Influence of starvation and feeding on the hepatopancreas of larvalHyas araneus (Decapoda,Majidae)

Zoea-1 larvae ofHyas araneus were kept under different nutritional conditions. Their midgut glands were investigated with a transmission electron microscope. The glandular epithelium consists of the cell types known from adult decapods. It is mainly the R-cell type that undergoes ultrastructural alterations which reflect nutritional conditions. R-cells of fed larvae are characterized by large lipid inclusions; after a certain period of food deprivation (point-of-no-return) the original ultrastructure cannot be reestablished. Refeeding results in large glycogen deposits in these cells.     

Growth patterns,chemical composition and oxygen consumption in early juvenileHyas araneus (Decapoda: Majidae) reared in the laboratory

Early (instar I and II) juveniles of the spider crabHyas araneus were reared under constant conditions (12 °C, 32‰S) in the laboratory, and their growth, biochemical composition, and respiration were studied. Every second day, dry weight (W), ash-free dry weight (AFW), and contents of ash, organic and inorganic carbon (C), nitrogen (N), hydrogen (H), protein, chitin, lipid, and carbohydrates were measured, as well as oxygen consumption. Changes in the absolute amounts of W. AFW, and C, N, and H during the moulting cycle are described with various regression equations as functions of age within a given instar. These patterns of growth differ in part from those that have been observed during previous studies in larval stages of the same and some other decapod species, possibly indicating different growth strategies in larvae and juveniles. There were clear periodic changes in ash (% of W) and inorganic C (as % of total C), with initially very low and then steeply increasing values in postmoult, a maximum in intermoult, and decreasing figures during the premoult phase of each moulting cycle. Similar patterns were observed in the chitin fraction, reaching a maximum of 16% of W (31% of AFW). Ash, inorganic C, and chitin represent the major components of the exoskeleton and hence, changes in their amounts are associated with the formation and loss of cuticle material. Consequently, a high percentage of mineral matter was lost with the exuvia (76% of the late premoult [LPM] ash content, 74% of inorganic C), but relatively small fractions of LPM organic matter (15% of AFW, 11% of organic C, 5–6% of N and H). These cyclic changes in the cuticle caused an inverse pattern of variation in the percentage values (% of W) of AFW, organic C, N, H, and biochemical constituents other than chitin. When these measures of living biomass were related to, exclusively, the organic body fraction (AFM), much less variation was found during individual moulting cycles, with values of about 43–52% in organic C, 9–10% in N, 6–9% H, 31–49% of AFW in protein, 3–10% in lipid, and <1% in carbohydrates. All these constituents showed, on the average, a decreasing tendency during the first two crab instars, whereas N remained fairly constant. It cannot be explained at present, what other elements and biochemical compounds, respectively, might replace these decreasing components of AFW. Decreasing tendencies during juvenile growth were observed also in the organic C/N and in the lipid/protein weight ratios, both indicating that the proportion of lipid decreased at a higher rate than that of protein. Changes were observed also in the composition of inorganic matter, with significantly lower inorganic C in early postmoult (2–4% of ash) than in later stages of the moult cycle (about 9%). This reflected probably an increase in the degree of calcification, i.e. in the calcium carbonate content of the exoskeleton. As a fraction of total C, inorganic C reached maximum values of 17 and 20% in the crab I and II instars, respectively. The energy content of juvenile spider crabs was estimated independently from organic C and biochemical constituents, with a significant correlation between these values. However, the former estimates of energy were, on the average, significantly lower than the latter (slope of the regression ≠1). Since organic C should be a reliable integrator of organic substances, but the sum of protein, lipid, chitin, and carbohydrates amounted to only 60–91% of AFW, it is concluded that the observed discrepancy between these two estimates of energy was caused by energy from biochemical constituents that had not been determined in our analyses. Thus, energy values obtained from these biochemical fractions alone may underestimate the actual amount of organic matter and energy. Respiration per individual in juvenile spider crabs was higher than that in larval stages of the same species (previous studies), but their W-specific values of oxygen consumption (QO₂) were lower than in conspecific larvae (0.6–2μg O₂·[mg W]⁻¹). QO₂ showed a consistent periodic pattern in relation to the moult cycle: maximum values in early postmoult, followed by a rapid decrease, and constant values in the intermoult and premoult phases. This variation is interpreted as an effect mainly of cyclic changes in the amounts of cuticle materials which are metabolically inactive. From growth and respiration values (both expressed in units of organic C), net growth efficiency, K₂, values may be calculated. In contrast to previous findings in larval stages, K₂ showed an increasing trend during growth of the first two juvenile instars ofH. araneus.     

