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UNIVERSIDADE ESTADUAL PAULISTA
CENTRO DE AQÜICULTURA
CAMPUS DE JABOTICABAL
Ciclo de muda e metabolismo durante o desenvolvimento
larval do camarão-da-amazônia Macrobrachium amazonicum
(Heller, 1862)
Moulting cycle and metabolism during larval development of
the Amazon River prawn Macrobrachium amazonicum
(Heller, 1862)
Liliam de Arruda Hayd
Bióloga
Jaboticabal – São Paulo
Fevereiro - 2007
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UNIVERSIDADE ESTADUAL PAULISTA
CENTRO DE AQÜICULTURA
CAMPUS DE JABOTICABAL
Ciclo de muda e metabolismo durante o desenvolvimento
larval do camarão-da-amazônia Macrobrachium amazonicum
(Heller, 1862)
Moulting cycle and metabolism during larval development of
the Amazon River prawn Macrobrachium amazonicum
(Heller, 1862)
Liliam de Arruda Hayd
Orientador: Prof. Dr. Wagner Cotroni Valenti
Co-orientador: Prof. Dr Daniel Lemos
Tese apresentada ao Programa de Pós
Graduação em Aqüicultura da UNESP,
como parte das exigências para a
obtenção do título de Doutor em
Aqüicultura
Jaboticabal - São Paulo
Fevereiro - 2007
ads:
Hayd, Liliam de Arruda
H412c Ciclo de muda e metabolismo durante o desenvolvimento larval
do camarão-da-amazônia Macrobrachium amazonicum (Heller, 1862).
– – Jaboticabal, 2007.
iv, 87 f. il.: 28 cm
Tese (doutorado) - Universidade Estadual Paulista, Centro de
Aqüicultura, 2007.
Orientador: Wagner Cotroni Valenti
Banca examinadora: Maria Célia Porltella, Margarete Mallasen,
Francisco de Assis Leoni, Laura Nakaghi
Bibliografia
1. Camarão de água doce-larva-ciclo de muda. 2. Camarão de
água doce-larva–composto nitrogenado. 3. Camarão-metabolismo. I.
Título. II. Jaboticabal-Centro de Aqüicultura.
CDU 639.512
Ficha catalográfica elaborada pela Seção Técnica de Aquisição e Tratamento da Informação -
Serviço Técnico de Biblioteca e Documentação - UNESP, Câmpus de Jaboticabal.
AGRADECIMENTOS
A DEUS, por ser o meu refúgio e fortaleza, socorro bem presente na angústia.
Ao meu querido orientador Prof. Dr. Wagner Cotroni Valenti pela oportunidade, apoio,
orientação, confiança, amizade, dedicação e grande paciência, principalmente na
finalização do trabalho. Sua orientação tem contribuído para meu crescimento científico
e pessoal.
Ao meu co-orientador Prof. Dr. Daniel E. L. Lemos do Instituto Oceanográfico – USP
pela orientação, sugestões, críticas, paciência e oportunidade de realizar o estudo
metabólico.
Ao Dr. Klaus Anger do Biologische Anstalt Helgoland – BAH – Alemanha, pela
orientação, sugestões, críticas construtivas e oportunidade de realizar o estudo do ciclo
de muda. Seu ritmo de trabalho e seu estilo de fazer ciência contribuíram muito para o
estabelecimento de bases essenciais para minha formação científica e pessoal.
Aos membros da Banca: Prof. Dr. Wagner C. Valenti, Profa. Dra Laura Nakaghi, Profa.
Dra Maria Célia Portella, Dra. Margarete Mallasen e Prof. Dr. Francisco A. Leoni pela
análise cuidadosa do trabalho e críticas construtivas.
À diretoria, coordenadores e funcionários da Pós-Graduação do Centro de Aqüicultura
da Unesp- CAUNESP, pelo carinho e atenção dispensados. Aos professores pelos
ensinamentos e amizades estabelecidas.
À Universidade Estadual de Mato Grosso do Sul – UEMS pela concessão do
afastamento. À CAPES programa PQI/CAPES/UEMS/CAUNESP (Proc. 137030) pela
bolsa de doutorado concedida.
Ao Deutscher Akadmischer Austauschdienst - DAAD, (Bonn, Germany; Grants
A/05/33827) pelo auxilio de Pesquisa na Alemanha e à FAPESP (Proc. 05/54276-0)
pelo auxílio no transporte dos animais do Brasil para a Alemanha.
Ao Setor de Carcinicultura – CAUNESP - Brasil e ao Biologische Anstalt Helgoland –
BAH - Helgoland - Alemanha pelo suporte técnico durante a realização dos
experimentos.
Ao Prof. Dr. Flavio Ruas e Ronaldo do laboratório de Patologia da UNESP/FCAV pela
concessão do equipamento Milique-Milipore durante a realização do experimento das
avaliações metabólicas.
À Profª. Dra. Laura
Nakaghi e Sr Orandi do laboratório de Morfologia e Fisiologia
Animal da UNESP/FCAV, pela concessão do material microscópico e pelo carinho e
atenção sempre dispensados.
Ao Prof. Dr. Antonio Ferraudo pela paciência e atenção demonstrado durante os meus
intermináveis questionamentos estatísticos.
Aos professores de estatística: Prof. Dr. Antonio Ferraudo, Prof Dr Gener e Prof. Dr.
Carlos Oliveira pelos auxílios prestados.
Aos professores: Dr. Jairo Casseta, Dr. Paulo e MSc Rosangela (SAERG) pelos auxílios
nos cálculos químicos.
Aos queridos amigos: Wagner, Patrícia, Janaina, Cristiana, Priscilinha, Breno, Virginia,
Antonio Francisco, Ineide, Marta Atique, Ana Paula, Severino, D Julieta, Ermínia,
Wanderly, Cléo, Marcio e Marcia por termos compartilhado momentos felizes em
Jaboticabal.
Aos queridos amigos: Wagner, Patrícia e Raíssa pelo carinho, apoio e pelos laços
fraternos estabelecidos. Vocês são muito especiais.
As inesquecíveis amigas: Patrícia, Cristiana, Janaina, Priscilinha e Virginia, pelo
carinho, cuidado, desabafos e paciência demonstrados durante todo este período
compartilhado na vida acadêmica e pessoal.
Aos queridos estagiários e amigos: Priscila Atique e Breno Donadon pelo grande
auxílio durante a realização dos experimentos.
Aos colegas do setor: Camilo, Bruno, Fabrício, Michellinha, Daniela, Alessandra,
Luciane, Luciana, Michelle, Mayra, Graziela, Gustavo, Dino, Karina, Mariana, Fabio,
Itanhaem, Marcelo, Fernando, Randy, José Mario, Frederico, Sebastian e John, pelo
convívio.
Aos técnicos Roberto e Valdecir pelos auxílios prestados no laboratório.
Aos amigos: Klaus, Uwe, Kathelen, Karen, Patricia, Mirna e Andréa pela atenção,
cuidados e carinho demonstrados durante minha estadia em Helgoland - Alemanha.
Ao querido amigo Klaus pela hospitalidade calorosa, atenção e grande contribuição ao
trabalho durante o meu retorno à Alemanha.
À minha querida família Norton e Letícia, que com seus cuidados especiais, paciência e
amor incondicional me deram o suporte emocional para vencer mais esta etapa.
Aos meus pais Osmar e Solange pelo grande amor, cuidados, amizade e incentivos, e a
minha irmã Liliane, pelo carinho e palavras de incentivo
Ao meu querido e amado vovô Ambrósio (in memorian) que sempre dizia que eu seria
uma doutora até mesmo antes de eu entender o significado desta palavra, e por ter me
mostrado o verdadeiro sentido de “ser amada”, você me faz muita falta.
Aos meus familiares por compreenderem minha ausência nos grandes e importantes
momentos festivos da nossa família e pelo estímulo contínuo.
Aos meus queridos amigos sul-matogrossenses pelos churrascos deliciosos e
inesquecíveis momentos de confraternização nas minhas raras aparições no nosso
Estado.
Aos queridos amigos e líderes espirituais: Elcio, Norma e Célia pelas orações, amizade
e atenção demonstrados durante todos estes anos.
As queridas amigas: Mitiko, Célia, Luciana e Onilda por toda a atenção, zelo, cuidado e
carinho dedicados a nossa eterna amizade.
Finalmente, a todos que de alguma forma contribuíram para a realização desse trabalho.
À DEUS
“
““
“Aquele que habita no esconderijo do Altíssimo, à sombra do onipotente
descansará. Direi do Senhor: Ele é o meu Deus, o meu Refúgio, a minha
Fortaleza e nEle confiarei”. Sl. 91:1
À minha querida e amada filha LETICIA, que soube compreender
minhas ausências em vários momentos importantes de sua vida
DEDICO
SUMÁRIO
Ilustration of list.............................................................................................................................
iii
Resumo............................................................................................................................................
01
CAPÍTULO 1 - INTRODUÇÃO GERAL………………………………….…...…………...… 03
1.1. REFERÊNCIAS......................................................................................................................
06
CAPÍTULO 2 – Ciclo de muda dos estágios larvais iniciais do camarão-da-amazônia,
Macrobrachium amazonicum, cultivado em laboratório (The moulting cycle of early life
stages of the Amazon
River prawn, Macrobrachium amazonicum, reared in laboratory).....
12
Resumo………………………………………………………...……….……………………….
12
Abstract....................................................................................................................................... 14
1. Introduction.............................................................................................................................
16
2. Material and methods.............................................................................................................
18
3. Results...................................................................................................................................... 19
4. Discussion................................................................................................................................ 22
5. References................................................................................................................................
25
CAPÍTULO 3 – Variação ontogenética do metabolismo durante os estágios iniciais do
camarão-da-amazônia Macrobrachium amazonicum (Heller, 1862) (Crustacea, Decapoda,
Palaemonidae) (Ontogenetic variation in metabolism during the early life stages of the
Amazon River prawn Macrobrachium amazonicum (Heller, 1862) (Crustacea, Decapoda,
Palaemonidae))…………………………………………….…………………..………………...
35
Resumo………………………………………………………………………………………… 35
Abstract....................................................................................................................................... 37
1. Introduction.............................................................................................................................
39
2. Material and methods.............................................................................................................
40
3. Results...................................................................................................................................... 43
i
4. Discussion................................................................................................................................ 45
5. References............................................................................................................................... 50
CAPÍTULO 4 – Efeito do nitrito no desenvolvimento e metabolismo das larvas de
Macrobrachium amazonicum (Effects of ambient nitrite on development and metabolism
of Macrobrachium amazonicum larvae).......................................................................................
60
Resumo………………………………………………………………………………………... 60
Abstract....................................................................................................................................... 62
1. Introduction............................................................................................................................ 64
2. Material and methods............................................................................................................ 65
3. Results......................................................................................................................................
70
4. Discussion................................................................................................................................ 71
5. References............................................................................................................................... 75
CAPÍTULO 5 – CONCLUSÕES ................................................................................................ 86
ii
ILUSTRATION OF LIST
Page
CHAP. 2
The moulting cycle of early life stages of the Amazon River prawn,
Macrobrachium amazonicum, reared in laboratory……….……...….…...
Figure 1 - Macrobrachium amazonicum, larval instar zoea I: A. early
postmoult (stage A), with spongy epidermal tissue structure, large
hemolymph-filled spaces (lacunae); B. intermoult (stage C), with
epidermal growth, tissue concentration along the inner surface of the
cuticle, reduced lacunar spaces; C. early premoult (stage D, substage D
0
),
with beginning epidermal retraction from the cuticle (apolysis); D.
intermediate premoult (substage D
1
), with advanced apolysis, beginning
epidermal invaginations at the setal bases; ap, apolysis; ep, epidermis; i,
invagination……………………………………………………………….....
32
Figure 2 - Macrobrachium amazonicum, larval instars zoea II (A-C) and
zoea III (D-F): A. zoea II, early postmoult (stage A); B. zoea II, late
postmoult (stage B), with advancing tissue concentration along the inner
surface of the cuticle; C. zoea II, intermediate premoult (substage D
1
), with
uropod formation (u) inside the telson of the zoea II; D. zoea III, postmoult
(stage B), with telson (T) and newly appearing uropod (U); E. zoea III,
intermediate premoult (substage D
1
); F. zoea III, late premoult (substages
D
2-4
), with formation of a thin new cuticle (nc); ap, apolysis; ep, epidermis;
i, invagination; ns, new setae………………………………………………...
33
Figure 3 - Macrobrachium amazonicum, moulting (ecdysis) of larval instar
zoea I to zoea II: A.-C., retraction of the telson from the old exoskeleton
(oe); D, shedding of the exuvia, appearance of the zoea II (ZII); E, zoea II
with shed exuvia; ch, typical dorsal chromatophore on larval
pleon………………………………………………………………………….
34
CHAP. 3
Ontogenetic variation in metabolism during the early life stages of the
Amazon prawn Macrobrachium amazonicum (Heller, 1862) (Crustacea,
Decapoda, Palaemonidae)……………...………………………...…………
Table 1 – Age, dry weight (DW), number of individuals, and
biomass:volume ratio in respirometers used to determine oxygen
consumption and ammonia-N excretion rates during early life stages of
Macrobrachium amazonicum at 30 ± 1°C and 10 salinity. Embryo, zoea I -
IX= larval stages, PL 1 = post-larvae, PL 7 and PL 14 = post-larvae with 7
and 14 days after metamorphosis, respectively. Results are expressed as
mean values ± SD. Number of replicates = 10.……………………….……...
56
Table 2 – Individual oxygen consumption and ammonia-N excretion rates
and atomic oxygen:nitrogen ratio (O:N) in early life of Macrobrachium
amazonicum at 30 ± 1°C and 10 salinity. ZI - IX= larval stages, PL1, PL7
and PL14 = post-larvae with 1, 7 and 14 days after metamorphosis,
respectively. Results are expressed as mean values ± SD, [number of
replicates]. ……………………………………………………………..…….
57
iii
Table 3 - Regression parameters obtained from relationships between dry
weight and oxygen consumption and dry weight and ammonia-N excretion
from embryo to PL 14 of Macrobrachium amazonicum (temperature=
30±1°C, 10 salinity from embryo to ZIX and 0 for PL1 to
PL14)………………………………………………………….……………...
58
Figure 1 - Weight-specific rates oxygen consumption (QO
2
) (A) and
ammonia excretion (B) during the early life stages of Macrobrachium
amazonicum at 30 ± 1°C. Results are expressed as means ± SD. (DW = dry
weight, Z = zoea stages; PL = post-larvae)...…………….…………………..
59
CHAP. 4
Effects of ambient nitrite on development and metabolism of
Macrobrachium amazonicum larvae………...…………………..…………
Table 1 - Age, dry weight (DW), number of individuals, and
biomass:volume ratio in respirometers used to determine oxygen
consumption and ammonia-N excretion rate during zoea (Z) I, III, VII and
IX stages of M. amazonicum at 30 ± 1 °C and 10 salinity. Results are
expressed as mean values ± SD. Number of replicates = 10……………..…..
81
Table 2 - Survival rate, productivity, weight gain and larval stage index
(LSI) (means ± standard deviation) obtained at the end of 17 days of
Macrobrachium amazonicum culture (30 ± 1ºC and 10 salinity) in different
ambient nitrite concentrations. Mean values in the same column with
different letters are significantly different (P < 0.05). N = 25……………….
81
Table 3 – Nitrite concentration, individual oxygen consumption (VO
2
) and
ammonia-N excretion rates in zoea (Z) I, III, VII and IX stages of
Macrobrachium amazonicum in 0, 0.4, 0.8 and 1.6 mg.L
-1
nitrite
concentrations at 30 ± 1°C. Results are expressed as mean values ± SD.
Number of replicats. = 6...................................................................................
82
Table 4 - Regression between dry weight and oxygen consumption and dry
weight and ammonia-N excretion from zoea I, III,VII and IX of
Macrobrachium amazonicum in different nitrite concentration 0 and 1.6
mg.L
-1
NO
2
-N at 30 ± 1°C and 10 salinity ...…………………….…………..
