CSIRO PUBLISHING

www.publish.csiro.au/journals/mfr Marine and Freshwater Research, 2008, 59, 987–997

Batch or trickle: understanding the multiple spawningstrategy of southern calamary, Sepioteuthis australis(Mollusca : Cephalopoda)

Matthew R. KuipersA,B,D, Gretta T. PeclC and Natalie A. MoltschaniwskyjA,B

ASchool of Aquaculture, Tasmania Aquaculture and Fisheries Institute, University of Tasmania,Private Bag 1370, Launceston, Tas. 7250, Australia.

BNational Centre for Marine Conservation and Resource Sustainability, Australian Maritime College,University of Tasmania, Locked Bag 1370, Launceston, Tas. 7250, Australia.

CMarine Research Laboratories, Tasmania Aquaculture and Fisheries Institute, University of Tasmania,Private Bag 49, Hobart, Tas. 7001, Australia.

DCorresponding author. Email: kuipersm@utas.edu.au

Abstract. Many cephalopods are ‘multiple spawners’; however, we know little about the timing and dynamics ofegg production. This has implications for the allocation of energy to reproduction, lifetime fecundity and subsequentrecruitment. The current study aimed to determine if Sepioteuthis australis (Quoy and Gaimard, 1832), which spawnsmultiple times, produces mature oocytes for deposition in a continuous trickle or in larger discrete batches. Throughouta spawning season, developmental stages were assigned to the ovaries of each female by combining macroscopic andhistological analyses of the oocytes. Half of the females (46%) showed a significant peak in oocytes at one of the maturationstages, indicating that females were developing eggs in batches. It was hypothesised that the remaining females were alsobatch spawning, given that the oviduct weights of the remaining females (54%) were high and the other measured biologicalcharacteristics were similar to those of the females showing a peak in oocyte stage. Average batch fecundity declined overthe 3-month spawning season, but total egg numbers in the ovary increased, suggesting that females might have depositedsmall batches more often during December. As reproduction requires large allocations of energy, understanding howfemales distribute reproductive effort throughout their lives is crucial to understanding the behaviour of populations,individuals and their offspring.

Additional keywords: cephalopod, egg, oocyte, ova, ovary, reproduction.

Introduction

For any species, spawning strategy plays a critical role in deter-mining the timing and mechanism of egg release, ensuring thatenough gametes develop to become viable reproductive adults.In many marine vertebrate and invertebrate species, the massrelease of gametes at specific times related to lunar cyclesensures predator swamping (Robertson 1991) and the dispersalof gametes away from the reef (Johannes 1978). Another formof bet-hedging shows species releasing gametes over severallunar cycles (Gladstone and Westoby 1988), ensuring temporalspreading of mortality risks.This strategy increases the chance ofoffspring experiencing favourable conditions and allows someoffspring to become viable individuals. Whichever strategy isadopted, a species should minimise the cost and maximisethe benefits in the environment they inhabit. As reproductiveinvestment requires a large amount of the female’s energy, under-standing the way in which individuals allocate reproductiveeffort over their lifetime is crucial to understanding the behaviourof both populations and individuals (Maxwell and Hanlon 2000),and the future of their offspring (Steer et al. 2004).

The short lifespans of many cephalopod species, typically1 year or less (Jackson and Choat 1992; Pecl 2001), means thattheir spawning strategy must ensure that these single genera-tion populations are maintained each year exclusively throughyoung-of-the-year recruits. Recruitment failure in any year willresult in a population crash as the current generation dies fol-lowing the completion of spawning activities in their short adultreproductive phase (Basson and Beddington 1993). Multiplespawning appears to be more widespread among cephalopodsthan previously thought (Harman et al. 1989), with the excep-tion of octopus. Several squid and sepiolid species produce largenumbers of eggs over much of the adult life time, for exam-ple, Loligo vulgaris (Lamarck, 1798) (Laptikhovsky 2000) andSepioteuthis australis (Quoy and Gaimard, 1832) (Pecl 2001).These species also produce large eggs, relative to the size of theadult female (Mangold 1987), with large yolk reserves requir-ing high levels of energy input by females. The reproductiveadvantage of multiple spawning is particularly evident in smallcephalopods that produce large eggs because they are physicallyunable to manage the lifetime production of oocytes in a single

© CSIRO 2008 10.1071/MF07200 1323-1650/08/110987

988 Marine and Freshwater Research M. R. Kuipers et al.

Table 1. Expected differences between trickle and batch spawning

Trickle Batch

Ovaries Numbers of oocytes evenly distributed in every stage A discrete batch of developing oocytes expected with a poolof development (with the exception of large of immature oocytesnumbers of immature oocytes)

Oviduct The same size relative to body size in all reproductively Either be empty (indicative of recently deposited batch)mature females or full (indicative of a batch ready to be deposited)

Population variation Little if any variation will be apparent between the Variation in oviduct mass, that is, females ready to spawnovaries and oviducts of females in the population will have large full oviducts compared with femalesover the spawning season that have recently spawned

batch (Sauer et al. 2002). Multiple spawning is also an advan-tage when producing eggs with large yolk reserves and whenreproduction is fuelled directly by ingested food and not storedreserves (Moltschaniwskyj and Semmens 2000). The ecologicaladvantages of maturing oocytes in batches allows for the pro-duction of high numbers of eggs over an extended period (Meloand Sauer 1999), thereby spreading the risk of mortality overtime (Sauer et al. 2002).

