbert, Adelman and Arnold1990). This is especially true in the case of squids (teuthids), where few studies have been conducted on the ontogeny of the brain. Nixon & Mangold (Reference Nixon and Mangold1996) described the development of the brain of the common octopus, Octopus vulgaris, by reviewing scattered data. According to this study, the neuropil of the subvertical and vertical lobes, which are parts of the visual and tactile memory centre, showed a marked increase in volume, but the neuropil of the anterior and posterior basal lobes, concerned with swimming at the time of planktonic paralarvae settling, showed a decrease in volume. In the European common cuttlefish Sepia officinalis, the postembryonic development of the vertical lobe (VL) complex is consistent with the development of learning and memory (Messenger, Reference Messenger1973, Reference Messenger1979; Dickel et al., Reference Dickel, Chichery and Chichery1997, Reference Dickel, Chichery and Chichery2001).

Among cephalopods, variations in the brain structure reflect not only the phylogenetic relationship but also the variety in habitat (Young, Reference Young1977; Maddock & Young, Reference Maddock and Young1987). For example, in the two benthic species, Amphioctopus fangsiao, which directly settles after hatching, and O. vulgaris, which enters a planktonic phase after hatching, the relative size of some brain regions differ during the early phases of life. In A. fangsiao, the brachial lobe, which is a region of the brain associated with adapting to benthic life, is relatively large at hatching, whereas in O. vulgaris, it remains small. In contrast, in O. vulgaris, the basal lobe system, which is a region of the brain that controls complicated body movements in the water column, is larger than that in A. fangsiao at hatching (Yamazaki et al., Reference Yamazaki, Yoshida and Uematsu2002).

The oval squid, Sepioteuthis lessoniana, is a nektonic squid and is widely distributed throughout the shallow waters of the Indian Ocean and the West Atlantic Ocean. Hatchlings of S. lessoniana are semi-planktonic and usually swim near the surface while actively feeding on small living prey (Segawa, Reference Segawa1987). Initially, S. lessoniana has a basic form of attacking, which leads to more specialized strategies, until 4–6 weeks of age when the adult pattern is apparent (Kier, Reference Kier1996). Sepioteuthis sepioidea, a species phylogenetically similar to S. lessoniana, forms well-structured schools and has social interactions within the shoals, where each member is arranged in a definite order with one individual acting as a sentinel (Moynihan & Rodaniche, Reference Moynihan and Rodaniche1982). Similar types of schooling behaviour appear in S. lessoniana up to two months after hatching, in which the squid may be able to recognize schoolmates (Sugimoto & Ikeda, Reference Sugimoto and Ikeda2012). This social behaviour by S. lessoniana must be formed along with the development of the brain, similar to the synchronization of the development of learning-memory and enlargement of the VL complex in S. officinalis (Dickel et al., Reference Dickel, Chichery and Chichery1997). However, related information has not been reported in social cephalopods such as squids. Thus, in this study we discuss similarities between these behavioural changes and brain development in S. lessoniana as a model for the process of acquiring particular behaviours under the control of the brain.

The developmental anatomy of the brain lobes of S. lessoniana during the embryonic phases has been investigated in detail (Shigeno et al., Reference Shigeno, Tsuchiya and Segawa2001a). According to Shigeno et al. (Reference Shigeno, Tsuchiya and Segawa2001a), although the fundamental development plan is common among the coleoids, the formation of the neuropil occurs earlier in S. lessoniana than in other species, such as O. vulgaris. This heterochronic difference in neuropil formation among cephalopod species seems to correlate with the mode of life. Shigeno et al. (Reference Shigeno, Tsuchiya and Segawa2001a) also described the development of the paralarval and juvenile brains in S. lessoniana, and reported that all lobes of the central brain increase in volume, especially in the supraoesophageal mass. However, this observation was based only on data from individuals at 3 and 10 days of age. Thus, detailed information on the developmental process of each brain lobe is still unavailable. This is because of the difficulty in rearing squids after hatching, as mentioned previously. In this study, we examined postembryonic brain development in captive reared S. lessoniana, focusing on the vertical lobe complex and the anterior basal lobe (BA). These two regions are concerned with visual recognition and integrating movements, respectively (Boycott, Reference Boycott1961); therefore, they directly control particular behaviours such as schooling and feeding.

