An Acad Bras Cienc (2020) 92(Suppl. 2): e20180560 DOI 10.

1590/0001-3675202020180560
Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences
Printed ISSN 0001-3765 I Online ISSN 1678-2690
www.scielo.br/aabc | www.fb.com/aabcjournal

BIOLOGICAL SCIENCES

Size at onset of sexual maturity in

Macrobrachium amazonicum (Heller, 1862)
phenotypes: an integrative approach

LUCAS R.P. PASCHOAL & FERNANDO J. ZARA

Abstract: The size at the onset of sexual maturity (SOM) was determined in four
hololimnetic populations of Macrobrachium amazonicum using morphometric,
physiological and functional criteria. Male prawns from two populations analyzed had
hypertrophied chelipeds and large body proportions, showing the presence of four
morphotypes, i.e. large-size phenotype (LS). However, the other two populations showed
smaller males without morphotypes, i.e. small-size phenotype (SS). The development
of sexual weapons in males modulated the mating system and SOM in this species. It
was verified that there is a synchrony between physiological and functional maturities
in SS males. On the other hand, functional maturity in LS males occurred after
physiological and morphological maturities. In females, we verified synchronization
between morphological and functional maturities. The results obtained indicated that,
in both sexes, physiological maturity anticipates the others criteria. There were no
differences between the sequential scheme of sexual maturity for female phenotypes,
being: physiological → functional ≅ morphological. However, the evaluation of sexual
maturity in males must take into account the phenotype of population, since that was
modulated by functional maturity. The sequential scheme for LS males is: physiological
→ morphological → functional, while for SS males it is: physiological = functional →
morphological.
Key words: Amazon River prawn, morphometric variation, size at maturity, reproduction.

INTRODUCTION Rica to Argentina. In Brazilian basins, it occurs in

estuarine and/or freshwater ecosystems from
Some prawn species of the genus Macrobrachium the state of Roraima to Paraná (Vergamini et al.
Spence Bate, 1868 can inhabit coastal and inland 2011, Pileggi et al. 2013).
waters (without marine influence), as is the case In estuaries, M. amazonicum has an
of the Amazon River prawn Macrobrachium amphidromous life history, where adults live
amazonicum (Heller, 1862). This species displays and reproduce in freshwater environments,
a great environmental adaptability and wide while their larvae develop in estuarine
intraspecific variability, with populations environments, returning to freshwater when
showing significant differences in morphological juveniles (Bauer 2013). In these environments,
and reproductive traits (Odinetz-Collart & Rabelo individuals of this species have higher
1996, Vergamini et al. 2011, Anger 2013, Hayd & dimensions (body size) than those recorded
Anger 2013, Augusto & Valenti 2016, Paschoal et for animals of inland populations. Furthermore,
al. 2019). The Amazon River prawn has a wide amphidromous females show high fecundity
distribution in the Americas, occurring from Costa values (maximum fecundity range: 1,344 - 7,417

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LUCAS R.P. PASCHOAL & FERNANDO J. ZARA SEXUAL MATURITY IN Macrobrachium amazonicum

eggs) when compared to females of inland species being variable between populations
populations (maximum fecundity range: 315 - (Pantaleão et al. 2012). Thus, it is necessary to use
4,264 eggs) (Maciel & Valenti 2009, Hayd & Anger several criteria to estimate the SOM, since this
2013, Pantaleão et al. 2018, Paschoal et al. 2019). information is crucial for the maintenance and
In addition, amphidromous populations have improvement of the Amazon River prawn stocks
a social hierarchy, where males with dominant (Moraes-Valenti & Valenti 2010). This species
morphotypes (GC1 and GC2) have selective is very important in artisanal fisheries and is
advantages over submissive morphotypes (TC highly consumed in northern and northeastern
and CC) (Moraes-Valenti & Valenti 2010, Ibrahim Brazil (Odinetz-Collart & Moreira 1993, Maciel
2011). However, some populations with such & Valenti 2009), and considered a species with
characteristics were recently recorded in lentic great potential for world aquaculture (New
environments of the Paraná Basin (southeastern 2005). Consequently, several studies have been
Brazil) (Pantaleão et al. 2014, Paschoal & Zara conducted in recent years in order to improve,
2017, 2018, Paschoal et al. 2019), indicating the maintain and increase the quality of stocks,
shift of amphidromy by the entirely freshwater larviculture and sustainable farming (Preto et al.
life cycle (i.e. hololimnia), due to the biological 2010, Henares et al. 2013, Maciel & Valenti 2014,
and physical impossibility of migrations to Dutra et al. 2016, Henry-Silva et al. 2015, Santos
estuaries (Anger 2013). et al. 2016).
The knowledge of the life history for different Despite its importance, information about
populations of M. amazonicum is fragmented sexual maturity in M. amazonicum is limited to
and has gaps to be filled as reproductive and morphometric relationships (relative growth)
morphological aspects, since it is focused on and the determination of morphometric
studies dealing with amphidromous populations maturity (see Odinetz-Collart & Rabelo 1996,
(Anger et al. 2009). In addition, the phenotypic Moraes-Riodades & Valenti 2004, Maciel &
plasticity of M. amazonicum populations is not Valenti 2009, Pantaleão et al. 2012, 2014). Thus,
yet fully understood (Anger 2013). Three types there is a gap related to physiological maturity
of phenotypes are recorded for the species: (i.e. maturation of the reproductive systems)
(a) large-size amphidromous populations, and for the species. Also, there were no studies
(b) large-size and (c) small-size hololimnetic analyzing functional maturity (i.e. ♂: capability
populations (Moraes-Riodades & Valenti 2004, to mate and copulate, ♀: to carry eggs) in M.
Vergamini et al. 2011, Pantaleão et al. 2012, 2014, amazonicum. In this context, the present study
2018, Paschoal & Zara 2017, 2018, Paschoal et al. analyzes the morphometric differences of four
2019). These phenotypic differences recorded for hololimnetic populations of M. amazonicum
this species are attributed to intrinsic factors showing distinct phenotypes and determines
(i.e. physiological and behavioral differences) the SOM in these populations, correlating
(Moraes-Riodades & Valenti 2004, Augusto & morphometric, physiological and functional
Valenti 2016) and/or the availability of nutritional criteria.
resources in different areas (Pantaleão et al.
2012, 2014).
The wide phenotypic and physiological
plasticity of M. amazonicum makes the size
at the onset of sexual maturity (SOM) of the