Multiple spawning occurs through the maturation of newgametes between spawning events (Harman et al. 1989)and might be evidenced by the presence of primary oogo-nia in the ovaries of females that have already oviposited(Moltschaniwskyj 1995). However, it is possible that in somespecies primary oogonia are not continuously produced, butbatches of eggs are released as mature oocytes developed froma stock of primary oogonia, of which a large percentage mightnever mature (Laptikhovsky and Nigmatullin 1993). Therefore,if a female produces multiple large batches or more frequentsmall batches, both would fall under the multiple spawning strat-egy as long as there was maturation of new gametes betweenspawning events. In contrast, species that produce a single batchof eggs fail to continue producing primary oogonia (Knipe andBeeman 1978; Lopes et al. 1997). How females coordinatethe process of primary oogonia formation, oocyte develop-ment, fertilisation and ovipositing remains a critical question incephalopod reproductive biology (Maxwell and Hanlon 2000).

There are several methods to determine the spawning strategyof a species, and they can be divided into direct and indi-rect methods. Direct methods involve observations by videorecording (Jantzen and Havenhand 2003) or by tagging andtracking individuals to determine how often and where spawn-ing occurs (Pecl et al. 2006). This is time consuming, expensiveand focuses on small numbers of individuals. Indirect meth-ods involve measuring hormone concentrations (Matsuyamaet al. 1994) or the analysis of gonads at both population- andindividual-based levels. Ovarian condition has been assessedusing several approaches, with varying degrees of complexity(Coward and Bromage 2002), including gross external appear-ance of the ovary, oocyte size distribution and the stage frequencydistribution of oocytes using histological sections (Coward andBromage 2002). The last two methods allow examination of thedynamics of oocyte maturation in the ovary. Using histologi-cal slides of egg stage frequencies alone might not provide anadequate picture of the numerical ratio of oocytes in differentdevelopment stages because of the need to have large numbers

of sections from throughout the ovary and the relatively smallnumber of oocytes captured in a single slide (West 1990), partic-ularly for a species with larger eggs. Size frequency distributionsof oocytes can allow large numbers of eggs to be measured fromthroughout the ovary, but the correlation between oocyte size andstage needs to be supported using histological evidence (West1990). Using a combination of both techniques will providean adequate picture of the relative numbers of oocytes in eachstage of maturation. Combining these approaches also allowsus to examine oocyte dynamics in tandem with whole-animalassessments of the reproductive system.

To understand the mechanisms controlling recruitment inshort-lived species, studies encompassing all stages of egg pro-duction and variability are required (Lowerre-Barbieri et al.1998), including egg deposition. Although the present studyprimarily looked at egg development, it was assumed that all eggsthat developed to maturity were deposited. Evidence suggeststhat mature southern calamary, S. australis, females activelymove about the spawning grounds for several months, poten-tially depositing eggs throughout this period (Pecl et al. 2006).We propose that there are two possible egg development strate-gies that female S. australis might be adopting. One possibilityis that females develop eggs in discrete batches with a periodof time (days to weeks) between each batch during which timethe next batch of eggs is maturing and females are allocatingenergy to provisioning of the eggs. Alternatively, females mightremain on the spawning beds for several months developing anddepositing small numbers of eggs each day or two, with smallamounts of energy allocated to egg production each day. Thestrategy of egg deposition adopted by females has the capacityto affect how females allocate energy to egg production, patternsof life-time fecundity, and survival. Producing eggs in batchesrequires more energy over short periods and potentially placesphysical strain on the individual, possibly increasing susceptibil-ity to predation. In contrast, females that trickle eggs slowly overan extended period might risk having smaller lifetime fecundityif mortality occurs early in their reproductive life.

