Reproductive Patterns and Energy Management Strategies of Females of the Fiddler Crab Leptuca Uruguayensis with Short Reproductive Seasons

Canadian Journal of Zoology

Reproductive patterns and energy management strategies of females of the fiddler crab Leptuca uruguayensis with short reproductive seasons

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2020-0129.R2

Manuscript Type: Article

Date Submitted by the 12-Oct-2020 Author:

Complete List of Authors: Marciano, Agustina; Consejo Nacional de Investigaciones Cientificas y Tecnicas, Instituto de Biodiversidad y Biología Experimental y Aplicada; Universidad de Buenos Aires Facultad de Ciencias Exactas y Naturales, DepartamentoDraft de Biodiversidad y Biología Experimental, Laboratorio de Biología de la Reproducción y el Crecimiento de Crustáceos Decápodos López-Greco, Laura; Consejo Nacional de Investigaciones Cientificas y Tecnicas, Instituto de Biodiversidad y Biología Experimental y Aplicada; Universidad de Buenos Aires Facultad de Ciencias Exactas y Naturales, Departamento de Biodiversidad y Biología Experimental, Laboratorio de Biología de la Reproducción y el Crecimiento de Crustáceos Decápodos Colpo, Karine; Consejo Nacional de Investigaciones Cientificas y Tecnicas, Instituto de Limnología Dr. Raúl A. Ringuelet, Universidad Nacional de La Plata

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Leptuca uruguayensis, fiddler crab, spawning pattern, number of Keyword: spawns per female, ovary and hepatopancreas reserves, capital breeding strategy, income breeding strategy

Reproductive patterns and energy management strategies of females of the fiddler crab Leptuca uruguayensis with short reproductive seasons

1. A. Marciano

Universidad de Buenos Aires, CONICET, Instituto de Biodiversidad y Biología

Experimental y Aplicada (IBBEA), Facultad de Ciencias Exactas y Naturales,

Departamento de Biodiversidad y Biología Experimental, Laboratorio de Biología de

la Reproducción y el Crecimiento de Crustáceos Decápodos, Buenos Aires,

C1428EGA, Argentina. E-mail: agustina.marciano@ bg.fcen.uba.ar

2. L. S. López Greco Universidad de Buenos Aires, CONICET,Draft Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA), Facultad de Ciencias Exactas y Naturales,

Departamento de Biodiversidad y Biología Experimental, Laboratorio de Biología de

la Reproducción y el Crecimiento de Crustáceos Decápodos, Buenos Aires,

C1428EGA, Argentina. E-mail: [email protected]

3. K. D. Colpo

Corresponding author

Instituto de Limnología Dr. Raúl A. Ringuelet, CONICET, Universidad Nacional de La

Plata, La Plata, 1900, Argentina

Boulevard 120 and 60, PB N° 712, La Plata, 1900, Argentina.

+54 221 4222775

Email: [email protected], [email protected]

Reproductive patterns and energy management strategies of females of the fiddler crab Leptuca uruguayensis with short reproductive seasons

A. Marciano, L.S. López Greco and K.D. Colpo

Reproduction is a costly process that depends on the management of available resources. Here, we aimed to understand the energetic strategies of females of the fiddler crab Leptuca uruguayensis (Nobili 1901), in a population with short reproductive seasons. For this, we developed an integrated approach to recognize the brooding time, spawning pattern modulated by female size, number of spawns per female, and content of reserves in the ovary and hepatopancreas. Based on the condition of the ovary and hepatopancreas,Draft the reproductive season was divided into three periods. In each of these periods, it was possible to record a spawning event, which was mainly represented by large females. Most of the females had one spawn during the breeding season, and only large females had two spawns, with an interval of approximately two months. We propose that L. uruguayensis presents a mixed capital-income breeding strategy associated with the female size and period of the reproductive season. We conclude that large females make the greatest reproductive effort for the population, because they can have two spawns, whereas medium and small females make a low contribution because they are still investing energy in somatic growth to increase fecundity in the next reproductive season.

Leptuca uruguayensis; fiddler crab; spawning pattern; number of spawns per female; ovary and hepatopancreas reserves; capital breeding strategy; income breeding strategy

Introduction

Reproduction is an essential process in an animal's life, which must be carried

out successfully to ensure the continuity of the species (Bell 1980; Salmon et al.

2001; Møller et al. 2010). This implies taking advantage of the environment and

minimizing risks (Reznick 1985; Barnes and Partridge 2003). Although species may

have different reproductive strategies, identifying the reproductive season and the

individuals of a population which reproduce contributes to predicting the quantity and

quality of the offspring produced (Reid 1987; Koivula et al. 2003; Harshman and

Zera 2007; Møller et al. 2010). The timing and length of the reproductive season are

affected mainly by the photoperiod and temperature. In crustaceans, for example, as

latitude increases, the reproductive season is usually restricted to the months with

highest temperature (Wehrtmann etDraft al. 2012; Faria et al. 2017), while, at low

latitudes, animals can reproduce continuously throughout the year and tend to have

more spawns per year (Lackey 1978; Kokita 2003; Castilho et al. 2007).

In female decapods, reproductive expenses are related mainly to

vitellogenesis and embryo care (Fernández et al. 2000; Taylor and Leelapiyanart

2001; Silva et al. 2007; Oliveira et al. 2011; Simpson et al. 2015; Bert et al. 2016),

two processes in which the hepatopancreas plays an important role, because it

contains the main glycogen reserve that supplies the energetic demands and

provides the ovary with essential nutrients for embryo development (Subramoniam

2011; Jimenez and Kinsey 2015; Wang et al. 2015; Colpo and López Greco 2018).