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MATERIALS AND METHODS subsamples were sexed by the (a) verification of

the secondary sexual characters: absence (♀) or
Sampling
presence (♂) of the appendix masculina on the
Monthly samples were collected from October
endopodites of the second pair of pleopods, (b)
2014 to December 2015, in Grande River (Paraná
verification of the positions of the gonopores:
Basin) at four sampling sites. Two points in the
openings in the coxopodites of the 3° (♀) or 5°
Marechal Mascarenhas de Morais Hydroelectric
(♂) pair of pereiopods, and (c) dissection of the
Power Station (HPS) reservoir - municipalities of
reproductive organs: ovaries (♀) and testes (♂)
Cássia (CAS - 20° 30’ 53.6’’ S, 46° 50’ 16.4’’ W) and
(Paschoal & Zara 2017).
São João Batista do Glória (SBG - 20° 39’ 43.5’’ S,
46° 32’ 5.8’’ W); another two points in the Furnas Measurement, morphotyping and
HPS reservoir - municipalities of Carmo do Rio morphometric maturity
Claro (CRC - 20° 57’ 20’’ S, 46° 9’ 14’’ W) and São
The specimens of M. amazonicum had body
José da Barra (SJB - 20° 43’ 10.6’’ S, 46° 11’ 26.4’’
structures measured with an analogic caliper
W) in the state of Minas Gerais, southeastern
(0.02 mm), according to Moraes-Riodades
Brazil.
& Valenti (2004) and Pantaleão et al. (2012,
Prawns were collected by active sampling,
2014): carapace length (CL), total length (TL),
a sieve (60 cm in diameter and 1.25 mm mesh)
width of the second abdominal pleura (2° PL),
was swept through marginal vegetation and
abdomen length (AL), major cheliped length
macrophyte banks for 30 minutes by one person;
(MCL), propodus length (PPL) and dactyl length
and passive collection, where six baited-traps
(DCL) (Fig. 1). For the measurement of MCL, PPL
(85 cm length x 35 cm width/height) were placed
and DCL, the right cheliped of the animals was
near the margin (0.5 - 2 m), baited with small
assessed. The fresh weight (W) of each individual
pieces of beef liver and removed after four
was obtained with an analytical scale (± 0.0001
hours. Immediately after capture, the animals
g).
were anesthetized by chilling (ten minutes in
Male prawns were visually divided into four
ice) and preserved in 70% ethanol (except
morphotypes: translucid claw (cheliped) (TC),
for animals used in histological analysis that
cinnamon claw (CC), green claw 1 (GC1) and green
were kept alive). After that, they were placed in
claw 2 (GC2). These morphotypes were classified
properly labeled bottles and transported to the
according to Moraes-Riodades & Valenti (2004)
laboratory.
and Pantaleão et al. (2014), based on size and
All animals collected during the monthly
color of the chelipeds, angles of the spines on
samplings were counted, and subsamples
the carpus and propodus and the pubescence
were obtained following Wenner et al. (1991): if
on dactyls. Subsequently, principal component
N ≤ 80 individuals were collected, all animals
analyses (PCA’s) were performed to separate
were analyzed; when 80 < N < 160 individuals
different morphological groups (morphotypes)
were collected, 80 individuals were randomly
in males for each population, using all body
selected to be sexed and measured. If 160 <
dimensions measured (log10) in these animals
N < 320 were collected, 50% of the specimens
(Sampedro et al. 1999).
were checked; and in the case of samples with
The morphometric maturity was estimated
N ≥ 321 individuals, 25% of the animals were
by the relative growth patterns of the species,
selected. Individuals randomly selected from the
which was determined by allometric equations

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Figure 1. General scheme for prawn size dimensions used in the morphometric analysis in Macrobrachium
amazonicum (modified from Pantaleão et al. 2014). 2° pl, width of the second abdominal pleura. al, abdomen
length. cl, carapace length. dcl, dactyl length. mcl, major cheliped length. ppl, propodus length. tl, total length.

Y = aXb (Huxley 1950). These equations were were performed with the software R version 3.3.1.
adjusted, having as independent variable (X) the (R CORE TEAM 2016).
CL and relating it to the other body dimensions
of the animal (dependent variables, Y). Values Histology and physiological maturity
of the allometric constant (b) were tested using For the determination of the physiological
the t-test, as H0:b = 1 (or 3 in the case of weight), maturity, all males and females of the subsamples
and used to determine the growth patterns of of each population had their reproductive
a specific body part in relation to CL. Equations systems dissected. The macroscopic morphology
were subsequently linearized (logy = loga* logx of the reproductive systems was analyzed under
- b). The morphometric relationships that stereomicroscope. The physiological SOM in
presented differences in the growth patterns the different populations was established by
were submitted to a non-hierarchical analysis the smallest CL recorded for the animals that
of K-means clustering, minimizing the variability reached the following criteria: (a) males with
within the groups and maximizing the variation spermatozoa in their testes and vasa deferentia
between them. The result of this analysis was (VD), and (b) females with oocytes in exogenous
refined by applying a discriminant analysis. The or secondary vitellogenesis (with yolk granules)
inflection point between the groups identified (Viau et al. 2006, Poljaroen et al. 2010, Soonklang
by the discriminant analysis was considered as et al. 2012, Zara et al. 2012, 2013, Nascimento &
the morphometric SOM, i.e. the smallest adult Zara 2013). The presence of spermatozoa in the
in population (Sampedro et al. 1999, Pantaleão testes and VD, as well as that of vitellogenic
et al. 2012). After the determination of groups, oocytes in the ovarian lobes, was verified by
differences between slopes (b) and intercepts smears of these structures (obtained from
(a) of lines at each development stage were animals preserved in ethanol) under light
compared using Covariance analysis (ANCOVA) microscope (Cuzin-Roudy & Amsler 1991,
(α = 0.05). All calculations and statistical analyzes Paschoal & Zara 2017, 2018). In order to confirm