MATERIALS AND METHODS

Egg collection and rearing of paralarvae

The egg capsules of Sepioteuthis lessoniana, which were spawned on a set net, were collected from Nago Bay, Okinawa Island, Japan. The egg capsules were transferred to the laboratory at the Department of Biology, Chemistry and Marine Sciences, University of the Ryukyus. The egg capsules were maintained in a square polystyrene tank (180 l) with a closed seawater system (dimensions, 726 × 1065 × 303 mm; filtration tank volume, 60 l, with a condenser and sterilizer). The hatchlings were moved to a circular tank (20 l) with a closed seawater system (Multihydense® Aqua Inc.; diameter, 300 mm; filtration tank volume, 180 l) and reared until 55 days of age. The duration through which all squid hatched extended to nine days. The date when the largest number of squid (35% of the total hatchlings) hatched was defined as day 0. Water temperature was maintained between 24.0°C and 25.0°C throughout the experiment. The salinity was adjusted to approximately 35 psu and pH was maintained above 7.8. Water quality and environmental factors recorded were as follows: temperature: 24.1–24.7°C; salinity 34.0–36.5 psu; and pH: 7.53–8.1 (note: the lowest pH value was measured for just two days; for the majority of the experiment, the pH was above 7.8). Squid up to 14 days of age were fed live prey (adult mysids Neomysis japonica, guppy fry Poecilia reticulata, and nauplii of the brine shrimp Artemia salina). Squid older than 14 days of age were fed frozen organisms (sakura shrimp Sergia lucens, anchovy Engraulis japonicus, and common prawn Palaemon paucidens) three times a day. Newly hatched squid were fed 4 times a day at about the same time each day.

Histological observations of the brain

A total of 38 squid were collected between three and 55 days post-hatching. These individuals were anaesthetized using a mixture of 2% ethanol in seawater, followed by measurements of the dorsal mantle length (ML) and wet body weight (BW). The squid were then killed by decapitation, and the brain was removed and fixed in 10% formalin in seawater. In cephalopods, the optic lobe is located behind the eyes, and the central part of the brain is located between the optic lobes. The central part of the brain surrounds the oesophagus and consists of two large masses, namely, the SPM and SBM, with several lobes (Young, Reference Young1971; Reference Young1974). Because we focused our interest with ontogeny on recognition and motor control, we did not observe the optic lobe, similar to previous studies (Dickel et al., Reference Dickel, Chichery and Chichery1997, Reference Dickel, Chichery and Chichery2001; Messenger, Reference Messenger1973, Reference Messenger1979; Nixon & Young, 1996; Yamazaki et al., Reference Yamazaki, Yoshida and Uematsu2002). The central part of the brain was extracted and dehydrated using a graded series of ethanol and then embedded in paraffin wax. Serial sagittal 10-μm-thick sections of the paraffin block were obtained and stained in Mayer's haematoxylin and eosin solution (HE).

Accordingly, in the current study, when we refer to ‘the brain’, we mean the central part of the brain of S. lessoniana.

Measurement of the brain and brain volume

We calculated the total volume of the brain and lobes as follows. The sagittal width of the brain was determined by summing the number of sections and multiplying by the thickness of the sections (10 µm). Then, 20–27 serial sections were selected with equal intervals for each individual (between 30–170 µm; these intervals were estimated on the basis of the number of sections). These sections were photographed using a digital camera (Canon EOS 5D Mark III) by viewing under a light microscope (Zeiss Axioplan2). The area of the lobe in a single section was measured using Image J® on a computer (Apple Macintosh). Areas of the lobes were integrated for the selected sections to estimate the volume of the lobes and the brain, according to the following formula:

$$V=0.01X \lpar a_{1}+a_{2}+a_{3}+\ldots +a_{n}\rpar$$

where, V = volume, X = distance between each selected section, and a = area of each selected section.