Proteins, lipids, and carbohydrates are fundamental to egg quality, which, in turn,

conditions progeny success (Holcomb et al. 2004), because these are structural

components and the substrate for the energy metabolism of oocytes and embryonic

tissue (Gardner 2001; García Guerrero et al. 2003; Rosa et al. 2005).

Another important issue in the energy investment for reproduction is how this energy is managed. Two main strategies called capital and income breeding have been proposed for different animals (Drent and Daan 1980; Berrigan 1991; Jönsson

1997; Brown and Shine 2002; Warner et al. 2008; Wessels et al. 2010; Bert et al.

2016; Williams et al. 2017). Capital breeders store energy that they will use during the reproductive season, whereas income breeders take the available energy from the environment at the time of reproduction (Jönsson 1997; Griffen 2017, 2018). The capital breeding strategy is more expensive, since producing reserve molecules has an additional energy cost. However, income breeders are most susceptible to environmental variations, because they directly depend on the food available

(Jönsson 1997; Griffen 2017, 2018). Several studies on this topic have been carried out in arthropods (Berrigan 1991; VarpeDraft et al. 2007; Zeng et al. 2014; Bert et al.

2016; Griffen 2018). In particular, brachyurans have been considered to be capital breeders, since they cover the costs of reproduction by means of an endogenous energy source (Ng et al. 2008; Zeng et al. 2014; Bert et al. 2016; Griffen 2018).

Nevertheless, some brachyuran species can present a mixed strategy (Alava et al.

2007; Brockerhoff and McLay 2011; Griffen et al. 2012; Zeng et al. 2014), usually having early spawns using accumulated reserves (capital strategy) and late spawns using the resources of the environment (income strategy). To do this, they depend on their physiological state and environmental conditions (Zeng et al. 2014), and those which manage to combine the strategies and use the advantages of both can increase the reproductive output of the population.

The fiddler crab Leptuca uruguayensis (Nobili 1901) inhabits temperate estuaries in Argentina, where its reproductive season is restricted to the four warmer months (Armendáriz and César 2006; Ribeiro et al. 2016; Colpo and López Greco

2017). In this region, the life expectancy of L. uruguayensis is two years (Spivak et

al. 1991), and thus individuals probably experience two reproductive seasons during

their lives. Although these reproductive patterns are known, there are other aspects

which remain unstudied or uncertain, and this hinders understanding of the

strategies used by the population to maximize the use of resources and ensure

successful offspring. Therefore, in the present study, we aimed to recognize the

different reproductive patterns of L. uruguayensis females, in order to understand the

energy management strategies in species with a narrow window for reproduction.

For this goal, we developed an integrated approach along the short reproductive

season of this fiddler crab to analyze the brooding time, spawning pattern modulated

by female size, number of spawns per female, and content of reserves in the ovary

and hepatopancreas. Considering Draftthat this population of L. uruguayensis has only

two short reproductive seasons during its life, we predicted that females manage the

energy budget to be able to have two or three spawns per reproductive season, and

postulated that multiple spawns are possible only if L. uruguayensis uses the

advantages of both the capital and income breeding strategies.

Material and Methods

Study area

The study was performed in Samborombón Bay, Río de la Plata estuary,

Argentina. The field work was carried out in a muddy sand area of approximately 3500

m2 in the intertidal zone of the Saladero canal (36°25'0.3”S – 56°57'11”W), in the

locality of General Lavalle, Buenos Aires province. The study area has about 25 m of

intertidal zone with low slope, which is uncovered every low tide (both spring and neap

tides). Leptuca uruguayensis inhabits the high intertidal zone, which extends over 8 to

10 m (Figure 1). In this estuary, the tides are semi-lunar and semi-diurnal, with amplitude of about 1 m in spring tides (Acha et al. 2008). The field work was performed during low tides on the days of neap tides.

Brooding time

The brooding time of L. uruguayensis was assessed during the 2017 - 2018 reproductive season. A total of 60 non-ovigerous adult females (carapace width -

CW > 6.5 mm; Hirose et al. 2013) were sampled on November 10th 2017 and

February 1st 2018. They were taken to the laboratory and were kept individually in plastic containers with 10 cm of sediment from the environment to allow females to burrow. The sediment was always moist at salinity between 12 and 20 ppm. The containers remained at room temperatureDraft (25 ± 1 °C) and summer photoperiod (14 h

L: 10 h D). The females were checked daily to record the day of spawning and of larval hatching. The interval between the two days was considered the brooding time.

Spawning pattern

The brooding time results were used to define the field sampling schedule to recognize the spawning pattern of L. uruguayensis. This allowed establishing an adequate time interval to carry out the field samplings and observe all the spawning events of the females. Sampling was performed every 14.7 ± 2.1 days from October

18th 2018 to February 27th 2019 (i.e. a total of nine sampling days; Table 1). Only the sampling of December 17th could not be performed due to weather conditions. On each sampling date, two trained persons collected ovigerous and non-ovigerous adult females randomly from the study area for 1 h. Since females of L.

uruguayensis remain in the burrow during the egg’s incubation period (Ribeiro et al.