Respiration and growth of Hyas araneus L. Larvae (Decapoda:Majidae) from hatching to metamorphosis

Rates of respiration and growth were measured for larvae of the spider crab Hyas araneus L., reared in the laboratory from hatching to metamorphosis. The moulting cycle was simultaneously monitored. In both zoeal instars individual respiration rate (R) increased as a linear function of time (t) of development, whereas growth, measured as dry weight (W), carbon (C), nitrogen (N), hydrogen (H), and energy content (E, calculated from C) followed a power function of t. Weight-specific respiration rate (QO₂) was in all instars maximum in early postmoult, and minimum in intermoult and early premoult. Zoea II and megalopa instars showed another conspicuous QO₂ increase during late premoult. Respiration (both R and QO₂)and growth of the megalopa could be described by non-linear (quadratic) functions of t. R and QO₂ during this larval stage were not correlated with W, but were controlled by events of the moulting cycle: R followed a similar pattern to QO₂ (minimum values in intermoult), whereas biomass of the megalopa changed conversely, with a maximum in intermoult and early premoult. The respiratory coefficient (i.e. the ratio of metabolic energy loss: energy gain by body growth) was far lower (<0.8) in the zoeal instars than in the megalopa (>5), suggesting a strongly reduced capability of energy conversion in the final larval stage of H. araneus.     

Developmental patterns of larval growth in the edible spider crab, Maja brachydactyla (Decapoda: Majidae)

The large edible spider crab Maja brachydactyla Balss, 1922, an overexploited coastal fishery resource in Galicia (NW Spain), is considered as a potential aquaculture candidate. Patterns of its larval growth were studied under controlled laboratory conditions (constant 18 ± 1 °C; 36‰ salinity; photoperiod ca. 12:12 h; lipid-enriched Artemia metanauplii provided as food). From hatching through complete larval development and metamorphosis to the first juvenile crab instar, changes in carapace size, dry weight (DW), ash content, elemental composition (carbon, hydrogen, nitrogen; CHN), and proximate biochemical composition (total proteins, lipids, carbohydrates; Pr, L, Ch) were measured in successive stages (zoea I, II, megalopa, crab I). Body size may be described as a linear function of the number of molting cycles, whereas the amounts of DW, CHN, Pr, L, and Ch per individual increased exponentially (3 to 9 fold). The highest growth rates were observed in L, C and H, the lowest in DW, Pr and N. As a consequence of these patterns, the C:N mass ratio as well as the fractions of L, C and H (in % of DW) increased significantly, while those of Pr and N decreased from 26% to 16% of DW. Throughout development, however, Pr remained the principal biochemical component of total DW. Positive correlations between biochemical and CHN data allow for estimates of Pr from N and of L from C values per individual. The patterns of larval growth observed in M. brachydactyla are, in general, similar to those previously described for other brachyuran crabs with a planktotrophic mode of larval development.     

