The settlement phase in the common octopus Octopus vulgaris: a complex transition between planktonic and benthic lifestyles

doi:10.21203/rs.3.rs-2148127/v1

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The settlement phase in the common octopus Octopus vulgaris: a complex transition between planktonic and benthic lifestyles

https://doi.org/10.21203/rs.3.rs-2148127/v1

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published 29 Mar, 2023

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Planktonic octopuses undergo a transitional period from a pelagic lifestyle to the predominantly benthic life of the juvenile stage, known as settlement, that is scarcely documented in the wild or captivity. In this work two generations of the common octopus, Octopus vulgaris, were reared in captivity and three different stages were defined for the settlement phase based on morphological, anatomical, and behavioural changes: pre-settlement or “tactile” stage, settlement, and post-settlement or “ninja” stage. Before settlement the swimming planktonic paralarvae are transparent with 65–80 chromatophores, iridophores covering eyes and digestive system, Kölliker organs, circular pupils, ~ 20 suckers, and mantle length (ML) bigger than total length (TL; ML/TL > 60%). The “tactile” stage (ML/TL from 65 − 55% and ~ 20–25 suckers) is marked by the onset of pre-settlement reflexes, where the late paralarvae touch the walls and bottom of the tank and start crawling clumsily. Morphologically, the paralarvae are transparent with increasing chromatophores and iridophores along the arms and the dorsal area of head and mantle. During the settlement stage (~ 55 − 48% ML/TL, and ~ 25–35 suckers), there is an exponential increase of chromatophores in the dorsal area and a marked change in behaviour, with paralarvae showing strong negative phototaxis, looking for shelter when disturbed. The skin is still transparent but new chromatic cells (leucophores) start to develop and the Kölliker organs are almost lost. During the post-settlement stage (~ 48 − 40% ML/TL, > 35 suckers) the chromatic cells keep increasing exponentially, giving a pale look to the skin. The early juveniles have horizontal pupils, with the “eye-bar” and display very fast and coordinated movements like “ninjas”. The start of the benthic phase is marked by the presence of skin sculptural components (papillae and cirrha) and the display of complex chromatic and body patterns.

Octopus vulgaris

settlement phase

metamorphosis

paralarva

juveniles

behaviour

merobenthic

The transition from paralarva to subadult in cephalopods does not involve a proper metamorphosis as found in many other marine invertebrates (Boletzky 1974). The changes occurring are subtle and concern the variation in growth rates, in body proportions or in the structure or function of specific organs (Robin et al. 2014). However, these changes are markedly different depending on the mode of life of the early stages within the family Octopodidae: holobenthic and merobenthic. The holobenthic strategy requires the production of few large eggs resulting in well-developed, benthic, adult-like hatchlings that share the same habitat with adults. Octopodid species producing eggs > 10–12% of adult mantle length fall within this category (Boletzky 2003). These early stages can be considered as juveniles or subadults, since they have all diagnostic morphological features generally used to define the species and actively use their arms as a mode of locomotion and food search (Villanueva and Norman 2008). The merobenthic strategy, on the other hand, corresponds to the production of numerous small eggs that hatch into swimming planktonic hatchlings, with low survival rates. Only octopodids producing eggs smaller than 8–10% of adult mantle length are merobenthic (Boletzky 2003). The hatchlings are not adult-like, characterized by short arms, a swimming behaviour based in jet propulsion and translucent bodies with few chromatophores in the ventral zone, called paralarvae. This merobenthic strategy is followed by up to 70 species within the family Octopodidae (see table 1, Villanueva and Norman 2008), and it is considered as the ancestral state in benthic octopuses (Ibáñez et al. 2012)