2016; Colpo and López Greco 2017), we dug 40 cm deep to sample them. In the

field, the carapace width (CW) of the sampled females was measured with a caliper

and the proportion of ovigerous females was estimated from the total amount of adult

females (CW > 6.5 mm) in the population. On each sampling date, non-ovigerous

adult females were taken to the laboratory.

Three cohorts of females can be recognized in one reproductive season:

small females (< 10 mm CW), medium females (10 - 12 mm CW), and large females

(> 12 mm CW). Females that hatched first reached the first reproductive season of

their life as medium females, while females that hatched later reached their first

reproductive season as small females. Both female cohorts reach the second

reproductive season as large femalesDraft (Spivak et al. 1991). The proportion of

ovigerous females was also estimated for each female cohort.

Number of spawns per female

To estimate the number of spawns per female, adult females were confined to

in situ mesocosms and monitored throughout the reproductive season. The

mesocosms were 54 x 29 x 35 cm plastic baskets with 5 mm perforations on all

sides to allow the water flow produced by tidal cycles (Figure 1). On the first

sampling date (October 18th 2018), eight mesocosms were randomly distributed in

the high intertidal zone, where L. uruguayensis predominates. The mesocosms were

filled with 30 cm sediment from the environment and buried in the field (Figure 1).

On the second sampling date (November 2nd 2018), 55 adult females without

eggs were collected, measured, and individually identified using labeled plastic

markers with different colors glued on the carapace (Figure 1). On this date, only

medium and large females were found in the field; therefore, the number of spawns was recorded only for these cohorts. Seven or eight females, as well as five mature males for mating, were added to each mesocosm. The crab density in the mesocosms was similar to that in the field (i.e. 70 crabs.m2) (Colpo unpublished data). Each mesocosm was covered by a plastic mesh (2 mm2) to prevent the crabs from escaping (Figure 1). On each sampling date, the mesocosm covers were lifted and the sediment inside was dug and carefully checked for females. The identity of retrieved females and their ovigerous or non-ovigerous status were recorded. The crabs were kept in the mesocosms throughout the experiment.

Condition of the ovary and hepatopancreas

On every sampling date, theDraft non-ovigerous adult females taken to the laboratory, were sacrificed after being cold-anesthetized for 15 min and then dissected to determine the wet gonadosomatic index (GSI = wet gonad weight / wet female weight) and the wet hepatosomatic index (HSI = wet hepatopancreas weight / wet female weight). The ovarian development was categorized based on GSI values, as follows: developed ovaries had a GSI greater than 8, developing ovaries had a

GSI between 8 and 4, and incipient ovaries had a GSI lower than 4. In addition, the content of reserves of both organs was biochemically determined from the second to the ninth sampling date. Total proteins and total lipids were determined in both organs, whereas glycogen concentration was measured in the hepatopancreas.

To obtain the minimum critical tissue mass to perform the biochemical analyses, the ovaries and hepatopancreas from five crabs were pooled. Each of these pools was considered a replicate. The ovary and hepatopancreas of females were stored at −20 °C until the biochemical analyses. To determine total protein

concentration, samples of ovary and hepatopancreas were homogenized in 4 : 1

volume (μL) : weight (mg) with 50 mM Tris-HCl buffer (pH 7.5), and then centrifuged

at 10,000 g for 30 min in a refrigerated centrifuge (4 °C). Supernatants were diluted

with distilled water, as follows: 1 : 20 for the ovary and 1 : 3 for the hepatopancreas

(volume : volume). Total proteins were determined by the Coomassie blue dye

method, in a spectrophotometer at 595 nm (Bradford 1976). Bovine serum albumin

was used as standard. Values were expressed as mg total proteins/g tissue. For

total lipid determination, samples of ovary and hepatopancreas were homogenized in

20 : 1 volume (μL) : weight (mg) of a mixture of chloroform and methanol (2 : 1

volume : volume), then mixed and centrifuged with 0.9% NaCl to separate the lipid

fraction. Total lipids were determined by the sulfophosphovanillin method according

to Folch et al. (1957) modified by FringsDraft et al. (1972) and measured with a

spectrophotometer at 530 nm. Extra-virgin olive oil diluted with absolute ethanol was

used to build the standard curve. Values were expressed as mg of total lipids/g

tissue. For glycogen determination, samples of hepatopancreas were digested by

boiling with alkaline solution (KOH 30%). Saturated SO4Na2, absolute alcohol, and

centrifugation were used to achieve glycogen precipitation. The resulting pellet was

dissolved with enough distilled water. Glycogen was determined following the

method described by van Handel (1965) and measured with a spectrophotometer at

620 nm. Rabbit liver (Fluka®) was used to build the standard curve. Values were

expressed as mg of glycogen/g tissue.

Statistical analysis

The sizes of the females that had 0, 1, or 2 spawns in the mesocosms were

compared by means of a one-way analysis of variance (ANOVA). The proportion of

ovigerous females between the field and the mesocosms was compared by means of a generalized linear model (GLM). Because in the mesocosms we tracked only medium and large females, such comparisons were performed with field females of the same cohorts. Two fixed-effect factors were used : location (field and mesocosms) and sampling (each sampling day with data for both locations).

Binomial distribution and “logit” as link function were implemented (Zuur et al. 2010).