A developmental study of serotonin-immunoreactive neurons in the larval central nervous system of the spider crabHyas araneus (Decapoda,Brachyura)

Larval development in crabs is characterized by a striking double metamorphosis in the course of which the animals change from a pelagic to a benthic life style. The larval central nervous system has to provide an adequate behavioural repertoire during this transition. Thus, processes of neuronal reorganization and refinement of the early larval nervous system could be expected to occur in the metamorphosing animal. In order to follow identified sets of neurons throughout metamorphosis, whole mount preparations of the brain and ventral nerve cord of laboratory reared spider crab larvae (Hyas araneus) were labelled with an antibody against the neurotransmitter serotonin. The system of serotonin-immunoreactive cell bodies, fibres and neuropils is well-developed in newly hatched larvae. Most immunoreative structures are located in the protocerebrum, with fewer in the suboesophaegeal ganglia, while the thoracic and abdominal ganglia initially comprise only a small number of serotonergic neurons and fibres. However, there are significant alterations in the staining pattern through larval development, some of which are correlated to metamorphic events. Accordingly, new serotonin-immunoreactive cells are added to the early larval set and the system of immunoreactive fibres is refined. These results are compared to the serotonergic innervation in other decapod crustaceans.     

Influence of temperature on larval survival, development, and respiration inChasmagnathus granulata (Crustacea, Decapoda)

Larvae of an estuarine grapsid crabChasmagnathus granulata Dana 1851, from temperate and subtropical regions of South America, were reared in seawater (32/%.) at five different constant temperatures (12, 15, 18, 21, 24 °C). Complete larval development from hatching (Zoea I) to metamorphosis (Crab I) occurred in a range from 15 to 24 °C. Highest survival (60% to the first juvenile stage) was observed at 18 °C, while all larvae reared at 12 °C died before metamorphosis. The duration of development (D) decreased with increasing temperature (T). This relationship is described for all larval stages as a power function (linear regressions after logarithmic transformation of bothD andT). The temperature-dependence of the instantaneous developmental rate (D ⁻¹) is compared among larval stages and temperatures using the Q₁₀ coefficient (van't Hoff's equation). Through all four zoeal stages, this index tends to increase during development and to decrease with increasingT (comparing ranges 12–18, 15–21, 18–24 °C). In the Megalopa, low Q₁₀ values were found in the range from 15 to 24 °C. In another series of experiments, larvae were reared at constant 18 °C, and their dry weight (W) and respiratory response to changes inT were measured in all successive stages during the intermoult period (stage C) of the moulting cycle. Both individual and weight-specific respiration (R, QO ₂) increased exponentially with increasingT. At each temperature,R increased significantly during growth and development through successive larval stages. No significantly differentQO ₂ values were found in the first three zoeal stages, while a significant decrease with increasingW occurred in the Zoea IV and Megalopa. As in the temperature-dependence ofD, the respiratory response to changes in temperature (Q₁₀) depends on both the temperature range and the developmental stage, however, with different patterns. In the zoeal stages, the respiratory Q₁₀ was minimum (1.7–2.2) at low temperatures (12–18 °C), but maximum (2.2–3.0) at 18–24 °C. The Megalopa, in contrast, showed a stronger metabolic response in the lower than in the upper temperature range (Q₁₀=2.8 and 1.7, respectively). We interpret this pattern as an adaptation to a sequence of temperature conditions that should typically be encountered byC. granulata larvae during their ontogenetic migrations: hatching in and subsequent export from shallow estuarine lagoons, zoeal development in coastal marine waters, which are on average cooler, return in the Megalopa stage to warm lagoons. We thus propose that high metabolic sensitivity to changes in temperature may serve as a signal stimulating larval migration, so that the zoeae should tend to leave warm estuaries and lagoons, whereas the Megalopa should avoid remaining in the cooler marine waters and initiate its migration to wards shallow coastal lagoons.     