83
Figure 1 – Relationship between survival rate (N = 25, F = 15.59, P < 0.05)
(A), productivity (N = 25, F = 10.91, P < 0.05) (B), weight gain (N = 25, F
= 26.64, P < 0.05) (C) and larval stage index (LSI) (N = 25, F = 138.87, P <
0.05) (D) and nitrite concentration. Figures over data-points indicate the
number of identical values……….
……………………………………………………
84
Figure 2 – Weight-specific rates oxygen consumption (A), ammonia-N
excretion (B) and O:N ratio (C) during the early life stages of
Macrobrachium amazonicum to different nitrite concentration. Results are
expressed as means ± SD. (I = zoea I, III = zoea III, VII = zoea VII and IX
= zoea IX stages)…………………………………………………………..…
85
iv
1
Resumo
O estudo teve por objetivo descrever o ciclo de muda e estudar o metabolismo
nas fases iniciais do desenvolvimento ontogenético de Macrobrachium amazonicum. O
trabalho está organizado em cinco capítulos. O capítulo 1 apresenta uma introdução
geral, apresentando os estudos inerentes à M. amazonicum e o programa de tecnologia
onde este estudo esta inserido. No capítulo 2 estão descritos os estágios do ciclo de
muda de M. amazonicum. As descrições foram determinadas e documentadas
fotograficamente em intervalos diários usando o telson como principal região de
referência e aplicando o sistema de classificação de Drach, observando as principais
mudanças que ocorrem na epiderme e na cutícula. O desenvolvimento é rápido (1-2 dias
ou 2-4 dias por instar larval a 29 e 21°C, respectivamente). Foram descritos os seguintes
estágios de muda, A/C (pós-muda/intermuda combinados), D (pré-muda) e E (ecdise).
Estima-se que a pós-muda/intermuda (A/C) ocupe cerca de 40-50% do total de duração
do instar enquanto que o período da pré-muda (D) requer mais que a metade do tempo
nas temperaturas experimentais. O capítulo 3 apresenta a descrição do metabolismo de
embriões, larvas e pós-larvas (PL com 1, 7 e 14 dias após a metamorfose) nas fases
iniciais do desenvolvimento ontogenético. O peso seco, consumo de oxigênio, excreção
de amônia total–N e taxa atômica O:N foram determinados. Os animais em estágio de
muda A/C foram separados conforme o estágio de desenvolvimento larval e colocados
dentro de câmaras respirométricas (30mL) por 2h para quantificar as taxas metabólicas.
Após este período, as amostras foram analisadas pela titulação de Winkler e método de
Koroleff para o consumo de oxigênio e nitrogênio amoniacal, respectivamente. As taxas
metabólicas foram expressas como taxas individuais e peso-específico. A taxa
individual de consumo de oxigênio e excreção de amônia nitrogenada aumentou durante
o desenvolvimento larval. Os valores de O:N apresentaram valores baixos, indicando
2
que em M. amazonicum predomina o metabolismo de proteína. O conhecimento das
taxas metabólicas irá subsidiar a interpretação de vários experimentos relativos à
larvicultura dessa espécie, bem como fornecerá informações importantes para o
dimensionamento adequado do sistema de aeração e dos filtros biológicos em sistemas
de larvicultura. O capítulo 4 apresenta um estudo do metabolismo e do desenvolvimento
das larvas em todos os estágios zoeas, submetidas em diferentes concentrações de
nitrito. A sobrevivência, produtividade, ganho de peso e índice de estágio larval (IEL),
foram avaliados. As larvas foram cultivadas em água com 0; 0,2; 0,4; 0,8 e 1,6mg.L
-
1
NO
2
-N, com 5 repetições para cada tratamento. As larvas foram mantidas em béqueres
de vidro de 600mL com 300mL de solução-teste em água salobra (salinidade 10‰),
aeração e temperatura constante de 30°C, com fotoperíodo de 12:12h claro:escuro. O
consumo de oxigênio e a excreção de amônia foram analisados em zoea I, III, VII e IX
expostos a 0; 0,4; 0,8 e 1,6mg.L
-1
NO
2
-N com 5 repetições para cada tratamento. O
experimento foi conduzido dentro de câmaras plásticas cilíndricas de 30mL.
Sobrevivência, produtividade, ganho de peso e IEL decresceram linearmente com o
aumento da concentração de nitrito no ambiente. Entretanto, não houve diferença
significativa entre as concentrações 0-0,8mg.L
-1
NO
2
-N em todos os parâmetros
avaliados. A concentração de 1,6mg.L
-1
NO
2
-N no ambiente, retarda o desenvolvimento
larval, reduz a sobrevivência, a produtividade, o ganho de peso, o IEL e afeta as taxas
metabólicas. A conclusão geral do trabalho foi apresentada no capítulo 5. Assim, as
informações obtidas nos experimentos fornecem subsídios importantes para o
desenvolvimento de sistemas e métodos de produção de pós-larvas de M. amazonicum.
3
CAPÍTULO 1
1. INTRODUÇÃO GERAL
A carcinicultura de água doce é uma forma lucrativa de produção de crustáceos
com baixo impacto ambiental (Valenti e Moraes-Riodades, 2004; New, 2005) e atende
aos preceitos da aqüicultura sustentável (Valenti, 2002; Valenti e Tidwell, 2006). Os
camarões de água doce ocupam posição inferior aos marinhos no mercado mundial. No
entanto, apresentam maior resistência a doenças, larvicultura mais simples,
independência da água salgada na fase de crescimento final e sistema de produção
compatível com pequenas propriedades (New, 2005).
As estatísticas de produção são difíceis de serem obtidas porque a maioria desses
crustáceos é produzida por pequenos proprietários rurais (Valenti e Moraes-Riodades,
2004). No entanto, sabe-se que a carcinicultura de água doce é um dos setores que mais
crescem no mundo (Valenti, 2002; New, 2005). Os dados estatísticos indicam que o
volume de Macrobrachium rosenbergii aumentou de 19.035 para 193.570 toneladas no
período de 1995 a 2004, sendo que a China aparece como principal produtor desta
espécie (FAO, 2006). Esses dados indicam que o setor tem apresentado grande
desenvolvimento, e somente a produção mundial de M. rosenbergii foi estimada em
193.570 toneladas em 2004 (FAO, 2006).
A carcinicultura de água doce no Brasil está baseada na espécie M. rosenbergii
(Valenti e Moraes-Riodades, 2004). O Brasil apareceu como o 6° produtor mundial em
2004 (FAO, 2006). Essa espécie é de origem asiática e apresenta características
biológicas que favorecem o seu cultivo. Porém, não existem estudos referentes ao
impacto de sua liberação nos ambientes naturais brasileiros. Isto é preocupante porque
além de ser uma espécie alopátrica competidora pode trazer microfauna associada que
pode ser disseminada no sistema aquícola brasileiro. Dessa forma, apesar de seu grande
4
potencial de cultivo em escalas comerciais, estudos com espécies nativas brasileiras
tornam-se necessárias.
No Brasil, ocorrem três espécies de camarão de água doce que apresentam
grande potencial para o cultivo, Macrobrachium carcinus, Macrobrachium acanthurus
e Macrobrachium amazonicum (Valenti, 1993). M. amazonicum se destaca por
apresentar grande distribuição geográfica, ocorrendo nas bacias do Orinoco, Amazonas,
São Francisco, Paraná, Paraguai, áreas costeiras do norte e nordeste da América do Sul,
Rio Paraguai (Cáceres e Descalvado) e rios, Miranda, Negro e Taboco (Magalhães,
2001). Devido a esta ampla distribuição, seu cultivo na maior parte do Brasil não
oferece riscos de introdução de espécies exóticas na natureza. Essa espécie vem sendo
amplamente explorada pela pesca artesanal na região Nordeste (Gurgel e Matos, 1984) e
nos estados do Pará e Amapá (Odinetz-Collart e Moreira, 1993). É bem aceita nos
mercados consumidores do Norte e Nordeste porque sua carne apresenta textura mais
firme e apresenta um sabor mais acentuado quando comparado com M. rosenbergii
(Moraes-Riodades e Valenti, 2001).
Os estudos existentes sobre M. amazonicum estão relacionados aos aspectos
ambientais e biologia pesqueira de populações naturais (Odinetz-Collart, 1991a,b;
Bialetzki et al., 1997), desenvolvimento larval (Guest, 1979a; Magalhães, 1985; Rojas
et al., 1990), crescimento relativo (Moraes-Riodades e Valenti, 2002), morfotipos de
machos (Moraes-Riodades e Valenti, 2004), morfofisiologia do hepatopâncreas (Papa et
al., 2004; Ribeiro, 2006), desenvolvimento gonadal (Bragagnoli e Grotta, 1995),
fecundidade (Odinetz-Collart e Rabelo, 1996; Da Silva, et al., 2004), densidade de
estocagem (Lobão et al., 1994; Vetorelli e Valenti, 2004), salinidade (Guest, 1979;
McNamara et al., 1983; Araújo, 2005), alimentação (Barreto e Soares, 1982; Araújo e
Valenti, 2005), cultivo em laboratório (Guest, 1979a; Hayd et al., 2004), fase de
5
crescimento (Moraes-Riodades, 2005; Kiyohara, 2006), qualidade de água (Moraes-
Riodades et al., 2006), transporte (Sperandio e Valenti, 2006) e viabilidade econômica
(Vetorelli, et al., 2006; Hayd et al., submetido).
No início do ano 2000, um programa de pesquisa multiinstitucional e
multidisciplinar visando à produção comercial de M. amazonicum foi iniciado (Valenti
et al., 2003) com o desenvolvimento de vários trabalhos sobre seu cultivo. O presente
trabalho está inserido nesse programa. Foram realizados estudos referentes ao ciclo de
muda e ao metabolismo das larvas dessa espécie. O trabalho foi dividido em cinco
capítulos, no entanto, os capítulos 2, 3 e 4 serão apresentados na forma de artigo
científico . No capítulo 2 é apresentado a descrição dos estágios do ciclo de muda de M.
amazonicum. As descrições são essenciais para padronizar a fase de muda das larvas
usadas nos experimentos, reduzindo assim a variabilidade fisiológica e bioquímica dos
sucessivos instares larvais. O capítulo 3 apresenta a descrição do metabolismo de
embriões, larvas e pós-larvas (PL com 1, 7 e 14 dias após a metamorfose) nas fases
iniciais do desenvolvimento ontogenético. O conhecimento das taxas metabólicas em
cada estágio de desenvolvimento irá subsidiar a interpretação de vários experimentos
relativos à larvicultura dessa espécie, bem como fornecerá informações imprescindíveis
para o dimensionamento do sistema de aeração e dos filtros biológicos em sistemas de
larvicultura e auxiliará nos cálculos de densidade de estocagem para o transporte de
larvas e pós-larvas. O capitulo 4 apresenta um estudo do metabolismo e do
desenvolvimento das larvas submetidas a um fator de estresse comum na larvicultura de
camarões, o teor de nitrito. Assim, o estudo teve por objetivo descrever o ciclo de muda
e estudar o metabolismo nas fases iniciais do desenvolvimento ontogenético de
Macrobrachium amazonicum, fornecendo informações importantes para o
desenvolvimento da tecnologia de produção de pós-larvas do camarão-da-amazônia.
6
1.1 - REFERÊNCIAS
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amazônia. Macrobrachium amazonicum em berçário I. Acta Scient. Animal
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amazonicum (Heller, 1862) (Decapoda, Palaemonidae) sob condições controladas
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Macrobrachium amazonicum (Heller) (Decapoda, Palaemonidae) in Leopoldo’s
inlet (Ressaco do Leopoldo) upper Paraná river, Porto Rico, Paraná, Brazil. Rev.
Bras. Zool. Curitiba. 14(2), 379-390.
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amazonicum (Heller, 1862) nos açudes públicos do nordeste brasileiro. Rev Nord.
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Macrobrachium amazonicum (Crustacea, Palaemonidae). Braz. J. Biol. 64(3A),
489-500.
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7
Guest, W.C., 1979. Palaemonid shrimp, Macrobrachium amazonicum, effects of
salinity and temperature on survival. Prog. Fish Cult. 41(1), 14-18.
Guest, W.C., 1979a. Laboratory life history of the palaemonid shrimp Macrobrachium
amazonicum (Heller, 1862) (Decapoda, Palaemonidae). Crustaceana. 37(2), 141-
152.
Gurgel, J.J.A., Matos, M.O.M., 1984. Sobre a criação extensiva do camarão-canela,
Macrobrachium amazonicum (Heller, 1862) nos açudes públicos do nordeste
brasileiro. In: Simpósio Brasileiro de Aqüicultura, 3. São Carlos. Anais... p. 295–
311.
Hayd, L.A., Vetorelli, M.P., Moraes-Riodades, P.M.C., Valenti, W.C., 2004. Seleção e
manejo de fêmeas ovígeras para larvicultura de Macrobrachium amazonicum
(Heller, 1862). In: I Congresso da Sociedade Brasileira de Aqüicultura e Biologia
Aquática. Anais... Vitória/ES. AQUIMERCO. 1(1), 393.
Hayd, L.A., Vetorelli, M.P., Martins, M.I.E.G., Valenti, W.C. Economic feasibility of
the Amazon river prawn farming to supply live bait shelfish market in Pantanal,
South America. (Submitted).
Kiyohara, F., 2006. Cultivo de Macrobrachium amazonicum (Heller, 1862) em cercados
e em tanques-rede durante a fase de berçário e a fase de crescimento final & cultivo
de Macrobrachium potiuna (Muller, 1880) em laboratório (Crustacea, Decapoda,
Palaemonidae). Universidade de São Paulo–USP. Instituto de Biociências,
Departamento de Zoologia da USP. São Paulo. 190p. Tese de Doutorado.
8
Lobão, V.L., Roverso, E.A., Lombardi, J.V., 1994. Influência da densidade inicial de
estocagem no desenvolvimento de Macrobrachium rosenbergii (De Man, 1879) e
Macrobrachium amazonicum (Heller, 1862) (Decapoda, Palaemonidae) em
laboratório. Bol. Inst. Pesca. 21, 11-17.
Magalhães, C., 1985. Desenvolvimento larval obtido em laboratório de palaemonideos
da região Amazônica I, Macrobrachium amazonicum (Heller, 1862) (Crustacea,
Decapoda). Amazoniana. 9(2), 247-274.
Magalhães, C., 2001. Caracterização da comunidade de crustáceos decapodos do
Pantanal, Mato Grosso do Sul, Brasil. Cap. 5. In: Conservation International. Rapid
Assessment Program. RAP Bulletin of Biological Assessment, 18. CD-Room.
McNamara, J.C., Moreira, G.S., Moreira, P.S., 1983. The effect of salinity on
respiratory metabolism, survival and moulting in the first zoea of Macrobrachium
amazonicum (Heller) (Crustacea, Palaemonidae). Hydrobiologia. 101, 239-242.
Moraes-Riodades, P.M.M., Valenti, W.C., 2001. Freshwater prawn farming in brazilian
amazonia shows potential for economic and social development. Global Aquacult.
Advocate. 4(5), 73-74.
Moraes-Riodades, P.M.C., Valenti, W.C., 2002. Crescimento relativo do camarão
canela Macrobrachium amazonicum (Heller) (Crustacea, Decapoda, Palaemonidae)
em viveiros. Rev. Bras. Zool. 19(4), 1169-1176.
Moraes-Riodades, P.M.C., Valenti, W.C., 2004. Morphotypes in male Amazon river
prawns, Macrobrachium amazonicum. Aquaculture. 236, 297-307.
Moraes-Riodades, P.M.C., Kimpara, J.M., Valenti, W.C., 2006. Effect of the Amazon
river prawn Macrobrachium amazonicum culture intensification on ponds
hydrobiology. Acta Limnologica Brasiliensia. 18(3), 2006.
9
Moraes-Riodades, P.M.C., 2005. Cultivo do camarão-da-amazônia Macrobrachium
amazonicum (Heller, 1862). (Crustacea, Decapoda, Palaemonidae) em diferentes
densidades, Fatores ambientais, biologia populacional e sustentabilidade econômica.
Jaboticabal. Centro de Aqüicultura da UNESP. 135p. Tese de Doutorado. Centro de
Aqüicultura da UNESP.
New, M.B., 2005. Freshwater prawn farming, global status recent research and a glance
at the future. Aquaculture Research. 36, 210-230.