Data analysis

To determine the effects of age on relative growth of the lobes or regions, we used the Kruskal–Wallis test for non-parametric data and one-way analysis of variance (ANOVA) for parametric data. The correlation between the relative growth of the lobes or regions and the body weight (BW) were determined using Pearson's correlation coefficient.

RESULTS

Post-embryonic development of body size in Sepioteuthis lessoniana

At day 3, ML and BW were 5.79 ± 0.62 mm (mean ± standard deviation (SD)) and 0.035 ± 0.01 g, respectively. At day 55, ML and BW were 27.54 ± 4.03 mm and 2.007 ± 0.78 g, respectively. After day 30, individual variability was clearly observed in both ML and BW (Figure 1). During the period between day 3 and day 55, the mean values of ML and BW increased approximately four times and 57 times, respectively.

Fig. 1. Post-embryonic growth in the mantle length and wet body weight of Sepioteuthis lessoniana. Mantle length black circle; wet body weight: black square. Symbols and lines indicate mean and standard deviation, respectively.

Identification of Sepioteuthis lessoniana brain

We identified the nervous system structure in S. lessoniana according to the descriptions of the embryonic brain for this species (Shigeno et al., Reference Shigeno, Tsuchiya and Segawa2001a) and comprehensive descriptions of Loligo (Young, Reference Young1974, 1976, Reference Young1977, 1979; Messenger Reference Messenger1979). The main constituents of the brain, SPM and SBM, can be divided into several lobes. A lobe is the basic invertebrate organization with an outer cell body layer and inner neuropil layer (Young, Reference Young1974, 1976, Reference Young1977, 1979; Messenger, Reference Messenger1979; Hochner et al., Reference Hochner, Shomrat and Fiorito2006).

In the SPM, we identified the VL complex (the VL, subvertical lobe, inferior frontal lobe, precommissural lobe and superior frontal lobe) and the basal lobe system (the anterior and posterior basal lobes) in the same morphology as mentioned in previous studies. For the SPM, we measured the volumes of neuropil of the VL, superior frontal lobe (FRS) and BA. The VL and FRS are parts of the VL system, which are located on the dorsal section of the brain and are associated with learning and memory (Young, Reference Young1958, Reference Young1965, Reference Young1971). In contrast, the BA, which is located in the ventral region of the SPM, forms a system of higher motor centres regulating the direction and speed of movements (Young, Reference Young1971). The SBM is a lower motor centre of the brain and is divided into the MSA, MSM and MSP (Young, Reference Young1974). For the SBM, we measured the volume of the neuropil of the MSA, MSM and MSP. The MSA regulates the actions of the arms and suckers. The MSM is involved with the intermediate and lower motor centres, controlling most of the movements of the animal. The MSP regulates the movements of the mantle and partly those of the viscera (Young, Reference Young1971).

Post-embryonic development of the brain volume in Sepioteuthis lessoniana

The total volume of the brain increased with age (Figure 2A, C, E). In the SPM and SBM, the neuropil area of the vertical and the superior frontal lobes increased more than those in the other brain lobes (Figure 2B, D, F).

Fig. 2. Sagittal sections of the post-embryonic developing brain of Sepioteuthis lessoniana at three days of age: (A, C); 30 days of age; (B, D); and 55 days of age; (E, F.) Bottom, schematic drawing of the whole brain (left) and the supraoesophageal mass. Vertical lobe (VL), superior frontal lobe (FRS), anterior basal lobe (BA), anterior suboesophageal mass (MSA), middle suboesophageal mass (MSM), and posterior suboesophageal mass (MSP). Staining: haematoxylin and eosin. Direction of the brain: top, dorsal; right, anterior. Scale bars: A, C, E, 500 µm (the whole brain); B, D, F, 100 µm (the supraoesophageal mass).

The total volume of the brain exponentially increased with age (Figure 3A). Considerable variation in brain volume was observed for individuals older than 30 days. To evaluate the ontogenetic changes in the brain, the relative volume of the brain as a percentage of BW was calculated. This resultant value showed a decrease with age (N = 38, F = 12.9, P < 0.0001; Figure 3B). The brain volume significantly increased with increasing BW and ML (BW: N = 38, r = 0.99, P < 0.0001: ML: N = 38, r = 0.98, P < 0.0001; Figure 4).