Neurogenesis in larval stages of the spider crab Hyas araneus (Decapoda,Brachyura): proliferation of neuroblasts in the ventral nerve cord

Larvae of the spider crab Hyas araneus were reared in the laboratory from hatching through to metamorphosis. Neurogenesis was recorded in the ventral nerve cord during development of successive larval stages, zoea 1, zoea 2, megalopa and crab 1. Proliferating cells were detected immunocytochemically after in vivo labelling with 5-bromo-2-deoxyuridine (BrdU) which, as a thymidine analogue, is incorporated into the DNA of dividing cells. Segmental sets of mitotically highly active neuroblasts were found in newly hatched larvae. A dorsal neuroblast, a ventral-median neuroblast, 3–6 anterior-ventral neuroblasts and 1–3 lateral neuroblasts could be distinguished in each thoracic ganglion. Significantly fewer neuroblasts were labelled in the suboesophageal ganglion as compared to the thoracic ganglia. The number of active labelled neuroblasts was high throughout zoea 1 and about 30% of zoea 2 development and then dramatically decreased towards premetamorphosis. In the newly moulted megalopa, only a reduced set of neuroblasts was labelled which ceased dividing within the first few days of megalopa development. There is an indication that, although most ganglion mother cells born by unequal division of neuroblasts may go through their final division at an early stage, certain clusters of ganglion mother cells obviously delay their final mitosis. These results are discussed with regard to neuronal integration which necessarily changes during the course of metamorphosis in relation to the altered behavioural repertoire of the larvae.     

Temperature and the larval development of Hyasaraneus L. (Decapoda : Majidae); extrapolation of laboratory data to field conditions

The larval development of the spider crab Hyas araneus L. was studied in the laboratory at different constant temperatures (2, 6, 12, and 18°C). Linear relationships between log temperature and log stage duration were described by means of regression equations. They were used in a simple simulation model predicting larval moulting and metamorphosis at different temperatures. Experiments were also carried out under temperature regimes which simulated field conditions. Correspondence between predicted data and those observed in these experiments was generally good. Based on these observations, a number of predictions was made on larval occurrence in the plankton, on changes in the composition of larval populations, and on the period and intensity of settlement near Helgoland (North Sea). The extent and quality of the effects of deviations from the average course of temperature were also estimated. The most important predictions were: (1) settlement of H. araneus takes place mainly from late May to mid-June; (2) assuming wide annual fluctuations in the average spring temperature (±2°C), a total settlement period ranging from late April to early July can be expected; (3) delay of larval development due to a decrease in temperature is stronger than the acceleration caused by an equally great increase; and (4) both delay and acceleration effects become weaker during the hatching season and from stage to stage.     

The larval development of Penaeus semisulcatus de Haan, 1850 (Decapoda, Penaeidae) reared in the laboratory

P. semisulcatus is a commercially important species of the ArabianSea. In the present studies the mature females were broughtfrom the sea to the laboratory for spawning. The spawned eggswere reared in laboratory conditions up to the post larval stage.Five naupliar, three protozoeal, three mysis and first postlarval stages were described. The diagrams were made with thehelp of camera lucida. The measurements were made with a micrometer.     

Laboratory studies on the larval growth and development of Nyctiphanes capensis (Euphausiacea)

Larvae of Nyctiphanes capensis Hansen were reared in the laboratoryunder different trophic conditions (i.e., algal, animal andmixed diets) from the second calyptopis stage to the first juvenilestage. It is demonstrated that diet is important in determininggrowth rates and development of the larvae. The highest growthrates were achieved on diets of Artemia nauplii mixed with eitherthe flagellate Tetraselmis or Pseudodiaptomus nauplii. Poorerdietary conditions such as with the diatom Phaeodactylum yieldedslower growth rates where additional moults were needed to completefurcilia development. Under favourable trophic conditions theintermoult period was 4–6 days and was not age dependent.Three dominant pathways of pleopod development were found inhealthy individuals.     