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the smears results, ten recently-collected 26 °C (± 0.5). For CAS and SBG populations,
individuals of both sexes from each population showing the presence of males with submissive
showing different macroscopical stages of sexual and dominant morphotypes (see below), and
development were anesthetized by chilling (-20 nocturnal and cryptic habits (L.R.P. Paschoal
°C / 5 min.) and dissected. After the dissection, & F.J. Zara personal data), the LS prawns were
the animals had fragments of their reproductive observed in the field for six hours (18:00 to 0:00
systems fixed in 4% buffered-paraformaldehyde h) at each monthly sample, for observation of
(24 hours). Subsequently, they were washed pair formation and copulation. For nocturnal
twice in the same 0.2 M sodium phosphate observations, LED spotlights (up to 5 m in
buffer (pH 7.2), dehydrated in an increasing range) were positioned near to the margins of
series of ethanol (70 to 95%), and embedded the aquatic environments. When the copulation
and included in glycol-methacrylate historesin success was verified, the pairs were immediately
Leica®. Blocks were cut in 4-5 μm sections with captured with a hand net (0.13 mm mesh). All
a rotary microtome. After the microtomy, the four morphotypes were examined with regard to
slides were stained with hematoxylin and eosin mating behavior and reproductive probabilities.
(H&E) for general histological description (Zara After the evaluation of the mating behavior in
et al. 2012, 2013). the populations, the functional SOM in males
was established by the smallest CL recorded for
Functional maturity the individual captured, showing the necessary
For M. amazonicum females, functional maturity characteristics for the successful copulation.
was determined by the smallest CL recorded The types of mating systems observed in M.
for ovigerous females captured in the field for amazonicum were analyzed and classified
each population (Viau et al. 2006). In males, according to Correa & Thiel (2003).
functional maturity was considered as the
ability to copulate and transmit ejaculate (Elner
& Beninger 1995). For this purpose, animals of RESULTS
the SJB and CRC populations were analyzed by Morphometric analysis, morphotypes and
ad libitum sampling in the laboratory (one set morphometric maturity
per population), in order to verify the mating A total of 14,375 individuals were collected during
behavior and copulation. Due to the gregarious the 15 months analyzed at the four sampling
behavior of these SS populations (L.R.P. Paschoal sites. Subsamples containing 5,335 animals were
& F.J. Zara personal data), 90 prawns of each analyzed: CAS population - 307 ♂ and 1,276 ♀, SBG
population (N: 180) were kept for a month in population - 664 ♂ and 559 ♀, SJB population -
six aquariums with dark bottoms (45 cm x 25 1,090 ♂ and 553 ♀, and CRC population - 441 ♂
cm x 30 cm), with basalt gravel as substrate and and 445 ♀. Table I summarizes the morphometric
macrophytes and fragments of rocks for shelter. variables measured for all individuals. Females
Each aquarium contained 15 females and 15 were larger and heavier than males in all
males. Male prawns had their cephalothorax populations, and they also possess larger body
marked with a mixture of fast-drying glue gel structures. The only exception was the chelipeds
(Super Bonder Power Flex Gel ®) and silver and their articles (propodus and dactyls) in
purpurin powder. Prawns were acclimated and males of CAS and SBG populations (Table I).
conditioned to a 12 hour photoperiod and The presence of morphotypes in males of these

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Table I. Mean (± standard deviation - SD), minimum (Min) and maximum (Max) values of the analyzed
morphometric variables of male and females of Macrobrachium amazonicum in populations with large (LS) and
small (SS) size phenotypes. 2° PL, width of the second abdominal pleura. AL, abdomen length. CAS, Cássia. CL,
carapace length. CRC, Carmo do Rio Claro. DCL, dactyl length. MCL, major cheliped length. PPL, propodus length.
SBG, São João Batista do Glória. SJB, São José da Barra. TL, total length. W, fresh weight.

    Males Females

Population
Variables Mean SD Min Max Mean SD Min Max
(phenotype)
CL (mm) 13.05 5.08 3.60 24.90 14.80 3.41 5.10 28.70
TL (mm) 42.10 14.00 12.10 76.40 48.23 10.13 17.20 85.90
AL (mm) 22.43 7.10 6.70 40.50 25.79 5.23 10.20 43.70
CAS 2° PL (mm) 5.08 1.73 1.30 9.22 7.06 1.75 2.00 12.10
(LS) MCL (mm) 31.63 20.31 10.20 108.90 28.33 8.16 7.00 79.40
PPL (mm) 8.20 7.24 1.80 39.00 6.24 2.27 1.30 25.20
DCL (mm) 3.91 3.39 0.80 19.80 2.95 1.06 0.90 11.90
W (g) 1.685 1.694 0.018 8.668 1.979 1.217 0.072 10.045
CL (mm) 9.02 2.56 4.20 23.50 10.59 3.08 4.50 19.10
TL (mm) 32.45 7.56 15.30 68.80 36.96 9.68 11.90 63.00
AL (mm) 17.72 3.90 8.10 34.70 20.10 5.20 8.80 33.60
SBG 2° PL (mm) 3.83 0.96 1.75 8.10 5.02 1.82 1.50 9.80
(LS) MCL (mm) 18.02 9.56 7.30 87.60 19.64 6.44 6.55 40.40
PPL (mm) 3.95 3.23 1.20 28.80 4.35 1.72 1.90 10.50
DCL (mm) 1.86 1.55 0.70 12.90 2.00 0.79 0.80 4.90
W (g) 0.586 0.629 0.039 6.001 0.967 0.786 0.043 4.581
CL (mm) 6.90 1.05 3.70 11.30 8.15 1.24 4.10 13.40
TL (mm) 25.33 3.52 11.90 40.70 28.55 4.34 10.09 48.90
AL (mm) 13.98 1.79 6.30 21.30 15.60 2.27 8.60 21.60
SJB 2° PL (mm) 2.89 0.44 1.30 4.70 3.90 0.88 1.80 7.60
(SS) MCL (mm) 13.32 2.36 5.60 28.20 15.39 2.63 5.10 23.30
PPL (mm) 2.72 0.44 1.50 5.40 3.33 0.56 1.40 5.10
DCL (mm) 1.27 0.20 0.70 2.20 1.49 0.25 0.65 2.70
W (g) 0.222 0.100 0.035 0.967 0.358 0.160 0.051 1.829
CL (mm) 7.31 1.14 4.10 10.50 8.65 1.72 4.10 14.30
TL (mm) 26.11 3.94 15.10 37.20 30.07 5.73 14.50 43.80
AL (mm) 14.55 2.43 8.20 26.80 16.43 3.14 8.00 24.00