When comparing the two strategies, it is expected that femalesspawning in batches would differ greatly from those using thetrickle spawning strategy. The most obvious biological charac-teristics that would differ between the two strategies would beevident in the oocyte dynamics in the ovaries of mature females(Table 1). These differences can be used as testable predictionsto easily identify the adopted strategy when looking at matureovaries. The present study addresses the question of multiple

Southern calamary spawning strategy Marine and Freshwater Research 989

spawning in S. australis from observations of ovarian maturationand differentiation of oocyte batches, using both histological andoocyte size frequency analyses. Sepioteuthis australis typicallylives for approximately 1 year or less (Pecl 2001) and individ-uals aggregate in the spring/early summer on shallow inshoreseagrass beds in southern Australia to court, mate and depositeggs (Moltschaniwskyj et al. 2002). Sepioteuthis australis is amultiple spawner (Pecl 2001), but it is not known if individualsdeposit small numbers of eggs frequently (e.g. daily) or largebatches of eggs less frequently (e.g. weekly or fortnightly). Wesought to determine if individuals spawned in batches or alter-natively trickled eggs continuously, and whether or not thesestrategies varied between individuals in a population.

Materials and methodsSample collectionCollection of female Sepioteuthis australis was from the easternshores of Great Oyster Bay (GOB), on the east coast of Tasma-nia (42◦07′34′′S, 148◦17′51′′E), which is a major spring/earlysummer spawning site for S. australis. From 1 September to31 November 2004, the Department of Primary Industries andWater closed GOB to all commercial and recreational S. australisfishing. Sampling occurred during the spring/summer spawningseason between August 2004 and January 2005, with samplescollected every 7–14 days using jigs on handlines by researchscientists from the University of Tasmania with the aid of com-mercial fishers operating under a research permit. From eachof the 11 individual sampling days, a maximum of 20 femalesquid were measured and kept for dissection. Biological infor-mation and ovary samples from the mid section of the ovariesfixed in a FAACC (10% formalin, 5% glacial acetic acid and1.3% calcium chloride) (10 mL H2CO, 5 mL CH3COOH, 1.3 gCaCl2 and distilled water to 100 mL) were obtained from a totalof 176 females. The biological information collected includeddorsal mantle length (± 0.5 cm), total bodyweight (± 1 g) andovary and oviduct weight (± 0.1 g). The average size of ovulatedeggs within each S. australis individual shows little variation(Pecl 2001); therefore, three oviduct eggs were measured (alongthe longest axis) to estimate the average ovulated egg size foreach individual.

Oocyte stagingThe size frequency distributions of oocytes in the ovary weredetermined from a subsample (∼100–500 developing oocytes)of fixed ovary tissue of known weight. Oocytes in the subsamplewere teased apart to isolate individual oocytes and photographedusing a Sony Cybershot digital 4.1 megapixel camera (SanDiego, CA, USA). A vernier calliper was included in each phototo provide a scale when calibrating the oocyte measurements.From the photos, the length and width of each oocyte was deter-mined using image analysis software, ‘SigmaScan Pro V5.0.0’(Systat, Chicago, IL, USA), and the volume of each oocyte wascalculated. Given that the oocytes are not spherical, egg volumewas considered to be a better descriptor of size than the lengthor width of the egg by itself.

The developmental stage of each oocyte was estimated fromits volume; this was validated by histological sectioning ofoocytes across a size range from two females at each sample

time. Before sectioning, the oocytes were photographed for sizedetermination (macroscopic image), and then sectioned at 5 µm,stained with haematoxylin and eosin, and mounted under DPXresin. Images of each histological section were captured usinga microscope 10× mounted camera ‘Jenoptik ProgRes C14’(Jenoptic AG, Jena, Germany) and ‘Adobe Photoshop CE v8.0’(Adobe, San Jose, CA, USA) (microscopic image). From eachfemale, 10–50 oocytes were sectioned and staged microscopi-cally to allow for a comparison with the macroscopic staging.Using the macroscopic and microscopic images, stages of oocytedevelopment (Sauer and Lipinski 1990) were assigned to differ-ent size classes of oocytes. Histological slides of the ovaries wereexamined for the presence of post-ovulatory follicles.

Oocyte development occurs continuously after sexual mat-uration has been reached (Lewis and Choat 1993); therefore,the size frequency distributions of oocytes in each maturitystage overlap. To minimise incorrect identification of oocytestages based on oocyte size, the five oocyte stages from Sauerand Lipinski (1990) were collapsed into three new stages;immature oocytes (<10 mm3, corresponding to Stages 1 and 2oocytes), maturing oocytes (10–20 mm3, corresponding toStage 3 oocytes) and vitellogenic oocytes (>20 mm3, corre-sponding to Stages 4 and 5 oocytes).

Oocyte stagesOviduct ova size is an important indicator of how much energya female has allocated to the production of each individual egg(McGrath and Jackson 2002). Although average egg size variesbetween females, egg size within a female is very constant (Pecl2001). For some of the females sampled, average ova size was notestimated, therefore a separate regression analysis examined therelationship between the percentage frequency of vitellogenicoocytes and the average ova size among individuals to see ifbatch size is related to average egg size within a female.