Molecular phylogeny and biogeographic history of the European Maja spider crabs (Decapoda, Majidae)

We have assessed for the first time the phylogenetic relationships and biogeographic history of the crabs of the genus Maja that inhabit European coasts: M. brachydactyla, M. crispata, M. goltziana and M. squinado. Using mitochondrial markers, we have recovered a well-resolved phylogenetic tree that supports a single origin for the European species, most likely from an Indo-West Pacific ancestor during the Early Miocene. In this phylogeny, M. goltziana appears as the basal European species, with a sister lineage bifurcating into an Eastern Atlantic (M. brachydactyla) and a Mediterranean (M. crispata and M. squinado) clade. We propose the Tethyan Seaway as the initial colonization route, although an entrance through South Africa cannot be discounted. The Eastern Atlantic/Mediterranean split seems to predate the Messinian salinity crisis, which, in turn, could have promoted the recent divergence within the Mediterranean. In addition, Pleistocene glaciations could explain the current diversity in the Eastern Atlantic Ocean, where a unique mitochondrial lineage is found. According to this, the genetic profile of South African crabs appears to belong to M. brachydactyla, questioning the validity of the putative species M. capensis.     

The effect of salinity and temperature on the larval development of Mithrax caribbaeus Rathbun, 1920 (Brachyura: Majidae) reared in the laboratory

Larvae of Mithrax caribbaeus were reared in the laboratoryin a factorial experiment employing three temperatures (22,25 and 28°C) and three salinities (32, 35 and 38). Survivaland duration of larval stages were recorded. Ovigerous femalesof M.caribbaeus were collected from the south-eastern coastof Margarita Island, Venezuela, and maintained in individualaquaria until hatching. Eggs from three of the females hatchedin the laboratory. Larvae from each hatching were subdividedinto groups of 10 and reared in plastic bowls containing 200ml filtered and UV-irradiated sea water at different temperature–salinitycombinations. Larvae were transferred daily to clean bowls withnewly hatched Artemia nauplii, and the number of molts and mortalitywithin each bowl was recorded. Complete larval development ofM.caribbaeus occurred under all experimental conditions. Salinityhad the greatest effect on percentage survival of each larvalstage and complete development up to the first crab stage. Thefirst zoeal stage exhibited the highest survival rate. Maximumsurvival for this stage occurred at 25°C, 32–35. Survivalin the second zoeal stage and the megalopa was affected onlyby salinity. Effects of temperature and salinity on survivaldecreased with advance in development. The duration of the twozoeal stages, the megalopa, and development to the first crabstage showed a gradual reduction with increasing temperature.Salinity showed an effect on the duration of zoeal stages butnot on the megalopal stage. Development from hatching to thefirst crab stage required 8–18 days, depending on thetemperature–salinity combination, and was inversely relatedto temperature, averaging 14.3 days at 22°C, 11.8 days at25°C and 9.2 days at 28°C.     

The complete larval development and first juvenile of the spider crab Inachus thoracicus Roux, 1830 (Brachyura: Majidae: Inachinae)

The larval (two zoeae and megalopa) and first crab stages ofthe majid crab Inachus thoracicus Roux, 1830 are described andillustrated from laboratory-hatched material obtained from anovigerous female collected on the western Mediterranean continentalshelf. The morphology of all the stages is compared with thatof the corresponding stages of other known species of the genusInachus. The larvae of I. dorsettensis, I. phalangium and I.thoracicus are morphologically similar, and differ in some characteristicsfrom I. leptochirus.     

