Scientific Note
ISSN 1678-2305 online version
BOLETIM DO INSTITUTO DE PESCA
Silva et al. Bol. Inst. Pesca 2019, 45(2): e428. DOI: 10.20950/1678-2305.2019.45.2.428 1/6
MOULTING CYCLE STAGES IN Macrobrachium rosenbergii BY
SETOGENESIS METHOD
ABSTRACT
This study had the objective of evaluating the duration of the moulting cycle stages of
Macrobrachium rosenbergii, using the method of setogenesis. The experiment was conduced in the
Prawn Farming Laboratory of the Federal University in Paraiba, in the town of Bananeiras, Paraiba,
Brazil. Fifteen prawns (5.7 ± 0.3 g) were distributed in 3 aquariums (60 x 30 x 40 cm), maintained
in natural photoperiod (12 x 12 hours), at a controlled temperature (28 °C), and feed was offered
3 times a day (8 a.m., 12 and 16 p.m.), throughout the experimental period. For identification, the
animals were marked with colored silicone rings on the ocular peduncle and with colored plastic
discs fixed on the carapace. From the setogenesis method, the prawns were classified into stage
A, B, C, D0, D1, D2 or D3 of the moulting cycle. Setogenesis was observed stereomicroscopically every
day, at the same time, 7 a.m., from the first ecdysis to the next ecdysis. The duration of the moulting
cycle was 27.7 ± 3.2 days, and the intermoult stage (C) was the longest (8.0 ± 2.38 days). Stage A
was the shortest (1 day), and the pre-moult stage (stages D) lasted 12.98+1.65 days. We concluded
that juvenile of M. rosenbergii have a diecdysis type moulting cycle with shorter intermoult than
pre-moult periods.
Key words: prawn farming; growth; ecdysis.
ESTÁGIOS DE MUDA EM Macrobrachium rosenbergii PELO MÉTODO DE
SETOGÊNESE
RESUMO
O presente estudo teve como objetivo avaliar a duração dos estágios de muda do
Macrobrachium rosenbergii, utilizando o método da setogênese. O experimento foi realizado no
Laboratório de Carcinicultura da Universidade Federal da Paraíba, em Bananeiras, Paraíba, Brazil.
Quinze camarões (5,7 ± 0,3 g) foram distribuídos em 3 aquários (60 x 30 x 40 cm), mantidos em
fotoperíodo natural, temperatura controlada (28 °C) e ração ofertada 3 vezes ao dia (8 a.m., 12 e
16 p.m.), durante todo o período experimental. Para identificação, os animais foram marcados
com anéis coloridos de silicone no pedúnculo ocular e com discos plásticos coloridos fixados à
carapaça. Os camarões foram classificados em estágio A, B, C, D0, D1, D2 ou D3 do ciclo de muda,
através do método da setogênese. A setogênese foi observada estereomicroscopicamente todos
os dias, às 7 a.m., no mesmo horário, a partir da primeira ecdise até a ecdise seguinte. Como
resultado, a duração do ciclo de muda foi 27,7 ± 3,2 dias, com o estágio de intermuda (C) sendo
o mais duradouro (8,00 ± 2,38 dias). O estágio A foi o mais breve (1 dia), e a fase de pré-muda
(os estágios D) durou 12,98 ± 1,65 dias. Conclui-se que juvenis de M. rosenbergii tem o ciclo de
muda do tipo diecdise com o período de intermuda menor que o de pré-muda.
Palavras-chave: carcinicultura; crescimento; ecdise.
INTRODUCTION
Within aquaculture activity, freshwater prawn farming has had a prominent position,
with high growth worldwide, mainly with the production of Macrobrachium prawn genus
(FAO, 2014). In this scenario, Macrobrachium rosenbergii is the main representative
species cultivated, with a production of 220,254 tons per year (FAO, 2014). In Brazil,
the introduction of M. rosenbergii occurred in 1977 through the Department of
Oceanography of the Federal University in Pernambuco (UFPE), which was able to
import post-larvae from Hawaii, USA. From the introduction into the country, the species
Marcos Antônio Sinésio da Silva
1
Bianca de Oliveira Ramiro
1
Marino Eugênio de Almeida Neto
2
Ricardo Romão Guerra
1
1
Universidade Federal da Paraíba – UFPB, Centro de
Ciências Agrárias, Programa de Pós-graduação em
Zootecnia, Rodovia PB-070, s/n, CEP 58397-000,
Areia, PB, Brasil. E-mail: ricardo@cca.ufpb.br
(corresponding author).