The term paralarva was defined by Young and Harman (1988) as “a cephalopod of the first post-hatching growth stage that is pelagic in near-surface waters during the day and that has a distinctively different mode of life from that of older conspecific individuals”. Under this definition only the merobenthic species will have paralarvae, and therefore only in this group of octopods would be possible to identify, characterize and quantify the changes that occur during the settlement phase, from the swimming-paralarvae to the crawling-juveniles. While in the holobenthic species this transitional phase between paralarval stages and juveniles is hardly identifiable or inexistent, merobenthic species do have profound changes that have been previously considered as true metamorphosis. One of these remarkable changes concerns the skin. The skin of planktonic paralarvae is almost transparent with only 65–80 mesodermal black pigment-filled chromatophores, or founder chromatophores, of which eight appear under the dorsal mantle covering the digestive gland (Packard 1985). During settlement, chromatophore number increases exponentially and new chromatic cells (iridocytes and leucophores) develop specially in the dorsal area, which will help them for camouflaging on the seafloor (Messenger, 2001). Mantle length is nearly double the arm length at the planktonic stage, but this ratio clearly diminishes during settlement owed to positive allometric arm growth (Villanueva and Norman 2008). When old paralarvae have grown to a size at which the arms attain a length like the length of the mantle, they switch to bottom life by progressively contacting the substratum (Villanueva et al. 1995). There are no skin sculptural components, apart from the Kölliker organs, in planktonic paralarvae (Nixon and Mangold 1996), which are common features in benthic juveniles, essential for camouflage and communication (Messenger 2001). Other morphological changes associated with settlement include the loss of Kölliker organs, the loss of the “lateral line system” and the loss of the oral denticles of the beaks (Villanueva and Norman 2008). Before settlement, the planktonic young octopodids swift their swimming existence with a new mode of locomotion that requires the coordinated action of the arms and suckers: crawling. The onset of this behaviour was defined as a “pre-settlement reflex” by Villanueva (1995) or “clinging” by Dan et al. (2021). Late paralarvae of O. sinensis in captivity showed diurnal clinging and nocturnal swimming, likely related with minimizing the exposure to visual predators (Dan et al. 2021).

Despite being one of the main transitions in octopus life cycle, the settlement phase has been narrowly addressed owed to two main facts: the technical difficulties in finding these organisms on the field and the lack of standardized protocols for rearing the paralarvae in captivity until settlement. The only works describing some morphological changes occurring in late paralarvae and early juveniles of O. vulgaris collected in the wild were those of Rees (1950), Packard (1985) and Nixon and Mangold (1996), Roura et al. (2019), while Sakaguchi et al. (1999) is the only work for O. sinensis. The growth of Octopus “vulgaris” during the planktonic phase and after settlement shown by Nixon and Mangold (1996) was based on the data obtained by Itami et al. (1963) in Japan. However, it is important to point out that the octopod cultivated by Itami and collaborators was not O. vulgaris, but O. sinensis (Gleadall 2016). This western Pacific octopod is raised at higher temperatures from 23 to 26.7ºC and has a shorter pelagic phase < 25 days (Itami et al. 1963; Okumura et al. 2005; Dan et al. 2021) than O. vulgaris that is raised at 21–23ºC and starts settling after 45 days (Villanueva 1995; Iglesias et al. 2004; Carrasco et al. 2006).

Few works have succeeded producing benthic juveniles from planktonic paralarvae in captivity (O. vulgaris: Villanueva, 1995; Iglesias et al. 2004; Carrasco et al. 2006; O. sinensis: Itami et al. 1963; Okumura et al. 2005; Dan et al. 2021; Robsonella fontaniana: Uriarte et al. 2010). Of these, Uriarte and collaborators studied the morphological changes in R. fontaniana for up to 120 days during the pelagic and benthic phases, while Dan et al. (2021) focused on the behavioural changes in swimming, clinging and shelter use during the onset of the settlement phase in O. sinensis from 10 to 28 days old. Works made in captivity with O. vulgaris have gone through the settlement to adulthood (Iglesias et al. 2004; Carrasco et al. 2006; De Wolf et al. 2011), but no reference to the changes occurring during this transitional phase were done apart from indicating the pre-settlement reflexes found in the late paralarvae (Iglesias et al. 2004; Carrasco et al. 2006). The only work that described morphological and behavioural changes during the settlement phase stopped at 60 days (Villanueva 1995), evidencing that this phase is a complex process with numerous and profound changes that needs to be addressed in detail.

In this work we studied two generations of O. vulgaris reared in captivity and describe the major changes undergone between the planktonic and benthic phases. We propose to divide the settlement phase into three different stages based on the main morphological, behavioural and ecological changes observed during this transition from the pelagic to the benthic mode of life.