Fig. 3. Post-embryonic growth of the brain volume (A) and the relative brain volume to wet body weight (B) of Sepioteuthis lessoniana. Symbols and lines indicate mean and standard deviation, respectively.

Fig. 4. Relationship between brain volume and body size expressed as mantle length (black circle) and wet body weight (white circle) of Sepioteuthis lessoniana. The wet body weight is shown on a logarithmic scale against brain volume on a logarithmic scale. The solid lines indicate regression lines (P < 0.001).

Development of brain lobes and brain regions

The relative volume of the VL and FRS developed more rapidly than the volume of the whole brain (BV) (VL/BV: F-measure (F) = 9.74, P < 0.0001; FRS/BV: F = 6.45, P < 0.001, respectively; Figure 5A). The relative volume of the BA appeared to grow nearly isometrically with the BV (F = 1.69, P = 0.15; Figure 5A). The relative volume (i.e. whole brain volume = 1) of the MSA and MSP developed more gradually than that of the BV (MSA/BV: F = 3.8, P < 0.01; MSP/BV: F = 4.87, P < 0.01, respectively; Figure 5B). Conversely, the relative volume of the MSM did not increase with age but grew isometrically with the BV (MSM/BV: F = 0.3, P = 0.95; Figure 5B).

Fig. 5. Growth in the relative volume of the brain regions to brain volume in Sepioteuthis lessoniana: (A) relative volumes of the vertical lobe (black bar), superior frontal lobe (dark grey bar), and anterior basal lobe (light grey bar); (B) relative volumes of the anterior (black bar), middle (dark grey bar), and posterior suboesophageal mass (light grey bar). Bars and lines indicate mean and standard deviation, respectively.

To compare the ontogeny of the brain lobes with the SPM, we calculated relative volumes of each lobe as a percentage of the SPM. From hatching to day 55, the VL and the FRS grew faster than the SPM (VL/SPM: F = 3.98, P < 0.001; FRS/SPM: F = 2.03, P < 0.01, respectively; Figure 6A). In contrast, the BA developed nearly isometrically with the development of the SPM (BA/SPM: F = 1.7, P = 0.15; Figure 6A). From day 39 to day 55, the relative volume of the VL tended to become a constant (Figure 6A). We also calculated the relative volume of each region of the SPM as a percentage of the SPM. The MSA developed more gradually than that of the SBM (MSA/SBM: hazard ratio (H) = 12.3, P < 0.1; Figure 6B). On the other hand, the MSM and MSP developed nearly isometrically with the development of the SBM (MSM/SBM: H = 9.52, P = 0.21; MSP/SBM: H = 9.17, P = 0.24; Figure 6B).

Fig. 6. Growth in the relative volume of the brain regions to the supraoesophageal mass (A) and suboesophageal mass (B) in Sepioteuthis lessoniana: (A) relative volume of the vertical lobe (black bar), superior frontal lobe (dark grey bar), and anterior basal lobe (light grey bar); (B) relative volume of the anterior (black bar), middle (dark grey bar), and posterior suboesophageal mass (light grey bar). Bars and lines indicate mean and standard deviation, respectively.

Compared to the growth of body size, the volume of each brain lobe and brain mass (MSA, MSM and MSP) increased significantly (Figure 7). However, the slope of the regression line was different for each region, which suggests a difference in the speed of development among the regions of the brain. The relative volume of the VL and the FRS to BV significantly increased with BW (VL/BV: r = 0.74, P < 0.001; FRS/BV: r = 0.71, P < 0.001; Figure 8B). In contrast, the relative volume of BA to BV showed a tendency to decrease, but there was no correlation between BA volume and BW (Figure 8A).

