MORPHOMETRIC CHANGES AND GROWTH RATE DURING EMBRYONIC DEVELOPMENT OF ROBSONELLA FONTANIANA I. Uriarte, O. Zuñiga, A. Olivares, V. Espinoza, V. Cerna, A. Farías, C. Rosas

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I. Uriarte, O. Zuñiga, A. Olivares, V. Espinoza, V. Cerna, et al.. MORPHOMETRIC CHANGES AND GROWTH RATE DURING EMBRYONIC DEVELOPMENT OF ROBSONELLA FONTANIANA. Vie et Milieu / Life & Environment, Observatoire Océanologique - Laboratoire Arago, 2009, pp.315- 323. ￿hal-03253776￿

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MORPHOMETRIC CHANGES AND GROWTH RATE DURING EMBRYONIC DEVELOPMENT OF ROBSONELLA FONTANIANA

I. URIARTE 1,2*, O. ZUÑIGA 3, A. OLIVARES 3, V. ESPINOZA 1, V. CERNA 1, A. FARÍAS 1,2, C. ROSAS 4 1 Instituto de Acuicultura, Universidad Austral de Chile, PO Box 1327, Puerto Montt, Chile 2 CIEN Austral, Puerto Montt, Chile 3 Facultad de Recursos del Mar, Universidad Antofagasta, Antofagasta, Chile 4 Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Sisal, Yucatán, Mexico * Corresponding author: [email protected]

EGGS ABSTRACT. – During paralarval development of the octopus Robsonella fontaniana, the man- EMBRYOS YOLK tle size is reduced in proportion to the total octopus length, whereas the organs more directly PERIVITELLINE PROTEIN involved in catching prey tend to increase in direct proportion to the total length. A morphomet- MORPHOMETRY ric and gravimetric study of R. fontaniana embryos was made in order to determine whether the OCTOPODS morphometric trends and exponential growth rates observed in the planktonic and benthic phas- ROBSONELLA FONTANIANA es begin during the embryonic development. Seven clutches, sampled during different seasons of the year, were studied under controlled conditions until hatching of the planktonic paralarvae. Yolk reserves and protein content in the perivitelline liquid were quantified to determine how the yolk absorption and the consumption of perivitelline protein are modulated during embry- onic development. Results demonstrate that during embryonic development the growth rate for total length, mantle length, and arm length is exponential, indicating that the type of growth described for paralarvae and juveniles begins during embryonic life. The morphometric changes and growth are related to an exponential reduction of egg yolk and a linear consumption of perivitelline protein.