Two generations of O. vulgaris juveniles were analysed in this study. The first one was obtained from eggs of a wild female collected at the Ría de Vigo with scuba diving (NW Spain), and the second one was obtained from the animals reared in captivity (F1). Six well-developed egg strings (stage XX) were transported to the Instituto de Investigaciones Marinas de Vigo (IIM-CSIC) in 5L recipients with natural sea water in dark conditions. Transport lasted 2 hours and the eggs were maintained in a flow-through tank (20×20x30cm) filled with seawater at 18ºC and ~ 36psu (practical salinity units). Egg strings were tied from sections of plastic pipe and immersed in the tank with enough horizontal current to keep the egg strings moving gently. After 5 days paralarvae hatched and the experiment begun.

The paralarvae were reared at a density of 5 paralarvae/L in 50L dark green fibre tanks provided with filtered seawater (1µm) and a central outlet with 500µm filter. An open water system with 150% renovation per day was used with mean water temperature 19ºC (18.1–20.5), salinity 35.4 (34,8-36.2) and a 14:10 h light cycle provided with LED lights. The bottom of the tanks was siphoned every day. Live diet consisted of sub-adult Artemia salina (1–3 mm TL) at a concentration of 0.1 − 0.05 ind/ml, cultivated at 25ºC with a phytoplankton mix. During settlement adult Artemia was offered as prey together with small fragments of frozen food. After settlement frozen food was offered everyday including mostly fish (Merluccius merluccius, Trachurus trachurus), but also mussels (Mytilus galloprovincialis) and crabs (Liocarcinus depurator), until around 5 months when it was modified to five times a week. The food rations were calculated as 20% of the octopus biomass estimated for each tank. Juveniles were placed in three glass tanks of 30L in an open water system with 200% renovation per day, mean water temperature 18ºC (17.8–18.3), salinity 35.7 (34,8-36.2) and a 14:10 h light cycle provided with LED lights. The bottom of the tanks was siphoned every day and prey remains were removed and weighted. Tanks were transparent with black plastic covering the sides, to allow recording the behaviours displayed during the settlement phase.

At day 153, the surviving specimens were transported to the facilities of Estación de Ciencias Marinas de Toralla (ECIMAT, Universidad de VIGO). Subadults and adults were maintained in 400L fiberglass tanks, at a density of 5–10 kg/m³, with an open water system with 400% renovation per day at 18ºC. One refuge per octopus − 40 cm long PVC pipes 35 cm in diameter - was placed inside the tanks. The tanks were cleaned every day, and food remains weighted daily. Once the females started laying eggs (around 15 months) they were isolated in individual 400L tanks with a 400% renovation and 18ºC. The PVC pipes were used as dens, were females spawned for 15–20 days between May and June 2019. Egg strings were attached on the upper side of the pipe, constantly cleaned, and oxygenated by gentle water jets from the female’s siphon. After 56 days the paralarvae started to hatch and a new culture experiment was settled in similar conditions to the first one but reducing paralarval density to 4 specimens/L and temperature to 18º. For the settlement phase, late paralarvae/early juveniles were placed in six grey tanks of 100L in an open water system with 200% renovation per day a mean water temperature 18ºC (17.8–18.3), salinity 35.7 (34,8-36.2) and a 14:10 h light cycle provided with LED lights. The bottom of the tanks was siphoned every day, prey remains removed and weighted.

Meristic data was obtained to the nearest 0.05 mm using a stereomicroscope (Nikon SMZ800) coupled to an image analysis software (NIS-Elements) from fresh and ethanol preserved paralarvae/juveniles: mantle length (ML) / total length (TL), as well as number of suckers. A percentage of shrinkage in mantle length (9.74%) caused by fixation was considered for those individuals stored in 70% ethanol (Villanueva 1995). Fresh weights were obtained to the nearest 0.01 g in alive animals that were returned to the tanks from day 45 onwards. All the individuals were weighted at the four month in both experiments. An interocular area in the head, defined by the 6 dorsal founder chromatophores above and between the eyes, was used to quantify chromatophore genesis during settlement. Body changes were photographed and recorded throughout the settlement stage.