A multiple regression was used to compare the percentage fre-quency of vitellogenic oocytes with ovary and oviduct weight andwith somatic condition. To determine if there were differences inthe mean percentage of vitellogenic oocytes and to determine ifa linear relationship through time existed, a regression analysiswith multiple values of the dependent variable for each valueof the independent variable was used (Sokal and Rohlf 1980).Data were examined for normal distributions and homogeneousvariances; transformations of the data were not necessary.

Ovary classesSeveral techniques, with varying degrees of complexity, can beused to assign an ovary to a specific reproductive maturity stage.In the present study, all females would be assigned to eitherStage 1 (because the most abundant oocyte stage present wasStage 1) or to Stage 5 (based on the most developed oocytestage and the macroscopic appearance of the ovary and oviduct).However, neither of these stages captures the dynamics of therelative frequency of different oocyte development stages in theovary. Therefore, the stage frequency distributions of oocytes inthe ovary were compared among all females sampled using aχ2-test of independence. This analysis was used to identifyfemales that had greater numbers of oocytes than expected(i.e. a peak) of a particular oocyte stage. Standardised residuals

990 Marine and Freshwater Research M. R. Kuipers et al.

Table 2. Estimates of egg deposition dates based on the appearance of the eggs

Stage Appearance of egg strands No. days eggs were depositedand embryos before SCUBA survey

1 Strands completely clear of biofouling 0–7and eggs yet to be clearly visible

2 Biofouling started and eggs clearly 8–21visible inside strand

3 Extensive biofouling and embryos 22–35have eye spots

were used to determine which oocyte maturation stages differedsignificantly between the observed and expected frequencies(Quinn and Keough 2003). Females were assigned to ovaryclasses based on whether they showed: no difference betweenthe observed and expected frequencies of any oocyte matura-tion stage (NP), a greater than expected number of immatureoocytes (Class I), a greater than expected number of maturingoocytes (Class II), or a greater than expected number of vitel-logenic oocytes (Class III). The numbers of females classifiedinto each of the three ovary classes were compared among themonths of capture using a χ2-test of independence.

The absolute size of the ovary and oviduct might not bea good indicator of morphological characters because it isoften heavily influenced by body size. Indices are also a poorway of presenting size-independent morphological measures(Moltschaniwskyj 2004), and instead residuals from a regres-sion of the morphological character, that is, ovary weight v.total bodyweight, were used as a size-independent measure ofovary and oviduct weight. Individuals with positive residualshave heavier ovaries for their body size, whereas individuals withnegative residuals have lighter ovaries for their body size. Resid-uals were also used to assess somatic condition (mantle weight v.total bodyweight). Biological characteristics (e.g. residuals ofsomatic condition, ovary size and oviduct size) of the femalesin each ovary class were compared using ANCOVA, with ovaryand body size as the covariates. The data used in the ANOVA,ANCOVA and regression analyses were checked for equal vari-ances using residual plots and plotted to check for normality;the data did not need to be transformed before analysis.

FecundityPotential fecundity, which was defined as the total number ofoocytes/eggs in a female at a given time, was estimated from thetotal number of oocytes in the ovary and the total number of eggsin the oviduct. The number of eggs in the oviduct was estimatedusing oviduct weight and average ovulated egg size (G. T. Pecl,unpubl. data):

Oviduct egg number = 135.30 + oviduct weight × 9.13+ max oviduct egg length (mm) × 16.87

The number of eggs in the ovary was estimated by scaling thetotal number of eggs in the known weight subset to the wholeovary:

Total no. oocytes in ovary = (weight of ovary/weight of subset)× no. oocytes in subset

The numbers of both oviduct eggs and ovary oocytes weresummed to give an estimate of potential fecundity. Using a mul-tiple regression, the relationship between potential fecundity andsquid weight and oviduct was determined. A separate regressionanalysis was used to examine the relationship between potentialfecundity and average oviduct ova size because of the differencein sample size.

Egg depositionEstimated densities of deposited eggs on the spawning groundprovided an estimate of egg production that could be used todescribe the reproductive output of the population at a given time.Temporal measures of egg deposition were then used to describewhat was going on throughout the spawning season at a popula-tion level. Each month during the spawning season, the density ofeggs deposited on the seagrass beds of eastern Great Oyster Baywas estimated at each of six sites using 20 10 m × 2 m transects(Moltschaniwskyj et al. 2002). Egg masses were allocated toone of three stages based on appearance (Moltschaniwskyj et al.2002). By combining the stage of the egg mass with the ratesof embryo development (Steer et al. 2003), it was possible toestimate when each egg mass had been deposited (Table 2). Thesize frequency distribution of egg masses was compared amongthe different deposition dates using a χ2-test of independence.