The variables of the ovary and hepatopancreas (GSI, HSI, total protein, total lipids and glycogen contents) were used in a non-metric multidimensional scaling

(nmMDS) to compare eight sampling days (from the second to the ninth sampling day) and detect variations throughout the reproductive season. In addition, a similarity percentage analysis (SIMPER) was performed to identify the most important variables which generateDraft the dissimilarity patterns observed among different sampling periods within the reproductive season. For both nmMDS and

SIMPER analyses, the Bray-Curtis coefficient was used as a similarity measure

(Clarke and Warwick 2001; Gotelli and Ellison 2004). A one-way ANOVA was performed to compare the ovary and hepatopancreas variables throughout the reproductive season.

For the ANOVA, the data were tested for normality and homoscedasticity with a QQ plot and Levene’s test, respectively. The Tukey’s test was applied when significant differences were found. All tests were carried out at the 95% significance level with RStudio Team (2015).

Results

Brooding time

Of the 60 females evaluated in the laboratory, only 15 became ovigerous. Of

these, six lost their eggs in the first 10 days, whereas, in the remaining nine females,

the clutch fully developed and hatched. The brooding time was estimated to be 21 ±

1.8 days.

Spawning pattern

A total of 684 females of L. uruguayensis were collected during the nine

samplings carried out. Of those, 552 were non-ovigerous and 132 were ovigerous.

These females were divided in three cohorts according to their sizes (small, medium,

and large females). The mean size and standard deviation for small females was 8.3

± 0.7 mm of CW, for medium females was 10.8 ± 0.5 mm of CW, and for large

females was 12.7 ± 0.8 mm of CW.Draft

On the first sampling date (October 18th 2018), we found no ovigerous

females in the population. The first ovigerous females were found on the second

sampling date, in early November (Figure 2). This confirms that we recorded the

beginning of the reproductive season. On the first four sampling dates (from October

18th 2018 to November 30th 2018), the percentage of small females did not exceed

5%. On the fifth sampling date (December 28th 2018), small females were found and,

at this moment, they represented 35% of adult females. On the sixth (January 14th

2019) and eighth (February 13th 2019) sampling dates, small females were

ovigerous (Figure 2). On the fifth (December 28th 2018), seventh (January 27th 2019)

and ninth (February 27th 2019) sampling dates, we found no ovigerous females in

the population (Figure 2).

Three spawning events were recorded in this reproductive season (Figure 3).

The first event occurred during the second and third sampling dates (November 02nd

and 30th), when 29.2% of the adult females were ovigerous, divided into 6.4%

medium females and 22.8% large females. The second spawning event was

observed on the sixth sampling date (January 14th 2019), when 55.4% of the females

sampled were ovigerous, divided into 18.7% small females, 11.2% medium females,

and 25.5% large females. Finally, the third spawning event was observed on the

eighth sampling date (February 13th 2019), when 30% of the female population was

ovigerous, divided into 8.6% small females, 4.3% medium females, and 17.1% large

females (Figure 3).

Number of spawns per female

The 55 females tracked in the mesocosms allowed us to estimate the number

of spawns that one female can haveDraft during the reproductive season. We found that

20% of the females (n = 11) never spawned, 31% (n = 17) had only one spawn, and

22% (n = 12) had two spawns during the reproductive season (Table 2). Additionally,

15 females (27%) died or were lost before ever spawning. The females that had two spawns were larger than those that had one or no spawns (one-way ANOVA, F =

10.59; df = 2; p < 0.001) (Table 2). For females that spawned twice, the average time between consecutive spawns was 58 ± 11 days. In the middle of the reproductive season, we recorded eight females that spawned once and molted in the mesocosms (i.e. we found labeled exuviae of females that were ovigerous on previous sampling dates). These females did not spawn again during the reproductive season and most of them were medium females (CW mean 11.05 ±

1.06 mm). This molt event was corroborated by the observation of some females with soft carapaces in the field.

The spawning events recorded for large and medium females in the field and

mesocosms were synchronous (Figure 4). The GLM showed no differences between

the proportion of ovigerous females recorded in the field and that of those recorded

in the mesocosms (LR Chiq = 1.9; df = 1; p = 0.168), indicating that the confinement

of the females in the mesocosm did not affect their reproductive performance (Figure

4). The GLM showed no difference in the proportion of ovigerous females between

the field and the mesocosms (LR Chiq = 116.5; df = 6; p <0.001), corroborating the

presence of three synchronized spawning events during the reproductive season

(Figure 4).

Condition of the ovary and hepatopancreas

The nmMDS analyses of theDraft ovary and hepatopancreas condition clustered the

sampling dates in three groups at 95% similarity (Figure 5). The first group included

the second, third and fourth sampling dates, which corresponded to the beginning of

the reproductive season (November 02nd, November 16th, and November 30th 2018).

The second group included the fifth and sixth sampling dates, which corresponded to

the middle period of the reproductive season (December 28th 2018 and January 14th

2019). Finally, the third group included the seventh, eighth and ninth sampling dates,

which corresponded to the end of the reproductive season (January 27th, February

13th, and February 27th 2019) (Figure 5). The SIMPER analysis showed that the main

variables that explained the dissimilarity among the three periods within the

reproductive season were the total lipids and glycogen content of the hepatopancreas

and the GSI. The total lipids and glycogen content of the hepatopancreas contributed

62% of the dissimilarity between the beginning and the middle of the reproductive

season, whereas the GSI and the content of total lipids in the hepatopancreas

contributed 54% of the dissimilarity between the beginning and the end of the reproductive season. The GSI and glycogen content in the hepatopancreas contributed 52% of the dissimilarity between the middle and the end of the reproductive season (Table 3).