CRC 2° PL (mm) 3.10 0.53 1.70 4.45 4.18 1.13 1.60 7.00
(SS) MCL (mm) 13.41 2.60 2.50 20.20 15.79 3.62 7.50 24.40
PPL (mm) 2.83 0.50 1.00 4.60 3.43 0.76 1.50 5.55
DCL (mm) 1.32 0.23 0.70 2.00 1.56 0.37 0.70 3.15
W (g) 0.263 0.117 0.042 0.846 0.446 0.244 0.046 1.345

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populations was confirmed by PCA’s, resulting in PCA’s (Fig. 2). Due to this, it was possible to
in a clear separation of morphological groups verify the presence of two phenotypes, referred
—
(Supplementary Material - Fig. S1). The first two here as large-size phenotype (LS) in CAS (CL = ♂:
—
axes of PCA’s retained most of the data variability 13.05 ± 5.08, ♀: 14.80 ± 3.41 mm) and SBG (CL = ♂:
and provided good explanations (CAS: 98.05%, 9.02 ± 2.56, ♀: 10.59 ± 3.08 mm) populations, and
—
SBG: 97.92%, SJB: 89.85% and CRC: 87.35%). Major small-size phenotype (SS) for SJB (CL = ♂: 6.90 ±
—
cheliped length (MCL) was the morphometric 1.05, ♀: 8.15 ± 1.24 mm) and CRC (CL = ♂: 7.31 ± 1.14,
variable with the largest contribution in Axis 1 ♀: 8.65 ± 1.72 mm) populations.
for CAS and SBG populations, while for SJB and For males of LS populations, the
CRC populations it was the total length (TL) relationship that best demonstrated a clear
(Appendix I). Linear regressions were adjusted difference between juveniles and adults, and
using the variable with the greatest contribution separated the morphotypes was MCL vs. CL. The

Figure 2. Dispersion of points for the morphometric relationship mcl vs. cl for Macrobrachium amazonicum males
in large-size phenotype populations (cas and sbg), showing the separation of morphotypes and the size at onset
of morphological maturity. For the small-size phenotype populations (sjb and crc) the morphometric relationship
tl vs. cl shows the morphological maturity, without the presence of morphotypes. cas, Cássia. CC, cinnamon claw.
cl, carapace length. crc, Carmo do Rio Claro. GC1, green claw 1. GC2, green claw 2. Juv, juveniles. Mat, matures. mcl,
major cheliped length. sbg, São João Batista do Glória. sjb, São José da Barra. TC, translucid claw. tl, total length.

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morphometric SOM in LS males was estimated CRC population these rates are quite similar
at 8.40 mm CL for CAS population, and at 7.40 (Appendix II). The allometric levels of these
mm CL for SBG population (Fig. 2 and Table II). populations are similar, showing that adult
The ANCOVA results showed that there were animals tend to invest less energy for body
significant differences for the intercepts (a) in all growth than juveniles (Appendix III).
morphotypes of LS populations. However, only Similar to LS males, LS females also
the morphotypes CC vs. GC1 in both populations invested energy on growth of the chelipeds.
showed differences in slopes (b) indicating that The relationship that best demonstrated the
the other morphotypes have similar relative differences between juveniles and adults was
growth rates when compared to each other MCL vs. CL. The morphometric SOM was recorded
(Appendix II). Additionally, it was possible to at 10.50 mm CL in CAS population, and at 10.30
demonstrate that only the GC1 morphotype mm CL in SBG population (Fig. 3 and Table II). The
showed positive allometry for MCL vs. CL results of the ANCOVA’s showed that for the SBG
relationship (Appendix III). Both LS populations females, the growth rates for chelipeds and their
showed similar relative growth parameters and articles are divergent along maturation, whereas
allometric levels, demonstrating the investment for CAS females the allometric coefficient rates
in sexual weapons (chelipeds and their articles) are similar (Appendix IV). Adult females of both
(Appendices II and III). On the other hand, in LS populations showed positive allometry for
SS males without morphotypes, the TL vs. CL chelipeds and their articles (Appendix V). In
relationship demonstrated the morphometric females of SS populations, the relationship that
SOM at 5.30 mm CL in SJB population, and at best demonstrated the sexual maturity was 2°
6.30 mm CL in CRC population (Fig. 2 and Table PL vs. CL. The morphometric SOM was recorded
II). The results of ANCOVA’s show that in SJB at 7.30 mm CL in SJB population, and at 8.30
population, immature and mature SS males mm CL in CRC population (Fig. 3 and Table II).
have different relative growth rates for almost In both populations, the body growth (TL) is
all morphometric relationships, whereas in similar between immature and mature females,

Table II. Morphometric (Morp.), physiological (Phys.) and functional (Func.) maturities values in both sexes of
Macrobrachium amazonicum in large-size (LS) and small-size (SS) phenotype populations, as well as the number
of ovigerous females captured under to the size estimated for morphometric maturity (OVG). CAS, Cássia. CRC,
Carmo do Rio Claro. N, number of animals. SBG, São João Batista do Glória. SJB, São José da Barra.