ResultsOocyte stagesThe percentage of vitellogenic oocytes in the ovary ranged from0.16 to 23.52%, and only 49% of this variability was explainedby oviduct weight (F3,169 = 2.90, P = 0.037). The percentage ofvitellogenic oocytes increased by 0.52% (s.e. = 0.03) with each1-g increase in ovary weight. Neither average oviduct ova sizenor somatic condition made a significant contribution towardsexplaining the variation in the percentage of vitellogenic oocytesin the ovary.

There was a significant difference in the mean percent-age of vitellogenic oocytes per female among sampling times(ANOVA; F10,162 = 4.59, P < 0.001). The percentage of vitel-logenic oocytes present in the ovaries declined by 4.8%(± 0.11 s.e.) per day during the spawning season (regression;F1,9 = 20.68, P < 0.001), and 69% of the variability in the per-centage of vitellogenic oocytes was explained by the date thefemales were caught (Fig. 1).

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Fig. 1. The linear relationship between the percentage frequency (± s.e.)of mature (Stage 4/5) oocytes and time during the spawning season.

Ovary classesIn the ovaries of every female caught, immature oocytes werepresent at much higher relative frequencies than the other oocytestages; however, when comparing all oocyte stages, peaks ofmaturing and mature oocytes were still clearly visible. Theoocytes in the ovary varied greatly in size (from <0.1 to 8 mm)and all females had most size classes present.There was evidencethat the oocyte stage frequency distributions differed among thefour different maturity stages (χ2

9 = 30.94, P < 0.001). Somefemales had more immature (Stage 1/2) oocytes and fewer matur-ing or mature oocytes than expected; these females were classi-fied as having Class I ovaries (Fig. 2). Females with relativelymore maturing (Stage 3) oocytes were classified as having ClassII ovaries, whereas females with more mature (Stage 4/5) oocyteswere classified as having Class III ovaries (Fig. 2). As immatureoocytes were present at much higher relative frequencies thanthe other oocyte stages, a peak in immature oocytes was expectedwhen running the analysis. The χ2 analysis showed which indi-viduals deviated from the expected. However, some females hada stage frequency distribution of oocytes that was not differentfrom that expected under the assumption of independence, andwere classed as having no dominant peak (NP) (Fig. 2).Across allfemales sampled throughout the spring spawning season, 46%showed a peak in the frequency of an oocyte stage, whereas theremaining 54% showed no dominant ovary stage (Fig. 3).

There were monthly differences in the frequency of femaleswith different ovary classifications (χ2

9 = 32.13, P < 0.001).Females caught during September had fewer individualswith Class I ovaries than expected, whereas those caught inOctober and December had more Class III and I ovaries,respectively, than expected (Fig. 4). No significant differences

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Fig. 2. The four ovary classes used to group females based on the stage fre-quency of oocytes. Each figure illustrates a frequency distribution of oocytesin an ovary with (a) no dominant peak (NP), and a high relative frequency of(b) Stage 1/2 oocytes (Class I), (c) Stage 3 oocytes (Class II) and (d) Stage4/5 oocytes (Class III). The ↑ and ↓ arrows indicate oocyte class percentagesthat have significantly more or less oocytes, respectively, in each stage thanexpected.

in the average oviduct residuals were found among the ovaryclasses (F3,54 = 0.51, P = 0.676).

FecundityThe potential fecundity of females caught during the spawn-ing season varied by a factor of 10, with between 1977 and20 302 oocytes and/or eggs per female. The potential fecundityof females was positively related to total weight (F2,98 = 16.40,P < 0.001), with 25% of the variation in fecundity explainedby total weight. Each gram of squid weight increased poten-tial fecundity by 7.66 (± 1.45) eggs. Oviduct weight did notsignificantly explain the variation in potential fecundity.

The average potential fecundity differed significantly amongdays (F7,93 = 2.43, P = 0.02), but the change over the spawningseason was not linear (F1,6 = 3.91, P = 0.10). As the ANOVAshowed a significant difference among means, but the post-hoc test was unable to determine where the difference was, weassumed that at least the smallest and the greatest mean valueswere significantly different from one another (Fig. 5); therefore,the average potential fecundity of females caught on 8 November2004 was 53% greater than the potential fecundity of femalescaught on 6 October 2004. No significant relationship was found

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Fig. 3. Percentage of females in each ovary class throughout the spawningseason.

between potential fecundity and average ova size (F1,99 = 1.45,P = 0.23).

Egg productionThere was a significant relationship between the average num-ber of eggs deposited across the spawning sites surveyed andthe average number of eggs in the oviduct of the females(F1,3 = 59.21, P = 0.005), and 94% of the variation in thedeposited eggs was explained by the number of eggs in the femaleoviducts.As the average number of eggs on the spawning groundincreased, the average number of eggs found in the oviductincreased by 143.36 (± 4.56). The potential fecundity of femalesdid not make a significant contribution toward explaining thevariation in eggs deposited on the spawning ground.