The GSI decreased 37.1% from the beginning to the middle of the reproductive season and 79.8% towards the end of it (Table 4). At the beginning of the reproductive season, most of the females showed developed ovaries (63.2%), whereas in the middle, the proportions of females with developed, developing, and incipient ovaries were similar. Finally, at the end of the reproductive season, most of the females (85.3%) had incipient ovaries (Table 4). The total protein content of the ovaries was 37.8% higher at the beginning than at the end of the reproductive season, whereas the total lipid contentsDraft of the ovaries did not vary during the reproductive season (Table 4). The HSI decreased by 13.2% between the beginning and the middle of the reproductive season, and then increased 42.4% at the end of it. The hepatopancreas total protein content showed no differences throughout the reproductive season, whereas total lipids decreased by 34.6% from the beginning to the end of the reproductive season. The glycogen content of the hepatopancreas decreased 59.4% in the middle of the reproductive season, but then increased at the end of it (Table 4).

Discussion

The reproductive patterns identified for L. uruguayensis allow us to propose that energy management strategies diverged for females of different sizes. During the short reproductive season of this species, we recorded three population spawning events, in which large females contributed importantly. However, contrary

to our prediction, females did not have two or three spawns during the reproductive

season. Most females had only one spawn, and only some of the large females

spawned twice at an interval of approximately two months. However, our results

confirmed that double spawns in the short reproductive season of this species were

possible due to the mixing of the capital and income breeding strategies.

Based on the observation of underground mating in another tidal creek of

Samborombón bay, Ribeiro et al. (2016) suggested that L. uruguayensis could have

five or six spawns per reproductive season. Considering a reproductive season of

about 120 days and an incubation period of 21 days, it would be possible to reach

six spawns per female if they spawned a new clutch immediately after hatching their

larvae. However, all dissected ovigerous females, with eggs at different stages of

embryonic development, had an incipientlyDraft developing ovary (Colpo and Marciano

unpublished data), suggesting that the ovary is not mature after larval hatching. This

indicates that L. uruguayensis females become receptive to males only after enough

energy reserves are accumulated to ensure the viability of a new egg clutch (Christy

and Salmon 1984). Since L. uruguayensis, in temperate estuaries, participates in two

reproductive seasons throughout its life (Spivak et al. 1991), our results suggest that

females have no more than three spawns during their lifetime: one spawn in their

first reproductive season, and a maximum of two spawns in the second. This finding

evidences the high costs of reproduction in a short reproductive season.

The GSI, which is usually used as an indicator of the reproductive status of

populations (Janz et al. 2001; Hasek and Felder 2005; Shafi 2012; Parker et al.

2018), showed maximum values for L. uruguayensis at the beginning of the

reproductive season, which was related to a high amount of reserves in the ovary

and to a great proportion of developed ovaries. These findings coincide with those

obtained in several other species with short reproductive seasons in temperate environments, which reach the maximum GSI value and greatest amount of reserves in the ovary just before the start of the reproductive season (Kyomo 1988; López

Greco and Rodríguez 1999; Yamaguchi 2001; Aristizabal 2007; Pérez et al. 2010;

Naderi et al. 2018)

Our previous studies about this population of L. uruguayensis showed that, in late winter (before the beginning of vitellogenesis), the hepatopancreas of adult females reaches maximum values of HSI (8.3 ± 1.4) and glycogen content (14.8 ±

4.4 mg / g tissue), indicating a high level of stored energy (Colpo and López Greco

2017, 2018). During spring, when vitellogenesis begins, these parameters decrease and reach values similar to those here found at the beginning of the reproductive season (HSI = 3.8 ± 1.3 and glycogenDraft content = 3.2 ± 1.9 mg / g tissue). These results would indicate that the energetic requirements and nutrients demanded for ovarian maturation at the beginning of the reproductive season are derived mainly from hepatopancreas reserves, supporting the first spawning event carried out by medium and large females. Therefore, we can propose that, at the beginning of the reproductive season, L. uruguayensis medium and large females depend on the capital breeding strategy (Figure 6).

In the middle of the reproductive season, the indicative variables of ovarian maturity declined and reached intermediate values as a result of the first spawning event (Antunes et al. 2010; Fortes et al. 2011). Moreover, in this period, the HSI, glycogen, and total lipids of the hepatopancreas reached the lowest values, a fact that suggests a depletion of the female energy reserves. At the middle of the period, the second spawning event occurred, being the greatest of the reproductive season, probably because all female sizes were involved. Medium and large females that had

their first spawn during this period probably used the capital breeding strategy, as had

occurred at the beginning of the reproductive season. In contrast, large females that

had their second spawn probably shifted their reproductive strategy to income

breeding. We believe that these females use the reserves to have an earlier spawn in

the reproductive season (capital strategy), and then feed and get energy from the

environment until the ovary matures again to have a second spawn in the reproductive

season (income strategy). We suggest the use of income strategy because, during the

reproductive season, the energy reserves in the hepatopancreas (HSI and glycogen

content) never reached the levels recorded in Colpo and López Greco (2017, 2018) in

pre-reproductive periods (Figure 6). Small females cannot store much energy because

their size is a limitation for the accumulation of reserves (Stephens et al. 2009) and,

consequently, for the clutch size Draft (Hines 1992; Ramirez Llorda 2002; Henmi 2003;

Swiney et al. 2012). Probably, no small females were found at the beginning of the

reproductive season because they had juvenile sizes. During the spring and the

beginning of the reproductive season, the small females would be growing to reach

the middle of the season with larger sizes, and then have their first spawn. However,

the growth process is expensive (Cox et al. 2010) and the energetic reserve indices

(HSI = 3.4 ± 1.7) and gonadal development indices (GSI = 3.8 ± 3.3) of the small

females were low on the first sampling date when they were recorded (December 28th

2018) (Figure 6). These low values indicate that small females did not have enough

stored energy to support vitellogenesis, suggesting they used the income breeding

strategy to have one late spawn, by taking advantage of available resources from the

environment (Williams et al. 2017; Griffen 2018).