INTRODUCTION one of the major factors influencing the initial feeding conditions of the hatchlings. Thus, evaluations of embry- Robsonella fontaniana (D’Orbigny, 1834) is a small- onic yolk reserves could constitute the basis for a quality sized pygmy octopus found off southern Chile and Argen- criterion for hatchlings. Such information could be useful tina. Some reproductive studies of R. fontaniana have for both aquaculture and fisheries management. shown that this species can easily spawn up to 2500 eggs Variations in individual growth influence many facets (Briceño-Jacques 2004, González et al. 2008). Morpho- of a cephalopod population such as its size and age struc- metric and gravimetric changes have also been described ture, reproductive dynamics, and hatchling survival rate. for R. fontaniana paralarvae and juveniles up to 160 days Ontogenetic changes in size and shape influence an ani- after hatching (Uriarte et al. 2009). Another study consid- mal’s locomotion as it grows. Villanueva et al. (1996) ered the effects of temperature and feeding on paralarvae observed strong morphometric changes in O. vulgaris (González et al. 2008) and the effect of diet on digestive paralarvae and juveniles that dramatically influenced their enzyme modulation during the paralarval phase (Pereda swimming capacities and, at the same time, their predato- et al. 2009). Given the advanced knowledge of the spe- ry abilities. However, no similar studies indicate how cies’ biological aspects, R. fontaniana could be used as a embryonic growth determines the exponential growth reference model for rearing other cephalopod species with curves observed in the first part of the cephalopod life planktonic phases (Gonzalez et al. 2008, Uriarte et al. cycle (Mangold 1983). In a previous study, Uriarte et al. 2008, 2009, Pereda et al. 2009). (2009) observed drastic morphometric changes in R. fon- Several authors have suggested that the transition from taniana related to the logarithmic growth of the mantle yolk utilization to active predation is a critical period in with respect to the total length, as compared to the organs the early life history of cephalopods that depends on the related directly to catching prey (e.g., arm length and eye embryonic yolk reserves (e.g. Boyle & Chevis 1992, Han- diameter), which grow linearly with respect to total lon & Wolterding 1989, Naef 1928, Vecchione & Hand length. Herein, we provide morphometric and gravimetric 1989, Vidal & Haimovici 1998), similar to the critical analyses of R. fontaniana embryos in order to determine period concept described for octopus paralarvae (Villan- whether the exponential growth curves observed in the ueva & Norman 2008). According to Boletzky (2003), the paralarvae during the planktonic phase begin during the volume of yolk remaining in the inner sac at hatching is embryonic phase of development. Yolk reserves and pro- 316 I. URIARTE, O. ZUÑIGA, A. OLIVARES, V. ESPINOZA, V. CERNA, A. FARÍAS, C. ROSAS tein content in the perivitelline liquid were quantified in mic: Y = a + b*ln(X), or allometric: Y = a*Xb; where Y is the an attempt to determine how the yolk absorption and the dependent variable and X is the independent variable). consumption of perivitelline protein are modulated dur- ing embryonic development and how they relate with embryonic growth. 2. Gravimetric analyses and quantification of the perivitell- ine protein. Seven clutches and their respective females were MATERIAL AND METHODS collected from their natural environment during the year 2007- 2008. Each clutch consisted of approximately 1700 eggs clus- R. fontaniana females with eggs were collected tered in strands of 40 eggs. Two samples of 30 eggs were taken between November 2007 and November 2008 at Hueihe at random from each of the seven clutches, every 30 days, dur- (41°52´S; 73°51´W), Region X, Chile. The octopuses and ing embryonic development prior to hatching; thus, a total of their spawn (attached to stones) were taken to the Marine 1220 eggs were sampled. These eggs were measured individu- Invertebrate Hatchery Laboratory of the Universidad Aus- ally for length with a caliper (± 0.1 mm) and weighed on a Sar- tral de Chile (HIM-UACH) in 70-L tanks of aerated sea torius analytical scale (± 0.0001 g). We calculated the age of the water. Once in the laboratory, the animals and stones with eggs from each clutch after hatching based on a 74-day-long eggs were placed in individual aerated tanks of sea water embryonic period; this value was empirically obtained from kept at 12°C and 30 psu salinity; the tanks were connected eggs laid in our laboratory by a gravid female and reared at 12ºC to a sea water re-circulation system. The animals were under maternal care. Each sampled egg was punctured and its placed in a semi-dark environment with a 12:12 h light: contents were emptied into a previously weighed Eppendorf dark photoperiod and were fed daily with white fish tube. Once emptied, the 30 egg contents were centrifuged at (Odontestes sp.) to keep them from eating their spawn. 1000 rpm at 4ºC for 10 min, after which the supernatant corre- Throughout embryonic development, the females sat on sponding to the perivitelline liquid of the 30 eggs was trans- and cared for the spawn. ferred to a new, previously weighed Eppendorf tube, whereas the precipitate corresponding to the yolk plus the embryo was 1. Morphometric analyses and yolk quantification on the left in the original tube. Two aliquots (10 µl each) were taken embryonic stages. We took samples (four eggs per day) from a from the perivitelline liquid and the soluble protein content was recent clutch using long forceps. These eggs were photographed, measured with a portable refractometer (Quimis, model Q-767- allowing us to study the embryonic anatomy over a period of 70 5 Serum Protein). Later, the perivitelline liquid and the days. A total of 320 eggs and their corresponding embryos were yolk+embryo were freeze-dried in a SAVANT freeze-dryer measured and photographed using a light microscope (Stemi (model Novalyphe-150) and weighed to determine their dry 2000-C) coupled to an AxioCam (ICc3; Zeiss) camera. The weight. We evaluated the growth rates of the eggs between the stages of embryonic development are defined according to Naef 30-day periods and related the change in perivitelline liquid pro- (1928) and the photos were used to determine morphometric tein concentrations to the egg size and age. relationships. For this, we measured egg length (EL), egg weight (EW), embryo total length (ETL), mantle length (ML), arm length (AL), and eye diameter (ED) during the same 70-day RESULTS period (until hatching). The yolk sac volume was estimated by superimposing standard geometric forms onto the shape of the Morphological relationships during embryonic yolk sac. The anterior yolk sac, also called the outer yolk sac, development was measured by superimposing ellipsoid, cylindrical, or spher- ical forms during embryonic development. The formulas used to Immediately after being laid (day 0), the central axis of determine the anterior yolk sac volume were: the clutch was visible and the chorionic filaments of each i) Cylindrical volume (CV) = π r2 H egg were wrapped around this (Fig.1A). In stage I, the ii) Spherical volume (SV) = 4/3 π r3 cleavage of the egg occurred at the animal pole where where r is the ratio of the cylinder base or the ratio of the sphere, there was no yolk, thus forming a discoblastula. The rest π is 3.1416, and H is the cylinder length. Yolk volumes were of the cytoplasm did not divide (day 8). The polar bodies multiplied by a density of 1.036 mg mm-3 to convert them to wet were found in the perivitelline space adjacent to the bor- weights (Vidal et al. 2002). der of the discoblastula (Fig. 1B). In stage VI, the rotary An exponential equation was used to relate EL, ETL, AL, movement of the gastrula was evident (day 15). The blas- ML, and ED with octopus age (t): toderm cells had migrated to cover more than 50 % of the morphological characteristic (mm) = a * ebt yolk surface (Fig. 1C). Once in stage IX (day 22), the where e is the base of the natural logarithm, the constants a and embryo had rotated 180º, with its cephalic part at the base b are the intercept and slope, and t is the age in days during of the egg. The vitelline and the cephalic portions of the embryonic development. Other models were also adjusted to the embryo could clearly be distinguished. In the latter por- morphological data as required (linear: Y = bX + a, or logarith- tion, the formation of the mantle could be observed, along