Sex Population (phenotype) N Morp. Phys. Func. OVG

CAS (LS) 307 8.40 5.70 14.40 -

SBG (LS) 664 7.40 4.60 15.20 -
Male
SJB (SS) 1,090 5.30 3.70 3.70 -
CRC (SS) 441 6.30 4.00 4.00 -
CAS (LS) 1,276 10.50 6.70 9.40 6
SBG (LS) 559 10.30 6.70 9.40 2
Female
SJB (SS) 553 7.30 5.00 7.10 1
CRC (SS) 445 8.30 5.00 7.40 12

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however they exhibit differences in abdominal Physiological maturity

pleura width (2° PL) growth rates during the The smallest size at the onset of physiological
maturation (Appendix IV). Adult females of the (histological) maturity recorded in LS males
SS populations presented positive allometry was 5.70 mm CL in CAS population, and 4.60
for the width of the second abdominal pleura mm CL in SBG population, whereas for the SS
(2° PL), while in the LS populations, this males it was 3.70 mm CL in SJB population,
condition was registered only in females of the and 4.00 mm CL in CRC population (Table II).
SBG population. In CAS population, females No anatomical or histological differences were
showed negative allometry for this relationship observed in the male reproductive system
(Appendix V). for studied populations or between the four
morphotypes, except for CC males that show a
very low concentration of spermatozoa in their

Figure 3. Dispersion of points for the morphometric relationship mcl vs. cl in immature and mature females of
Macrobrachium amazonicum in large-size phenotype populations (cas and sbg), and 2° pl vs. cl for small-size
phenotype populations (sjb and crc). 2° pl, width of the second abdominal pleura. cas, Cássia. cl, carapace length.
crc, Carmo do Rio Claro. Juv, juveniles. Mat, matures. mcl, major cheliped length. sbg, São João Batista do Glória.
sjb, São José da Barra.

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testes (Supplementary Material - Fig. S2). In M. easily noted in the distal region of the VD, the
amazonicum, the reproductive system consists ejaculatory ducts (ED) (Fig. 4e-f ). This final
of a pair of testes and vasa deferentia (VD) portion of VD is much dilated (i.e. bulbs of the
connected to the gonopores of the coxopodites ED), which have thicker musculature than the
of the fifth pair of pereiopods (Fig. 4a-b). The other regions (Fig. 4e-f ) and the androgenic
testes exhibit thick anterior lobes arranged over glands are arranged perpendicularly to the
the hepatopancreas or perigastric organ (Fig. 4a), muscular layer on ED (Fig. 4e). These glands are
while the posterior lobes are thinner, located well-developed in mature animals, whereas in
below the heart. These organs are connected immature animals they are extremely reduced
to the VD, which are arranged laterally in the or absent.
cephalothorax, connecting to the gonopores of The smallest size at the onset of
the fifth pair of pereiopods (Fig. 4b). In mature physiological maturity recorded for females of
animals, the testes and VD are whitish and quickly the two LS populations was 6.70 mm CL, while for
identified macroscopically (Fig. 4a-b). These the two SS populations it was 5.00 mm CL (Table
organs in immature animals are thin, colorless II). The physiological maturity is marked by the
and cannot be distinguished macroscopically. colorimetric and volumetric transformation of
Testes smears from immature animals showed the ovaries throughout their development and
only spermatogonia and the spermatozoa were progressive accumulation of yolk granules in the
absent in VD. In physiologically mature males, it cytoplasm of oocytes (Fig. 5a-i). The immature
is noted by histology that the testes are formed ovaries are colorless or translucent before
by several seminiferous tubules grouped in reaching physiological maturity (Fig. 5a). These
lobules with cells in spermatogenesis and organs are characterized by the presence of
spermiogenesis (Fig. 4c-d), and the VD are many oogonia, previtellogenic oocytes (meiosis)
filled by secretion and spermatozoa (Fig. 4e-f). and oocytes in primary vitellogenesis without
Testes smears and histology in mature animals mature yolk granules (Fig. 5b-c). At this stage,
show typical tack-shaped spermatozoa with a the oocytes in primary vitellogenesis show
very prominent spike filling the seminiferous a large rounded nucleus with few blocks of
tubules, whereas only a small peripheral area heterochromatin dispersed and one or two
of the tubules shows spermatogonia (Fig. 4c- well evident nucleoli. The cytoplasm is strongly
d). The seminiferous tubules are surrounded basophilic and may have small vesicles or
by accessory (Sertoli) cells and involved by acidophilic dilatations that characterize the
connective tissue (Fig. 4d). The VD is divided into endogenous yolk (Fig. 5b-c). When females attain
three regions: proximal, medial and distal, these the physiological maturity, they show for the
regions are differentiated only by the increase first time the greenish coloration of the mature
of the vessel caliber. All regions show the same ovaries, a macroscopic evidence of sexual
histological aspect, vary from squamous to cubic maturity in this species (Fig. 5d). This color is
epithelium with basal nuclei enveloped laying due to the presence of oocytes in exogenous
on the musculature (Fig. 4e-f). Inside the lumen (secondary) vitellogenesis, with mature yolk
of VD in physiologically mature animals, a large granules accumulated in the cytoplasm (Fig. 5e-
amount of centrally compacted spermatozoa f). At the beginning of this stage, large follicular
is surrounded by secretion forming a single cells surround the secondary vitellogenic
sperm mass involved by acidophilic secretion, oocytes. The nucleus becomes smaller, due to

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LUCAS R.P. PASCHOAL & FERNANDO J. ZARA SEXUAL MATURITY IN Macrobrachium amazonicum

Figure 4. a. Dorsal view of the male reproductive system in Macrobrachium amazonicum, showing the testes with
their thick anterior lobes (black arrow) arranged over the hepatopancreas. b. Lateral view of the cephalothorax
of a male, showing the vas deferens (black arrow) opening into the gonopopore of the coxopodite of the fifth
pereiopod (white arrow). C. General aspect of the seminiferous tubules of the testis, with a large quantity
of spermatozoa filling the lumen and a small peripherical area containing spermatogonia. d. An isolated
seminiferous tubule filled with the tack-shaped spermatozoa in the lumen. e-f. Longitudinal section of the distal
region of the vas deferens filled with spermatozoa surrounded by secretion. This region has a thick muscular
layer and shows large amounts of centrally compacted spermatozoa. 1st, first pleopods. AG, androgenic gland. ep,
epithelium. Ct, connective tissue. G, genital pore. Hp, hepatopancreas. ml, muscle. s, secretion. Sg, spermatogonia.
Spz, spermatozoa. ST, seminiferous tubules. T, testes. Vd, vas deferens.