We did not expect molts during the short reproductive season of L.

uruguayensis, because we supposed that females would devote all the energy to

reproduction (Kobayashi and Archdale 2017) and because, in some brachyuran crabs, reproduction and molting are discrete processes (Adiyodi 1968; Raviv et al.

2008), especially considering the narrow window for reproduction. Most females that molted in the mesocosms were medium sized and belonged to the same female cohort that had only one spawn during the reproductive season. Therefore, medium females spawned early in the reproductive season, using the capital breeding strategy. After that, they molted and returned to the growing process (Figure 6) to reach larger size and increase their fecundity in the next reproductive season (Hines

1992; Ramirez Llorda 2002; Swiney et al. 2012).

The spawning event at the end of the reproductive season was produced primarily by small females that had their first spawn, and by large females that had their second spawn (mesocosm information).Draft Presumably, as we suggested before, these females followed an income breeding strategy to have their spawns. At the end of the reproductive season, most of the females in the population showed few signs of ovarian maturity. Usually, total lipid and protein reserves in the ovary decrease toward the end of the reproductive process (Antunes et al. 2010; Fatima et al. 2013; Sacristán et al. 2017). In the present study, we found that, at the beginning of the reproductive season, when the capital breeding strategy would be predominant, the protein content in gonads was higher than at the end of reproductive season, when the income breeding strategy would be prevalent, and that the lipid content remained stable throughout the time. This pattern might indicate that the amount of protein that females transfer to the eggs would vary according to the reproductive strategy used. This might result in decreased egg quality at the end of the reproductive season, as already recorded in other species of crustaceans that have successive clutches using the income energy obtained during the reproductive

process (Varpe et al. 2007; Bert et al 2016; Griffen 2018). Since most L.

uruguayensis females had finished the reproductive process after the middle of the

reproductive season, the energetic demand decreased, and the values of HSI and

glycogen content in the hepatopancreas increased at the end of the reproductive

season. However, total lipid reserves did not increase at this moment, probably

because they are more expensive to generate and take longer to accumulate

(Sánchez-Paz et al. 2006).

Based on all the above, we propose that L. uruguayensis presents a mixed

capital-income breeding strategy (Williams et al. 2017) for the use of reserves

related to the female size and the period of the reproductive season. At the onset of

their reproductive life, when they are young and still allocate energy for somatic

growth, small and medium femalesDraft have only one spawn during their first

reproductive season (Figure 6). In the second reproductive season of their lives,

large females do not spend energy in growing and can have their first spawn at the

beginning of the reproductive season, using the capital breeding strategy. After zoea

hatching, these large females leave the burrow, start eating to get energy and take

about 60 days to have a second spawn, using the income breeding strategy (Figure

6). The advantage of using a mixed breeding strategy, is that it allows minimizing the

costs of the specific features of each strategy, and increasing the reproductive output

of the population, optimizing all energetic resources (Varpe et al. 2009; Wessels et

al. 2010; Williams et al. 2017).

The results of this work evidence how crabs manage the available energy

when the window of optimal conditions to reproduce is narrow. Leptuca

uruguayensis females had fewer spawns than we expected during the breeding

season, a fact that shows the high costs of reproduction. However, they applied a

strategy that maximizes the use of resources to ensure a successful reproductive process.

Acknowledgments

This study is part of Agustina Marciano postgraduate scholarship in Consejo

Nacional de Investigaciones Científicas y Técnicas (CONICET) and PhD dissertation in Facultad de Ciencias Exactas y Naturales of Universidad de Buenos Aires (FCEN

- UBA), Argentina. LSLG received partial financial support from the Agencia Nacional de Promoción Científica y Tecnológica (PICT 2016 project 0759), CONICET (PIP

2015-2017- 11220150100544) and UBACYT (2018-2021-20020170100021BA).

We thank Gabriel Rosa (Departamento de Biodiversidad y Biología

Experimental, FCEN-UBA) and CarlosDraft Marciano for their help in the building of the mesocosms. We also thank all our colleagues from Instituto de Biodiversidad y

Biología Experimental y Aplicada (IBBEA) and Instituto de Limnología “Dr. Raúl

Ringuelet” (ILPLA) and collaborators from General Lavalle for field work support. We thank Liane Stumpf (IBBEA, CONICET-UBA) for her collaboration in the biochemical analysis. We thank the reviewers, which comments improved this manuscript.

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Table 1: Fiddler crab Leptuca uruguayensis sampling schedule of the 2018/2019 reproductive season in Samborombón Bay, Río de la Plata estuary, Argentina.