Vie Milieu, 2009, 59 (3-4) GROWTH AND DEVELOPMENT OF ROBSONELLA FONTANIANA EMBRYOS 317

Fig. 1. – Embryo development of R. fontaniana. A, Egg cluster at day 0; ca is central axis, chf are chorionic filaments. B, Stage I at day 8; d is discoblastula, pb are the polar bodies. C, Stage VI at day 15; bc are blastoderm cells and y is yolk surface. D, Stage IX at day 22; vp is vitelline portion, cp is cephalic portion, m is mantle, and r is retina. E, Stage XI at day 26; vs is vitelline sack, ra are rudimentary arms, e is eye, and m is mantle. F, Stage XV at day 50; chr are chromatophores, ivs is internal yolk sac and evs is external yolk sac. G, Stage XIX at day 61; evs is external yolk sac, chr a are arm chromatophores, and chr m are mantle chromatophores. H, Stage XX at day 71, ecd is a complete developed embryo, and ee is an empty egg.

Vie Milieu, 2009, 59 (3-4) 318 I. URIARTE, O. ZUÑIGA, A. OLIVARES, V. ESPINOZA, V. CERNA, A. FARÍAS, C. ROSAS

Fig. 2. – R. fontaniana clutch selected for morphometric analy- ses: relationships observed in 88 eggs between A, increased total egg length (EL) and egg age, B, increased egg weight (EW) and egg age, C, egg weight (EW) and egg length (EL), all for the entire period between laying and hatching. R2 is the coefficient of determination and P indicates the significance of the coeffi- cient of regression. with the palleo visceral complex and the optical complex with the pigmentation of the retina (Fig. 1D). Later, in stage XI (day 26), the vitelline sac and the body of the embryo were clearly distinct. The rudimentary arms, the eye over the portion of the cranial complex, and the man- Fig. 3. – R. fontaniana clutch selected for morphometric analy- tle were easily identified (Fig. 1E). The first chromato- ses: relationships observed between different embryonic organs phores, both in the ventral and dorsal portions of the and embryo age: A, increase in total embryo length (ETL); B, embryo, were formed in stage XV (day 50). At this point, increase in mantle length (ML); C, increase in arm length (AL); and D, increase in eye diameter (ED). R2 is the coefficient of the internal yolk sac was also organized in relation to the determination and P indicates the significance of the coefficient displacement of the external yolk (Fig. 1F). In stage XIX, of regression. the embryo had turned for the second time (day 61). The outer yolk sac was small and some content could remain between the arms, each of which had four chromato- guished on the back of the mantle (Fig. 1G). Once the phores. Eight rows of chromatophores could be distin- embryo reached stage XX, it had completed its develop-