The population biology and genetics of the deep-sea spider crab,Encephaloides armstrongi Wood-Mason 1891 (Decapoda: Majidae)

Numerous specimens of the majid spider crab, Encephaloides armstrongi, were sampled from six stations (populations) between 150 and 650 m depth, on the continental slope off the coast of Oman. This extended the known geographic and bathymetric range of E. armstrongi, which is now known to occur along the continental margins of the northern Indian Ocean from the western coast of Burma to the coast of Oman. This band-like distribution is contiguous to the oxygen minimum zone in this region.The biology and genetics of populations of Encephaloides armstrongi separated by depth were studied. The overall sex ratio of the E. armstrongi sampled was male-biased (p less than 0.01; 3.3 males: 1 female; S_o = 0.538). However, sex ratio varied both between populations (p less than 0.01) and between size classes of crabs. Size frequency analysis indicated that the male and female crabs consisted of at least two instars, one between 6 and 16mm carapace length and one between 16 and 29 mm carapace length, which probably represented the terminal (pubertal) moult for most individuals. Accumulation of female crabs in the terminal instar probably caused the variation of sex ratio with size classes. Some male crabs grew to a larger size (up to 38 mm carapace length), possibly as a result of maturity at later instars.Length frequency distribution was significantly different between sexes (one-way ANOVA p less than 0.001). Within sexes, length frequency distributions varied between different populations. In both male and female Encephaloides armstrongi the individuals from a single population located at 150 m depth were significantly smaller than individuals at all other stations and were considered to represent a juvenile cohort. For female crabs no other significant differences were detected in length frequency between populations from 300 m to 650 m depth. Significant differences in length frequency were detected between male crabs from populations between 300 and 650 m depth.Horizontal starch gel electrophoresis was used to detect six enzyme systems coding for eight loci for individuals sampled from each population of Encephaloides armstrongi. Genetic identity (I) values between populations of E. armstrongi (I = 0.98-1.00) were within the normal range for conspecific populations. Observed heterozygosity (H_o = 0.080-0.146) was lower than expected heterozygosity (H_e = 0.111-0.160), but in the normal range detected for eukaryotic organisms.F-statistics were used to analyse between population (F_ST) and within population (F ) genetic structure. For both male and female E. armstrongi significant genetic differentiation was detected between the population located at 150 m depth and all other populations. Analyses of F_IS and F_ST, excluding the 150 m population indicated that for female E. armstrongi there was no significant structuring within or between populations. For male E. armstrongi significant heterozygote deficiencies were detected within populations and significant genetic differentiation between populations.The most likely explanations for the observations of the present study are: the population of Encephaloides armstrongi located at 150 m depth represented a juvenile cohort that is genetically distinct from deeper populations; female E. armstrongi formed a single population between 300 m and 650 m depth in the sampling area; male E. armstrongi were from two or more genetically distinct populations which are represented by different numbers of individuals at stations between 300 m and 650 m depth. This caused the observed significant differences in morphology (size distribition) and allele frequencies of male populations. It is likely that E. armstrongi exhibits gender-biased dispersal and that the crabs collected between 300 m and 650 m depth formed spawning aggressions. This also explains the bias in sex ratio of individuals sampled in the present study.     

Influence of starvation on larval development of Carcinus maenas L. (Decapoda : Portunidae)

Lethal and sublethal effects of particular starvation events were investigated in larvae of Carcinusmaenas L. Mean survival times of continuously starved zoeae-1 were approximately twice the normal stage duration (12, 18, 25°C), and both increased with falling temperatures. At 6°C zoea-1 was unable to develop to stage-2. No larva retained the ability for successful further development if starved for half the stage duration time and was then refed. The zoea-1 larvae had to feed for at least 20 % of the normal stage duration for some larvae to moult to zoea-2. Some initial feeding was necessary to start zoea-1 development. Beyond a certain point of energy and accumulation of reserves development of the larvae seems to continue regardless of feeding rates. The demands for larval feeding correspond very well with the larval moulting cycle. Larvae of C. maenas proved to be well adapted to natural shortage of food.     