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the process of compaction promoted by the yolk had functional maturity simultaneously to
granules. These granules are acidophilic and histological maturity (Table II). Besides that,
few lipid droplets are detected at this stage. The males with small sizes (CL ≥ 3.70 mm) had a well-
previtellogenic oocytes are still easy to find in developed and functional appendix masculina,
the ovary (Fig. 5e). At the end of maturation, the corroborating the functional maturity. On the
ovaries occupy a large area of the coelomic cavity other hand, copulations in LS populations were
in the cephalothorax and exhibit an intense observed only after the pair formation in field.
greenish coloration (Fig. 5g). At this stage, the Thirty couples were captured and evaluated
oocytes are well-developed and have a larger in CAS population, while in SBG population 20
diameter when compared to the previous stages couples were analyzed. It was possible to verify
(Fig. 5h-i). The main feature of these oocytes is that only males with dominant morphotypes
the cytoplasm filled with acidophilic mature yolk (GC1 and GC2) were able to copulate, although
granules and many lipid droplets. In this ovarian submissive males (TC and CC) were more
stage, the oocytes are surrounded by flattened abundant in both populations (morphotypes
follicular cells and are well adhered to each ratio: 4 TC: 3 CC: 2 GC1: 1 CG2). GC1 and GC2 males
other, producing a more compact appearance were observed near to the margin (up to 2 m)
(Fig. 5h-i). in aquatic environments with sandy sediment,
and placed themselves behind or beside (in
Functional maturity parallel) of receptive females. Dominant males
In all studied populations, ovigerous females protected and guarded females before and
were captured during the 15 months of study, after copulation, and it was common to observe
indicating a pattern of continuous reproductive agonistic behaviors (fights) between males with
activity. The smallest size recorded for ovigerous hypertrophied chelipeds. During these fights, the
females in SS populations was 7.10 mm CL in male elevated its pereiopods and the anterior
SJB and 7.40 mm CL in CRC, whereas for LS portion of the body, and attacked its opponent
females the smallest ovigerous had 9.40 mm CL with the dactyls of the second pair of chelipeds.
in both populations. It is noteworthy that these Commonly, this event was short ranging from
–
females carrying eggs with eye pigmentation, three to fifteen seconds (X : 12 ± 3 seconds). Sneak
i.e. fertilized eggs. Functional maturity was copulation strategy was not observed in the LS
close to the morphometric maturity, with populations during the 15 months of sampling.
few ovigerous females captured under to the The mating system of these populations has the
size estimated for morphological maturity “neighborhoods of dominance” pattern (sensu
(Table II). Laboratory mating (copulation) Correa & Thiel 2003). The smallest size of males
experiments carried out with SS populations in LS populations captured after the observed
were characterized by the “pure search” pattern copulation was 14.40 and 15.20 mm CL for CAS
(sensu Correa & Thiel 2003), with no signs of and SBG, respectively (Table II).
agonistic behavior among males. The animals
tended to concentrate in one area and there
were no pair formation. Males of this phenotype

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Figure 5. a. Dorsal view of the immature, colorless and small-sized ovary (white arrow) in Macrobrachium
amazonicum. b-c. Cross-section of immature ovary with large quantity of previtellogenic and primary vitellogenic
oocytes showing a strongly basophilic cytoplasm with small unstained vesicles (black arrows). d. Dorsal view of
the ovary at the onset of sexual maturity. These organs occupy a small proportion in the cephalothorax and have
a greenish coloration. e-f. Secundary vitellogenic oocytes showing mature acidophilic yolk granules (black arrows)
accumulated in the cytoplasm. These oocytes are surrounded with large and ovoid follicular cells. G. Dorsal view
of the mature ovary. These organs occupy a large area at cephalothorax and have an intense greenish coloration.
H-I. Mature oocytes showing nucleus in the central region surrounded by many acidophilic yolk granules and lipid
droplets. The follicular cells of these oocytes are flattened. fc, folicular cell. ld, lipid droplet. n, nucleus. of, ovarian
folicle. Pvo, previtellogenic oocytes. Vo1, primary vitellogenic oocyte. Vo2, secondary vitellogenic oocyte. y, mature
yolk granules.

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DISCUSSION of sexual weapons during the passage to the

morphotype GC2, and after entering in this final
The populations of Macrobrachium amazonicum morphotype they cease the somatic growth,
analyzed in this study showed very distinct allocating energy only for the maintenance
morphological and reproductive characteristics, of metabolic processes (Moraes-Riodades &
indicating the great environmental adaptability Valenti 2004, Augusto & Valenti 2016). On the
and wide phenotypic plasticity of the species. other hand, the increase of chelipeds size in
Intraspecific variation of the reproductive females from LS populations would be related
strategy and SOM maximizes the reproductive to the optimization of the reproductive events,
success of the species in different aquatic as mating choices, protection and maternal care
environments (Odinetz-Collart 1991). for hatching and spawning (Viau et al. 2006).
In two of the four populations analyzed, Currently, only the study of Pantaleão et
both adult males and females exhibited large al. (2014) found morphotypes in males of M.
body proportions, and these animals spent amazonicum from inland populations (Tietê
energy for the growth of chelipeds (i.e. large- River Basin, São Paulo - southeastern Brazil),
size phenotype). In addition, the presence of being the present work the second record of this
morphotypes in males of these populations condition. These same authors estimated the
was verified through the analysis of this morphometric SOM in males with morphotypes
morphometric variable (MCL). In general, males at 8.8 mm CL, a higher value than recorded here.
of the genus Macrobrachium have higher However, the functional maturity analyzed here
body proportions than females, since they in populations with morphotypes was recorded
tend to invest more energy for somatic growth with significantly higher values, showing that
and the development of robust chelipeds neither the physiological nor the morphological
(i.e. sexual weapons), increasing the success maturities actually express the moment when
in intra/interspecific competitions (Correa the LS males are able to copulate and transfer
& Thiel 2003, Moraes-Riodades & Valenti their gametes and/or defend females and
2004, Paschoal & Zara 2018). The pronounced territories. Regarding females, there are no
sexual dimorphism in M. amazonicum with the studies evaluating morphometric maturity in
presence of social hierarchy confers adaptive populations with dominant morphotypes from
advantages for dominant males (GC1 and inland waters or using the same methodology
GC2), such as: more success at obtaining food used here, which hinders comparison with other
resources and dominating territories (Correa populations. However, the morphometric SOM
& Thiel 2003, Ibrahim 2011), and access to in LS females is similar among the studied
females available for mating, as observed in the populations.
present study. Thus, animals with larger sizes The absence of morphotypes, the lower
and robust sexual weapons would have greater energy investment for somatic growth and,
reproductive success, explaining the positive consequently, the smaller body sizes were
allometric growth for MCL registered only for the main characteristics of males from the
the GC1 morphotype. Since the morphotype other two studied populations (i.e. small-size
development is sequential, it could be raised phenotype). However, the females of these
that animals with GC1 morphotype will populations invest energy for the growth of
concentrate more energy for the acquisition the second abdominal pleura. This pattern is

