Hatching rate and diapause duration in eggs of Paracartia latisetosa (Copepoda: Calanoida)

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1 Hatching rate and diapause duration in eggs of Paracartia latisetosa (Copepoda: Calanoida) GENUARIO BELMONTE * AND ANNA CARMELA PATI MARINE BIOLOGY STATION, DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES AND TECHNOLOGIES, CO.N.I.S.MA. U.O. UNIVERSITY OF LECCE, ECOTEKNE, I LECCE, ITALY *CORRESPONDING AUTHOR: genuario.belmonte@unile.it Received April 14, 2006; accepted in principle September 25, 2006; accepted for publication October 24, 2006; published online December 5, 2006 Communicating editor: R.P. Harris Paracartia latisetosa is a seasonally dominant copepod species in Mediterranean coastal waters. This species is well known for the production of diapause eggs which allow it to over-winter. Diapause eggs are morphologically distinguishable (they have long spines on the chorion) from subitaneous eggs (which are smooth or only have short spines). From September 2003 to December 2004, eggs produced by 329 adult females (collected biweekly and incubated for 24 h) were maintained under constant conditions until hatching. Subitaneous eggs were produced on all sampling dates and hatched during the first week after laying. Diapause eggs were produced from September to December and they hatched after a period of obligatory rest (from 54 to 202 days). Variability of life history traits (egg production, hatching rate, diapause duration) have been interpreted as an adaptation of the species to high environment variability. Regression analysis showed that the diapause duration was positively correlated with photoperiod length at the time of collection of the adults. As a result of this range in resting time, the majority of diapausing eggs hatched in February March. This fact, as they are not influenced by external stimuli, suggests that the timer of diapause duration of the eggs is under parental control. INTRODUCTION Dormancy is a well-known phenomenon which allows for survival during unfavourable periods. Many populations of marine Calanoida in temperate waters vary in abundance on a seasonal basis. In coastal areas the production of resting eggs is common (see Mauchline, 1998, for a list of species). During adverse periods, such species may completely disappear from the plankton and their year after year re-occurrence in a given area is due to the re-activation of dormant stages under favourable environmental conditions (Grice and Marcus, 1981). The features of copepod dormancy vary in relation to many internal (taxon, ontogenetic stage) or external factors (latitude, physico-chemical parameters) (see the reviews of Dahms, 1995; Williams-Howze, 1997). Embryonic diapause within the egg envelope is common among the Centropagoidea whether freshwater or marine (Alekseev, 1990). In contrast to freshwater species, many marine Centropagoidea produce diapause eggs morphologically distinguishable from the subitaneous ones (e.g. Sazhina, 1968; Belmonte, 1992, 1997; Santella and Ianora, 1990; Ianora and Santella, 1991). The different morphology justifies their identification as products of a different genetic programme (a developmental conversion) in the real sense of the term diapause (Andrewartha and Birch, 1971). In light of this context, resting eggs should not be considered as responsible for their entrance into doi: /plankt/fbl064, available online at # The Author Published by Oxford University Press. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org