Odinetz-Collart, O., 1991a. Stratégie de reproduction de Macrobrachium amazonicum
en Amazonie Centrale (Decapoda, Caridea, Palaemonidae). Crustaceana. 61(3), 253-
270.
Odinetz-Collart, O., 1991b. Tucurui dam and the populations of the prawn
Macrobrachium amazonicum in the Lower Tocantins (PA-Brasil), a four years study.
Archiv fur Hidrobiologie. 122(2), 213-228.
Odinetz-Collart, O., Moreira, L.C., 1993. Potencial pesqueiro do camarão
Macrobrachium amazonicum na Amazônia Central (Ilha do Careiro). Amazoniana,
12(3/4), 399–413.
Odinetz-Collart, O., Rabelo, H., 1996. Variation in egg size of the freshwater prawn
Macrobrachium amazonicum (Decapoda, Palaemonidae). J. Crustacean Biol.
Laurence. 16(4), 684-688.
Papa, L.P., Vicentini, I.B.F., Ribeiro, K., Vicentini, C.A., Pezzato, L.E., 2004.
Diferenciação morfotípica de machos do camarão de água doce Macrobrachium
amazonicum a partir da análise do hepatopâncreas e do sistema reprodutor. Acta
Scient. Animal Sciences. 26(4), 463-467.
10
Ribeiro, K., 2006. Aspectos estruturais do hepatopâncreas, desenvolvimento ovocitário
e caracterização hormonal de fêmeas de Macrobrachium amazonicum durante as
fases de maturação gonadal. Jaboticabal. Centro de Aqüicultura da UNESP. 85p.
Tese de Doutorado. Centro de Aqüicultura da UNESP.
Rojas, N.E.T., Lobão, V.L., Barros, H.P., 1990. Métodos de manutenção de larvas de
Macrobrachium amazonicum HELLER, 1862 (Crustacea, Decapoda,
Palaemonidae). Bol. Inst. Pesca, 17(único), 15-26.
Sperandio, L., Valenti, W.C., 2006. Transportation of Amazon river prawn
Macrobrachium amazonicum juveniles in different biomass densities. In: AQUA
2006, Linking Traditio & Tecnology Highest Quality The Consumer, 2006,
Florence- Italy. AQUA 2006-Linking Traditio & Tecnology Highest Quality The
Consumer. Florença, World Aquacult. Society, 2006. v. 01.
Valenti, W.C., 1993. Freshwater prawn culture in Brazil. World Aquaculture, Baton
Rouge, 24(1), 29-34.
Valenti, W.C., 2002. Situação atual, perspectivas e novas tecnologias para produção de
camarões de água doce. In: Congresso de Aqüicultura. XII Simpósio Brasileiro de
Aqüicultura. Campus II da Escola de Agronomia/ UFG. Anais... Goiânia, ABRAQ,
P. 99-106.
Valenti, W.C., Franceschini-Vicentini, I.B., Pezzato, L.E., 2003 The potential for
Macrobrachium amazonicum culture. In: World Aquaculture 2003, 2003, Salvador.
Book of Abstracts. Salvador. World Aquacult. Society. p. 804.
Valenti, W.C., Moraes-Riodades, P.M.C., 2004. Freshwater prawn farming in Brazil.
Global Aquacult. Advocate. 7(4), 52-53.
11
Valenti, W.C., Tidwell, J.H., 2006. Economics and management of freshwater prawn
culture in Western Hemisphere In: Leung. P. S. and Engle. C. (Ed.) Shrimp Culture,
Economics. Market and Trade. Oxford. Blackwell Science. 416pp.
Vetorelli, M.P., Valenti, W.C., 2004. Post-larvae productivity of amazon river prawn
Macrobrachium amazonicum stocked at different densities. In: 3 Brazilian
crustacean congress and the crustaceana society meeting. 2004.
Anais…Florianopolis/SC. 1(1), 129.
Vetorelli, M., Valenti, W.C., Martins, M.I.E.G., 2006. Viabilidade econômica da
produção de pós-larvas do camarão-da-amazônia Macrobrachium amazonicum em
sistema fechado dinâmico, estocados em diferentes densidades. In: II Congresso da
Sociedade Brasileira de Aqüicultura e Biologia Aquática.. 2006. Anais… Bento
Gonçalves/RS. Aquaciência 1 (1), 18.
12
CAPÍTULO 2 (submitted in Aquaculture)
Ciclo de muda dos estágios larvais iniciais do camarão-da-amazônia,
Macrobrachium amazonicum, cultivado em laboratório
The moulting cycle of early life stages of the Amazon River prawn, Macrobrachium
amazonicum, reared in the laboratory
Resumo
O ciclo de muda das larvas do camarão-da-amazônia, Macrobrachium
amazonicum foi estudado em laboratório. Usando o telson como a principal região de
referência e aplicando o sistema de classificação de Drach, foram observadas a
epiderme e a cutícula duas vezes ao dia e documentadas as principais mudanças
estruturais tais como, a retração dos tecidos da epiderme da cutícula e o
desenvolvimento setal. O desenvolvimento é rápido (1-2 dias ou 3-4 dias por instar
larval a 29 e 21°C, respectivamente), o tegumento larval é fino, e como as mudanças do
tegumento são muito rápidas, permitiram classificar o ciclo de muda em três principais
estágios, A/C (estágio de pós-muda/intermuda combinados), D (pré-muda) e E (ecdise).
Esses períodos poderiam ser morfologicamente divididos em subestágios; entretanto,
não forneceriam um sincronismo definitivo para suas transições. Em pós-muda inicial
(estágio A), a cutícula é fina e absorve água, de modo que o corpo da larva se expande
rapidamente, alcançando a forma e o tamanho final. Os tecidos epidermais mostram
neste estágio uma estrutura esponjosa com numerosas lacunas. Durante a pós-muda
final (estágio B) e durante a intermuda (estágio C)
, a
cutícula larval é reforçada, quando
a epiderme mostra um condensamento crescente com redução dos espaços lacunares e
crescimento conspícuo do tecido. A pré-muda inicial (subestágio D
0
) começa com uma
13
retração da epiderme da cutícula, denominada de apolise, que é inicialmente visível na
base da seta. Durante a pré-muda intermediária (subestágio D
1
) ocorre a invaginação da
epiderme, conduzindo a uma ampliação substancial da superfície da epiderme, iniciando
a formação de novas setas e apêndices (setogênese, morfogênese). Subseqüentemente,
uma nova cutícula fina é secretada na superfície da epiderme (subestágios D
2-4
). A
muda (estágio E) é um processo muito rápido, durando somente alguns minutos. Inicia
dorsalmente com a ruptura cuticular entre o cefalotórax e o abdome, seguidos por uma
retração rápida do abdome do exoesqueleto velho, e eventualmente, retirando as partes
anteriores da cutícula velha. O período combinado da pós-muda/intermuda (estágio
A/C) ocupou cerca de 40-50% do total de duração do instar nas temperaturas
experimentais, e a pré-muda (estágio D) requereu mais da metade do tempo. Como o
crescimento larval ocorre predominantemente durante a pós-muda/intermuda, e este
último é considerado como a fase mais estável do ciclo de muda, sugerimos que as
mensurações fisiológicas e bioquímicas comparando sucessivos instares larvais sejam
realizadas perto do fim deste período, isto é, cerca de 30-40% do ciclo de muda.
Palavras-chaves: Ciclo de muda, Larva, Macrobrachium, Camarão de água doce
14
Abstract
The moulting cycle was studied in laboratory-reared larvae of the Amazon River
prawn, Macrobrachium amazonicum. Using the telson as main reference region and
applying Drach’s classification system, we checked twice daily the epidermis and
cuticle and documented major structural changes such as the retraction of epidermal
tissues from the cuticle and setal development. Rapid development (1-2d or 3-4d per
larval instar at 29 and 21°C, respectively), a thin and little structured larval integument,
and gradual rather than abrupt integumental changes allowed for only a coarse
classification of the moulting cycle with three principal stages, A-C (postmoult and
intermoult stages combined), D (premoult), and E (ecdysis). These periods could be
morphologically further divided into substages; however, without providing a definite
timing for their transitions. At early postmoult (stage A), the cuticle is still thin and
water is taken up, so that the larval body expands and rapidly attains its final size and
shape. The epidermal tissues reveal at this stage a spongy structure with numerous
lacunae. During later postmoult (stage B) and throughout intermoult (stage C), the
larvae reinforce the cuticle, while the epidermis shows an increasing condensation with
reduced lacunar spaces and conspicuous tissue growth. Early premoult (substage D
0
)
begins with a retraction of the epidermis from the cuticle (apolysis), which is first
visible at the setal bases. During intermediate premoult (substage D
1
), epidermal
invaginations take place, leading to a substantial enlargement of the epidermal surface
and initiating the formation of new setae and appendages (setogenesis, morphogenesis).
Subsequently, a thin new cuticle is secreted on the epidermal surface (substages D
2-4
).
Moulting (stage E) is a very short process, which usually takes only a few minutes. It
begins dorsally with a cuticular rupture between the cephalothorax and the pleon,
followed by a rapid retraction of the pleon from the old exoskeleton, and eventually,
15
shedding the anterior parts of the old cuticle. The combined postmoult-intermoult
period (stages A-C) took at both experimental temperatures ca. 40-50% of total instar
duration, while the premoult period (stage D) required slightly more than one half of the
time. As larval growth is known to occur predominantly during postmoult and
intermoult, and the latter is generally considered as the metabolically most stable phase
within the moulting cycle, we suggest that physiological and biochemical measurements
comparing successive larval instars should be carried out near the end of this period, i.e.
at ca. 30-40% of the moulting cycle.
Keywords: Moulting cycle, Larvae, Macrobrachium, Freshwater prawn
16
1. Introduction
Growth and development of crustaceans appear to be discontinuous processes
associated with successive moults (Hartnoll, 2001). Using histological and other
morphological methods, Drach (1939) described in great detail moult-related
anatomical changes occurring regularly in the integument of adult edible crabs, Cancer
pagurus. Based on variations in the hardness of the cuticle as well as in epidermal and
cuticular structures, he proposed a classification system for the moulting cycle with five
principal stages (A-E) and numerous substages. This system was later further elaborated
by Skinner (1962) and Drach and Tchernigovtzeff (1967), and it has been used in
numerous studies on adult Decapoda and other crustaceans (Charmantier-Daures and
Vernet, 2004).
Besides in anatomy and morphology, the moulting cycle implies also changes in
behaviour, physiology and biochemistry, including cyclic activities of an antagonistic
hormonal control system (for review, see Skinner, 1985; Chang, 1995; Charmantier-
Daures and Vernet, 2004). Hence, knowledge of the course of the moulting cycle stage
is highly important for the understanding of various aspects of crustacean biology,
including physiology and biochemistry (e.g. Spindler-Barth, 1976; Chang, 1995;
Ahearn et al., 2004; Gaxiola et al., 2005), behaviour (Thompson and McLay, 2005;
Mikami, 2005), food requirements (Mantelatto and Christofoletti, 2001; Giménez et al.,
2002; Schmidt et al., 2004), reproduction (Diaz et al., 2003; Tarling and Cuzin-Roudy,
2003; de Lestang and Melville-Smith, 2006), accumulation dynamics of toxic
substances (Bondgaard and Bjerregaard, 2005; Norum et al., 2005), as well as fisheries
and aquaculture of commercially important species (Ziegler et al., 2004; de Oliveira et
al., 2006; Brylawski and Miller, 2006).
17
In basic scientific investigations as well as in applied research including
crustacean aquaculture, all such cyclical changes have therefore implications for the
experimental design and for the evaluation of data obtained from either laboratory or
field studies. While the moulting cycle has extensively been studied in adult Decapoda
and other crustaceans, it is much less known for larval stages, mostly due to practical
problems related to small body size, a thin and hardly structured integument, short
moulting cycles, and restricted availability of materials with precisely known age within
a moulting cycle (for review, see Anger, 2001).
In Brazil, the Amazon River prawn, Macrobrachium amazonicum Heller 1862 is
commercially fished (Odinetz-Collart, 1993) and has also a high potential for
aquaculture (Kutty et al., 2000; Kutty, 2005; New, 2005). Technologies for hatchery
and grow-out are presently under development (see New and Valenti, 2000; New,
2005). Populations of this species live in both freshwater and brackish estuarine habitats
(Moreira et al., 1986; Magalhães and Walker, 1988; Odinetz-Collart, 1991a, b; 1993;
Bialetzki et al., 1997). M. amazonicum shows an extended type of larval development
with ca. 9-11 free-swimming stages (Guest, 1979; Magalhães, 1985), requiring at
optimal rearing conditions (30°C, 10‰ salinity, Valenti, unpubl. data) about 18-21 days
from hatching to metamorphosis. The larval physiology of M. amazonicum has
experimentally been studied by only a few authors. McNamara et al., (1983) and
Zanders and Rodriguez (1992) described effects of temperature and salinity on
respiration rates of the two earliest zoeal stages, and Moreira et al., (1986) studied
effects of salinity on the upper thermal limits for their survival. In these investigations,
however, changes during the moulting cycle have not been considered. The present
study provides the first information about the course of the moulting cycle in larval
Amazon River prawn, M. amazonicum.
18
2. Material and methods
Ovigerous females of M. amazonicum were obtained from the Aquaculture
Center (CAUNESP) broodstocks, at the São Paulo State University, Brazil. They
originated from the northeastern Brazilian state of Para (01°13’S 48°17’W). Prawns
were transported to the Helgoland Marine Biological Laboratory, Germany, where they
were maintained in individual aquaria (30L) with freshwater, aeration, constant
temperature 29°C, and artificial 12:12h daylight:darkness regime. The adults were fed
daily with fish meat and grated carrots.
After hatching, ca. 500 larvae were mass-reared in gently aerated 1L glass
beakers filled with brackish water (10‰ salinity) kept at two constant temperatures
(21°C and 29°C), under the same conditions of light. The larvae were fed daily with
freshly hatched Artemia sp nauplii, the water was changed, and dead individuals were
removed. The successive larval stages (in order to avoid confusion with the term
“stage” in the context of the moulting cycle, from hereon referred to as larval “instars”,
for review of terminology, see Anger, 2001) were microscopically identified using the
morphological description provided by Guest (1979). When moults occurred, the larvae
were separated according to their instar, so that each rearing beaker contained
exclusively individuals being in the same instar and with the same age within a given
moulting cycle.
Samples of 3-5 larvae were taken twice daily from the cultures and examined
under a BH2-NIC (Olympus) photo-microscope equipped with differential interference
contrast. Changes in the epidermal structures were recorded and photographically
documented. We used primarily the larval telson as the reference region, and
additionally the uropods in advanced stages (from zoea III) (cf. Anger, 1983, 2001),
because these body parts are thin and transparent, so that changes occurring in the
19
epidermis and cuticle could easily be seen. As all larval stages presented in principle the
same sequence of anatomical modifications, we describe in this paper the moulting
cycles only for the two earliest instars (zoea I-III).
3. Results
The duration of development through successive larval instars depended greatly
on temperature. While the average time for each moulting cycle varied from ca. 3-4
days at 21°C, only 1-2 days were required at 29°C. Although late instars (from zoea V)
tended to develop at a slightly slower speed than the earliest ones, the duration of the
moulting cycle was generally too short to allow for a high temporal resolution of
sampling and microscopical examination. As another problem preventing a precise
moult-staging, the larval integument was found to be thin and little structured, and
morphological changes were often indefinitive. Thus, transitions between stages and
substages could not be identified with a comparably high accuracy and a precise timing
as in Drach’s classical system (elaborated for adult crabs with thick and multi-layered
structures). We therefore decided to combine the principal stages of postmoult (A-B)
and intermoult (C), where gradual rather than abrupt changes occurred. After the
combined stages A-C, premoult (stage D) and the moulting process (E) could be
identified. Although our description presents also some details including those of the
important premoult substages D
0
and D
1
, a precise schedule of their timing cannot be
provided.
The course of the moulting cycle was similar in all successive larval instars. We
therefore show here only some typical integumental changes, using micrographs taken
from the telson and uropods of the first three zoeal instars as examples (see Figs. 1, 2).