Fig. 7. Relationships between wet body weight (WBW) and the volume of the vertical lobe (VL, black circle), superior frontal lobe (FRS, black square), anterior basal lobes (BA, diamond), anterior (MSA, black triangle), middle (MSM, reverse triangle), and posterior suboesophageal mass (MSP, and reverse black triangle). The brain volume and wet body weight are shown on a logarithmic scale. The solid lines indicate regression lines (P < 0.001). Regression line and correlation coefficient (R): logVL = −0.23 + 0.9logWBW, R = 0.98; logFRS = −0.61 + 0.84logWBW, R = 0.98; log BA = −0.45 + 0.62logWBW, R = 0.93; logMSA = −0.31 + 0.82logWBW, R = 0.97; logMSM = 0.23 + 0.69logWBW, R = 0.97; logMSP = 0.27 + 0.8logWBW, R = 0.87.

Fig. 8. Relationships between wet body weight and relative volume of the vertical lobe (VL, black circle), superior frontal lobe (FRS, white square), basal lobes (BA, white diamond) (A), anterior (MSA, black circle), middle (MSM, white square), and posterior suboesophageal mass (MSP, white diamond) (B) of oval squid. The wet body weight is shown on a logarithmic scale. The solid lines indicate regression lines (P < 0.001). Regression line and correlation coefficient (R): log VL/BV = 5.13 + 1.82logWBW, R = 0.84; logFRS/BV = 1.99 + 0.44logWBW, R = 0.74; logMSA/BV = 4.45 + 1.08logWBW, R = 0.67.

The relative volume (the whole brain volume = 1) of the MSA increased slowly with increasing BW (r = 0.69, P < 0.001; Figure 8B). Conversely, the relative volumes of the MSM and MSP did not increase with increasing BW (Figure 8B).

DISCUSSION

This study is the first to document the ontogeny of the brain in squid (teuthids), aside from octopus and cuttlefish, during the post-hatching phase. Here, we demonstrated that the brain volume of Sepioteuthis lessoniana hatchlings increased exponentially with age until day 55, and the increase in brain volume had a positive relationship with the development of body size during the post-embryonic phase. In Amphioctopus fangsiao, the brain volume markedly increases along with the growth of the mantle during months 1–3 after hatching (Yamazaki et al., Reference Yamazaki, Yoshida and Uematsu2002). Since the relationship between the brain and body weight tends to follow an allometric equation at various taxonomic levels (Lande, Reference Lande1979), it must be applicable to the brain developmental pattern in cephalopods after hatching. The developmental pattern of the brain of S. lessoniana with age is, however, different from that of other invertebrates, such as the sea hare Aplysia. In Aplysia, although there was relatively little change in the brain neuropil volume during its early juvenile stage (stages 10–11), an approximately 10-fold increase in neuropil volume was observed during the late juvenile stages (stages 12E–12L) (Cash & Carew, Reference Cash and Carew1989). In the fruit fly Drosophila, the growth pattern of the total CNS mass shows a sigmoid curve during the larval and pupal periods (Power, Reference Power1952). Further, an exponential growth up to 124 hours after hatching was observed and could be described as metamorphic degeneration. This growth pattern of Drosophila up to 124 hours after hatching is similar to that observed in our study in S. lessoniana. However, similar to other cephalopods, S. lessoniana do not have a true larval stage, which means they do not experience a distinct metamorphosis. Thus, the development of the brain in S. lessoniana is likely to maintain an exponential pattern beyond day 55.

The proportion of the brain volume to BW developed with negative allometry in S. lessoniana. Because the number of cells in the brain of coleoid cephalopods is more than one hundred million, it is possible to compare the brains of cephalopods and vertebrates (Packard, Reference Packard1972). There are additional similarities between the cephalopod and vertebrate brains, namely, the hippocampus, VL (Young, Reference Young1991), cerebellum (Hobbs & Young, Reference Hobbs and Young1973), peduncle lobe (Gleadall, Reference Gleadall1990), pituitary (Wells & Wells, Reference Wells and Wells1969), and vestibulo-oculomotor and statocyst-oculomotor (Budelmann & Tu, Reference Budelmann and Tu1997). In fish, the brain generally grows in a negative allometric relationship to the body (Brandstätter & Kotrschal, Reference Brandstätter and Kotrschal1990). This developmental pattern is very similar to that of squid, as indicated in the present study. In terms of the quantitative developmental pattern of the brain, cephalopods and vertebrates are similar. Thus, there may be additional analogies between cephalopods and vertebrates.