2 diapause (they are produced with this unchangeable destiny). Rather they are considered able to choose the moment of their re-activation, which is stimulated by environmental (external) factors (Kasahara et al., 1974; Uye, 1985; Marcus, 1987) just as it occurs in other taxa (Alekseev, 1990). This sensitivity is active only after the end of the obligatory diapause period (the refractory phase). From this point of view, there are similarities with quiescence eggs (for terminology differences see Grice and Marcus, 1981), which, in contrast, are not programmed to require a resting period but can rest if unfavourable conditions occur. This maternal role, however, could be even more extended (e.g. on the diapause duration and the re-activation timing) than that generally assumed, relegating the external stimuli to an interfering role (e.g. as re-activation inhibitors) affecting the already programmed re-activation time. This interpretation has not received attention but it is suggested by some results not fitting the expected pattern, such as the finding of different diapause duration in eggs produced by co-specific females from different seasons (Marcus, 1987; Ianora, personal communication). Paracartia latisetosa (Acartiidae, Centropagoidea, Calanoida) is abundant in the Mediterranean Sea, particularly in confined brackish waters (Belmonte and Potenza, 2001) where a strong hydrographic variability results in sharp fluctuations in abundance (Belmonte et al., 1989; Comaschi-Scaramuzza et al., 1995; El Shabrawy and Belmonte, 2004), until the complete disappearance during winter, a period which is passed as resting eggs (Belmonte, 1992; Fanelli et al., 1992). In the present study, the features of both egg production and rest duration of P. latisetosa diapause eggs are investigated with the aim of clarifying whether a maternal role exists in the adaptation to an environment with high abiotic variability. METHOD Zooplankton sampling was carried out biweekly from 30 September 2003 to 20 January 2004 and from 4 May 2004 to 28 December 2004 in the Acquatina brackish water lake ( lat. N; long. E; on the Italian coast of the Adriatic Sea) where P. latisetosa is a seasonally dominant species. The lake has an area of about 45 ha, a maximum depth of 1.8 m (maximum daily tidal excursion, 35 cm), a salinity range 27 36, and a temperature range C (Scalzo et al., 1994). Zooplankton was collected with a 200-mm mesh net. Adult females were sorted after light anaesthetization with 10% MgCl 2 solution (maximum 3 min) by a hand pipette, about 1 h after collection. For each sampling date, 36 females (or all those present) of P. latisetosa were reared individually in multi-well plates (each well of 3 ml volume, with transparent bottom) for 24 h in Acquatina 50-mm filtered water. Eggs laid after 24 h were collected under the inverted microscope (magnification, 100), and incubated, separately from each mother, under constant conditions (temperature, 148C + 18C, and photoperiod, 12D:12L; conditions representing an equinox situation under which eggs could not distinguish the autumn from the spring) in filtered water. The eggs were checked daily under the inverted microscope until hatching. After the first two weeks, the remaining un-hatched eggs were checked weekly. Abortive eggs degenerated in a few days from their laying. The one-factor ANOVA test was utilized in order to establish the significance of differences in both production type and hatching percentage of eggs. Linear regression analysis was applied to investigate correlation between diapause length of resting eggs and photoperiod at the time when they were produced. RESULTS Female P. latisetosa produced eggs on all sampling dates when they were present in the lake (they were absent during the winter). During the whole investigation period, 329 females were reared, 229 of them (69.6%) produced eggs. A total of 1037 eggs were obtained. The average egg production was eggs female 21 day 21 ( eggs fecund female 21 day 21 ). Egg production varied considerably during the year (from eggs female 21 day 21 in May 2004 to eggs female 21 day 21 in August 2004) (Fig. 1). Three morphological types of eggs were produced (Table I): eggs with a smooth chorion, eggs with short spines, and eggs with long spines, named smooth, brush, and spiny types, respectively. The production and the hatching percentage of brush eggs were higher than both smooth and spiny eggs (Table I). Both smooth and brush eggs which did not hatch were observed to be discoloured and to disintegrate in a short time; these were presumed to be unfertilized or otherwise nonviable (abortive). The majority of both smooth and brush eggs hatched during the first week of incubation; so they were considered to be subitaneous eggs. Instead, hatching of spiny eggs occurred after an incubation of at least 54 days; thus these eggs were considered to be diapausal i40