20
The process of ecdysis is documented with photos showing the moult from the zoea I to
the zoea II instar.
3.1. Stages A-C combined (postmoult-intermoult)
Stage A (early postmoult). Immediately after hatching from the egg (zoea I) or
moulting (later instars), the cuticle is thin and wrinkled, and the larval body is
completely soft (ascertained by probing with a delicate forceps). Microscopical
examination revealed a spongy epidermal tissue structure with numerous large and
irregularly shaped lacunar spaces. Figures 1A and 2A illustrate this condition for the
telson of the zoeal instars I and II, respectively. Within a few minutes during and after
ecdysis, the larva takes up water, so that the integument is rapidly stretched, previously
invaginated setae and appendages are evaginated, wrinkles disappear, and the body
attains its final size and shape (cf. below, Figs. 3D, E).
Stage B (late postmoult). The cuticle becomes more rigid, and the epidermal
tissues begin to concentrate along the inner surface of the cuticle (as examples, see
telson of the zoea II, Fig. 2B, uropod of the zoea III, Fig. 2D).
Stage C (intermoult). Both the reinforcement of the cuticle and the condensation
of the epidermis tissues continue to a maximum, accompanied by a gradual reduction of
lacunar spaces and conspicuous tissue growth (see zoea I, Fig. 1B).
The average duration of stages A-C combined was ca. 2.5d at 21°C and slightly
less than 1d at 29°C, representing ca. 40-50% of the total time of the moulting cycle.
3.2. Stage D (premoult)
Substage D
0
(early premoult). Substage D
0
, the onset of the premoult period, is
well characterized by the beginning retraction of the epidermal matrix from the cuticle.
This conspicuous process (termed apolysis, Jenkin and Hinton, 1966) is first visible at
21
the bases of the terminal setae of the telson (Fig. 1C), proceeding only later through
other body regions and appendages.
Substage D
1
(intermediate premoult). The beginning of this substage is indicated
by the occurrence of epidermal infoldings or invaginations (Figs. 1D, 2C, 2E). This
internal enlargement of the tissue surface is a prerequisite for morphological
reconstruction processes (morphogenesis) including the lengthening of already existing
setae (setal growth), the formation of new setae (setogenesis, Fig. 2F), and the
appearance of other completely new organs (organogenesis). During these sub-surface
reconstruction processes in the epidermal tissues, the cuticle (i.e. the external shape and
size of the larval body) does not change. As an example, Figure 2C shows the formation
of uropods. These appendages appear externally only after the moult to the zoea III
instar (Fig. 2D), but they can already be seen inside the telson of the zoea II instar (Fig.
2C).
Substages D
2-4
(late premoult). When morphogenesis has been completed, a very
thin new cuticle is secreted on the surface of new epidermal structures such as setae and
appendages, while the gap between the old and the new cuticle increases (Fig. 2F). No
resorption processes or other changes in the cuticular layers, as observed in adult crabs
(Drach, 1939), were found during this period, so that no further distinction between
substages D
2-4
of Drach’s classification system was possible. The average duration of
complete Stage D was ca 3-4 days at 21°C and about 1d at 29°C, corresponding to ca.
50-60% of the moult-cycle duration.
3.3. Stage E (ecdysis)
The moulting process (ecdysis) takes normally at most a few minutes. It is
initiated by larval pumping movements, followed by a dorsal rupture of the cuticle
22
between the cephalothorax and the pleon. First, the telson is retracted from the old
exoskeleton (Figs. 3A-D). Subsequently, the larva sheds also the anterior parts of its
exuvia, so that a new larval instar appears, and a new moulting cycle begins (Fig. 3E).
4. Discussion
The course of epidermal and cuticular changes observed during moulting cycles
of larval Amazon River prawn, Macrobrachium amazonicum, is generally similar to
those previously described for other decapod crustacean larvae (Freeman and Costlow,
1980; McNamara et al., 1980; Anger, 1983, 1984). Comparison with adult life-history
stages (e.g. Drach, 1939; Peebles, 1977; Dexter, 1981) is difficult, because technical
constraints such as very short moult-cycle duration and a thin and little structed larval
integument do not allow for a comparably high resolution in the description of larval
moulting cycles (for review, see Anger, 2001).
For practical purposes in applied research including crustacean aquaculture,
however, the lack of numerous precisely defined substages may not be a serious
problem, as an identification of a few major moult stages should be sufficient to allow
for a separation of fairly homogeneous materials taken from large cultures, e.g. for
subsequent experiments, physiological measurements, or biochemical analyses. Our
study may therefore provide a simple and practical guide for the identification of the
major moult stages in larval M. amazonicum, aiding to the selection of materials for
studies of larval metabolism, growth, biochemical composition, or other relevant
aspects of crustacean aquaculture.
Various structures can be used for moult-staging in larval decapods. Freeman
and Costlow (1980) used for this purpose the antennae of larval mud crab
(Rhithropanopeus harrisii), while Anger (1983, 1984) used mainly the telson, but
additionally also pereiopods, antennae, and dorsal spines, to describe changes in the
23
integumental structures of larval spider crab (Hyas araneus). In early juvenile crayfish
(Parastacoides tasmanicus, Astacus leptodactylus) and penaeid prawns (Penaeus
esculentus), the uropods were most commonly used, at least in addition to the telson
(Mills and Lake, 1975; Herp and Bellon-Humbert, 1978; Smith and Dall, 1985). In the
present work, the description of the moulting stages was principally based on
microscopical examination of the larval telson, because this body part is plane, thin and
transparent, so that changes in the epidermal tissues can be easily observed. Similar
observations were made in a study by McNamara et al., (1980) with larvae of a
congener, the shrimp Macrobrachium olfersii. The uropods appear in these palaemonid
species only from the zoea III instar, providing an additional reference region for
studies of later larval development.
Tayamen and Brown (1999) proposed criteria for the evaluation of larval quality
in Macrobrachium rosenbergii, based on characteristics of body coloration, setation,
muscles, swimming behaviour, etc. An opaque appearance of the muscles in the pleon,
for example, along with sluggish behaviour, represent a poor condition. However, the
same characteristics occur also during early postmoult, when the larval body is limp and
the larvae tend to sink towards the bottom due to weak swimming activity. A “healthy”
colour and behaviour, e.g. a positive response to light, are rapidly recovered during later
postmoult and intermoult. This shows that the moulting cycle must be taken into
consideration when the health condition of shrimp larvae is evaluated.
The same applies to larval feeding and growth, which cease from late premoult
through ecdysis and early premoult, re-starting only in late postmoult or intermoult
(Anger, 2001). Stage C seems to be a period without dramatic structural and metabolic
changes, characterized only by substantial tissue growth. During the premoult period,
by contrast, the rates of growth and feeding decrease, while numerous structural
24
changes occur in the integument, and the mass-specific metabolic rate increases (Anger,
2001). As stage C is the metabolically and morphologically most stable period within
the moulting cycle, physiological experiments or biochemical analyses comparing
successive larval instars of a species, or equivalent instars of different species, should
preferably be conducted during this phase. In the larvae of M. amazonicum, the most
suitable reference point for such comparisons may thus be found at ca. 30-40% of total
moult-cycle duration, i.e. near the end of stage C and shortly before the transition to
substage D
0
, when apolysis occurs.
Besides intrinsic hormonal control factors, extrinsic variables such as
temperature, food, water chemistry, or photoperiod may affect the moulting cycle in
crustaceans (Chang, 1995; Ismael and New, 2000; Kulum and Ku, 2005). Also in the
present study, it was accelerated by higher temperature (29° vs. 21°C), shortening the
absolute time spans for each stage of the moulting cycle. Preliminary observations
suggested, however, that variation in temperature might change also the temporal
proportions of individual stages within the moulting cycle, with an apparently
increasing duration of stages A-C combined in relation to stage D. However, further
studies with a substantially enhanced temporal resolution of sampling and
microscopical observation are necessary to evaluate the extent and significance of such
an effect. Also, successive larval instars might differ in their response to environmental
factors, which requires more detailed studies with various instars exposed to differential
experimental conditions.
Acknowledgments
This research was supported by CAPES/PQI (Brasília, Brazil, Grant 137030),
Deutscher Akademischer Austauschdienst (DAAD, Bonn, Germany, Grant
25
A/05/33827), and FAPESP (São Paulo, Brazil, Grants 05/54276-0). The first author
would like to thank the Biologische Anstalt Helgoland, Helgoland, Germany, and the
Crustacean Sector of the CAUNESP, Brazil, for technical support. Thanks are also due
to U. Nettelmann and K. Walther for technical assistance in the laboratory.
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32
D
i
A
ep
B
ep
C
ep
ap
ep
ap
D
i
Figure 1 – Telson of Macrobrachium amazonicum, larval instar zoea I: A. Early postmoult
(stage A), with spongy epidermal tissue structure, large hemolymph-filled spaces (lacunae); B.
intermoult (stage C), with epidermal growth, tissue concentration along the inner surface of the
cuticle, reduced lacunar spaces; C. early premoult (stage D, substage D
0
), with beginning
epidermal retraction from the cuticle (apolysis); D. intermediate premoult (substage D
1
),
with advanced apolysis, beginning epidermal invaginations at the setal bases; ap, apolysis; ep,
epidermis; i, invagination.
33
B
ep
A
ep
U
T
ep
D
C
u
ep
ap
i
ns
nc
ap
F
U
T
i
i
ap
E
Figure 2
- Macrobrachium amazonicum, larval instars zoea II (A-C) and zoea III (D-F): A. zoea II, early
postmoult (stage A); B. zoea II, late postmoult (stage B), with advancing tissue concentration along the
inner surface of the cuticle; C. zoea II, intermediate premoult (substage D1), with uropod formation (u)
inside the telson of the zoea II; D. zoea III, postmoult (stage B), with telson (T) and newly appearing
uropod (U); E. zoea III, intermediate premoult (substage D1); F. zoea III, late premoult (substages D2-4),
with formation of a thin new cuticle (nc); ap, apolysis; ep, epidermis; i, invagination, ns, new setae.
34
A
oe
ep
B
ep
oe
D
oe
ZII
E
ZII
oe
C
ch
oe
Figure 3 -
Macrobrachium amazonicum, moulting (ecdysis) of larval instar zoea I to zoea II: letter A
until C., retraction of the telson from the old exoskeleton (oe), epiderme (ep); chromatophore (ch);
letter D, shedding of the exuvia, appearance of the zoea II (ZII); letter E, zoea II with shed exuvia; ch,
typical dorsal chromatophore on larval pleon.
35
CAPÍTULO 3 (Aquaculture Research)
Variação ontogenética do metabolismo durante os estágios iniciais do camarão-da-
amazônia Macrobrachium amazonicum (Heller, 1862) (Crustacea, Decapoda,
Palaemonidae)
Ontogenetic variation in metabolism during the early life stages of the Amazon
River prawn Macrobrachium amazonicum (Heller, 1862) (Crustacea, Decapoda,
Palaemonidae)
Resumo
O peso seco, o consumo de oxigênio e a taxa de excreção de amônia-N foram
determinados em embriões, larvas (zoea=Z) (ZI a ZIX) e pós-larvas (PL) com 1, 7 e 14
dias após a metamorfose (PL1, PL7 e PL14) em Macrobrachium amazonicum. Animais
em estágios de pós-muda/intermuda (A/C) foram classificados conforme o estágio de
desenvolvimento, e colocados nas câmaras respirométricas (ca. 30mL) por 2h para
quantificar as taxas metabólicas. Após esse período, as análises foram realizadas com
titulação de Winkler para o oxigênio e método de Koroleff para a determinação da
amônia-N. As taxas metabólicas foram expressas como individual e peso-específico. A
relação atômica O:N foi calculada. As taxas individuais do consumo de oxigênio e
excreção de amônia-N aumentaram durante o desenvolvimento larval. Os valores
médios do consumo de oxigênio variaram de 0,10±0,02 a 4,02±1,10µg.ind
-1
.h em
embrião e PL14, respectivamente e não diferiram de embrião a ZIV e ZV a ZIX
(P>0,05). A taxa de consumo de oxigênio foi significativamente maior em zoea e
embrião (P<0,05). A taxa de excreção de amônia individual variou de 0,0090±0,0039
em embrião a 1,0413±0,2492µgNH
3
-N.ind.
-1
h em PL14 e não diferiram entre embrião-
36
ZIV e ZV-ZIX (P>0,05), mas diferem entre PL1-PL14 (P<0,05). Os maiores
incrementos na excreção de amônia individual foram observados entre ZIV–ZV, ZIX–
PL1 e PL7–PL14. As taxas de excreção peso-específico apresentaram dois grupos,
embrião–ZII (P>0,05) e ZIII–PL14 (P>0,05). O menor valor encontrado foi em embrião
(0,17±0,07mgNH
3
-N.gPS
-1
.h) e os maiores foram observados em ZV e PL1 (0,65±0,25
e 0,64±0,27mgNH
3
-N.gPS
-1
.h, respectivamente). Foram observados baixos valores de
O:N, variando de 3,0 a 10,0, mostrando que em M. amazonicum ocorre o predomínio do
metabolismo de proteína. O valor de b obtido da relação consumo individual de
oxigênio/peso seco na análise de regressão foi 0,8 e foi significativamente menor que 1
(P<0,05) e da excreção individual/peso seco foi 1,1, e não diferiu de 1 (P>0.05). A
associação com as mudanças morfológicas e comportamentais, tais como a atividade
natatória, formação de novos apêndices (urópodos, pleópodos, pereópodos, etc.), o
tamanho do corpo, o decréscimo na relação superfície:volume, a estratégia de
alimentação e a transição de um estágio de zoea planctônico a pós-larva bentônico é
discutida.
Palavras-chaves: Larva, Macrobrachium amazonicum, Metabolismo, Excreção de
nitrogênio, Consumo de oxigênio, Pós-larva.
37
Abstract
Dry weight, oxygen consumption and total ammonia-N excretion rate were
determined in embryos, larvae (zoea=Z) (ZI to ZIX) and post-larvae (PL) with 1, 7 and
14 days after metamorphosis (PL1, PL7 and PL14) of Macrobrachium amazonicum.
Animals in postmolt-intermolt (A-C) stages were sorted according to their
developmental stages, and placed into respirometric chambers (ca. 30mL) for 2h to
quantify metabolic rates. After this period, analyses were carried out in end-point
samples by Winkler’s titration for oxygen and Koroleff’s method for ammonia-N
determination. Metabolic rates were expressed both as individual and dry mass-specific
oxygen consumption and ammonia-N excretion rates. Atomic O:N ratio was also
calculated. Individual rates of oxygen consumption and ammonia-N excretion increased
throughout larval development. Average values of oxygen consumption varied from
0.10±0.02 to 4.02±1.10µg.ind
-1
.h in embryo and PL14, respectively and did not differ
from embryo to ZIV and from ZV to ZIX (P>0.05). Post-larval oxygen consumption
rates were significantly higher than in zoea and embryo (P<0.05). Individual ammonia-
N excretion rates varied from 0.0090±0.0039 in embryo to 1.0413±0.2492µgNH
3
-
N.ind.
-1
h in PL14 and did not differ among embryo-ZIV and ZV-ZIX (P>0.05), but
differ among PL1–PL14 (P<0.05). The highest increments in individual ammonia-N
excretion were observed between ZIV–ZV, ZIX–PL1 and PL7–PL14. Weight–specific
excretion rates presented two groups, Embryo–ZII (P>0.05) and ZIII–PL14 (P>0.05).
The lowest value was found in embryo (0.17±0.07mgNH
3
-N.gDW
-1
.h) and the
maximum value was observed in ZV and PL1 (0.65±0.25 and 0.64±0.27mgNH
3
-
N.gDW
-1
.h, respectively). Low O:N values were observed, ranging from 3.0 to 10.0,
which showed that M. amazonicum has a protein dominated metabolism. The b value
obtained in the individual oxygen consumption versus dry weight regression analysis
38
was 0.8 and was significantly lower than 1 (P<0.05) and individual excretion versus dry
weight was 1.1, which did not differ from 1 (P>0.05). The association with
morphological and behavioral changes, such as swimming activity, new appendages
(uropods, pleopods, pereopods, etc.) formation, body size, a decrease in the
surface:volume ratio, feeding strategy and transition from a planktonic zoea stage to a
benthic post-larval stage is discussed.