This work was carried in two different projects (AQUOPUS and OCTOBLUE) with the aim to increase the survival and enhance the zootechnical aspects of the culture of O. vulgaris in captivity and, therefore, experiments do not fall under Directive 2010/63/EU of the European Parliament and of the Council, of 22 September 2010 on the protection of animals used for scientific purposes. Nonetheless, the experimental procedures were supervised and approved by an ethics committee at the different institutions (IIM for AQUOPUS and University of Vigo for OCTOBLUE projects). The authors minimized the number of animals euthanized and maximized the sampling of animals that were moribund and appropriate methods for anaesthesia and euthanasia were used. Paralarvae/early juveniles were anesthetized by immersion in a 1.5% ethanol-seawater solution at room temperature (18ºC) for 1 min. Afterwards, the specimens were immersed in 2% MgCl₂ at 8ºC for 5 minutes and finally immersed in 3.5% MgCl₂ filtered seawater solution at 8ºC for 10 min as a way of euthanasia (Fiorito et al. 2015).

Octopus paralarvae turn into fully benthic juveniles in less than three months at a temperature between 18–19ºC. The paralarvae hatch with 3 uniserial suckers and start to add suckers from day 6 onwards in two rows at the terminal tip of the arm in a proximodistal direction. Figure 1 shows the incorporation of suckers and the decrease in ML/TL ratio during the first four months of life in O. vulgaris. During the planktonic phase (Fig. 2a) the arms of the paralarvae start to grow and the ML/TL decreases from an initial 80% to ~ 60%, a figure that can be used as a proxy to mark the end of the planktonic phase, which is around day 45 and the paralarvae have ~ 20 suckers (ranging from 10–27).

At this moment the paralarvae have a cuttlefish-look, swimming with the arms pointing downwards. Late paralarvae swim with very precise movements, shifting directions with ease. When they are illuminated from outside of the tank the numerous iridocytes developing in the skin reflect the light giving a silvery look to the paralarvae (Fig. 2b). The shift from the pelagic environment of the paralarvae to the benthic mode of life of the adults requires the development of a new set of physiological adaptations and coordinated movements that takes around 30–45 days, which is called the settlement phase. Three different stages can be distinguished during the settlement phase (Fig. 3) based on behavioural, anatomic, and morphologic changes: pre-settlement (~ d45-60), settlement (~ d60-75) and post-settlement (~ d75-90).

During the pre-settlement stage or “tactile stage” (~ d45-60), the transparent paralarvae begin to switch between swimming and crawling, attaching to the bottom and walls of the tank in a clumsy way, i.e. without coordinated movements of the arms. During this stage numerous attacks were observed using the wall of the tank to catch the Artemia, where the paralarvae remained attached until they returned to swimming to feed on it. Similarly, when the paralarvae are disturbed while attached to any surface they come back to swimming displaying a very intense red colour, rather than hiding or crawling. Numerous paralarvae were also observed during this stage with the arms attached to the water-air interface. Anatomically, ML/TL ratio varies from 65 − 55% and they have ~ 20–25 suckers (Fig. 1). The paralarvae have increased the number of chromatophores along the length of the arms (Fig. 2b), but these are still absent in the dorsal area of the head and mantle (Fig. 3.1). The new chromatophores start filled with yellow pigments that gradually become darker (melanophores). The fresh weight of these paralarvae ranged between 80–150 mg and the ML ranged from 6 to 7.4 mm.

The settlement stage (~ d60-75) can be identified by the reclusive behaviour of the pre-juveniles within the refuges provided. At this point they display better arm coordination, and the early juveniles use them to crawl and hide inside of the shelters. When disturbed outside of the shelter they don’t swim but crawl and use jet scape (only going backwards) looking for dark zones of the tank. When disturbed in the shelter they direct water fluxes to the origin of disturbance and change from transparent to dark red (Fig. 4a). Most of the time they are inside of the shelters (Fig. 4b), except at night when they cautiously look for food nearby. At this point, the length of the arms equals that of the mantle (~ 55 − 48% ML/TL), and they have ~ 25–35 suckers (Figs. 1 and 2c, d). Chromatophore genesis starts to speed up in the dorsal area during this stage (Fig. 3). The musculature and skin are transparent and Kölliker organs can still be observed imbibed in the skin of the pre-juveniles (Fig. 2c, d). During this stage a loss of weight was observed, ranging from 70 to 150 mg fresh weight, and the ML recorded ranged from 6.3 to 8.2 mm.