3 G. BELMONTE AND A. C. PATI j DIAPAUSE DURATION IN PARACARTIA LATISETOSA Fig. 1. Types and number of eggs produced during the investigation period by reared females. From after 20 January 2004 to 4 May 2004 (excluded) sampling was not carried out. From 23 November 2004 to 28 December 2004, the population of P. latisetosa was absent from the water column. (Table II). Spiny eggs which were not viable disintegrated during the incubation period. Four brush-like eggs (0.81% of the total brush eggs) hatched after 21 days of incubation. Paracartia latisetosa produced subitaneous eggs (smooth and/or brush) on all sampling dates when the females were present in the population, whereas diapause eggs were produced only in autumn (September early January), before the species disappears from the water column (Fig. 1). In both the sampling periods, the production of diapause eggs occurred under a short-day regime, that is when the photoperiod varied from about 12L : 12D (September) to 9L : 15D (early January). The peak of diapause egg production occurred in October (Fig. 1). The conversion in the production of egg types (i.e. from subitaneous to diapause eggs) was never complete (it did not affect all the females of the same generation), and both egg types were present even in the same clutch. During the whole study period, 30.40% of females did not produce eggs within 24 h after collection, 49.54% produced only subitaneous eggs, 12.46% produced only diapause eggs and 7.60% of females produced either subitaneous or diapause eggs. During the autumn, however, a higher fraction (up to 63%, depending on sampling date) of reared females produced diapause eggs (Fig. 2). Different types of eggs were produced by the same female within the 24-h incubations. In fact, clutches differed with respect to egg type composition: most of them contained eggs having the same morphology, but almost 23% of clutches were mixed, containing two or three types of eggs, subitaneous and/or diapausal. The number of eggs produced by each female varied, depending on egg type: females which produced both subitaneous and diapause eggs were more prolific ( eggs fecund female 21 day 21 ) than females which produced only subitaneous ( eggs Table I: Total number, average reared female 21 day 21, production period and hatching percentage of different egg types produced by Paracartia latisetosa Egg types Total number (and percentage) of laid eggs on all sampling period Average eggs reared Production period Hatching female 21 day 21 percentage Smooth eggs 161 (15.53%) 0.49 Always (when the species is present) Brush-like eggs 646 (62.29%) 1.96 Always (when the species is present) Spiny eggs 230 (22.18%) 0.70 Autumn Hatching percentages refer to total eggs produced. i41

4 Table II: Hatching percentage and period of incubation of different egg types produced by Paracartia latisetosa Egg type Incubation days After 21 days From 54 to 202 days Smooth eggs (%) 99 1 Brush-like eggs (%) Spiny eggs For hatching percentage in several periods see Fig. 5 Hatching percentages refer to total eggs hatched for each egg type. fecund female 21 day 21 ) or diapause eggs ( eggs fecund female 21 day 21 ) (ANOVA, P ¼ 0.02) (Fig. 3, Table III). However, the average of subitaneous eggs laid by females producing both subitaneous and diapause ones ( eggs fecund female 21 day 21 ) was significantly (P ¼ ) lower than that ( eggs fecund female 21 day 21 ) of females producing only subitaneous eggs (Fig. 4, Table IV). The hatching of diapause eggs began around the middle of January and ceased at the beginning of May (in the first year of investigation) or April (in the second year). A total of 230 diapause eggs were obtained, which had a hatching success of 51.74%. None of them hatched before 54 days. The resting period varied from 54 to 202 days, but 83% of hatched diapause eggs had a resting period of days (Fig. 5). The regression analysis (Fig. 6, Table V) showed that the rest duration (expressed as days) of diapausing eggs was positively correlated (P ¼ 0.01) with photoperiod length (expressed as minutes of light day 21 ) existing at the time when they were produced. DISCUSSION Both the polymorphism and the different functional roles of the eggs in P. latisetosa are already known (Belmonte, 1992). Other Calanoida species producing resting eggs generally show morphological differences correlated with the destiny of eggs (e.g. Sazhina, 1968; Ianora and Santella, 1991; Belmonte and Puce, 1994; Belmonte, 1997), probably due to an evolutionary pressure which requires a spiny surface to survive in bottom sediments (Belmonte et al., 1997). Production of diapause eggs in autumn suggests the existence, for P. latisetosa, of a switching date (sensu Hairston and Munns, 1984) which is the moment when females start to also produce diapause eggs, probably responding to the decrease in photoperiod. Marcus Fig. 2. Typologies of females reared depending on produced eggs. From after 20 January 2004 to 4 May 2004 (excluded) sampling was not carried out. From 23 November 2004 to 28 December 2004, the population of P. latisetosa was absent in the water column. i42