Keywords: Larvae, Macrobrachium amazonicum, Metabolism, Nitrogen excretion,
Oxygen consumption, Post-larvae.
39
1. Introduction
The Amazon River prawn Macrobrachium amazonicum has a large geographic
distribution in South America, occurring in Brazil, Colombia, Venezuela, Peru,
Equador, Bolivia, Paraguay and Argentina (Holthuis, 1952; Pettovelo, 1996; Bialetzki
et. al., 1997). Some populations live in interior areas very far from the sea and complete
life cycle in freshwater. On the other hand, the populations that occur close to the coast
are dependent of brackish water for larval development (Kutty et al., 2000). The larvae
hatch as a typical free-swimming palaemonid zoea and pass through nine zoeal stages
(Guest, 1979). They are planktonic, swimming actively, tail first, upside-down within
the water column and exhibit positive phototaxy. After metamorphosis, post-larvae are
miniature of adult prawns and swim with the dorsal side uppermost assuming a more
benthic lifestyle. Both larvae and post-larvae are mainly omnivorous (Araújo & Valenti,
2005).
M. amazonicum presents high potential for aquaculture (New, 2005). Some
characteristics, which may contribute to the rearing feasibility, are fast larval
development (18-21 days) in recirculating systems (30±1°C and 10‰ salinity), and high
growth and survival rates in hatchery, nursery and grow-out phases (Valenti, unpubl.
data). Furthermore, meat has a firmer texture and a more marked taste when compared
with M. rosenbergii, hence being better accepted by consumers (Moraes-Riodades &
Valenti, 2001).
There are some studies on M. amazonicum wild populations (Odinetz-Collart,
1991a,b; Bialetzki et al., 1997), male morphotypes (Moraes-Riodades & Valenti, 2004),
reproduction (Odinetz-Collart & Rabelo, 1996; Da Silva et al., 2004), hatchery systems
(Lobão et al., 1994), salinity in culture systems (Guest, 1979; McNamara et al., 1983;
Zanders & Rodriguez, 1992), feeding (Barreto & Soares, 1982; Araújo & Valenti,
40
2005), economic viability as baits (Hayd, et al., submitted-a). However, studies related
with metabolic rates in M. amazonicum are few reduced. Favaretto et al. (1976) studied
the oxygen consumption in adults collected from rivers, McNamara et al. (1983)
described the effects of salinity on respiratory metabolism, survival and moulting in
zoea I and II and Zanders & Rodriguez (1992) evaluated the effect of temperature on
respiratory metabolism of M. amazonicum. None included the evaluation of oxygen
consumption and ammonia-N excretion rates from embryo to post-larvae.
Measuring metabolic rates is essential to assess energy requirements for larval
development (Lemos & Phan, 2001). Accordingly the authors emplaized that, oxygen
consumption and ammonia excretion are probably the main physiological parameters to
be evaluated. In addition, information on metabolism and the processes related to the
use of energy throughout the ontogenetic development is necessary to understand the
ecologic role of the species and to improve culture practices. It allows the establishment
of the adequate density for larvae and post-larvae transportation and to dimension the
biofilters used in recirculating hatchery systems. Thus, the objective of this study was to
evaluate metabolic rates in early life stages of hatchery reared M. amazonicum through
the simultaneous measurement of oxygen consumption and ammonia-N excretion rates.
2. Material and methods
Experimental animals
Embryo, larvae and post-larvae of M. amazonicum were obtained from
broodstock formed by wild animals captured in Northeast Para, Brazil (01°13’25”S
48°17’40”W) in 2001. Females carrying eggs in advanced stages of embryonic
development (approximately 2h before hatching) were collected. This phase of egg
incubation is characterized by slow movements of females and the presence of visible
41
eyes inside the transparent eggs. Animals were disinfected in formaldehyde solution at
20ppm for 30min. and then kept in a hatching tank at 70 ind.m
-2
and 8‰ salinity. It was
provided with aeration, heating system and artificial substrates. Some females had eggs
extracted for embryos metabolism and dry weight determination.
After hatching, larvae were stocked at 100 ind.L
-1
in 120 L cylindrical tanks
provided with mechanical and biological filter and artificial heater. Water temperature
was kept at 30°C and 10‰ salinity until metamorphosis to post-larvae (PL). Larvae was
fed on freshly hatched Artemia nauplii in zoea (Z) II to ZIV (3-7 nauplii. mL
-1
.day).
After this stage, they were fed on inert diet (25µg.mL
-1
.day) (see Mallasen & Valenti,
1998 for diet composition) plus Artemia nauplii (8 to 10 nauplii.mL
-1
.day), added to the
tanks twice daily until metamorphosis. Post-larvae were transferred to 1000 L tanks at 5
PL.L
-1
and fed on peletized diet twice a day (3–5g.day
-1
.tank Fri-Ribe Fri-Acqua
Camarão 35 LD). In this study, PL1 was the stage immediately after metamorphosis
while PL7 and PL14 were post-larvae with 7 and 14 days after metamorphosis, reared in
freshwater at 30±1°C.
Oxygen consumption and ammonia-N excretion determination
Cylindrical plastic containers with approximately 30mL sealed with silicon
tablets (Lemos & Phan, 2001; Lemos et al., 2003) were used as respirometric chambers
to incubate animals for oxygen consumption and ammonia-N excretion measurement.
Chambers were individually identified and the exact volume was gravimetrically
determined. An orifice of 1.5mm in the center of the cover enabled the elimination of
air bubbles from inside the chamber during closure. Respirometers were hermetically
closed inside 1000mL beakers filled with water (10‰ salinity) to avoid formation of
bubbles inside the containers. After being closed, inside water was isolated from air by
42
a plastic tablet 1.7cm diameter silicon seal so that the tension between orifice water and
the silicon seal could block respirometer inside water and air gas exchange.
Only larvae and post-larvae in the postmolt–intermolt (A-C) (Hayd et al.,
submitted or see chapter 1) and in the same development stage were used. The number
of individuals used inside respirometer was determined according to individual dry
mass (Table 1). The biomass:volume ratio (B:V) was calculated by dividing the total
dry mass of individuals by the chamber volume and varied from 16.9 to 85.6µg.mL
-1
(Table 1). Embryos, ZI to ZIX, PL1, PL7 and PL14 were kept in the sealed
respirometric chambers samples for 2h. Seawater and Milli-Q (Millipore) fosh water at
10‰ salinity were used as control respirometer without animals. Samples and controls
were kept in water bath at 30±1°C. After incubation, the tablet was removed, and water
was sampled through a plastic canula and stored in glass syringes chemically calibrated
(syringes plus plastic nozzles).
Variation in oxygen and ammonia-N nitrogen contents was calculated by the
difference between values obtained in sample (with animals) and control (no animals)
units. Dissolved oxygen in water at the end of every test was determined by Winkler
analysis method adapted to small volumes (Fox & Wingfield, 1938) and was never
lower than 80% of saturation. For ammonia-N analysis, the method described by
Koroleff (1983) in separate water samples was used. Oxygen consumption and
ammonia-N excretion were expressed as individual (µg.ind.
-1
.h) and dry-mass specific
(mg.g
-1
DW.h) rates. Salinity effect was corrected using the factor 1.06 (Koroleff, 1983).
Atomic O:N (oxygen respired to nitrogen excreted) ratios were calculated by dividing
the number of gram-atom of consumed oxygen by the number of gram-atom of nitrogen
excreted (N.O
-1
) in each developmental stage (Mayzaud & Conover, 1988).
43
Individual dry weight (DW) was determined by a groups of 10 (embryo and
ZIX) or 6 (PL) individuals gently rinsed with distilled water, dried with filter paper and
separated prior to weight determination with eight replicates. After 48h at 70°C, dry
samples were weighed on a Mettler Toledo AT21 analytical balance, at the nearest 1µg.
Statistical analysis
The results were subjected to analyses of detection and exclusion of outliers
(Statistica software, v. 6.0) with coefficient 1.5. Total excluded data was always lower
than 20% of data obtained for each stage (Statistica Software, v.6). The final number of
replicates is in Table 2. Normality was tested using the Shapiro-Wilk test and
Homocedasticity by Levene’s (using SAS 9.0 software). Differences among means
were tested by one-way ANOVA followed by Duncan's multicomparison test. Data of
the metabolic rates (oxygen consumption and ammonia excretion, in µg.ind.
-1
.h) and dry
weight (µg) were logarithmically adjusted by regression analysis (Statistica Software,
v.6). Slopes were compared to 1 using a t-test according to Zar (1999). Differences were
considered significant at P<0.05.
3. Results
Individual dry weight (DW) increased throughout the ontogenetic development
(Table 1) and varied significantly in different ages and developmental stages (P<0.05).
The highest increases in subsequent stages were verified between the ZIV to ZV
(118.03%) and PL1 to PL7 (99.52%), while the lowest value was found between ZIX
and PL1 (5.41%).
Individual oxygen consumption rate (VO
2
) showed four groups during the
developmental stage, Embryo–ZIV, ZV–ZIX, PL1–PL7 and PL14, and into these
groups there was no significant difference (P>0.05) (Table 2). Embryo presented the
44
lowest value (0.10±0.02µg.ind
-1
.h) while PL14 presented the highest one
(4.02±1.10µg.ind
-1
.h) and differ to all others stages (P<0.05) (Table 2). The weight-
specific oxygen consumption (QO
2
) varied during development and showed four
groups, Embryo, ZI–ZIV, ZV–ZVI, ZVII–PL14, and into these intervals there was no
significant difference (P>0.05), except in PL1 (Fig. 1A). Higher mean values were
found among ZI–ZIV and it decreased in subsequent stages, except in PL1 (Fig. 1A).
Individual ammonia-N excretion did not differ among embryo-ZIV and ZV-ZIX
(P>0.05), but differ among PL1–PL14 (P<0.05) (Table 2). The minimum observed
value was 0.0090±0.0039µgNH
3
-N.ind.
-1
.h
in embryo, and the maximum,
1.0413±0.2492µgNH
3
-N.ind.
-1
.h
in PL14 (Table 2). The highest increments in
individual ammonia-N excretion were observed between ZIV–ZV, ZIX–PL1 and PL7–
PL14. Weight–specific excretion rates presented two groups, Embryo–ZII (P>0.05),
ZIII–PL14 (P>0.05) (Fig. 1B). Stages ZIII, ZV and PL1, presented high standard
deviation (SD). Similarly to oxygen consumption, the lowest value was found in
embryo (0.17±0.07mgNH
3
-N.gDW
-1
.h), however the maximum value was observed in
ZV and PL1 (0.65±0.25 and 0.64±0.27mgNH
3
-N.gDW
-1
.h, respectively) (Fig. 1B).
Atomic O:N ratio were generally low, ranging from 3 in ZVII and PL14 to 10 in
embryo and ZI (Table 2).
Relationships between metabolic rates and dry weight (µg) showed high
coefficients of determination (r
2
>0.8). The slope obtained for individual oxygen
consumption/dry weight relationship was 0.8 and was significantly lower than 1
(P<0.05), while the b value for individual excretion/dry weight relationship was 1.1,
which did not differ significantly from 1 (Table 3).
45
4. Discussion
The weight of M. amazonicum increases during development stages. This pattern
was also observed for Macrobrachium rosenbergii larvae (Stephenson & Knight, 1980;
Agard, 1999; Ismael et al. 2001), and for other caridean larvae, such as Sesarma rectum
(Anger & Moreira, 2004), Crangon crangon and Crangon allmanni (Criales & Anger,
1986). The weight increase during larval and later developmental stages clearly
influences the weight-metabolic rate (Vernberg et al. 1981), since larvae show an
increase in individual oxygen consumption rate (VO
2
) and a decrease in weight-specific
oxygen consumption (QO
2
) (Anger, 2001). The VO
2
values increased significantly in
successive developmental stage of M. amazonicum (P<0.05). This increase may be
related with the different kinds the swimming activities, energy lost and with the gain
weight during the course of development.
The QO
2
values varied significantly through M. amazonicum ontogenetic
development (P<0.05). The highest values occurred in the first stages (ZI–ZIV) and the
lowest in the latter ones, except in PL1. High QO
2
at ZI–ZIV may be related to the
noticeable natatorium activities due to lower size, which spend more energy for
maintenance in water column, and thus, consuming more oxygen. The lower values in
the last stages may be due to profound developmental changes (Anger, 2001), a
disproportionate increase of metabolically inactive tissues, such as skeletal materials
and fat reserves and by overlaying external ecological factors (Anger, 2001) and a
decrease in relation surface:volume. Besides, in this period larvae show a bigger size
and loose less energy for maintaining in water column during planctonic phase, and
after the transition to benthic phase after metamorphosis in post-larvae (PL1).
Decreasing in QO
2
during the last larval stages was also observed in M. rosenbergii
larvae (Agard, 1999) in crabs Chamagnathus granulate (Ismael et al. 1997) and Hyas
46
araneus (Anger & Jacobi, 1985) and in the penaeidae shrimp Farfantepenaeus
paulensis (Lemos & Phan, 2001). The higher QO
2
rates in PL1 may be due to
osmorregulatory response and possible stress due to little time in acclimation at
respirometer. This physiological stress may indicate PL1 needs longer acclimation time
when transferred from brackish water to freshwater in nursery tanks. According to
Agard (1999) Macrobrachium rosenbergii larvae were moved by 1‰ salinity per hour
and Anger (pers. com.) suggested transferring from 10‰ salinity for 5‰, but staying in
this salinity for 4h and than transfering for lower salinity during 2 days to avoid the
mortality in subsequent stages.
Individual ammonia-N excretion rates increased during larval development
except in ZVI and ZVIII. This fact may be related with the higher larval variability
observed in larvae started in ZV. Present results showed three phases in excretion rates,
embryo–ZIV, ZV–ZIX and PL1–PL14. In the first phase larvae uses internal energy
reserves from yolk (Embryo–ZII) and after ZII a small quantity of the exogenous food.
However, there is yolk until ZIV (Araújo and Valenti, submitted). Therefore, larvae
present little dependence on exogenous feed. In the second phase (ZV–ZIX) larvae
started fed on inert diet plus Artemia nauplii and sometimes cannibalistic behavior were
observed in the tanks (pers. observ.). Thus, exogenous feed is essential. In the last phase
(PL1–PL14) there is an increase in individual ammonia excretion rates due to an
increase in larvae weight and consequently in the ingested food. In addition, there are
changes in the animal feeding strategy and habitat, assuming a more benthic lifestyle
and omnivorous habit. Weight–specific excretion rates also varied throughout
developmental stages (P<0.05). However, stages ZIII, ZV and PL1 presented higher
standard deviation (SD). In ZIII and ZV this fact may be associated with the beginning
of feeding on the Artemia nauplii and inert food, respectively. On the other hand, in PL1
47
may be related with the different life strategies after metamorphosis when larvae
changed the planktonic to benthonic habit and the fast transference in ambient of 10‰
salinity to freshwater, described above. Likewise, increasing in weight-specific
ammonia-nitrogen excretion was observed in freshwater prawn M. rosenbergii larvae
when occured a decreasing in salinity (Agard, 1999). Thus, our results suggested longer
time for acclimation when PL1 is transferred from hatchery to the nursery freshwater
tanks. Nevertheless, decrease in metabolic rates during the later stages has been
reported for crustaceans (Chu & Ovsianico-Koulikowsky, 1994; Agard, 1999; Lemos &
Phan, 2001).
Information about metabolic rates is very important to aquaculture. This may
help for determination of densities during transportation of larvae and post-larvae and
for dimensioning the biofilter in recirculating systems. Our data showed the mean
values of the 0.3µg.larva
-1
.h for ammonia-N excretion, this represent a load of about
7.2µg.larva
-1
.dia, indicating consequently a total ammonia excretion of 7,200µg.dia
-1
for
1000 larvae. Thus, our results may be used for future calculus of larvae and early post-
larvae to transport and biofilter dimensioning of M. amazonicum. Biofilter volume of
the larval culture tanks has varied from 4 to 20% (Valenti et al. 1998) related with size
of hatchery tank. This size was based on the daily maximum expected ammonia-
nitrogen load in the larval culture system of M. rosenbergii and the bacterial carrying
capacity of the filter with crushed coral media being used (Valenti & Daniels, 2000).