Vie Milieu, 2009, 59 (3-4) GROWTH AND DEVELOPMENT OF ROBSONELLA FONTANIANA EMBRYOS 319

Fig. 4. – R. fontaniana clutch selected for morphometric analy- ses. Decrease in external egg yolk (EY) throughout the period of embryonic development. R2 is the coefficient of determination and P indicates the significance of the coefficient of regression.

ment (day 71). It no longer had an external yolk sac and it was ready to hatch (Fig. 1H). The length of the egg (EL) and its weight (EW) increased exponentially throughout the embryonic devel- opment (Fig. 2A, 2B). The weight of the egg was related allometrically to its length, with a slope of 2.41 (Fig. 2C). An exponential relationship was obtained between embryo total length (ETL) and age (Fig. 3A). Similarly, exponential relationships were observed between ML and age and AL and age (Fig. 3B, 3C). Eye diameter showed a logarithmic curve with age; the maximum eye diameter for embryos was around 0.45 mm (Fig. 3D). The yolk volume decreased exponentially during embryonic development (Fig. 4) such that, a few days prior to hatching, only 10 % of the external yolk remained available. The length of the embryo was related exponentially to egg length (Fig. 5A). Both the mantle length (ML) and the arm length (AL) were related exponentially to the embryo length (Figs. 5B, 5C). The AL/ML ratio also showed a linear relationship with the total length of the embryo (Fig. 5D). ML, as a proportion of ETL, increased with age between day 43 and day 54, reaching 76 to 92 % of ETL. Subsequently, the ML proportion declined and then remained at 73 % (days 57 to 64). Prior to hatching (day 69), ML was reduced to 67 % of ETL; this proportion was maintained until hatching (Fig. 6A). In contrast, arm length (AL) as a proportion of ETL decreased with age; values were high on day 43 (23 % of ETL) and low from Fig. 5. – R. fontaniana clutch selected for morphometric analy- days 50 to 57 (on average, 16 % of ETL). After day 57, ses: relationship between A, embryo total length (ETL) and the AL in proportion to ETL returned to its initial value total egg length (EL); B, mantle length (ML) and embryo total length (ETL); C, arm length (AL) and embryo total length (around 23 %), remaining stable until hatching (Fig. 6B). (ETL); and D, ratio arm length/mantle length (AL/ML) and embryo total length (ETL). R2 is the coefficient of determination Gravimetric characterization and P indicates the significance of the coefficient of regression.

The eggs varied in size between the seven clutches observed and between the developmental stages within a this implied a variation between 2.9 and 8.0 mg. These single clutch, ranging from 2.4 to 4.7 mm. In wet weight, two variables were related through an allometric equation

Vie Milieu, 2009, 59 (3-4) 320 I. URIARTE, O. ZUÑIGA, A. OLIVARES, V. ESPINOZA, V. CERNA, A. FARÍAS, C. ROSAS

Fig. 7. – Decrease in perivitelline protein (PP) in relation to egg length (EL) for seven R. fontaniana clutches. Each point is an average of perivitellin protein (2 to 7 samples) per egg sizes between 2.8 and 4.3 mm. R2 is the coefficient of determination and P indicates the significance of the coefficient of regression.