2
Universidade Federal da Paraíba – UFPB, Centro de
Ciências Humanas, Sociais e Agrárias, Departamento de
Ciência Animal, Rua João Pessoa, s/n, CEP 58220-000,
Bananeiras, PB, Brasil.
Received: July 20, 2018
Approved: December 21, 2018
MOULTING CYCLE STAGES…
Silva et al. Bol. Inst. Pesca 2019, 45(2): e428. DOI: 10.20950/1678-2305.2019.45.2.428 2/6
showed an excellent ability to adapt to the climatic conditions
(Valenti and Cavalcanti, 1998). Afterwards, much research was
developed with this species, mainly in the 1980s. However, in
the 1990s the intensity of scientific studies and advances reduced
significantly with increased cultivation of marine species.
One of the areas of study on M. rosenbergii prawn that has
advanced little is on the physiology of their moulting cycle.
Moulting, or ecdysis, is one of the most important physiological
aspects of crustacean life (Corteel et al., 2012). This physiological
process directly or indirectly impacts the lives of these animals,
mainly feeding, reproduction, metabolism, behavior and sensitive
acuity (Passano, 1960; Santos et al., 2014; Barbieri et al., 2017).
Moulting for crustaceans represents the possibility of completing
normal growth processes. This occurs cyclically every time the
organism is prepared to increase in length and weight. At that
moment, the old exoskeleton is discarded, giving place to the new
exoskeleton, which was already ready below the previous one, but
still very flexible and vulnerable (Saravanan and Kamalam, 2008;
Corteel et al., 2012). The new exoskeleton will then stiffen until
it has the consistency and hardness of the previous exoskeleton.
During this process the body of the prawn tends to absorb water
to promote the expansion of the new exoskeleton, which when
stiffening will shape a body slightly larger than the one existing
before the ecdysis. Finally, the water absorbed will give rise
to tissue synthesized by the animal in the following moments,
consolidating the increase of volume and weight of the animal
(Chang, 1995; Hayd et al., 2008; Barbieri et al., 2013).
Once the new exoskeleton of shrimp is formed before discarding
the old one, for all the effects that the moult cycle exerts on these
animals, it was observed the necessity and importance of predicting
and average length of the moulting cycle (Corteel et al., 2012).
One of main moulting cycle effects on the prawn culture systems
is on feed consumption. By the time the molt occurs, begins the
physiological abstinence. That biological phenomenon implies
in reasonable feed leftovers on the culture systems, deteriorating
the water quality and raising the production costs. Therefore, the
predictability of when the most of the cultivated population will be
in molting could bring important technological advances to prawn
culture (Almeida Neto and Freire, 2007). Thus, several methods
have been developed to observe the duration and stages of the
moultling cycle (Saravanan and Kamalam, 2008; Corteel et al.,
2012), for example: histological observation of the integument
(Silva et al., 2018), measurement of gastroliths and/or regeneration
of pereiopods and observation of the setal development of the
appendices (setogenesis). The most used and widespread moulting
cycle observation method is the setogenesis method, mainly because
it is fast and neither cause stressful situations to the animal nor
mutilations, even with repeated observations. The setogenesis is
based on the observation of the degree of development of the setal
structures, such as the setae themselves (external protuberances
in appendages such as uropods, pleopods and antigens), setae
cones, setae bases, and setae nodules, as well as the formation
of the new exoskeleton, as the new epicuticle and the epidermal
line of the appendices (Robertson et al., 1987; Chan et al., 1988;
Rusaini and Owens, 2011).