Daniels et al., (1992) showed an example for calculating the amount of crushed coral
needed into the biofilters based on empirically data of 30µg.larva
-1
.dia (Valenti &
Daniels, 2000). Our data indicated that for M. amazonicum ammonia load is lower than
the recommended for M. rosenbergii and therefore biofilters may be lower too.
48
The O:N ratio is accepted as indicator of the metabolism substrate for energy
production (Anger, 2001). The index varies with the N content of the diet, reflecting the
biochemical composition of the used energy reserves (Mayzaud & Conover, 1988). O:N
ratio in the range 3 and 16 indicates predominant protein catabolism, while higher
values show an increasing utilization of lipids and/or carbohydrates. Present results
showed O:N values, ranging from 3 to 10 indicating a protein dominated metabolism.
The decrease trend in O:N of M. amazonicum from ZIII on may be associated with the
exhaustion of the lipid reserve (yolk) in the first zoea stages. This pattern was also
observed for early larvae of the palaemonid prawn M. rosenbergii (Agard, 1999) and
penaeidae shrimps Metapenaues ensis (Chu & Ovsianico-Koulikowsky, 1994).
The general relationship between organism size and metabolic rates is described
by the simple allometric equation, M = a W
b
(Bertalanffy, 1957), where M is the
metabolic rate per unit time, W is the body weight, and a and b are constants. This
equation compares individuals of the same species at different ages or sizes.
Bertalanffy’s classification showed three metabolic types according with relationship
between metabolic rate and body size. In the first, metabolic rate is proportional to the
surface or the 2/3 power of the body mass (b=0.67). In second, metabolic rate is
proportional to the body mass (b=1) and in the last type, metabolic rate is intermediate
between proportionality body mass and surface (0.67<b<1). Thus, the mass exponent b
generally varies between ca. 0.67 and 1.0, indicating that the metabolic rate is
proportional to the animal’s body surface area or to its volume. Variation in b may be
caused by changes in the relation between body shape and volume, in the proportions of
living protoplasm and metabolically inert components of biomass (Anger, 2001). In the
present study, the value of b in oxygen consumption logarithmic regression analysis was
0.8 below 1 (P<0.05), and the parameter represents the respiration rate of a unit animal
49
indicating that the increase in oxygen consumption rate was lower than growth in the
studied period. This value may indicate favorable conditions to larval development
(Lemos & Phan, 2001). On the other hand, the regression for ammonia excretion
showed that metabolic rate is proportional to the body mass during the development,
because the values are close to 1. The value of b<1 has been also registered in other
decapod, such as 0.904 for M. rosenbergii larvae (Stephenson & Knight, 1980) and
0.628 for juveniles (Nelson et al. 1977), 0.06 for Pagurus criniticornis larvae (Vernberg
et al. 1981) and 0.668 for adults (Shumway, 1978), 0.67 for Menippe mercenaria larvae
(Mootz & Epifanio, 1974) and 0.697 for penaeid shrimp F. paulensis in early stages
(Lemos & Phan, 2001).
Results obtained in the present study may be useful in developing and
optimizing rearing techniques of M. amazonicum, such as the determination of the
proportions biofilter:rearing tank size, aeration volume and stocking density in culture
tanks or in transport bags. Further complementary metabolic studies, such as elemental
and biochemical composition, activity of digestive enzymes and energy content should
be performed to elucidate the adaptative value of the metabolic changes during larval
development and to subsidize the formulation of a suitable diet for larvae rearing.
Acknowledgements
This research was supported by the CAPES/PQI (Brasília, Brazil, Grants
137030) and FAPESP (São Paulo, Brazil, Grants 05/54276-0). The authors are grateful
to the P. Atique and B. Donadon for technical assistance in the laboratory. D. Lemos
appreciate funding by FAPESP (05/50578-2) and CNPq/SEAP (504031/03-1).
50
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temperature and salinity on the metabolic rate of juvenile Macrobrachium
rosenbergii (Crustacea, Palaemonidae). Comparative Biochemistry and Physiology
56A, 533-537.
New, M.B. (2005). Freshwater prawn farming, global status, recent research and a
glance at the future. Aquaculture Research 36, 210-230.
Odinetz-Collart, O. (1991)a. Stratégie de reproduction de Macrobrachium amazonicum
en Amazonie Centrale (Decapoda, Caridea, Palaemonidae). Crustaceana 61(3),
253-270.
Odinetz-Collart, O. (1991)b. Tucurui dam and the populations of the prawn
Macrobrachium amazonicum in the lower Tocantins (PA-Brasil), a four years study.
Archive fur Hidrobiologie 122(2), 213-228.
Odinetz-Collart, O. & Rabelo, H. (1996). Variation in egg size of the freshwater prawn
Macrobrachium amazonicum (Decapoda, Palaemonidae). Journal of Crustacean
Biology Laurence 16(4), 684-688.
Pettovelo, A.D. (1996). First record of Macrobrachium amazonicum (Decapoda,
Palaemonidae) in Argentina. Crustaceana 69, 113–114.
Shumway, S.E. (1978). Osmotic balance and respiration in the hermit crab, Pagurus
bernhardus, exposed to fluctuating salinities. Journal of Marine Biology Association
U.K. 58, 869–876.
55
Stephenson, M.J. & Knight, A.W. (1980). Growth, respiration and caloric content of
larvae of the prawn Macrobrachium rosenbergii. Comparative Biochemistry and
Physiology 66A, 386-391.
Valenti, W.C., Mallasen, M. & Silva, C.A. (1998). Larvicultura em sistema fechado
dinamico. In: Carcinicultura de água doce, tecnologia para a produção de
camarões. Valenti, W. C (Ed.). Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP), São Paulo e Instituto Brasileiro do Meio Ambiente e dos
Recursos Naturais Renováveis (IBAMA), Brasília. p. 112–139.
Valenti, W.C. & Daniels, W.H. (2000). Recirculation hatchery systems and
management. In: New. M.B., Valenti. W.C. (Eds.). Freshwater prawn culture. The
farming of Macrobrachium rosenbergii. Londres. Blackwell. Oxford. p. 69-90.
Vernberg, W.B., Moreira, G.S. & McNamara, J.C. (1981). The effect of temperature on
the respiratory metabolism of the developmental stages of Pagurus criniticornis
(Dana) (Anomura, Paguridae). Marine Biology Letters. 2, 1-9.
Zanders, I. P. & Rodríguez, J.M. (1992). Effects of temperature and salinity stress on
osmoionic regulation in adults and on oxygen consumption in larvae and adults of
Macrobrachium amazonicum (Decapoda, Palaemonidae). Comparative
Biochemistry and Physiology 101A(3), 505-509.
Zar, J.H. (1999). Bioestatistical analysis. Prentice Hall, New Jersey, 663pp.
56
Table 1 – Age, dry weight (DW), number of individuals, and biomass:volume ratio in
respirometers used to determine oxygen consumption and ammonia-N excretion rates
during early life stages of Macrobrachium amazonicum at 30±1°C and 10‰ salinity.
Embryo, zoea I-IX=larval stages, PL1=post-larvae, PL7 and PL14=post-larvae with 7
and 14 days after metamorphosis, respectively. Results are expressed as mean
values±SD. Number of replicates=10.
Stage Age ¹ DW (µg) Individuals/
respirometer
Biomass:volume
(µg.mL
-1
)
Embryo
-0.1 71.7 ± 0.6 32 76.5
Zoea I
0.5 58.6 ± 0.3 12 23.4
Zoea II
2 63.7 ± 0.6 12 25.5
Zoea III
3 84.3 ± 1.2 6 16.9
Zoea IV
5 118.3 ± 2.9 6 23.7
Zoea V
7 258.0 ± 2.6 4 34.4
Zoea VI
8 301.3 ± 2.5 4 40.2
Zoea VII
9 433.0 ± 7.8 3 43.3
Zoea VIII
11 551.0 ± 24.1 2 36.7
Zoea IX
14 665.7 ± 16.2 2 44.4
PL1
18 701.7 ± 66.1 1 23.4
PL7
25 1400.0 ± 58.6 1 46.7
PL14
32 2569.4 ± 216.7 1 85.6
¹-days after hatching
57
Table 2 – Individual oxygen consumption and ammonia-N excretion rates and atomic
oxygen:nitrogen ratio (O:N) in early life of Macrobrachium amazonicum at 30±1°C and
10‰ salinity. ZI-IX=larval stages, PL1, PL7 and PL14=post-larvae with 1, 7 and 14
days after metamorphosis, respectively. Results are expressed as mean values±SD,
[number of replicates].
Developmental O
2
consumption NH
3
-N excretion O:N
Stage (µgO
2
.ind.
-1
h
-1
) (µgNH
3
-N.ind.
-1
h
-1
)
Embryo
0.10 ± 0.02 [8] d 0.0090 ± 0.0039 [6] e 10.0 [6]
Zoea I
0.20 ± 0.02 [9] d 0.0171 ± 0.0058 [9] e 10.0 [9]
Zoea II
0.20 ± 0.04 [12] d 0.0219 ± 0.0082 [10] e 8.0 [10]
Zoea III
0.36 ± 0.05 [11] d 0.0387 ± 0.0185 [6] e 8.0 [6]
Zoea IV
0.45 ± 0.11 [19] d 0.0644 ± 0.0255 [21] e 6.0 [21]
Zoea V
0.68 ± 0.16 [9] c 0.1689 ± 0.0523 [10] d 4.0 [10]
Zoea VI
0.74 ± 0.19 [19] c 0.1138 ± 0.0510 [13] d 6.0 [13]
Zoea VII
0.92 ± 0.21 [11] c 0.2739 ± 0.0250 [5] d 3.0 [5]
Zoea VIII
1.21 ± 0.23 [9] c 0.1931 ± 0.0408 [11] d 5.0 [11]
Zoea IX
1.37 ± 0.36 [16] c 0.3036 ± 0.0667 [4] cd 4.0 [4]
PL1
2.79 ± 0.81 [14] b 0.4460 ± 0.1735 [16] c 5.0 [16]
PL7
2.90 ± 0.72 [12] b 0.6487 ± 0.1467 [10] b 4.0 [10]
PL14
4.02 ± 1.10 [11] a 1.0413 ± 0.2492 [6] a 3.0 [6]
58
Table 3 - Regression parameters obtained from relationships between dry weight and
oxygen consumption and dry weight and ammonia-N excretion from embryo to PL14 of
Macrobrachium amazonicum. (temperature=30±1°C, 10‰ salinity from embryo to ZIX
and 0‰ for PL1 to PL14).
Log a b r² n
Individual O
2
consumption (µgO
2
.
ind
-1
h
-1
) -2.2 0.8 0.88 143
Individual NH
3
-N excretion (µgNH
3
-N.ind
-1
h
-1
) -3.7 1.1 0.86 118
The values a and b are constants in the equation Log M=a+bLogDW, where,
M=individual metabolic rate and DW=dry weight (µg). r²=determination of coefficient
and n=number of observations.
59
0
1
2
3
4
5
6
Emb
r
yo
ZI
ZII
ZII
I
ZI
V
ZV
ZVI
ZVI
I
Z
V
III
ZIX
PL 1
PL 7
PL
1
4
QO
2
(mgO
2
g
-1
DW h
-1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Emb
r
y
o
ZI
ZI
I
Z
I
II
ZIV
ZV
ZV
I
Z
V
II
ZVI
II
ZIX
PL
1
PL 7
PL
1
4
Weight specific excretion
(mgNH
3
N g
-1
DWh
-1
)
Figure 1
– Weight-specific rates oxygen consumption (QO
2
) (A) and ammonia
excretion (B) during the early life stages of
Macrobrachium amazonicum
at 30±1°C.
Results are expressed as means±SD. (DW=dry weight, Z=zoea stages, PL=post-larvae).
A
B
c
a
a
a
bc
b
c
c
c
a
c
c
a
bc
bc
ab
a
b
c
a
b
a
b
a
b
c
a
a
b
ab
a
a
ab
60
CAPÍTULO 4 (Aquaculture international)
Efeito do nitrito no desenvolvimento e metabolismo das larvas de Macrobrachium
amazonicum
Effects of ambient nitrite on development and metabolism of Macrobrachium
amazonicum larvae
Resumo
O efeito das concentrações de nitrito no desenvolvimento e metabolismo das
larvas de
Macrobrachium amazonicum
foi estudado no laboratório. A taxa de
sobrevivência, produtividade, ganho de peso e índice de estágio larval (IEL), consumo
de oxigênio e excreção de amônia e desenvolvimento larval foram avaliados. As larvas
foram submetidas às seguintes concentrações de nitrito na água: 0; 0,2; 0,4; 0,8 e
1,6mg.L
-1
NO
2
-N. Cada tratamento teve 5 repetições. O consumo de oxigênio e a
excreção de amônia foram analisados em zoea (Z) I, III, VII e IX expostas a 0; 0,4; 0,8
e 1,6mg.L
-1
NO
2
-N com 5 repetições para cada tratamento. O primeiro experimento foi
conduzido em béqueres de vidro de 600mL com 300mL de solução-teste com salinidade
10‰, temperatura constante de 30°C e fotoperíodo 12:12h claro:escuro. O segundo
experimento foi conduzido em câmaras cilíndricas plásticas de 30mL. A sobrevivência,
a produtividade, o ganho de peso e o índice de estágio larval (IEL) decresceram
linearmente com o aumento da concentração de nitrito no ambiente. Entretanto, não
houve diferença significativa entre as concentrações de 0 a 0,8mg.L
-1
NO
2
-N em todos
os parâmetros avaliados. O consumo individual de oxigênio (VO
2
) aumentou
significativamente durante o desenvolvimento larval em todas as concentrações de
nitrito (
P
0,05). A relação entre a taxa metabólica e a taxa individual peso-específico
(QO
2
) variou significativamente durante o desenvolvimento ontogenético (
P
0,05).
61
Altos valores de QO
2
foram encontradas nos estágios iniciais (ZI e ZIII) e decresceram
nos estágios subseqüentes (ZVII e ZIX). A excreção de amônia individual (NH
3
-N)
mostrou uma tendência de aumento significativo durante a maioria dos estágios de
desenvolvimento nas diferentes concentrações de nitrito (
P
0,05). A taxa de excreção
peso–específico de amônia também variou significativamente durante o
desenvolvimento larval (
P
0,05). A tendência de aumento começou nos estágios
iniciais e decresceu nos estágios subseqüentes. Observou-se baixos valores de O:N,
variando de 3,7 a 15,9. As taxas de O:N mostraram que em larvas de
M. amazonicum
predomina o catabolismo de proteínas. A taxa metabólica individual apresentou
b
<1,
indicando que o aumento da taxa de consumo de oxigênio e excreção de amônia foi
menor que o crescimento no período estudado. A concentração de 1,6mg.L
-1
NO
2
-N no
ambiente, retarda o desenvolvimento larval, reduz a sobrevivência, a produtividade, o
ganho de peso, o IEL e afeta as taxas metabólicas de larvas de
M. amazonicum
. Por
outro lado, níveis abaixo de 0,8mg.L
-1
NO
2
-N podem ser considerados seguros para a
larvicultura dessa espécie.
Palavras-chaves: Excreção de amônia, Larva,
Macrobrachium
, Nitrito, Consumo de
oxigênio.
62
Abstract
The effects of ambient nitrite concentration on development and metabolism of
Macrobrachium amazonicum
larvae were studied in laboratory. Survival rate, productivity,
weight gain, larval stage index (LSI), oxygen consumption, ammonia excretion and larval
development were evaluated. Larvae were reared in water with nitrite concentration of 0,
5
0.2, 0.4, 0.8 and 1.6mg.L
-1
. Each treatment had five replicates. Oxygen consumption and
ammonia excretion were also analyzed in zoea (Z) I, III, VII and IX exposed to 0, 0.4, 0.8
and 1.6mg.L
-1
NO
2
-N and each treatment was conducted in five replicates. The first
experiment was carried out in 600mL glass beakers filled with 300mL of test solution at
10‰ salinity, constant temperature 30°C and 12:12h daylight:darkness regime. The second
10
experiment was carried out in 30mL cylindrical plastic chambers. Survival, productivity,
weight gain and LSI rate decreased linearly with increasing ambient nitrite concentration.