Fig. 6. – R. fontaniana clutch selected for morphometric analy- ses: A, variations in mantle length as a proportion of embryo total length (ML/ETL); and B, variations in the arm length as a proportion of embryo total length (AL/ETL), both throughout embryonic development. Different letters indicate significant differences in the ratio ML/ETL or AL/ETL between different embryo ages (Tukey HSD test; P < 0.05). Fig. 8. – Variations in perivitelline protein (PP) as a function of the days of embryonic development for five R. fontaniana clutches of known ages. n = 2 samples per clutch at two differ- with a slope of 1.53. The eggs had specific growth rates 2 -1 ent ages. R is the coefficient of determination and P indicates of 0.0035 day (± 0.0011; n = 5) for length and of 0.0096 the significance of the coefficient of regression. day-1 (± 0.0021; n = 5) for wet weight, with no significant differences between clutches. The protein content in the perivitelline liquid declined exponentially from 0.19 to embryological studies of pre-hatching and hatched indi- 0.04 mg protein µL-1 (Fig. 7) with increased egg size. On viduals; these studies were reviewed by Boletzky (1977, the other hand, the protein content in the perivitelline liq- 1989, 1994). However, to date, little of the data available uid decreased linearly (Fig. 8) with the increasing age of has been used in an attempt to describe the allometric the embryos. changes occurring during embryonic development in cephalopods. During organogenesis, the cephalic organs (central DISCUSSION nervous system and eyes) are the first conspicuous organs to appear in embryos because they are largely formed In this study, we have added a new growth phase in from ectodermic layers (Boletzky 2003). In the present the life cycle of cephalopods – embryonic development – study, cephalic organs were observed after day 22 (Stage during which the growth rate for total length, mantle IX, Fig. 1D) and well-defined eyes after day 26 (Stage XI, length, and arm length is also exponential, indicating that Fig. 1E). Our results for eye diameter indicate that, during the type of growth described for paralarvae and juveniles embryonic development, the eyes grow following a loga- begins during embryonic development. rithmic curve, suggesting that the cephalic organs form The growth curve described for cephalopods is char- from ectodermic invaginations and grow rapidly at the acterized by an initial exponential-type phase that lasts beginning of embryonic development until organogenesis from the planktonic stage (where it exists) to the juvenile is completed. After that, during the planktonic post-hatch- phase. After this, the growth is power-type until attain- ing stage, the growth of the cephalic organs follows an ment sexual maturity (Van Heukelem 1973, Mangold exponential curve (Uriarte et al. 2009). This suggests that 1983, Semmens et al. 2004, Leporati et al. 2007). Most the cephalic organs, once completed, follow a growth rate morphometric information on embryos comes from similar to that of the arms or mantle. According to Villan-