In setogenesis method, the moultling cycle was initially divided
into five main stages: Stage A (Recent postmoult), Stage B
(Late postmoult), Stage C (Intermoult), Stage D (Premoult) and
Stage E (Ecdysis) (Passano, 1960; Smith and Dall, 1985). Later,
Chan et al.(1988) described the setogenesis in penaeid shrimp
Litopenaeus vannamei by subdividing it into seven stages: Stage A
(Recent postmoult), Stage B (Late postmoult), Stage C (Intermoult),
Stages D0 and D1 (Recent premoult) and Stages D2 and D3
(Late premoult), and stage E (Ecdysis) was not described, since
it is an event that lasts a few seconds.
Among the biological aspects of prawn that are affected by the
moulting cycle, two main ones, feeding and reproduction, can
be highlighted. Regarding feeding, some authors, such as Smith
and Dall (1985) and Chan et al. (1988) reported that there are
moments in the life of prawn in which food consumption stops or
decreases. This is extremely important in the producer management
of the species. This cyclical process of non-feeding of prawns was
called physiological abstinence, and may be related to the fact
that in the process of exoskeleton detachment, some structures
such as the mouth, esophagus and part of the stomach cease to
be completely functional (Almeida Neto and Freire, 2007). These
organs have a layer of chitin in continuation to the outer layers,
which detaches along with the old exoskeleton at the moment of the
moult, preventing the organs to continue performing their normal
functions (Ceccaldi, 1987). On the other hand, prawn reproduction
is also strongly affected by the moulting cycle. In L. vannamei,
copula, and consequent transfer of spermatophores to the female,
is only effective when it occurs at moments of intermoulting,
with the exoskeleton rigid (Ostrensky and Barbieri, 2002). While
in M. rosenbergii it is the reverse, the copula and consequent
transfer of spermatophore to the female telic only occurs after
the nuptial moult, in the moments of recent postmoult, with the
flexible exoskeleton (Valenti and Cavalcanti, 1998).
As mentioned above, the molting cycle interferes with several
faults in the production system, such as feeding, breeding and
development. Therefore, the present study aimed to evaluate
the duration of the moult cycle, and its stages, in juveniles of
M. rosenbergii, through the setogenesis method. Thus, it can subsidize
studies aimed at adopting new strategies in food management, as
well as generating basic information for studies on the digestive,
reproductive and behavioral physiology of the species.
MATERIAL AND METHODS
Experimental design
The experiment was carried out in the Laboratory of Prawn Farming
of the Agricultural College “Vidal de Negreiros” (CAVN), Center
for Human, Social and Agricultural Sciences (CCHSA), Federal
University of Paraíba (UFPB), in the municipality of Bananeiras,
Paraíba, Brazil, and at the Histology Department of the UFPB, Areia,
Paraíba, Brazil. Fifteen juvenile Macrobrachium rosenbergii were
used with 5.7 ± 0.3 g. The prawns were distributed in 3 aquariums
(60 cm x 30 cm x 40 cm). Each aquarium had the bottom covered
by biological plaques, a layer of fiberglass wool and 5 cm of fine
sand, from bottom up. And each one had a small pump blowing
MOULTING CYCLE STAGES…
Silva et al. Bol. Inst. Pesca 2019, 45(2): e428. DOI: 10.20950/1678-2305.2019.45.2.428 3/6
air constantly through porous stones. Above each aquarium a
biofilter was installed with a layer of fiberglass wool, one 5 cm
layer of coarse sand and one 3 cm layer of fine sand, respectively.
The water circulation through the biofilters was through suction
pumps submerged in each experimental aquarium. This way, the
aquarium water was circulated continuously throughout the biofilters
at the rate of 150 L h
-1
. The temperature was maintained at 28 °C
through thermostats inserted into each aquarium. The ammonia
analysis was carried out every 3 days with the ALCON Ltda
kit for the analysis of toxic ammonia. The aquariums were kept
under natural photoperiod, 12 x 12 hours and the prawns were
fed 3 times a day throughout the experimental period (8:00 a.m.,
12:00 and 4:00 p.m.) at a rate of 30% of the biomass per day;
with commercial feed for marine prawns containing 30% crude
protein. One hour after feeding, the leftover feed was removed
from the aquariums by siphoning.