Sampling Date

1st sampling October 18th 2018

2nd sampling November 02nd 2018

3rd sampling November 16th 2018

4th sampling November 30th 2018

Sampling not done December 17th 2018

5th sampling December 28th 2018

6th sampling January 14th 2019

7th sampling January 27th 2019

8th samplingDraftFebruary 13th 2019

9th sampling February 27th 2019

Table 2: Number and average size of the fiddler crab Leptuca uruguayensis females

from the mesocosms that had 0, 1, or 2 spawns and time of occurrence in the

reproductive season.

3rd 4th 5th 6th 7th 8th 9th Sampling Nov Nov Dec Jan Jan Feb Feb dates 16 30 28 14 27 13 27

Number of CW (mm) n spawns per mean ± SD females female

0 11.35 ± 1.09a 11

6 x

1 11.61 ± 1.03a 4 Draft x

7 x

8 x x

2 x x 2 13.04 ± 0.74b 1 x x

1 x x

Note: Values (mean ± SD) with different letters are significantly different (p < 0.05).

Table 3: SIMPER analysis results indicating the contribution of the ovary and hepatopancreas variables that explained more than 50% of dissimilarity among the three periods within the short reproductive season of the fiddler crab Leptuca uruguayensis.

Periods within the Variable Variables reproductive season Contribution (%)

Hepatopancreas glycogen 36.2 Beginning and middle Hepatopancreas total lipids 26.2

Cumulative % = 62.4

GSI* 34.0 Beginning and end Hepatopancreas total lipids 19.9

Draft Cumulative % = 53.9

GSI 35.0 Middle and end Hepatopancreas glycogen 17.0

Cumulative % = 52.0

*GSI = gonadosomatic index.

Table 4: Comparison of ovary and hepatopancreas variables of the non-ovigerous

females of the fiddler crab Leptuca uruguayensis during the beginning (November),

middle (December and January), and end (February) of the reproductive season.

Periods within the reproductive season ANOVA Variables Beginning Middle End df F p n

GSI* 8.9±4.2a 5.6±4.6b 1.8±2.8c 2 66.3 <0.001 459

%Developed † 63.2 27.1 7.9 - - - - Ovary %Developing ‡ 16.8 33.6 6.8 - - - -

§ %Incipient 20 39.3 85.3 - - - - Draft Total proteins 108.3±49a 98.2±30.6ab 67.4±27.9b 2 4.7 0.014 49 (mg/g tissue)

Total lipids 43.3±17.1a 39.9±10.2a 42±11.6a 2 0.1 0.868 33 (mg/g tissue)

HSI|| 3.8±1.3b 3.3±1.5c 4.7±1.9a 2 26.9 <0.001 428

Total proteins 19.5±7.8a 20.4±5.6a 16.83±6.1a 2 0.9 0.401 45 (mg/g tissue)

Hepatopancreas Total lipids 55.5±15.5a 36.4±17.8ab 36.3±17.4b 2 5 0.016 25 (mg/g tissue)

Glycogen 3.2±1.9a 1.3±0.6b 2.3±1.4ab 2 8.4 <0.001 50 (mg/g tissue)

*GSI = gonadosomatic index.

†Developed ovary (GSI ≥ 8).

‡Developing ovary (8 > GSI ≥ 4).

§Incipient ovary (GSI < 4).

||HSI = hepatosomatic index.

Note: One-way ANOVA results are provided. Values (mean ± SD) with different letters are significantly different (p < 0.05).

Draft

Figure 1: Study area and mesocosms used to study the fiddler crab Leptuca

uruguayensis in Saladero canal, Río de la Plata estuary, Argentina. (a) General view

showing the extension of high intertidal zone (HIZ) inhabited by L. uruguayensis and

the arrangement of some mesocosms in the experimental area together with a

protective device. (b) View of a mesocosm buried and filled with sediment from the

environment with the tracked fiddler crabs. In the detail one male and one labeled

female. Scale bar = 10 mm. (c) View of a mesocosm covered by plastic mesh (2

mm2) held by a wooden frame, after incorporation of fiddler crabs.

Figure 2: Fiddler crab Leptuca uruguayensis spawning pattern. Percentage of

ovigerous and non-ovigerous females for each size class (carapace width in mm),

plotted for each sampling date. N-Ov,Draft number of non-ovigerous females and Ov,

number of ovigerous females for each sampling date.

Figure 3: Proportion of ovigerous females of each cohort of the fiddler crab Leptuca

uruguayensis found in the field throughout the reproductive season. Small females

(black; CW < 10 mm), medium females (gray; 10 - 12 mm CW), and large females

(white; CW > 12 mm). CW: carapace width.

Figure 4: Proportion of medium (10 - 12 mm CW) and large (CW > 12 mm) ovigerous

females of the fiddler crab Leptuca uruguayensis found in the field (dashed line) and

in the mesocosms (full line) for each sampling date. No differences were found in the

proportion of ovigerous females between the field and the mesocosms (p = 0.142).

Different letters indicate a significant variation in the proportion of ovigerous females

among the sampling dates (p < 0.05). CW: carapace width.

Figure 5: Non-metric multidimensional scaling (nmMDS) based on ovary and

hepatopancreas variables of the fiddler crab Leptuca uruguayensis. Three groups

were determined at 95% similarity (Bray Curtis coefficient): the first group clustered

the first three sampling dates (beginning of the reproductive season); the second

group clustered the fifth and sixth sampling dates (middle of the reproductive

season); and the third group clustered the last sampling dates (end of the

reproductive season).