Egg masses of all sizes were observed throughout the season,although no large masses (>300 eggs) were present in Septem-ber (Fig. 6). There was evidence that the sizes of the egg massesdiffered in relative abundance throughout the spawning season(χ2

10 = 21.18, P = 0.020). Early in the season there were signif-icantly more small egg masses present than expected, whereasno large egg masses were present (Fig. 6). During the middleof the spawning season there was an increase in the number ofegg masses, but medium-sized egg masses (151–200 eggs) wererelatively more frequent. In December, late in the spawning sea-son, the total number of masses had declined and there weresignificantly more small egg masses than expected (Fig. 6).

Discussion

Sepioteuthis australis on the east coast of Tasmania aggre-gate on the same spawning grounds every spring/early summer,with females producing multiple batches of eggs and spend-ing a significant part of their adult life reproducing (Pecl 2001;Moltschaniwskyj and Steer 2004). The present study found that

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Fig. 4. Frequency of individuals in eachof the four ovary classes caught in Septem-ber, October, November and December.The ↑ and ↓ arrows indicate ovary classesthat had more or less females, respectively,than expected.

S. australis adopts a reproductive strategy whereby multiple dis-crete batches of eggs mature and are deposited throughout anindividual’s reproductive lifetime. We found large numbers ofovulated eggs in the oviduct, suggesting that females accumu-lated ovulated eggs in the oviduct and were not depositing smallnumbers of eggs frequently. Given that the biological and ener-getic requirements for trickle and batch spawning differ, thebiological characteristics of females, such as somatic condition,ovary size and oviduct size, should differ dramatically amongindividuals using the two strategies.As no significant differenceswere found between the biological characteristics of individu-als, it appears that these individuals were all using the samereproductive strategy. When comparing the oviduct weight offemales in the four different maturity classes, a cycle of matura-tion, ovulation and deposition was clearly evident (Fig. 7). Basedon the development of oocytes in the ovaries and the weight ofthe oviduct, individuals from the spawning population could beidentified as being at one of four stages within the cycle (Fig. 7).

Assigning spawning strategiesFemales seemed to recruit oocytes from a large group of imma-ture oocytes that were always more prevalent than matureoocytes. It is unclear whether all immature oocytes will even-tually mature and ovulate ready for deposition or if most of theimmature oocytes observed in the ovary will not mature and

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Fig. 5. The average potential fecundity (± s.e.) of females on each sam-pling occasion throughout the 2004 spawning season. Means with differentletters are significantly different from one another.

therefore will not be deposited (Maxwell and Hanlon 2000).A significant proportion (46%) of the females exhibited apeak in oocytes in one of the three oocyte development stages(immature/re-starting, maturing and mature). This suggests thatthese females were producing discrete batches of oocytes, andit is likely, based on the extensive time mature females spendmoving between spawning beds in the general spawning area(Pecl et al. 2006), that multiple batches were being developed.As these females spawn multiple times and the aggregation hadno defined synchrony, each individual on the spawning groundcould have been at a different reproductive stage at any giventime. In the present study, all females were collected in areaswhere egg deposition was occurring. Therefore, it is likely thatthe females caught were either about to deposit eggs, were caughtwhile actively depositing eggs or had just finished egg deposi-tion, which explains why most females caught (54%) fell intothe NP classification. As only females on the spawning bedswere caught during the present study, other components of thefemale population have been included. The females not includedmight be batch spawning individuals that have left the spawningground either to die or to prepare their next batch of oocytes forspawning. The rest of the females on the spawning grounds, notdepositing eggs, could possibly have been seeking a mate.

There was little evidence of synchronicity in oocyte matu-ration and ovulation at the population level, with females atall stages of oocyte batch production present throughout thespawning season. Individuals making up the aggregation arefrom different cohorts and have experienced different envi-ronmental conditions before arriving on the spawning grounds(Moltschaniwskyj and Pecl 2007). Therefore, spawning aggre-gations of squid might not necessarily consist of well-definedor stable populations; rather, new individuals with different

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0

Fig. 6. Frequency of egg masses of different size classes, small (0–150eggs), medium (151–300 eggs) and large (>300 eggs). The ↑ and ↓ arrowsindicate dates that had more or less, respectively, egg masses in each sizeclass than expected.

life-history characteristics might move into the spawning aggre-gation. As all individuals entering the spawning aggregationshowed variation in life-history characteristics (Pecl et al. 2004),it was not possible to assign similarly developed females togroups because numerous factors could be influencing the pro-duction of oocytes and the rate or timing of development. Forexample, juveniles foraging and growing during a time when theabundance of zooplankton is high might have a higher fecunditythan others in the population (Boletzky 1986), owing to rapidgrowth and a larger size.