Observations on the moulting stages
For identification, the animals were marked with colored
silicone rings on the ocular peduncle and with colored plastic
discs fixed on the carapace. The first moult of each prawn was
awaited, and then certified when the exoskeleton with the marking
of the carapace released in the water was observed. This moment
was considered as day “0” for this prawn, and, from that day on,
it was observed daily, always at 2:00 p.m. This same procedure
was repeated for the other prawns under experimental conditions.
Thus, through the method of setogenesis, the animals had
their stage of the moult cycle characterized and determined daily
stereomicroscopically according to Corteel et al. (2012) adapted
methodology. With the next moult, after day “0”, the animals
were removed from the aquarium and this was counted as the
last day of observation in this animal. That is, each prawn had its
stage of moulting cycle characterized daily, by a complete cycle
of moult, from one ecdysis to another, with 15 moulting cycles
observed in 15 different prawns.
Statistical analysis
At the end of the experimental period and tabulation of data
(days), statistical analysis was carried out by using the statistical
software program Statistic version 13.0. The data were submitted
to a descriptive statistical analysis to evaluate the means, variances,
deviations and standard errors of the total duration of the moult
cycle and its stages.
RESULTS
From the observations of setogenesis in the endopods of
the uropods, we were able to identify and characterize seven
stages of the moulting cycle for juvenile freshwater prawn
Macrobrachium rosenbergii: A, B, C, D0, D1, D2 or D3 (Figure 1).
In stage A, immediately after moulting, the setal structures were
still in formation; with the absence of the setal cones in most
setae; absence of the inner cone in all setae; setal bases not very
evident and the epidermis completely filling the setal bases
(Figure 1B). Stage B was characterized by the setal cones in
most setae, as well as the development of the inner cone in some
setae; the setal bases were more evident with denser setal nodules
and partial filling of the setal base by the epidermis (Figure 1C).
In the intermoult stage (stage C), all structures of the exoskeleton
were already formed and evident, with emphasis on the internal
cone present in all setae and the epidermal line formed parallel
to the sternum nodules (Figure 1D). The Stage D0, first stage of
prawn pre-moult, was identified by the maximum retraction of the
epidermis and its detachment from the old exoskeleton (apolysis)
(Figure 1E). Stage D1, was marked by the appearance of the new
setae (“setogenesis”) (Figure 1F). For stage D2, we noticed the
presence of the new epicuticle, with the new more visible setae
(Figure 1G). Finally, stage D3, immediately before ecdysis, was
characterized by the complete formation of the new (invaginated)
setae and the high proximity of the new epicuticle with the old
setal nodules (Figure 1).
The duration of the complete moulting cycle was 27.7 ± 3.2 days
on average, and the intermoult stage (C) was the longest
(8.00 ± 2.38 days). The post-moult phase (adding stages A and B)
was 6.76 ± 1.96 days, while the pre-moult phase (adding stages
D) lasted 12.98 ± 1.65 days (Table 1).
Table 1. Moulting cycle stages in Macrobrachium rosenbergii by setogenesis method.
Variables
Average ± Standard deviation (days)
Variance Standard error CV* (%)
Intermould duration 27.76 ± 3.29 10.85 0.9139 11.85
Intermoult stage - A 1.00 ± 0.00 0.00 0.0000 0.00
Intermoult stage - B 5.76 ± 1.96 3.85 0.5440 34.02
Intermoult stage - C 8.00 ± 2.38 5.66 0.6602 29.75
Intermoult stage - D0 4.69 ± 1.65 2.73 0.4583 35.18
Intermoult stage - D1 3.92 ± 0.95 0.91 0.2646 24.23
Intermoult stage - D2 2.30 ± 0.48 0.23 0.1332 20.86
Intermoult stage - D3 2.07 ± 0.27 0.07 0.0769 13.04
*CV= coefficient of variation.