Vie Milieu, 2009, 59 (3-4) GROWTH AND DEVELOPMENT OF ROBSONELLA FONTANIANA EMBRYOS 321 ueva & Norman (2008), during paralarval stages, the pro- The spring, summer, and autumn clutches did not differ portion of the octopus mantle is greater than that of the significantly in their growth parameters during embryonic arms. This shows that, during the first planktonic stages development, which was done under standardized condi- of growth, the organs located in the mantle cavity are tions (12 ºC), so that the clutch selected for the first more important than the arms. Results from the present description was representative of the development of this study demonstrate that, although arms appear during early species. embryonic development (Boletzky 1984), the organs The entire R. fontaniana embryonic development took located in the mantle cavity determine the form in which 71 to 76 days, until the paralarvae hatched. The clutch the embryonic morphology is modulated. In fact, similar selected for the description contained recently laid and equations were obtained for the relationship between fertilized eggs, allowing us to describe the stages from the embryo total length vs. age (ETL = 0.38 e0.03 Age) and man- onset of segmentation (stage I) through the completion of tle length vs. age (ML = 0.30 e0.03 Age), indicating that, dur- development (stage XX). The embryos rotated twice: ing embryonic development, the total length is strongly once at 19 days (stage IX) and again at 61 days (stage influenced by mantle growth. According to Boletzky XIX). This is similar to observations for other octopuses (2003), once the organogenesis of the cephalic organs (e.g. Ignatius & Srinivasan 2006; Kaneko et al. 2006). starts, dramatic changes occur in the embryonic body, Such embryonic rotations in octopods correspond to dif- resulting in an increasingly compact arrangement of the ferent phenomena. The first rotation, once dubbed blas- organs, which take up their definitive position in the tokinesis, is due to cilia on the external part of the yolk developing animal. An exponential increment in oxygen sac that while changing direction of beat cause the embryo consumption by Sepia apama embryos could offer evi- to rotate around its longitudinal axis. The second rotation, dence of strong energy mobilization occurring during the which occurs at the end of the embryonic development, exponential growth curve of this developmental stage compensates for this earlier inversion and grants the (Cronin & Seymour 2000). embryo a better position for hatching (Boletzky 1971, In the present study, during embryonic development, Lenz 1997). the proportion of R. fontaniana mantle and arms changed In the present work, the hatching period for R. fontani- with age, with early mantle cavity growth followed by ana lasted an average of 4 to 5 days at 12 ºC. The hatch- later arm growth. In fact, the mantle cavity reached nearly ing period for a single clutch of eggs can be short (i.e., 95 % of the total embryonic length at day 54, indicating hours) or may last a few days or a period of weeks. It can that, during that development time, the energy was chan- be influenced by factors such as the length of the incuba- neled almost exclusively to organogenesis. In contrast, tion period of the eggs, the incubation temperature, and the proportion of the arms during embryonic development the species (Villanueva & Norman 2008). O. luteus hatch- was between 15 and 24 %, peaking just before hatching. es during 10 days at 18.2 to 20 ºC (Arakawa, 1962) and At this time, it seems that the post-hatching planktonic from 4 to 7 days at 21 to 27 ºC (Caveriviere et al. 1999), stage moved on to the second phase of development, in whereas O. vulgaris needs 9 days at 17 to 19 ºC (Iglesias which the arms were the priority before settlement (Uri- et al. 2004). For O. laqueus, on the other hand, 75 % of arte et al. 2009). the individuals that hatch do so within one hour on the R. fontaniana egg sizes fluctuated between 3.13 and same day (Kaneko et al. 2006). 3.77 mm length in the clutch used for the descriptions and The egg yolk was reduced exponentially during embry- between 2.4 and 4.7 mm (3.5 and 6.8 % of adult ML, onic development. Interestingly, the slope of these curves respectively) in the seven clutches used for gravimetric was negative (- 0.03), as compared to the slopes for the characterization (N = 1220). These eggs are considered to embryo total length and mantle length curves (0.03). This be small-type eggs (Boletzky 1977, Boletzky et al. 2002), indicates that the decreasing velocity of yolk consump- comparable with those of the species O. bimaculatus tion was a direct consequence of the embryonic growth. (Hochberg et al. 1992) and O. mimus (Zuñiga et al. 1997); The linear consumption of perivitelline protein during they fall within the range reported by Boletzky et al. embryonic development could indicate that this protein is (2002) for merobenthic octopodids (smaller than 10 % of being used in morphogenesis. For gastropods, the perivi- adult ML). Variations in egg size can be explained by telline fluid is consumed by the developing embryo as an water uptake through the corion, the growth in length and energetic and structural source because it is mainly com- weight throughout embryonic development, and differ- posed of lipoprotein (Garin et al. 1996), principally a ences between clutches. Nevertheless, the influence of lipoglycocarotenoprotein protecting the egg from oxida- environmental factors and other characteristics of the tion (Dreon et al. 2004). The use of the protein in the females (age, size) were not measured in this work. The perivitelline fluid has not been documented previously in range of growth rates observed for the eggs from different octopuses. clutches, both in size and in weight, perfectly encompass- According to those results, the use of yolk can be con- es the growth observed in the eggs of the only clutch used sidered as a source of energy for octopus development, for the morphological and morphometric descriptions. and probably yolk and perivitelline protein contribute

Vie Milieu, 2009, 59 (3-4) 322 I. URIARTE, O. ZUÑIGA, A. OLIVARES, V. ESPINOZA, V. CERNA, A. FARÍAS, C. ROSAS with protein and phospholipid for new cells during mor- ments to J Hernández & R Miranda, who provided technical phogenesis. So, the yolk and the protein in the perivitell- support. ine fluid could be used to monitor environmental effects or the egg quality on cephalopod clutches. There is an inverse relationship between temperature REFERENCES and length of embryonic development in marine poikilo- therms, so when cephalopods are distributed at lower Arakawa KY 1962. An ecological account on the breeding temperatures, their development times should be greater behaviour of Octopus luteus (Sasaki). Venus 22: 176-180. (Boletzky 1994, Johnston 1990, Naef 1928, Rocha et al Boletzky Sv 1971. Rotation and first reversion in the Octopus 2001). For tropical species, reports show development embryo: a case of gradual reversal of ciliary beat. 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