Figure 6: Schematic summary of the lifetime of females of the fiddler crab Leptuca

uruguayensis and its management of energy and breeding strategies. The scheme is

divided into the periods of the annual reproductive cycle of L. uruguayensis in

temperate conditions (Colpo and LópezDraft Greco 2017, 2018). Post RS/LA indicates the

post-reproductive season and the low activity periods that occur mainly in autumn and

winter, Pre RS indicates the pre-reproductive season (spring) when vitellogenesis

begins, and RS indicates the reproductive season. There are three RSs, each divided

into three periods (the beginning (B), the middle (M) and the end (E)): the hatching

RS, in which L. uruguayensis hatch, the first RS, in which the adult stage begins, and the second RS, in which the reproductive life ends. Each line pattern indicates the different life periods and the process that probably demands the most energy allocation: larval period of approximately 34 days (Rieger 1996), growing period, and reproductive seasons, when the spawns occur by use of the capital or income breeding strategy. GSI and HSI represent, respectively, the mean and standard deviation of the gonadosomatic index and hepatosomatic index recorded on the sampling date previous to the spawning event, for each female cohort. High levels of stored energy were recorded in the hepatopancreas (HSI = 8.3 ± 1.4 and glycogen content = 14.8 ±

4.4 mg / g tissue) of medium-sized and large females in the periods before

vitellogenesis begins (Colpo and López Greco 2017, 2018). In the first RS, L.

uruguayensis can produce one only spawn. Females that hatched first reached the

first RS as medium-sized females, while females that hatched later reached the first

RS as small females. Both female cohorts reach the second RS as large females and

can produce one or two spawns during the second RS.

Draft

Figure 1: Study area and mesocosms used to study the fiddler crab Leptuca uruguayensis in Saladero canal, Río de la Plata estuary, Argentina. (a) General view showing the extension of high intertidal zone (HIZ) inhabited by L. uruguayensis and the arrangement of some mesocosms in the experimental area together with a protective device. (b) View of a mesocosm buried and filled with sediment from the environment with the tracked fiddler crabs. In the detail one male and one labeled female. Scale bar = 10 mm. (c) View of a mesocosm covered by plastic mesh (2 mm2) held by a wooden frame, after incorporation of fiddler crabs.

85x83mm (600 x 600 DPI)

Draft

Figure 2: Fiddler crab Leptuca uruguayensis spawning pattern. Percentage of ovigerous and non-ovigerous females for each size class (carapace width in mm), plotted for each sampling date. N-Ov, number of non- ovigerous females and Ov, number of ovigerous females for each sampling date.

181x162mm (600 x 600 DPI)

Figure 3: Proportion of ovigerous females of each cohort of the fiddler crab Leptuca uruguayensis found in the field throughout the reproductive season.Draft Small females (black; CW < 10 mm), medium females (gray; 10 - 12 mm CW), and large females (white; CW > 12 mm). CW: carapace width.

85x49mm (600 x 600 DPI)

Figure 4: Proportion of medium (10 - 12 mm CW) and large (CW > 12 mm) ovigerous females of the fiddler crab Leptuca uruguayensis found in the Draftfield (dashed line) and in the mesocosms (full line) for each sampling date. No differences were found in the proportion of ovigerous females between the field and the mesocosms (p = 0.142). Different letters indicate a significant variation in the proportion of ovigerous females among the sampling dates (p < 0.05). CW: carapace width.

85x50mm (600 x 600 DPI)

Draft

Figure 5: Non-metric multidimensional scaling (nmMDS) based on ovary and hepatopancreas variables of the fiddler crab Leptuca uruguayensis. Three groups were determined at 95% similarity (Bray Curtis coefficient): the first group clustered the first three sampling dates (beginning of the reproductive season); the second group clustered the fifth and sixth sampling dates (middle of the reproductive season); and the third group clustered the last sampling dates (end of the reproductive season).

85x59mm (600 x 600 DPI)

Figure 6: Schematic summary of the lifetime of females of the fiddler crab Leptuca uruguayensis and its management of energy and breeding strategies. The scheme is divided into the periods of the annual reproductive cycle of L. uruguayensis in temperate conditions (Colpo and López Greco 2017, 2018). Post RS/LA indicates the post-reproductive season and the low activity periods that occur mainly in autumn and winter, Pre RS indicates the pre-reproductiveDraft season (spring) when vitellogenesis begins, and RS indicates the reproductive season. There are three RSs, each divided into three periods (the beginning (B), the middle (M) and the end (E)): the hatching RS, in which L. uruguayensis hatch, the first RS, in which the adult stage begins, and the second RS, in which the reproductive life ends. Each line pattern indicates the different life periods and the process that probably demands the most energy allocation: larval period of approximately 34 days (Rieger 1996), growing period, and reproductive seasons, when the spawns occur by use of the capital or income breeding strategy. GSI and HSI represent, respectively, the mean and standard deviation of the gonadosomatic index and hepatosomatic index recorded on the sampling date previous to the spawning event, for each female cohort. High levels of stored energy were recorded in the hepatopancreas (HSI = 8.3 ± 1.4 and glycogen content = 14.8 ± 4.4 mg / g tissue) of medium-sized and large females in the periods before vitellogenesis begins (Colpo and López Greco 2017, 2018). In the first RS, L. uruguayensis can produce one only spawn. Females that hatched first reached the first RS as medium-sized females, while females that hatched later reached the first RS as small females. Both female cohorts reach the second RS as large females and can produce one or two spawns during the second RS.

181x91mm (300 x 300 DPI)