The post-settlement stage also called the “ninja stage” (~ d75-90) can be identified from a behavioural point of view by the highly coordinated movements of the juveniles, that can “jump” in any direction and move very fast. This stage is marked by a profuse production of pigmented cells (chromatophores, iridocytes and leucophores) that gives the early juveniles a faint pale look. The increased number of chromatophores at the end of this stage allows them to start displaying basic camouflage. Despite the pupil is circular during the planktonic phase and pre-settlement, from ~ 80 days (> 35 suckers, ~ 48 − 40% ML/TL) the pupil starts to develop a horizontal pupillary response, an adaptation to a benthic mode of life (Fig. 4b). Another feature is the pigmentation of the eyelid, which is responsible for the creation of the “eye-bar” (Fig. 4b), but at this stage it does not cover the full length of the eye as in benthic juveniles (Fig. 4c). The early juveniles are very voracious and skilled hunters from now on, able to catch prey larger than themselves. A sharp increase in weight was observed during this stage with specimens ranging from 140 to 240 mg.

Finally, in the benthic phase the juveniles keep developing pigmented cells and are fully capable of camouflaging. They also develop the neural control of the skin musculature to create skin sculptural components like cirrha and dorsal papillae (Fig. 4c). From a behavioural point of view, the settled juveniles display two main cryptic postures when moving outside of the shelter. The most common during the first month of benthic life is a compressed posture that resembles a “stone” (first observed at day 88), where the juveniles have all the arms packed against the body and use the 4th pair of arms to move in short pulses (Fig. 5). This body pattern is very similar to the “zebra crouch” described in Packard and Sanders (1971), but without the colour transverse stripes (chevron pattern). The other pattern, less common than the “stone”, is the flamboyant posture (sensu Packard and Sanders 1971) that resembles a detached “alga” (Fig. 5). This body pattern was observed for the first time at day 92 when the juveniles collected for sampling were returned into the tank. They adopt this deceiving body pattern to swim through the water column until they reach the bottom, where the display is maintained for a while, usually turning into “stones” to look for shelter. They start to do the “cleaning manoeuvre” (first day observed 93 days-old) and seem to interact with other conspecifics with “passing clouds”, changing the colour unilaterally and doing sucker displays (defined in Packard and Sanders 1971). As mentioned in the post-settlement stage octopus juveniles are voracious predators, that accept and ingest unfrozen food with ease.

Cannibalism was registered throughout the settlement phase, especially during the settlement and post-settlement stages, accounting for 26% and 14% of the deaths in the first and second experiments, respectively. Some attacks were observed always directed from bigger and faster specimens (“ninja” juveniles) harassing smaller ones inside of the shelter, that tried to protect themselves with withdrawal manoeuvres showing the underside of the arms and dark red colouration. These interactions occurred very fast (less than a minute) and most of them ended with the smaller ones losing the tip of an arm or several. Depending on the extent of the damage and the health of the specimens attacked, these interactions resulted in the death of the smaller ones. Apart from the interactions observed, others happened without notice leaving no trace of the victims but the digestive gland.

In this work we have defined three distinct stages within the settlement phase and their associated morphological, anatomical, and behavioural adaptations, which are graphically shown on Fig. 5. The duration of the different phases (planktonic, settlement and benthic) and the 3 stages defined for the settlement phase (pre-settlement, settlement, and post-settlement) are the result of the specific rearing conditions followed in this study and are by no means fixed values in terms of days. It is expected that paralarvae reared at higher temperatures will have shorter planktonic phases and start the settlement earlier, since somatic growth is largely increased with higher temperatures during the planktonic stage, when cephalopods grow exponentially (Forsythe, 2004). Villanueva (1995) obtained settled juveniles of O. vulgaris from day 47 to 54 at 21.2ºC (from 19–23ºC), Iglesias et al. (2004) from day 40 at 22.5ºC (from 19 − 6–22.9ºC) and Carrasco et al. (2006) observed the first pre-settlement reflexes from day 40, and by day 52 they observed them crawling at 21.2ºC (from 19.3º to 22.6ºC). Despite the different rearing conditions, it is noticeable that the paralarvae need to gain a certain size to start with the settlement phase. Settled O. vulgaris paralarvae had between 5.7 to 7.5 mm DML (Villanueva 1995; Carrasco et al. 2006), which agrees with this work, despite the fact of being cultured at lower temperatures. The onset of clinging behaviour (pre-settlement reflexes) in Octopus sinensis late paralarvae start at smaller sizes (5.2–6 mg dry weight, Dan et al. 2020; 16 suckers, Dan et al. 2021), but are settled at similar sizes (5.7–7 mm DML and 21–27 suckers per arm, Itami et al. 1963). In this work, we did not observe day/night differences during the “tactile” stage as it occurs with O. sinensis in captivity, where late paralarvae alternate diurnal clinging / crawling with nocturnal swimming. This behaviour was observed until 12.2–15 mg dry weight (~ 85–105 mg fresh weight, considering that dry weight is around 7 times lower), when the early juveniles stop swimming and exhibit strong negative phototaxis and reclusive behaviour, both characteristics of the settlement stage defined herein.