Seasonal population variationAlthough there was no population synchrony, there were signif-icantly more females with a peak in mature oocytes present inOctober, indicating that individuals were either ready to ovulateor were in the process of ovulating with heavy oviducts. During

994 Marine and Freshwater Research M. R. Kuipers et al.

Heaviestoviduct

Lightestoviduct

Heaviestovary

Ovulating ova –

Not laying

Ovulation ending–

Starting to layeggs

Ovulated

Lightestovary

Laying eggs–

New oocytesdeveloping

–Fully laid

II

I

III

NP

Fig. 7. The proposed cycle of ovulation and egg laying (deposition) for female Sepioteuthis australis derivedfrom the relationship of the oviduct ovary and the percentage of vitellogenic oocytes. The bold text indicates theintermediate stages between the beginning and end of both laying and ovulating. Female squid were classifiedby the dominant oocyte stage in the ovary, immature oocytes (Stage I), maturing oocytes (Stage II), matureoocytes (Stage III) and no dominant stage (NP). Stage I: females with only immature oocytes present had thelightest ovaries, but had relatively heavy oviducts, suggesting that these females had finished ovulating, but hadnot yet deposited all their ovulated eggs. Stage II: females with a batch of maturing oocytes had relatively heavyovaries and relatively light oviducts, suggesting that a new clutch of oocytes was ready for ovulation. Stage III:females that have mature oocytes have the heaviest oviducts and the heaviest ovaries, suggesting that theseindividuals are ovulating oocytes, but have yet to start egg deposition. Stage NP: females that showed no signof a peak in any oocyte stage and are thought to be in the intermediate stage where ovulation, deposition andoocyte maturation occurred simultaneously, which was why both oviduct and ovary weights were intermediatein size.

the 2004 spawning season, most of the eggs were deposited frommid-October to mid-November (N.A. Moltschaniwskyj, unpubl.data), which could partly explain this peak. There was a reason-able correlation (r2 = 0.6–0.7) between the CPUE (catch per uniteffort) estimates of spawning biomass and the number of eggson the spawning ground (Ziegler et al. 2007). If this relationshipis accurate, high numbers of females would be found betweenmid-October and mid-November (because this was the periodwhere most eggs were deposited), with the number of femalesdecreasing after mid-November. Given that the percentage ofvitellogenic oocytes decreased, but the total numbers of oocytesin the ovaries increased, this suggests that females might have

been producing smaller batches of eggs more frequently as thespawning season progressed.

Individuals that might have previously spawned could beentering or returning to the spawning grounds for their consec-utive spawning events. These individuals might be less likely toproduce large batches of eggs, which occurs with hake, becauseprevious spawning reduces the number of eggs available forfuture spawning events (Osborne et al. 1999). All histologicalslides were examined for the presence of post-ovulatory folli-cles; however, none were found in the tissue that we examined.Therefore, determining the time interval between subsequentspawning events was not explored.

Southern calamary spawning strategy Marine and Freshwater Research 995

The large number of females with Class I ovaries in Decem-ber seems unusual as the spawning aggregation for S. australispeaks during September to December (Moltschaniwskyj andPecl 2007). However, the spawning aggregation is made upof a succession of microcohorts (Moltschaniwskyj and Pecl2007), indicating that new individuals constantly move into thespawning area. Therefore, it is possible that females caught inDecember were not necessarily spawning their last batch beforedeath, but might have been spawning one of their first batches.Why the spawning aggregation disappears after December is notknown. As batch spawning requires large amounts of energy,the timing of egg development might be related to food avail-ability, possibly explaining why spawning aggregations formduring spring/summer (Maxwell and Hanlon 2000; Jackson andMoltschaniwskyj 2001). Female S. australis derive their energyfor reproduction primarily from food not body stores (Ho et al.2004), therefore as the food abundance around the spawninggrounds decreases, individuals might spawn elsewhere.

Although new individuals constantly enter the spawningaggregation during the spawning season (Moltschaniwskyj andPecl 2007), the present study found that these individuals differin their reproductive characteristics, in particular, the percentageof vitellogenic oocytes in females decreased over the spawningseason. This suggests that although the population is consis-tently being renewed, the spawning capacities of the later cohortssteadily decrease as the spawning season progresses. Effectivebiological management of spawning aggregations might includelonger closures during the early stages of the spawning season,combined with shorter, less frequent closures as the season pro-gresses. This would allow individuals from all microcohorts tospawn before capture, and allow females present at the start of thespawning season and with greater spawning capacity to spawnfor longer.