5 G. BELMONTE AND A. C. PATI j DIAPAUSE DURATION IN PARACARTIA LATISETOSA Fig. 3. Comparison of egg production of females depending on the different egg types produced. (1980) reported that female Labidocera aestiva produced subitaneous eggs under a long-day regime (18L : 6D), and diapause eggs under a short-day regime (8L : 16D). Females are probably triggered to switch the type of egg production after experiencing a specific number of progressively shorter day lengths. This mechanism is reported to be responsible for the onset of diapause in several other Crustacea (Alekseev, 1990). However, temperature (Marcus, 1982a), overcrowding (Ban, 1992), and food availability (Zeller et al., 2004) may also modify the photoperiod effect. The production of diapause eggs in P. latisetosa seems to be linked to the female perception of progressively shorter photoperiods. The maternal perception could even occur very early in development of females, and Ban (1992) reported that egg type production in Eurytemora affinis was affected by environmental cues ( photoperiod and temperature) experienced by females at the nauplius stage (copepodids were not sensitive to environmental variations). What is typical of P. latisetosa is the high variability in egg type production. Even when females perceive the signal to produce resting eggs, they continue to produce the subitaneous ones, with both egg types present in the same generation, on the same day, and even in the same individual clutch. Also features of each egg type (morphology, viability, hatching time) are not rigid but show an intermediate range of values. This probably allows the species to be well adapted to environments which undergo extreme variability in time and/or space (such as confined brackish waters). Owing to their ability to produce both diapause and subitaneous eggs at the same time, female P. latisetosa do not face the dilemma of continuing to produce subitaneous eggs (betting on the present) or switching to dormant eggs (betting on the future), as occurs in other One factor ANOVA Table III: ANOVA of comparison among number of eggs produced by different groups Groups Count Sum Average Variance Eggs by females producing only subitaneous eggs Eggs by females producing both subitaneous and diapause eggs Eggs by females producing only diapause eggs Source of variation SS Df MS F P F crit Among groups Within groups Total Abbreviations. SS, Sum of squares; Df, Degrees of freedom; MS, Mean square; F, calculated F-ratio (variance among treatments/variance within treatments ratio); P, level of significance; F crit, critical value of F. i43

6 Fig. 4. Comparison of subitaneous eggs produced by females which produce only subitaneous eggs and females which produce both subitaneous and diapause eggs. species (see Avery, 2005, for Acartia hudsonica). They distribute the results of their reproductive effort over a long period, ensuring fitness betting even on the edges (sensu Philippi and Seger, 1989). It is also not known whether the females which produce both types of eggs are programmed to always produce heterogeneous clutches or whether the mixed clutches represent a transition phase in the switching from subitaneous to diapause egg production (Marcus, 1982b). However, we did not find a single group and/or period in which all females produced resting eggs, nor a progressive increase in numbers of females producing only resting eggs. Hence, we must accept as a rule that each P. latisetosa female can produce both egg types in autumn. Apart from the obvious difference of age at hatching period, between subitaneous and diapause eggs, diapause eggs also differ from one another with regard to their age at the hatching period; there were, in fact, 148 days difference from the first to the last hatched diapause egg. So identical experimental conditions did not affect all diapausing eggs similarly. This fact suggests that individual differences exist within diapause eggs produced at the same date, in internal timing mechanisms which maintain the diapause. The variability of hatching time in diapause eggs within the same clutch has an important evolutionary significance, because offspring will not emerge at the same time in the future (De Stasio, 2004). Diapause eggs were laid together with subitaneous eggs and incubated under the same experimental conditions, but they had a different fate, evidently because they were programmed differently. It is generally believed that, at least for the copepodid stage, the mechanism controlling diapause is a combination of internal cues (in the form of a timing mechanism, such as One-factor ANOVA Table IV: ANOVA of the comparison reported in Fig. 4 Groups Count Sum Average Variance Subitaneous eggs produced by females producing only subitaneous eggs Subitaneous eggs produced by females producing both subitaneous and diapause eggs Source of variation SS Df MS F P F crit Among groups Within groups Total For abbreviations, see Table III. i44