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typical in caridean shrimps that adopted the other species of the genus Macrobrachium
“pure search” mating system, where males that (Chow et al. 1982, Sagi et al. 1988, Mossolin &
do not protect or defend females invest energy Bueno 2002, Poljaroen et al. 2010, Soonklang et
for the production of gametes by shifting the al. 2012). In both sexes of M. amazonicum, the
energy that would be used for body growth, physiological maturity anticipates the others
while females accumulate energy for the growth maturities and is attained at small sizes (CL)
of the brood chamber (abdomen and pleuras) within the analyzed populations. This shows
optimizing the fecundity and protection of that in these animals the physiological and
embryos (Bauer 2004, Paschoal et al. 2013, 2016). morphological maturities are not synchronized,
Pantaleão et al. (2012) also analyzed and they need their reproductive systems
the morphometric aspects of small-size fully functional before the pubertal molt. This
phenotype of the Amazon River prawn from pattern is similar to that of other species of the
inland environments (Tietê River Basin, São genus Macrobrachium (Chow et al. 1982, Sagi
Paulo - southeastern Brazil). As recorded here, et al. 1988, Mossolin & Bueno 2002, Poljaroen
these authors did not register morphotypes in et al. 2010, Soonklang et al. 2012) and some
males, correlating the absence of morphotypes brachyurans (Zara et al. 2012, 2013, Nascimento
in SS populations to the fish predation and/ & Zara 2013). However, it is the opposite of that
or to the adoption of “pure search” mating recorded for some aeglids (Viau et al. 2006)
system. Pantaleão et al. (2012) estimated the and penaeid shrimps (Heckler et al. 2013),
morphometric SOM for this species at 4.26 where the morphological maturity anticipates
mm CL for males and 5.39 mm CL for females. the physiological maturity. In M. amazonicum,
These values are lower than those recorded males become physiologically mature before
in the present study. It can be verified that females, and this may be associated with the
the morphometric maturity estimated here, short lifespan of male prawns. Gavio et al. (2006)
when compared to the other criteria of and Paschoal et al. (2016) suggested that the
maturity (physiological and functional) shows caridean males reach their optimal reproductive
higher values, overestimating the SOM in M. condition at small sizes and are capable to
amazonicum. Males of SS populations are mature copulate with larger females, but they disappear
and functional even with small sizes, thus the from the population due to the low longevity,
analysis of sexual maturity in M. amazonicum while females with longer life spans remain in
should take into account the general phenotype the population and continue to grow.
of the population. The wide variability of SOM Only males with GC1 and GC2 morphotypes
values would justify the use of the physiological were able to copulate. Also, the sneak copulation
maturity in populations without morphotypes, was not observed in the LS populations of M.
because this is similar to functional maturity. amazonicum. Ra’anan & Sagi (1985) suggested
The male and female reproductive systems that the high frequency of large prawns in
of M. amazonicum show the anatomical populations favors small sneaking males, since
organization similar to that commonly described dominant males will be occupied with fights and
for gonochoric caridean shrimps (Sandifer territorial protection, which consumes a large
& Lynn 1980, Bauer 2004). Histologically, it is amount of energy and time. However, if the
possible to verify that the reproductive systems frequency of dominant males is low (as observed
of the Amazon River prawn are similar to the in the present study), their relative reproduction

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LUCAS R.P. PASCHOAL & FERNANDO J. ZARA SEXUAL MATURITY IN Macrobrachium amazonicum

advantage over the submissive males increases, palaemonid shrimps, as suggested by Paschoal
promoting a reproductive-dominance hierarchy et al. (2013, 2016).
(i.e. only dominant males are capable to mate
and copulate). The mating system that best fits
the LS populations of M. amazonicum would CONCLUSIONS
be the “neighborhoods of dominance” (sensu The use of morphometric data showed to be
Correa & Thiel 2003), which would explain the a useful tool for the determination of distinct
high values of functional maturity recorded for phenotypes and population stocks in M.
males of these populations. Dominant males amazonicum. The three criteria used for the
(GC1 and GC2 morphotypes) of these populations evaluation of sexual maturity showed that, in
have hypertrophied chelipeds and it is possible both sexes, physiological maturity anticipates
to observe that these animals protect and guard the others and that males reach sexual maturity
females during the mating period. Moreover, in smaller sizes than females in all criteria,
these males destined a smaller portion of except for functional maturity in males of LS
energy to sperm production, while a greater populations. The development of chelipeds
portion is directed to somatic growth and the (sexual weapons) in males, in addition to
production and maintenance of sexual weapons, change the mating strategies in M. amazonicum,
increasing the fitness and improving the success modulated the SOM in this species, since LS
of males in intraspecific competitions (Moraes- prawns needed robust chelipeds for pairing
Riodades & Valenti 2004, Paschoal & Zara 2018). and copulation success. Thus, the sequential
In contrast, males from the SS populations scheme of sexual maturity in M. amazonicum
invest much less energy for somatic growth may be summarized as: ♀ - physiological →
leading to absence of sexual weapons. However, functional ≅ morphological, ♂LS - physiological
they attain physiological and functional → morphological → functional, and ♂ SS -
maturities at smaller sizes when compared to physiological = functional → morphological. The
animals of LS populations, indicating that the sexual maturity in M. amazonicum varies from
energy that would be spent on growth is better one criterion to another, as well as between the
used in reproductive events, such as sperm two evaluated phenotypes, so we suggest that
production (Paschoal et al. 2016, Paschoal & the best criterion used to estimate the SOM for
Zara 2018). Additionally, SS males are gregarious, the maintenance and preservation of the Amazon
without display both agonistic behavior and River prawn stocks is that registered with the
female guard. Thus, the mating system of SS highest value of CL. The information presented
populations has the “pure search” pattern here may also be used in the evaluation and
(sensu Correa & Thiel 2003). In all populations of maintenance of farmed specimens and breeders,
M. amazonicum analyzed, few ovigerous females improvement of stocks, assessment of temporal
(i.e. functionally mature) were captured under to series of breeders, as well as may be used in
the size estimated for morphometric maturity, studies involving the population biology of the
indicating that there is synchronization between species in freshwater environments.
morphological and functional maturities in
females. Thus, the use of relative growth or the Acknowledgments
evaluation of the smallest ovigerous female This study was financed in part by the Coordenação
is indicated to estimate the SOM in female de Aperfeiçoamento de Pessoal de Nível Superior