However, there was no significant difference among larvae reared at concentration ranging
from 0–0.8mg.L
-1
NO
2
-N in all parameter evaluated. Individual respiration rates (VO
2
)
increased significantly during larvae development in all ambient nitrite concentration
15
(
P<0.05
). The relation between metabolic rate and individual dry weight (QO
2
) varied
significantly through the ontogenetic development (
P<0.05
). Higher means in QO
2
were
found in first larval stages (ZI and ZIII) and decreased in subsequent stages. Individual
ammonia-N (NH
3
-N) showed a significant increasing tendency during the majority of
developmental stages with different nitrite concentration (
P<0.05
). Weight–specific
20
excretion rates of ammonia also varied significantly throughout developmental stages
(
P<0.05
). Increase of tendency starting in the first stages and decreased in subsequent
stages. It was observed low O:N values, ranging from 3.7 to 15.9. O:N ratio showing that
M.
amazonicum
larvae has a protein-dominated metabolism. Individual metabolic rates
presented
b
<1, indicating that the increase in oxygen consumption rate and ammonia
25
63
excretion was lower than the growth in the studied period. In conclusion, increasing ambient
nitrite up to 1.6mg.L
-1
NO
2
-N delays larval development reduces survival, productivity,
weight gain, LSI and affects metabolism rates of the
M. amazonicum
larvae. On the other
hand, levels below 0.8mg.L
-1
NO
2
-N and this range may be safe for
M. amazonicum
hatchery.
5
Keywords: Ammonia excretion, Larvae,
Macrobrachium
, Nitrite, Oxygen consumption.
64
1. Introduction
Freshwater prawn farming has been recognized as one of the aquaculture segments
that has shown larger growth (New, 2005; Kutty, 2005). The production of
Macrobrachium
rosenbergii
expanded rapidly between 1995 and 2004 from 19.035 to 193.570 tones (FAO,
2006). This rapid global development may be probably because freshwater prawn farming is
5
a way of producing crustaceans with low environment impact (New et al
.
2000; Moraes-
Riodades and Valenti, 2001) and high environmental sustainability (Valenti and Tidwell,
2006).
In Brazil, production of
M. rosenbergii
is about 500mt.year
-1
(New, 2005).
Additionally, a large Brazilian group is working on the culture of an indigenous species, the
10
Amazon river prawn
Macrobrachium amazonicum
, which have high potential for small-
scale farms in the Amazonia (Valenti and Moraes-Riodades, 2004; New, 2005) and western
Pantanal region, Brazil (Hayd et al
.
submitted-a).
This species presents fast larval
development, with 9 free-swimming zoea stages (Guest, 1979), and a larval cycle of 18 to
21 days in dynamic closed systems (in temperature of 30±1°C and 10‰ salinity). In
15
addition, presents high growth and survival rates in semi-intensive grow-out ponds (Moraes-
Riodades and Valenti, 2007).
M. amazonicum
hatchery is conducted in intensive systems, with high stocking
densities (100-140 larvae.L
-1
) (Vetorelli et al. 2006). Thereby, increasing the load of
nitrogenous compounds and other toxic metabolites may changes water quality such as
20
increase ammonia and nitrite concentration (Das et al
.
2004; Madison and Wang, 2006).
These inorganic nitrogen are the most important pollutants in aquaculture systems (Koo et
al.
2005). Recirculating water through biological filters lows ammonia and nitrite levels due
to nitrification process which convert ammonia to nitrate via nitrite (Valenti and Daniels,
2000; Timmons
et al
.
2002). Nitrite is formed from ammonia oxidation and can be
25
65
accumulated in aquatic systems due to imbalances of nitrified bacterial activity (Das et al.
2004; Sowers et al. 2004; Koo
et al
.
2005; Madison and Wang, 2006). In crustaceans,
ambient nitrite reduces their thermal tolerance and induces methaemocyanin formation,
causes hypoxia in tissues and diminishes the respiration efficiency (Alcaraz and Carnegas,
1997; Timmons et al.
2002).
5
In general, high values of nitrite in the tank water is due to imbalance in nitrification
process at biological filters (Valenti and Daniels, 2000; Timmons et al
.
2002; Jensen, 2003;
Madison and Wang, 2006). Several authors recommend levels below 0.1mg.L
-1
of the nitrite
for
M. rosenbergii
hatchery (Correia et al
.
2000; New, 2002). Otherwise, levels around 1–
2mg.L
-1
did not stress larvae (Armstrong et al. 1976; Mallasen and Valenti, 2006).
10
Nevertheless, the tolerance limits and mechanisms of nitrite action in caridean development
and metabolism are almost unknown (Mallasen and Valenti, 2006). Hence, establishing
security levels of ambient nitrite for hatchery systems and knowing its effects on
metabolism of
M. amazonicum
is very important. The aim of the present study was to
investigate the effects of ambient nitrite on development and physiological changes of
M.
15
amazonicum
larvae under laboratory conditions.
2. Material and methods
Experimental animals
Larvae of
M. amazonicum
were obtained from broodstock formed by wild animals
captured in Northeast Para, Brazil (01°13’25”S 48°17’40”W) in 2001. Females carrying
20
eggs in advanced stages of embryonic development (approximately 2h before hatching),
were collected. This phase of egg incubation is characterized by slow movements of females
and the presence of visible eyes inside the transparent eggs. Animals were disinfected in
66
formaldehyde solution at 20ppm for 30min. and then kept in a hatching tank at 70 ind.m
-2
and 8‰ salinity. It was provided with aeration, heating system and artificial substrates.
Larval development test
In this test, the effects of nitrite concentration on survival, productivity, weight gain
and larval development were evaluated. Tested concentrations treatments were, 0, 0.2, 0.4,
5
0.8 and 1.6 mg.L
-1
NO
2
–N; each treatment was conducted in five replicates. All test solutions
were prepared by dissolving sodium nitrite in brackish water (10‰ salinity), according to
methods presented at APHA (1998). General methodology was adapted from Mallasen and
Valenti (2006) and is summarized below.
Larvae were reared in 600mL glass beakers filled with 300mL of test solution gently
10
aerated at 10‰ salinity, constant 30°C, and subjected to 12:12h daylight:darkness regime.
Beakers were placed inside black trays to minimize light reflections and prevent larvae
crowding around luminous points, due to positive phototactism. Fifteen newly-hatched
larvae were carefully washed out with the test solution, acclimated for 2h and transferred to
each beaker. Larvae were fed freshly hatched
Artemia
nauplii from ZII to ZIV (4-6
15
nauplii.mL
-1
). After this stage, they were fed on inert feed (3.3 to 13.2µg.mL
-1
) and
Artemia
nauplii (8 to 12 nauplii.mL
-1
), added to the beakers every day until metamorphosis. Food
residues were siphoned 3h after feeding.
Every 24h brackish water was changed for maintaining constant water quality and
the tested nitrite concentration. Temperature and salinity were monitored twice a day
20
(measured with a Yellow Springs Instruments, YSI 63) while dissolved oxygen (measured
with Yellow Springs Instruments YSI 52 oxymeter) and pH (measured with Yellow Springs
Instruments YSI 60 pHmeter) were measured three times a week. Ammonia and nitrite
levels were determined twice a week before water replacement, according to methods
67
presented at APHA (1998). Ammonia and nitrite analyses were performed using a Hach DR
2000 spectrophotometer. Larval stages were identified according to Guest (1979).
At the end of the experiment, larvae and post-larvae were counted, weighed, and
observed under a stereomicroscope (Leica MZ6) to determine larval stage. Larval stage
index (LSI) was estimated using the equation of Manzi et al. (1977),
LSI=( Si x ni). N
-1
,
5
where Si is larval stage (i=1-9), ni=number of larvae in stage Si and N=total number of
larvae observed.
Larvae were sampled of the hatchery tank to estimate initial dry weight (IDW). At
the end of this study, the surviving larvae and post-larvae were used to determine final dry
weight (FDW). Weight gain was then given by the difference between FDW and IDW. To
10
determine dry weight, the prawns were briefly rinsed in distilled water, placed in pre-
weighted cartridges and dried for 48h at 70ºC. Then, they were transferred to a desiccator
for 2h and weighed on a Mettler-Toledo Model AT21 analytical balance, at the nearest 1µg.
Experiment was finished when around 80% of the larvae of one replicate of any tested
treatment metamorphosys to post-larvae. The test followed a completely randomized design
15
with five nitrite concentrations (treatments) and five replicates. Data of the variables
(survival, productivity, weight gain and LSI) and ambient nitrite concentration (mg.L
-1
)
were described as a linear function, Y=
a
+
b
X, where Y is the variables and X is ambient
nitrite concentration tested.
Constants
a
and
b
denote elevation and slope, respectively.
Mean water temperature was 30°C and did not differ among beakers. Dissolved oxygen was
20
above 7mg.L
-1
and the pH ranged around 7.8. Ammonia concentration (NH
3
-N) mean value
was 0.4±0.1mg.L
-1
and nitrite (NO
2
-N) was very close to the nominal values of the
treatments.
Data were subjected to one-way ANOVA followed by Tukey test and linear regression
analysis. All measured values of each variable at each phase were entered into the regression
25
68
analysis (
P<0.05
). Statistics analyses were performed with the Statistical Analysis System
(SAS Intiture Inc., version 8.0). Values expressed as percentages were square root arcsine
transformed prior to analysis, although they are presented as non-transformed for easier
interpretation. Significance level was set at
P<0.05
.
Oxygen consumption and ammonia-N excretion determination
5
Oxygen consumption and ammonia-N excretion were determined in larvae at stages
I, III, VII and IX subjected to ambient nitrite concentrations of 0, 0.4, 0.8 and 1.6mg.L
-1
to
detect possible alterations in larval metabolism when ambient nitrite increases. Treatments
were conducted in six replicates. These selected larval stages and nitrite levels were defined
from the results obtained for the larval development test. First, larvae were sampled of the
10
120L cylindrical tanks provided with mechanical and biological filter and artificial heater,
with10‰ salinity and 30°C and were acclimated in beaker with water at different nitrite
concentration during two hours. After acclimating, larvae at postmolt-intermolt (A-C) stages
(Hayd et al., submitted or see chapter 1) were selected and placed into respirometric
chambers (
ca
. 30mL) for 2h to quantify metabolic rates.
15
Cylindrical plastic containers with approximately 30mL sealed with silicon tablets
(Lemos and Phan, 2001, Lemos et al
.,
2003) were used as respirometric chambers to
incubate animals for oxygen consumption and ammonia-N excretion measurement.
Chambers were individually identified and the exact volume was gravimetrically
determined. An orifice of 1.5mm in the center of the cover enabled the elimination of air
20
bubbles from inside the chamber during closure. Respirometers were hermetically closed
inside 1000mL beakers filled with water (10‰ salinity) to avoid formation of bubbles inside
the containers. After being closed, inside water was isolated from air by a plastic tablet
1.7cm diameter silicon seal so that the tension between orifice water and the silicon seal
could block respirometer inside water and air gas exchange.
25
69
The number of individuals used inside respirometer was determined according to
individual dry mass (Table 1). The biomass:volume ratio (B:V) was calculated by dividing
the total dry mass of individuals by the chamber volume and varied from 17.3 to 48.3µg.mL
-
1
(Table 1). ZI, III, VII and IX were kept in the sealed respirometric chambers samples for
2h. Seawater and Milli-Q (Millipore) fosh water at 10‰ salinity were used as control
5
respirometer without animals. Samples and controls were kept in water bath at 30±1°C.
After incubation, the tablet was removed, and water was sampled through a plastic canula
and stored in glass syringes chemically calibrated (syringes plus plastic nozzles).
Variation in oxygen and ammonia-N nitrogen contents was calculated by the
difference between values obtained in sample (with animals) and control (no animals) units.
10
Dissolved oxygen in water at the end of every test was determined by Winkler analysis
method adapted to small volumes (Fox and Wingfield, 1938) and was never lower than 80%
of saturation. For ammonia-N analysis, the method described by Koroleff (1983) in separate
water samples was used. Oxygen consumption and ammonia-N excretion were expressed as
individual (µg.ind.
-1
.h) and dry-mass specific (mg.g
-1
DW.h) rates. Salinity effect was
15
corrected using the factor 1.06 (Koroleff, 1983). Atomic O:N (oxygen respired to nitrogen
excreted) ratios were calculated by dividing the number of gram-atom of consumed oxygen
by the number of gram-atom of nitrogen excreted (N.O
-1
) in each developmental stage
(Mayzaud and Conover, 1988).
Individual dry weight (DW) was determined by groups of 10 larvae at ZI, ZIII, ZVII
20
and ZIX with eight replicates. Individuals
were gently rinsed with distilled water, dried with
filter paper and separated prior to weight determination. After 48h at 70°C, dry samples
were weighed on a Mettler Toledo AT21 analytical balance, at the nearest 1µg.
The results were subjected to analyses of detection and exclusion of outliers
(Statistica software, v. 6.0) with coefficient 1.5. Total excluded data was always lower than
25
70
10% of data obtained for each stage (Statistica Software, v.6). The test was a 4x4 factorial
design with four nitrite concentrations and four larval stages. Normality was tested using the
Shapiro-Wilk test and Homocedasticity by Levene’s (using SAS 9.0 software). Differences
among means were tested by two-way ANOVA followed by Duncan's multicomparison test.
Data of the metabolic rates (oxygen consumption and ammonia excretion, in µg.ind.
-1
.h
-1
)
5
and dry weight (µg) in 0 and 1.6mg.L
-1
NO
2
-N were logarithmically adjusted by regression
analysis (Statistica Software, v.6). Slopes were compared to 1 using a t-test according to Zar
(1999). Differences were considered significant at
P<0.05
.
3. Results
Larval development test
10
M. amazonicum
larvae developed until metamorphosis at 0, 0.4, 0.8 and 1.6mg.L
-
1
NO
2
-N. Survival, productivity, weight gain and larval stage index decreased linearly with
increase ambient nitrite concentration (Fig. 1). However, there were no significant
differences in all parameters at concentration ranging from 0–0.8mg.L
-1
NO
2
-N (
P>0.05
),
whereas they were significantly lower at 1.6mg.L
-1
NO
2
-N (
P<0.05
) (Table 2).
15
Oxygen consumption and ammonia excretion determination
Individual dry weight (DW) increased throughout the ontogenetic development
(Table 1) and varied significantly in different ages and developmental stages (
P<0.05
).
Individual oxygen consumption rates (VO
2
) increased significantly during development
larvae (
P<0.05
) in all ambient nitrite concentration (Fig. 2B; Table 3). ZI presented the
20
lowest value and into these stage there was no significant difference in all nitrite
concentrations (
P>0.05
), while ZIX presented the highest one (
P<0.05
) (Table 3). The VO
2
values increasing in ZIII and ZIX at 1.6mg.L
-1
NO
2
-N, and differ significantly from 0mg.L
-
1
NO
2
-N (
P<0.05
) (Table 3). The weight-specific oxygen consumption (QO
2
) in different
71
ambient nitrite concentration showed significant variation throughout development (
P<0.05
)
(Fig. 2A); ZI and ZIII presented the highest rates, contrasting with lower values in the later
stages ZVII and ZIX (Fig. 2A). Nitrite concentration at 1.6mg.L
-1
NO
2
-N increased the QO
2
significantly in ZI, ZIII and ZIX.
Individual ammonia-N excretion increased while weight-specific ammonia excretion
5
decreased throughout the larval development (
P<0.05
) (Table 3). The stages ZI, ZVII and
ZIX showed significant differences in individual and weight-specific ammonia excretion
between 0 and 1.6mg.L
-1
NO
2
-N (
P<0.05
); excretion increased in ZI and ZIX and decreased
in ZVII from 0 to 1.6mg.L
-1
NO
2
-N (Table 3).