Fresh weight at the pre-settlement stage was the better descriptor to predict survival through the settlement phase. Fresh weight is easy to sample, it does not require anaesthesia, and minimise the handling stress caused to the octopus. Counting suckers and measuring DML in alive individuals requires anaesthesia and may not be accurate because the end of the arms is coiled and get attached to the crystal, thus complicating the sampling and increasing the handling stress. Our observations suggest that animals > 110–120 mg are better prepared for surviving the major changes occurring during settlement, irrespective of age. Octopuses that display pre-settlement reflexes at weights < 100 mg have fewer chances to survive the settlement stage, where they need to hunt in the bottom. This new hunting field, completely different from the water column, requires a new set of skills that involves the development of certain brain lobes to coordinate the movements of arms and suckers (Nixon and Mangold 1996). Until these neural networks are developed, the early juveniles are not very skilled benthic hunters and too heavy to be swimming and so, the energy expenditure is high and resulted in ~ 10–15% weight loss and lower growth rates during the pre-settlement and settlement stages, which coincides with the observations made by Villanueva (1995).

Obvious morphological changes during growth are characterised by discontinuities in relative growth that highlight crucial limits in stages of development, and the first discontinuity seems to coincide with the transition from paralarva to subadult (Young and Harman 1988). These changes in growth have been documented in squid families including changes in body proportions in the Cranchiidae (Voss 1980) and changes in chromatophore patterns in the Onychoteuthidae (Young and Harman 1988), but also in octopods. This non-growth phase has also been observed in O. maya juveniles during the first 10 days post-hatching (Moguel et al. 2010). Despite being an holobenthic species with direct development and no planktonic phase, early O. maya go through a transition period characterized by changes in morphology, physiology and behaviour named “post-hatching” phase where they adapt to the benthic environment / juvenile phase. This post-hatching phase would be somehow equivalent to the pre- and settlement stages defined in this work since O. maya hatchlings show necto-benthic behaviour (the “tactile stage” defined herein also known as pre-settlement reflex or clinging) and the arms are proportionally shorter than the mantle (equivalent to ML/TL < 50% shown in this study).

Holobenthic species like O. bimaculoides and O. maya have juveniles that hatch with 70–121 mg (Forsythe and Hanlon 1988; Ibarra-García et al. 2018; Briceño et al. 2010), which is very similar to the weights recorded for the late paralarvae at the end of the planktonic phase before settlement (80–120 mg). In the first year of experiments, the average fresh weight of the surviving juveniles was 2.71 ± 0.68 g at 120 days (1.69–6.12 g; n = 8). However, in the second year an enormous dispersion was observed in the weights sampled at day 118 (average 380 mg, 100–2600 mg, n = 81). The difference in size recorded (that included specimens at the settlement and post-settlement stages and benthic juveniles) can be mostly attributed to the rearing conditions, since the first experiment was carried at 19ºC and the second at 18ºC. Temperature could be the main factor driving these differences in growth (Forsythe, 2004), as well as the tiny differences of size at hatching (Pecl et al. 2004), since small differences in initial weight are amplified through time in the same way for all individuals, (i.e. despite growing at the same rate, Briceño et al. 2010). The intrinsic variability observed in size-at-age data reflects individuals experiencing different conditions (like food availability, temperature variations), different metabolisms and or owed to their different abilities to hunt or process the ingested food (Forsythe and Hanlon 1988). In our study, the large dispersion in sizes registered in experiment 2 was the main cause of the cannibalism recorded during the settlement and benthic phases, were larger animals attacked conspecifics < 50% of their own weight. Weight management during the benthic phase is mandatory to reduce cannibalism and avoid aggressive behaviours as observed in other cultured species as O. maya (Moguel et al. 2010).