Fecundity of batch spawnersAs female oviduct weight was positively related to the num-ber of eggs deposited on the spawning ground, we hypothesisedthat batches of ovulated eggs were not being held for long inthe oviducts, but were deposited soon after ovulation. Matureoocytes found in wild-collected squid might only represent themature egg production of one or several days (Bello and Deickert2003). Many mature oocytes in females could suggest that eggshave not been laid in the last few days, whereas very few matureoocytes might be indicative of an immediate post-spawning sit-uation, that is, females that have spawned a few hours beforecollection (Bello and Deickert 2003). Although there was a rela-tionship between squid weight and fecundity, only 25% of thevariation in fecundity among females was explained by their totalweight. The sample size (100) used in the regression was alsoreasonably high, thus increasing the regression’s capacity to finda significant relationship. The weak relationship between fecun-dity and squid weight, that is, between the number of matureoocytes and squid weight, suggests that the actual reproductiveoutput of smaller females might not be a function of body size.However, it is important to note that 75% of the variation infecundity was unexplained and other factors need to be consid-ered, in particular, the frequency of spawning events and previousegg batches, both of which are difficult to measure.

Potential fecundity is the total number of oocytes in a femaleat one given time and is not a good representation of true fecun-dity because it is not certain whether all oocytes continue todevelop for later fertilisation or if they remain undeveloped forthe egg laying period (Maxwell and Hanlon 2000). In addi-tion, the measurements of undeveloped oocytes in the presentstudy might have been underestimated as it was not possibleto measure very small oocytes (0.1–0.7 mm) because they weretoo fragile to separate from the stroma without being damaged.The continued production or presence of primary oogonia in theovaries of females that have spawned is not a consistent fea-ture of loliginid squid, with some species having no primaryoogonia present following spawning (Knipe and Beeman 1978;Lopes et al. 1997), although they might be present in others(Moltschaniwskyj 1995). In Loligo pealeii, the mean number oflaid eggs exceeds the number of remaining oocytes and eggsby a factor of three, showing how unreliable potential fecun-dity can be at estimating true fecundity (Maxwell and Hanlon2000). Alternatively, a large percentage of oocytes might neverbe intended for laying and potential fecundity might overestimatetrue fecundity in some species (Laptikhovsky and Nigmatullin1993). As females in the present study were producing eggsin batches, only batch fecundity can be estimated and whetherthe potential fecundity underestimated or overestimated the truefecundity cannot be determined.

Trends in egg depositionEgg deposition, like fecundity, differed during the spawning sea-son; it was greatest during the middle of the season and reduced atboth ends of the season. Relatively more small egg masses weredeposited at the start and end of the spawning season, probablyas small egg masses, suggestive of small batches. This wouldbe expected at the start and end of the season, when individualsare starting and ceasing egg deposition respectively. Seasonalchanges in spawning biomass will also play an important rolein egg deposition because fewer individuals are expected at thebeginning and end of the spawning season (Moltschaniwskyj andPecl 2003). Egg masses might have eggs from multiple mothers(van Camp et al. 2004); however, females only add to recentlylaid masses because no older masses have been observed withnewly deposited eggs (N. A. Moltschaniwskyj, pers. obs.). Thissuggests that high numbers of small egg masses at the beginningand end of the season indicate that few females were present onthe spawning grounds at these times or that the batches producedby these females are much smaller than the subsequent batches.Both female fecundity and the number of eggs deposited werereduced at the start and end of the spawning season, suggestingthat females were not ready to spawn as soon as they arrived atthe spawning grounds. It is possible that courtship and matingwere important processes taking place at the spawning groundsbefore egg deposition.

Identifying that S. australis deposits discrete batches of eggswith periods of time between egg deposition and egg maturationhas implications for both our understanding of energy allocationto reproduction and also to management of the fishery. However,we are still hampered by a lack of knowledge about the repro-ductive history of an individual, in particular, the number andsize of oocytes in each batch, as well as the number of batches

996 Marine and Freshwater Research M. R. Kuipers et al.

and the time between spawning events. Thus, following individ-uals throughout their lifespan will be necessary, either by activetracking of animals (Semmens et al. 2007) or laboratory-basedexperiments (Maxwell and Hanlon 2000).

AcknowledgementsWe are indebted toTy Hibberd and to the commercial fishers who volunteeredtheir assistance in the collection of animals. This research was supportedby the Australian Research Council (LP0347556), DPIWE, the TasmanianRecreational Fishery Trust and the Tasmanian Commercial Calamary Fish-ers.All squid were taken under DPIWE permit number 5021 and this researchcomplies with the current collection laws and code of animal ethics of theUniversity of Tasmania. Thanks also to the referees who provided valuablecomments and suggestions during the publishing process.

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Manuscript received 25 October 2007, accepted 12 August 2008