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LUCAS R.P. PASCHOAL & FERNANDO J. ZARA SEXUAL MATURITY IN Macrobrachium amazonicum

- Brazil (CAPES) - Finance Code 001. We thank CAPES ELNER RW & BENINGER PG. 1995. Multiple reproductive
program Ciências do Mar II (CIMAR II #1989/2014 proc. strategies in snow crab, Chinoecetes opilio: physiological
23038.004309/2014-5) for the great support to FJZ during pathways and behavioral plasticity. J Exp Mar Bio Ecol
this study. FJZ also thanks the São Paulo Research 193: 93-112.
Foundation (FAPESP) program JP 2005#04707-5 and Biota GAVIO MA, ORENSANZ JM & ARMSTRONG D. 2006. Evaluation of
(Intercrusta #2018/13685-5) and Conselho Nacional de alternative life history hypotheses for the sand shrimp
Desenvolvimento Científico e Tecnológico (CNPq PQ2# Crangon franciscorum (Decapoda: Caridea). J Crust Biol
30331/2017-6). We thank Dr. Fernando L. Mantelatto, Dr. 26: 295-307.
Rogério Costa, Dr. João Pantaleão and Dr. Abner Carvalho
Batista for the constructive reviews and suggestions HAYD L & ANGER K. 2013. Reproductive and morphometric
that improved the manuscript. This study complied with traits of Macrobrachium amazonicum (Decapoda:
Brazilian regulations (MMA-ICMbio, license #47653-1 to Palaemonidae) from the Pantanal, Brazil, suggests initial
LRPP, and MMA-ICMbio, permanent license #34587 to FJZ). speciation. Rev Biol Trop 61: 39-57.
HECKLER GS, SIMÕES SM, LOPES M, ZARA FJ & COSTA RCD. 2013.
Biologia populacional e reprodutiva do camarão sete-
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PASCHOAL LRP, OLIVEIRA LJF, ANDREOLI GC & ZARA FJ . 2019.
Reproductive biology of Macrobrachium amazonicum

An Acad Bras Cienc (2020) 92(Suppl. 2)  e20180560  18 | 19 

LUCAS R.P. PASCHOAL & FERNANDO J. ZARA SEXUAL MATURITY IN Macrobrachium amazonicum

SOONKLANG N, WANICHANON C, STEWART MJ, STEWART P, LUCAS R.P. PASCHOAL1,2

MEERATANA P, HANNA PJ & SOBHON P. 2012. Ultrastructure https://orcid.org/0000-0003-2461-4675
of differentiating oocyte and vitellogenesis in the giant
fresh water prawn, Macrobrachium rosenbergii (de Man). FERNANDO J. ZARA1,3
https://orcid.org/0000-0002-7664-7674
Microsc Res Tech 75: 1402-1415.
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VERGAMINI FG, PILEGGI LG & MANTELATTO FL. 2011. Genetic Programa de Pós-Graduação em Ciências Biológicas
variability of the Amazon River prawn Macrobrachium (Zoologia), Universidade Estadual Paulista/
amazonicum (Decapoda, Caridea, Palaemonidae). Contr UNESP, Instituto de Biociências de Rio Claro, Av.
Zool 80: 67-83. 24A, 1515, 13506-900 Rio Claro, SP, Brazil
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Faculdade de Tecnologia Nilo de Stéfani/
VIAU VE, LÓPEZ-GRECO LS, BOND-BUCKUP G & RODRIGUEZ
FATEC, 14883-130 Jaboticabal, SP, Brazil
EM. 2006. Size at the onset of sexual maturity in the
3
anomuran crab, Aegla uruguayana (Aeglidae). Acta Zool Universidade Estadual Paulista/UNESP, Faculdade
87: 253-264. de Ciências Agrárias e Veterinárias de Jaboticabal,
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Castellane, s/n, 14884-900 Jaboticabal, SP, Brazil
South Edisto rivers in South Carolina. South Carolina
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Correspondence to: Lucas Rezende Penido Paschoal
ZARA FJ, GAETA HH, COSTA T, TOYAMA MH & CAETANO FH. 2013.
E-mail: lucasrezende20@gmail.com
The ovarian cycle histochemistry and its relationship
with hepatopancreas weight in the blue crab Callinectes
danae (Crustacea: Portunidae). Acta Zool 94: 134-146. Author contributions
Each author have contributed significantly to the intellectual
ZARA FJ, TOYAMA MH, CAETANO FH & LÓPEZ-GRECO LS. 2012.
content of the work, specifically: Dr. LRPP - Conceptualization,
Spermatogenesis, spermatophore and seminal fluid
Data sampling, analysis and interpretation, Writing original
production in the adult blue crab Callinectes danae
draft preparation, Writing-Reviewing and Editing, and Dr. FJZ
(Portunidae). J Crust Biol 32: 249-262.
- Conceptualization, Supervision and Writing-Reviewing last
version.

SUPPLEMENTARY MATERIAL
Figure S1. and Figure S2.
Appendices I - V.

How to cite
PASCHOAL LRP & ZARA FJ. 2020. Size at onset of sexual maturity in
Macrobrachium amazonicum (Heller, 1862) phenotypes: an integrative
approach. An Acad Bras Cienc 92: e20180560. DOI 10.1590/0001-
3675202020180560.

Manuscript received on June 4, 2018;

accepted for publication on November 6, 2018

An Acad Bras Cienc (2020) 92(Suppl. 2)  e20180560  19 | 19