Settlement phase: a metamorphosis between planktonic and benthic lifestyles?

A wide range of marine invertebrates have a pelago-benthic lifecycle that includes planktonic larval and benthic adult phases. The transition between these morphologically and ecologically distinct phases is known as metamorphosis and typically occurs when the competent larva detects an environmental cue via species-specific sensory system (Jackson et al. 2002). The morphogenetic cues are species-specific and range from food sources, microbial films, conspecifics, or particular benthic substrata (reviewed in Zimmer and Butman 2000). This ability to discriminate and respond to signals associated with different benthic substrata apparently ensures that larvae settle in a habitat that is suitable for juvenile growth and survival. The transition from the planktonic to the benthic lifestyle in merobenthic cephalopods is a complex process rarely seen in the wild driven by poorly understood factors. Although the factors that may be acting during the settlement phase are difficult to evaluate and far from the aim of this work, it is likely that certain chemical cues present in the benthos may act as a trigger inducing the changes described during the settlement phase (Fig. 5).Such contact with an inductive environmental cue would occur during the “tactile” or pre-settlement stage, when the late planktonic paralarvae of O. vulgaris start to contact with the benthos, before transitioning from the pelagic to the benthic habitats. Essentially, the transparent planktonic paralarvae will have to turn into fully benthic juveniles that use their coloured skin and texture for camouflaging and cryptic concealment.

The morphological changes include the development of a very complex skin with innumerable chromatic components (chromatophores, iridophores/reflector cells and leucophores), associated epithelial musculature and motoneurons to control them all (Packard 1985). When Octopus vulgaris settles, the rate of chromatophore genesis into the empty dorsal mantle field rapidly overtakes the rate of recruitment into the ventral that results in higher densities of smaller chromatophores dorsally than ventrally (Fig. 3). Packard (1985) mentioned that “the dorsal spurt in chromatophore genesis at the end of the planktonic phase is so dramatic as to hint at something like metamorphosis. It is as if the skin were waiting for its owner to settle on the sea floor before bringing out the fine-grain dress that is going to serve for the rest of its life and replace the coarse-grain set of extrategumental spots (on the surface of the viscera) that served during the transparent planktonic phase”.

The anatomical changes include the distal growth of the arms and the incorporation of new suckers. The patterns of ontogenic allometry suggest that during the first 20 days, growth is concentrated in the mantle area of the paralarvae and later shifts to the arms (Villanueva 1995), resulting in a decrease of the ML/TL ratio (Figs, 1 and 5). Naef (1923) reported Kölliker organs on a benthic juvenile measuring 10 mm ML collected in the Bay of Naples. Some authors suggest that the loss of Kölliker organs might be involved in the appearance of the first dermal papillae (Naef 1923), while other suggest that they may form the generative centres of the patches in the skin (Packard and Hochberg 1977). From our experience, we agree with Packard 1988 that these organs are adaptations for the pelagic environment, which may act as a defence since the bristles of these organs resembles the tiny spines of a cactus. There are far more Kölliker organs covering the skin of the planktonic paralarvae (Fig. 2a) than dermal papillae in juveniles and adults. Moreover, as the paralarvae start the settlement stage the number of Kölliker organs clearly diminishes and the few ones left are embedded by the new skin (as shown in Fig. 2c), and totally disappear from the surface of the benthic juveniles. This also suggest that these organs may not be involved in the creation of papillae or the patches in the skin, but more studies are needed to contrast it. The loss of these organs during the settlement phase would reinforce the idea that O. vulgaris experience a metamorphosis, since some anatomical features of the planktonic paralarva disappear during settlement.