Characteristics of palinurids (Decapoda; Crustacea) in larval culture

Complete development of phyllosomas wasaccomplished in Palinurus elephas, Panulirus japonicus, Jasus edwardsii and J. verreauxi. Larval development differsbetween species in duration and moult frequency aswell as morphology. The highest percent survival was obtained during the phyllosoma stage forJ. verreauxi. Phyllosomas of P. elephas were the mostdifficult to cultureregardless of the fact that they hatch at arelatively advanced stage. Culture experiments werecarried out to improve the percent survival of P. elephas. First instars of P. elephasexhibited vigorous predation upon the larvae ofJapanese sandfish Arctoscopus japonicus.Duration of the first instar was shorter at 12.8days when reared at 18 °C and percent survival wasapproximately 30%. Better results were obtainedfor first instars cultured in water inoculated withChaetoceros sp. and fed enriched Artemianauplii. Phyllosomas of J. edwardsii and P. japonicus were cultured without particulardifficulty. Most distinguishing features of P. elephasis the heavily setose six or seven longspines on the distal edges of the scaphognathite ofthe second maxillae while only four long spines arefound in other species. These facts suggest thatphyllosomas of P. elephas are initiallyplankton feeders and from first instar becomerapacious predators.     

Mating and role of seminal receptacles in the reproductive biology of the spider crab Maja squinado (Decapoda, Majidae)

The reproductive biology of the spider crab Maja squinado was analyzed based on monthly samples from an 18-month study carried out in Galicia (NW Spain) and laboratory experiments holding primiparous and multiparous females in captivity with and without males. The seminal receptacles of adult females were analyzed and their relationship with the presence and developmental stage of the eggs and the gonad maturity stage was determined. Gonad maturation in primiparous females began one or two months after the pubertal moult. Females having gonads in an advanced stage of development made their appearance in December and the first spawning took place in mid-winter or early spring. The percentage of ovigerous females from March to September was ∼75%. As the incubation period progressed, the ovaries became mature again in order to carry out the next spawning. Under experimental conditions the breeding cycle started earlier in multiparous females, during their second yearly cycle, than in primiparous ones. After mating, female spider crabs store sperm in seminal receptacles and this sperm is used in the fertilization of eggs immediately prior to spawning. The analyses of seminal receptacles consisted of the estimation of fullness and the number of differentiated sperm masses. The number of masses ranged between 0 and 6 in field samples (median for females with stored sperm=1) and was positively correlated with fullness. Differences in colour and volume of individual masses showed that, at least in some cases, females carried out successive matings with long intervals in between. This storage mechanism allowed females to fertilize successive broods without remating (as was also shown under experimental conditions). Juvenile females from shallow waters did not have developed seminal receptacles which indicated that mating was not possible until the onset of maturity. Postpubertal females in shallow waters (August to October), including animals participating in aggregations, always showed empty receptacles. The seasonality of receptacle fullness showed that mating involved hard-shelled females and occurred in deep water during the autumn migration from juvenile habitats or in the wintering habitats, during the last stages of gonad maturation (November to February). After fertilization ovigerous females continued to store sperm, but the volume was lower than in non-ovigerous females. Mating may occur in ovigerous females, particularly in the final period of incubation, because in females with broods almost ready to hatch, both new and older sperm masses were seen in the receptacles (distinguished by colour and size). The fullness of the receptacles decreased both in ovigerous and non-ovigerous females in the final phase of the annual breeding cycle (August–October), however, some sperm was still available. In the laboratory, mating was observed, and no courtship nor postcopulatory guarding was recorded. The analysis of receptacles from laboratory experiments indicated that primiparous and multiparous females showed differences in the seasonality of mating in the first phase of the breeding cycle (September–January), related to differences in the timing of gonad maturation and hatching. Mating occurred in the final stages of gonad maturation, a short time before hatching, and matings were detected in ovigerous females. Multiple matings were also evident, to a greater extent than in the field, probably due to the higher availability of males. Females underwent over four successive spawnings in the laboratory without having to recopulate, and the incubation lasted on the average from 40 to 58 days (∼18 and 16°C respectively) and the mean duration between hatching and the next spawning was 3.4 days. It is estimated that most females carry out three successive spawnings during the annual cycle.