In the present study, we clearly showed that the post-embryonic development of S. lessoniana brain proceeds differently in each lobe or brain region: (1) the volume of the VL, FRS, and MSA rapidly increased compared to that of the whole brain; and (2) the BA, MSM, and MSP of S. lessoniana do not show obvious developmental changes in relative volume. In S. lessoniana, the VL system (FRS and VL) develops very gradually, starting from the late embryonic stage (stage 28) (Shigeno et al., Reference Shigeno, Tsuchiya and Segawa2001a). In some species of cephalopods, it is known that the development of behaviours and changes in lifestyle with growth are also related to the post-embryonic development of the brain lobes (Messenger, Reference Messenger1973; Nixon & Mangold, Reference Nixon and Mangold1996; Dickel et al., Reference Dickel, Chichery and Chichery1997; Shigeno et al., Reference Shigeno, Kidokoro, Thuchiya, Segawa and Tamamoto2001b; Yamazaki et al., Reference Yamazaki, Yoshida and Uematsu2002). Sepioteuthis lessoniana form a school from 20 days post-hatching and complete the arrangement, similar to that of an adult school, at the age of two months (Sugimoto & Ikeda, Reference Sugimoto and Ikeda2012). Volumetric development of the VL and FRS may correlate with the ontogeny of some intellectual behaviours, such as learning and memory, similar to that observed in Sepia officinalis (Messenger, Reference Messenger1973; Dickel, 2004), or the formation of social interactions, such as schooling behaviour (Moynihan & Rodaniche, Reference Moynihan and Rodaniche1982; Sugimoto & Ikeda, Reference Sugimoto and Ikeda2012). Squids are visual predators that prey after judging whether the size of the prey is appropriate (LaRoe, Reference LaRoe1971; Boletzky & Hanlon, Reference Boletzky and Hanlon1983). Sepioteuthis lessoniana shift their method of hunting as they develop from hatchlings to adults at the age of 1–2 months (Kier, Reference Kier1996). Because the MSA of squids is associated with controlling the arm movements while catching prey (Young, Reference Young1975) and the VL complex is involved in the visual learning and memory, development in the MSA and VL, as shown in this study, may explain the shift in feeding behaviour in S. lessoniana. The tendency of convergence observed in VL development from 39 to 55 days may also be concerned with the shift in these behaviours using visual information.

The BA, MSM and MSP are involved in the movements of cephalopods. The basal lobe system is the higher motor centre and is associated with the control of movements in cephalopods (Young, Reference Young1971). The MSM and MSP are involved in majority of the movements of cephalopods, such as those of the fin, mantle, and funnel (Young, Reference Young1975). Sepioteuthis lessoniana hatches with a relatively large body size and a fully developed nektonic lifestyle, compared to species that hatch as a planktonic form, such as Todarodes pacificus. Sepioteuthis lessoniana boldly prey on the adults of the mysis Neomysis japonica just after hatching (Cho & Ohshima, 1961). Shigeno et al. (Reference Shigeno, Tsuchiya and Segawa2001a) reported that neuropil formation of the BA, MSM, and MSP occurs earlier than that of the VL complex in S. lessoniana. This fact may relate to the readiness of feeding ability during post-hatching in S. lessoniana.

The manner of post-hatching brain development in S. lessoniana (squid) shares some aspects with that of cuttlefish and octopus. In this study, we focused on the VL, FRS, and BA involving high-order function. The SPM includes other lobes, which involve different functions, with the exception of VL, FRS, and BA. The excluded parts appeared to be relatively small as the squid grew, although the VL and FRS became dramatically larger than the other lobes (see Figure 2). Whether these excluded lobes are concerned with the species-specific features of S. lessoniana, such as the development of sociality and schoolmate identification, must be investigated.

ACKNOWLEDGEMENTS

We thank H. Higa for assisting in squid egg collection and all laboratory members for their help in squid rearing.

FINANCIAL SUPPORT

We thank the 21st Century COE programme entitled ‘The Comprehensive Analyses on Biodiversity in Coral Reef and Island Ecosystems in Asian and Pacific Regions’ of the University of the Ryukyus Monbukagakusho and a Grant-in-Aid for Scientific Research (C), Project No. 20580207, MEXT to Y.I. for financial assistance.