In the intersection between morphological/anatomical and behavioural changes is the development of the nervous system, both central and peripherical, responsible for the new set of skills (e.g. crawling, camouflaging and body patterning) that “appear” throughout the settlement phase. Skin patterning result from neural control of radial muscles that expand the pigment sac of each chromatophores (Hanlon 1999). At the pre-settlement and settlement stages there is no specific patterning, and they can only shift from transparent to dark red (Fig. 4a), as it occurs during the planktonic phase. Specific patterning like the eye-bar (Fig. 4b, c) results from the selective neural excitation of groups of chromatophores, and it happens from the post-settlement stage into the benthic phase, when the skin has developed numerous chromatophores and their corresponding nervous connections. Concurrently, the new musculature developing in the skin enables settled octopuses to create texture in the skin, like the cirrha or dorsal papillae (Fig. 4c). These new sets of skills are reflected in the development of specific areas of the brain like the brachial, vertical, subvertical, subfrontal and optical lobes (Nixon and Mangold 1996). Furthermore, the brachial lobes are also involved in the control of the new suckers and their new functions like prey search, olfaction and tactile responses (Nixon and Mangold 1996). The development of the brachial lobes is responsible for the coordinated movements needed for crawling during the settlement and the post-settlement stages that are characterized by “ninja” movements, which are very fast and precise movements that require complex neural control. Complicated body patterns of juvenile octopus like the “alga” or the “stone” (Fig. 5) result from the interaction of chromatic, textural, postural and locomotor responses that seem very basic at the end of the post-settlement stage and become very precise at the beginning of the benthic stage.

Summarizing, the settlement phase in O. vulgaris does not imply a major modification of the body plan like in other molluscs (e.g. gastropods, bivalves), but requires profound modifications at morphological, anatomical and behavioural levels that, altogether, could be considered as a metamorphosis. Independently of being considered as a true metamorphosis or a “para-metamorphosis” (in line with the discrimination made by Young and Harman, 1988 to separate the terms “larva” and “paralarva”), the settlement phase can be divided in three different stages based on the modifications schematically shown in Fig. 5, which prepare the young octopods for their benthic lifestyle. Further research is needed to determine the environmental cues that drive this pelago-benthic transition, as well as the genetic and physiological basis of the changes observed.

Compliance with Ethical Standards

This study was financed by Armadora Pereira S.A. and CDTI funding to develop the projects AQUOPUS (IDI-20170704, 2017-2019, Instituto de Investigaciones Marinas de Vigo, IIM-CSIC) and OCTOBLUE (IDI-20190479, 2019-2021, ECIMAT, Universidad de Vigo). This work was carried with the aim to increase the survival and enhance the zootechnical aspects of the culture of O. vulgaris in captivity and, therefore, experiments do not fall under Directive 2010/63/EU of the European Parliament and of the Council, of 22 September 2010 on the protection of animals used for scientific purposes. Nonetheless, the experimental procedures (culture density, prey density, water quality, tank designs) were supervised and approved by an ethics committee at the different institutions (IIM for AQUOPUS and University of Vigo for OCTOBLUE projects).

Acknowledgements

We thank Dr. Ángel González for scientific assistance during both projects and the technical assistance of Lourdes Nieto, Rubén Chamorro and Xoán Martínez from IIM-CSIC. We are grateful to Damián Costas, Noelia Costoya, Arantxa Martínez, Victoriano Álvarez, Micael Vidal, Alberto García, Roberto Gómez, Enrique Poza, Rosana Rodríguez, Sergio González, Juán José Rodríguez, María Pérez and Alberto Álvarez for their technical assistance at ECIMAT. We would also like to thank Dr. Jesús Troncoso (Universidad de Vigo) and Dr. Pablo Sánchez (Universidad de Alicante). Special thanks to José Irisarri and Jade Irisarri for their underwater support.

Funding: This study was financed by Armadora Pereira S.A. and CDTI funding to develop the projects AQUOPUS (IDI-20170704, 2017-2019) and OCTOBLUE (IDI-20190479, 2019-2021).

Competing interest: The authors have no relevant financial or non-financial interests to disclose.

Author contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Alvaro Roura and Alexandra Castro. The first draft of the manuscript was written by Alvaro Roura and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability: The datasets generated are available on the supplementary table.

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The settlement phase in the common octopus Octopus vulgaris: a complex transition between planktonic and benthic lifestyles

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Settlement phase: a metamorphosis between planktonic and benthic lifestyles?

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