7 G. BELMONTE AND A. C. PATI j DIAPAUSE DURATION IN PARACARTIA LATISETOSA Fig. 5. Percentage of diapause eggs hatched during different 15-day periods of incubation. Fig. 6. Linear regression analysis between dormancy, days of diapausal eggs and photoperiod for the day of their day production. Sampling dates are reported between brackets. i45

8 Regression statistics Table V: Statistics and ANOVA of regression analysis reported in Fig. 6 R multiple R squared R squared adjusted Standard error Observations 8 ANOVA Df SS MS F P Regression Error For ANOVA abbreviations, see Table III. hormone levels or critical lipid content) and external ones such as a shift in the photoperiod (Miller et al., 1991; Hirche, 1996). The positive correlation between rest duration of diapause eggs and photoperiod at the time when they were produced, combined with the constant laboratory incubating conditions (which exclude the influence of external stimuli on the eggs), suggests that dormancy length is (at least partly) under the control of the mother. Several studies have shown a significant maternal effect on production of diapause eggs (for copepods, see Grice and Marcus, 1981; Hairston and Munns, 1984; Ban, 1992; Avery, 2005), but an effect on hatching has only been shown in non-copepod Crustacea (see De Meester et al., 1998, for Daphnia, and Van Dooren and Brendock, 1998, for Anostraca). The wide individual variability of this feature (up to 96 days difference in diapause duration of eggs from the same generation of females) probably masked this effect, but our data allow us to affirm that each female produces resting eggs with an average diapause duration. To conclude, the synchronous hatching of most diapause eggs produced in different periods and incubated under constant conditions (so without variation of external stimuli) suggests that diapause duration in P. latisetosa is under parental control (even a paternal role cannot be excluded with the present state of knowledge). In fact, the adults not only represent the stage which perceives the stimuli inducing the production of diapause eggs, but they also set the timer of diapause duration in each egg by means of a mechanism which could differentiate distribution of information ( possibly in the form of metabolite concentration) concerning the hatching time for each egg. External stimuli, at this point, do not promote egg hatching, but can only inhibit their re-activation (so acting on the resting duration). The possible prolongation of resting after the end of the refractory phase, should not be considered to be diapause but simply quiescence. ACKNOWLEDGEMENTS This research was supported by the Ministero per le Politiche Agricole e Forestali MiPAF ( project Nuove fonti alimentari per un acquacoltura sostenibile ricerca di nuove specie da utilizzare come alimento vivo per larve di pesci n86c34). REFERENCES Alekseev, V. R. (1990) Diapausa Rakoobraznykh: Ekologicheskiye i Fiziologicheskiye Aspeckty [Diapause in Crustaceans: Ecological and Physiological Aspects]. Nauka press, Moscow, 143 pp. (In Russian). Andrewartha, H. G. and Birch, L. C. (1971). Diapause. In Andrewartha, H. G. (ed.), The Distribution and Abundance of Animals. University of Chicago press, Chicago, pp Avery, D. E. (2005) Induction of embryonic dormancy in the calanoid copepod Acartia hudsonica: proximal cues and variation among individuals. J. Exp. Mar. Biol. Ecol., 314, Ban, S. (1992) Effects of photoperiod, temperature, and population density on induction of diapause egg production in Eurytemora affinis (Copepoda: Calanoida) in Lake Ohnuma, Ohkkaido, Japan. J. Crustac. Biol., 12, Belmonte, G. (1992) Diapause egg production in Acartia (Paracartia) latisetosa (Crustacea, Copepoda, Calanoida). Boll. Zool., 59, Belmonte, G. (1997) Resting eggs in the life cycle of Acartia italica and A. adriatica (Copepoda, Calanoida, Acartiidae). Crustaceana, 70(1), Belmonte, G. and Potenza, L. (2001) Biogeography of the family Acartiidae (Calanopida) in the Ponto-Mediterranean Province. Hydrobiologia, 453/454, Belmonte, G. and Puce, M. (1994) Morphological aspects of subitaneous and resting eggs from Acartia josephine (Calanoida). Hydrobiologia, 292/293, i46

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