References

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Figure 0

Fig. 1. Post-embryonic growth in the mantle length and wet body weight of Sepioteuthis lessoniana. Mantle length black circle; wet body weight: black square. Symbols and lines indicate mean and standard deviation, respectively.

Figure 1

Fig. 2. Sagittal sections of the post-embryonic developing brain of Sepioteuthis lessoniana at three days of age: (A, C); 30 days of age; (B, D); and 55 days of age; (E, F.) Bottom, schematic drawing of the whole brain (left) and the supraoesophageal mass. Vertical lobe (VL), superior frontal lobe (FRS), anterior basal lobe (BA), anterior suboesophageal mass (MSA), middle suboesophageal mass (MSM), and posterior suboesophageal mass (MSP). Staining: haematoxylin and eosin. Direction of the brain: top, dorsal; right, anterior. Scale bars: A, C, E, 500 µm (the whole brain); B, D, F, 100 µm (the supraoesophageal mass).

Figure 2

Fig. 3. Post-embryonic growth of the brain volume (A) and the relative brain volume to wet body weight (B) of Sepioteuthis lessoniana. Symbols and lines indicate mean and standard deviation, respectively.

Figure 3

Fig. 4. Relationship between brain volume and body size expressed as mantle length (black circle) and wet body weight (white circle) of Sepioteuthis lessoniana. The wet body weight is shown on a logarithmic scale against brain volume on a logarithmic scale. The solid lines indicate regression lines (P < 0.001).

Figure 4

Fig. 5. Growth in the relative volume of the brain regions to brain volume in Sepioteuthis lessoniana: (A) relative volumes of the vertical lobe (black bar), superior frontal lobe (dark grey bar), and anterior basal lobe (light grey bar); (B) relative volumes of the anterior (black bar), middle (dark grey bar), and posterior suboesophageal mass (light grey bar). Bars and lines indicate mean and standard deviation, respectively.

Figure 5

Fig. 6. Growth in the relative volume of the brain regions to the supraoesophageal mass (A) and suboesophageal mass (B) in Sepioteuthis lessoniana: (A) relative volume of the vertical lobe (black bar), superior frontal lobe (dark grey bar), and anterior basal lobe (light grey bar); (B) relative volume of the anterior (black bar), middle (dark grey bar), and posterior suboesophageal mass (light grey bar). Bars and lines indicate mean and standard deviation, respectively.

Figure 6

Fig. 7. Relationships between wet body weight (WBW) and the volume of the vertical lobe (VL, black circle), superior frontal lobe (FRS, black square), anterior basal lobes (BA, diamond), anterior (MSA, black triangle), middle (MSM, reverse triangle), and posterior suboesophageal mass (MSP, and reverse black triangle). The brain volume and wet body weight are shown on a logarithmic scale. The solid lines indicate regression lines (P < 0.001). Regression line and correlation coefficient (R): logVL = −0.23 + 0.9logWBW, R = 0.98; logFRS = −0.61 + 0.84logWBW, R = 0.98; log BA = −0.45 + 0.62logWBW, R = 0.93; logMSA = −0.31 + 0.82logWBW, R = 0.97; logMSM = 0.23 + 0.69logWBW, R = 0.97; logMSP = 0.27 + 0.8logWBW, R = 0.87.

Figure 7

Fig. 8. Relationships between wet body weight and relative volume of the vertical lobe (VL, black circle), superior frontal lobe (FRS, white square), basal lobes (BA, white diamond) (A), anterior (MSA, black circle), middle (MSM, white square), and posterior suboesophageal mass (MSP, white diamond) (B) of oval squid. The wet body weight is shown on a logarithmic scale. The solid lines indicate regression lines (P < 0.001). Regression line and correlation coefficient (R): log VL/BV = 5.13 + 1.82logWBW, R = 0.84; logFRS/BV = 1.99 + 0.44logWBW, R = 0.74; logMSA/BV = 4.45 + 1.08logWBW, R = 0.67.