As a general trend, developmental was characterized by low O:N ratio. The highest
10
value (18±0.6) was observed in ZI at 0mg.L
-1
NO
2
-N, which strongly decreased at 0.4, 0.8
and 1.6mg.L
-1
NO
2
-N (Fig. 2C). The slope obtained for individual oxygen consumption/dry
weight relationship at 0 and 1.6mg.L
-1
NO
2
-N were 0.63 and 0.60, respectively, and both was
significantly lower than 1 (
P
<0.05) (Table 4), while the
b
value for individual excretion/ dry
weight relationship at 0 and 1.6mg.L
-1
NO
2
-N were 0.71 (
P
<0.05) and 1.23, respectively, and
15
the latter one was significantly higher than 1 (Table 4).
4. Discussion
Complete larval development of
M. amazonicum
occurs in nitrite from 0 to 1.6mg.L
-1
NO
2
–N. However, survival, productivity, weight gain and larval stage index decreased as
nitrite concentration increased. Similar results have also been obtained for
M. rosenbergii
20
larvae
(Mallasen and Valenti, 2006) with added nitrite in concentration between 0 to
16mg.L
-1
NO
2
–N. This reduction may be related to physiological processes, such as the
changes in haemocyanin provoked by nitrite (Sowers et al. 2004) and may reflect in the
oxygen consumption (Harris et al
.
1997).
72
Generally, oxygen consumption increased with increasing the nitrite concentration
from 0-0.8 to 1.6mg.L
-1
NO
2
-N. Similar results were obtained for ZII of
M. rosenbergii
by
Mallasen and Valenti (2006). It suggests that tested nitrite levels did not disrupt significantly
oxygen transport and larvae may be allocated energy to adjust physiological mechanisms
against the toxic effect when in high nitrite concentration, thus reducing growth and
5
development. Therefore, it may increase culture cycles and decreased productivity and
individual size in commercial hatchery. The weight-specific oxygen consumption (QO
2
)
varied significantly through the ontogenetic development (
P<0.05
). Higher means in QO
2
were found in first larval stages (ZI and ZIII) and decreased in subsequent stages (ZVII and
ZIX), regardless nitrite concentration. Similar result was obtained in chapter 2. Thus, larvae
10
did not changed the metabolic pattern during the ontogenetic development with the addition
of nitrite in water. This tendency in QO
2
during growth may be a consequence of a
disproportionate increase of metabolically inactive tissues (Anger, 2001) and/or the
exponential increases in the weight of the larval exoskeleton during the development of
decapods crustaceans (Anger and Ismael, 1997). The same tendency was observed for
M.
15
amazonicum
larvae in a former papers (see chapter 2; Zanders and Rodriguez, 1992),
Macrobrachium rosenbergii
(Agard, 1999), the spider crabs
Hyas coarctatus
larvae (Jacobi
and Anger, 1985) and
Hyas araneus
larvae (Anger and Jacobi, 1985) during the larval
development.
Individual ammonia excretion showed an increasing tendency during the larval
20
development while weight-specific ammonia excretion rates were high in the first stages and
decreased in the latter ones. This is similar to the QO
2
and general patterns find by
crustacean larvae. Nitrite effect varied among larval stages. Weight-specific ammonia
excretion strongly increased with nitrite concentration in ZI. The mean obtained at 0mg.L
-
1
NO
2
-N is compatible with the value obtained in chapter 2 in normal conditions. Therefore,
25
73
the higher values obtained for 0.4, 0.8 and 1.6mg.L
-1
NO
2
-N may be really due to nitrite
effect. This indicate that ambient nitrite increase protein catabolism in lecithotrophic phase.
It is corroborated by the great decrease in O:N ratio observed by 0.4, 0.8 and 1.6mg.L
-1
NO
2
-
N in zoea I.
In ZIII, ammonia excretion did not changed with nitrite concentration. However QO
2
5
significantly increased at 1.6mg.L
-1
NO
2
-N. This indicate an increase in catabolism of lipid
which is corroborated by the increase in O:N ratio. In ZIII, larvae start exogenous feed and
fed on freshly hatched
Artemia
nauplii, which is rich in lipid. Therefore larvae may change
metabolic process. Similar results was observed in
Penaeus japonicus
fed with this
microcrustacea (Lemos and Rodrigues, 1998).
10
In ZVII, ammonia excretion decreased with nitrite concentration, while QO
2
did not
changed. It indicates that protein metabolism decreased, while general metabolism did not
change probably due to catabolism of lipid. This stage is characterized by a very large
ingestion of
Artemia
nauplii (Maciel, 2007), which may increase of the lipid as source of
energy.
15
In Z IX, ammonia excretion significantly increased from 0 to 0.4mg.L
-1
NO
2
-N, while
QO
2
only increased at 1.6mg.L
-1
NO
2
-N. It indicates that ambient nitrite increased protein
catabolism, but the proportion protein:lipid as energy source did not change.
In this study, the slope in both oxygen consumption/dry weight relationship at 0 and
1.6mg.L
-1
NO
2
-N were lower than 1 (
b
<1). These values suggest a decrease in weight–
20
specific metabolic rates throughout development in the studied period, and that ambient
nitrite presents low effect on oxygen consumption. Slope lower than 1 has been already
obtained for
M. amazonicum
early development stages (see chapter 2).
Like this, the overall
patterns of metabolism of
amazonicum
larvae is similar to other larval decapods studied,
such as,
M. rosenbergii
larvae (0.904) (Stephenson and Knight, 1980), and
M. rosenbergii
25
74
juveniles (0.628) (Nelson
et al.,
1977). On the other hand, the
b
value for individual
excretion/ dry weight relationship at 0 and 1.6mg.L
-1
NO
2
-N were 0.71 (
P
<0.05) and 1.23,
respectively, and the latter one was significantly higher than 1. In several studies with
decapod larvae,
b
values above the 1 were usually associated with physiological stress
(Anger, 2001). Therefore, the present study indicated that 1.6mg.L
-1
NO
2
-N produce
5
physiological stress, which cause changes in excretion patter. This fact was also showed in
caridean shrimp
Palaemon serratus
larvae subjected to stress of temperature and salinity
(Yagi et al., 1990), in brachyuran crab
Cancer irroratus
larvae (Johns, 1981) and lobster
Homarus americanus
larvae (Capuzzo and Lacaster, 1979) when exposed to unfavorable
temperatures.
10
Results of the present study indicated that during the lecithotrophic phase of
M.
amazonicum
, ambient nitrite concentration decrease the lipid and increase the protein
catabolism. On the contrary, when larvae started the exogenous feeding ambient nitrite
increases the lipid as source of energy. In addition, growth rate, survival, productivity and
weight gain of larvae are negatively affected by 1.6mg.L
-1
NO
2
-N. Therefore, maintaining
15
moderate intensity of the nitrite concentration, lower than 0.8mg.L
-1
in hatchery tanks water
is safe for
M. amazonicum
larvae development. However, further physiological studies focus
on the effect of the nitrite concentration ranged between 0.8–1.6mg.L
-1
NO
2
-N is necessary
to determine the optimum range in hatchery system.
Acknowledgments
20
This research was supported by the CAPES/PQI (Brasília, Brazil, Grants 137030)
and FAPESP (São Paulo, Brazil, Grants 05/54276-0). The authors are grateful to Dr C.
Oliveira and N. H. Rego for consultation with statistic analysis. Thanks are also due to the
P. Atique and B. Donadon for technical assistance in the laboratory.
75
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81
Table 1
– Age, dry weight (DW), number of individuals, and biomass:volume ratio in
respirometers used to determine oxygen consumption and ammonia-N excretion rate
during zoea (Z) I, III, VII and IX stages of
M. amazonicum
at 30±1°C and 10‰ salinity.
Results are expressed as mean values±SD. Number of replicates=10.
Larval
Stage
Age ¹ DW (µg) Individuals.
respirometer
-1
Biomass:volume
(µg.mL
-1
)
ZI
0.5 60.0 ± 1.2 12 24.0
ZIII
3.0 86.3 ± 1.7 6 17.3
ZVII
9.0 483.0 ± 8.5 3 48.3
ZIX
14.0 700.3 ± 19.2 2 46.7
¹-days after hatching
5
Table 2 -
Survival rate, productivity, weight gain and larval stage index (LSI)
(means±standard deviation) obtained at the end of 17 days of
Macrobrachium amazonicum
culture (30±1ºC and 10‰ salinity) in different ambient nitrite concentrations. Mean values
in the same column with different letters are significantly different (
P<0.05
). N=25.
10
Nitrite (mg.L
-
1
) Survival (%)
Productivity (PL.L
-
1
)
Weight gain (µg) LSI
0
88.0 ± 5.6 a 26.8 ± 2.4 a 627.6 ± 4.6 a 8.8 ± 0.1 a
0.2
82.8 ± 3.7 a 20.8 ± 2.3 a 626.7 ± 5.4 a 8.8 ± 0.0 a
0.4
81.4 ± 5.6 a 20.0 ± 2.4 a 620.2 ± 24.8 a 8.8 ± 0.1 a
0.8
80.0 ± 4.9 a 19.2 ± 8.3 a 601.8 ± 16.0 a 8.7 ± 0.1 a
1.6
65.6 ± 3.0 b 9.4 ± 2.8 b 545.0 ± 14.6 b 8.2 ± 0.0 b
82
Table 3 –
Nitrite concentrations, individual oxygen consumption (VO
2
) and ammonia-N
excretion rates in zoea (Z) I, III, VII and IX stages of
Macrobrachium amazonicum
in 0, 0.4,
0.8 and 1.6mg.L
-1
nitrite concentrations at 30±1°C and salinity 10‰. Results are expressed
as mean values±SD. Number of replicates=6.
Larval Nitrite Oxygen consumption Ammonia–N excretion
Stage NO
2
(mg.L
-1
)
µgO
2
.ind.
-1
.h
µg
NH
3
-N.ind.
-1
.h
ZI
0.0 0.19 ± 0.02 a 0.011 ± 0.005 a
0.4 0.18 ± 0.02 a 0.032 ± 0.010 b
0.8 0.18± 0.03 a 0.032 ± 0.007 b
1.6 0.23 ± 0.05 ab 0.041 ± 0.004 b
ZIII
0.0 0.34 ± 0.02 bc 0.035 ± 0.003 b
0.4 0.36 ± 0.04 bc 0.034 ± 0.007 b
0.8 0.38 ± 0.03 cd 0.038 ± 0.006 b
1.6 0.50 ± 0.06 d 0.038 ± 0.008 b
ZVII
0.0 0.83 ± 0.12 e 0.187 ± 0.028 d
0.4 0.76 ± 0.08 e 0.159 ± 0.017 cd
0.8 0.81 ± 0.08 e 0.130 ± 0.013 c
1.6 0.85 ± 0.17 e 0.117 ± 0.018 c
ZIX
0.0 1.08 ± 0.08 f 0.197 ± 0.050 d
0.4 1.19 ± 0.12 f 0.278 ± 0.044 e
0.8 1.12 ± 0.07 f 0.258 ± 0.114 e
1.6 1.53 ± 0.24 g 0.240 ± 0.059 e
5
83
Table
4
- Regression between dry weight and oxygen consumption and dry weight and
ammonia-N excretion from zoea I, III,VII and IX of
Macrobrachium amazonicum
in
different nitrite concentration 0 and 1.6mg.L
-1
NO
2
-N at 30±1°C and 10‰ salinity.
0 mg.L
-1
NO
2
-N
Log a b r² n
Individual O
2
consumption (µgO
2
.
ind.
-1
.h) -1.70 0.63 0.95 20
Individual NH
3
–N excretion (µgNH
3
-N.ind.
-1
.h) -2.70 0.71 0.90 20
1.6 mg.L
-1
NO
2
-N
Individual O
2
consumption (µgO
2
.
ind.
-1
.h) -1.60 0.60 0.83 20
Individual NH
3
–N excretion (µgNH
3
-N.ind.
-1
.h) -4.10 1.23 0.90 20
The values a and b are constants in the equation Log M=a+bLogDW, where, M=individual metabolic rate and
DW=dry weight (µg). r²=coefficient of determination and n=number of observations. 5
84
Y = 87.27 - 13 X
r
2
= 0.73
60
70
80
90
100
0 0.4 0.8 1.2 1.6
Survival (%)
Y = 24.95 - 9.50 X
r
2
= 0.63
5
10
15
20
25
30
35
0 0.4 0.8 1.2 1.6
Productivity (PL.L
-1
)
Y = 636.52 - 53.72 X
r
2
= 0.81
520
540
560
580
600
620
640
660
0 0.4 0.8 1.2 1.6
Weight gain (µg.L
-1
)
Y = 8.96 - 0.43 X
r
2
= 0.84
8.2
8.4
8.6
8.8
9.0
0 0.4 0.8 1.2 1.6
Nitrite (mg.L
-1
)
LSI
B
D
Figure 1
– Relationship between survival rate (N=25, F=15.59,
P
<0.05) (A),
productivity (N=25, F=10.91,
P
<0.05) (B), weight gain (N=25, F=26.64,
P
<0.05) (C)
and larval stage index (LSI) (N=25, F=138.87,
P
<0.05) (D) and nitrite concentration
.
Figures over data-points indicate the number of identical values.
3
2
2
2
3
2
2
2
2 2
3
3
2
3
4
3
2
2
2
5
2
2
C
3
A
2
2 2
4
2
2
3
85
0
1
2
3
4
5
6
I III VII IX
mgO
2
.gDW
-1
.h
-1
0
0.4
0.8
1.6
mg.L
-1
NO
2
-N
mg.L
-1
NO
2
-N
mg.L
-1
NO
2
-N
mg.L
-1
NO
2
-N
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
I III VII IX
mgNH
3
-N.gDW
-1
.h
-1
0
2
4
6
8
10
12
14
16
18
20
I III VII IX
Zoea stage
O:N
B
A
C
Figure 2
– Weight-specific rates oxygen consumption (A), ammonia-N excretion (B)
and O:N ratio (C) during the early life stages of
Macrobrachium amazonicum
to
different nitrite concentration. Results are expressed as means±SD. (I=zoea I, III=zoea
III, VII=zoea VII and IX=zoea IX).
d
bc
d
c
f
bc
e
f
f
f
f
f
f
a
bc
b
g
b
b
a
d
bc
cd
bc
cd
de
e
e
ef
d
d
d
86
CAPÍTULO 5
CONCLUSÕES
1
. Macrobrachium amazonicum
apresentou os estágios de muda,
A/C
(pós-muda/intermuda
combinados),
D
(pré-muda) e
E
(ecdise). Esta classificação foi baseada no grau de
homogeneidade do citoplasma, retração da epiderme e no estágio do desenvolvimento das
5
setas do telson.
2. O estágio
A/C
é uma fase muito curta em que a larva absorve água, a cutícula é secretada
e ocorre o crescimento e a condensação do tecido da epiderme. O estágio
D
é caracterizado
pelo início da retração da epiderme da nova cutícula, chamada “apolise”. Esta ocorre
inicialmente na base da seta do telson em todos os estágios larvais, sendo o período principal
10
da formação da seta (setogênese). O estágio E é um processo muito rápido (alguns minutos),
e inicia-se com a abertura entre o cefalotórax e o abdome.
3. A pós-muda/intermuda (A/C) ocupa 40-50% do total de duração do instar e a pré-muda
(D) requer mais da metade do tempo nas temperaturas experimentais.
4. O índice de condição larval (IEL) proposto por Tayamen and Brown, (1999) para avaliar
15
a qualidade larval de
Macrobrachium rosenbergii
não pode ser aplicado às larvas de
M.
amazonicum
, porque algumas características negativas atribuídas às larvas de
M. rosenbergii
ocorrem em larvas sadias no período inicial de pós-muda/intermuda (A/C) em
M.
amazonicum.
5. O conhecimento e a identificação dos estágios do ciclo de muda são importantes na
20
seleção das larvas que serão utilizadas em experimentos, reduzindo, assim, a variabilidade
das condições fisiológicas do animal e ajudará a entender o padrão de crescimento desta
espécie.
87
6. A taxa individual de consumo de oxigênio e excreção de amônia nitrogenada aumenta
com a elevação da massa corporal durante o desenvolvimento larval. A taxa metabólica
peso-específico diminui com o aumento da biomassa. As variações metabólicas observadas
são decorrentes de um desenvolvimento caracterizado por várias transformações
morfológicas externas e comportamentais, durante o desenvolvimento ontogenético de
M.
5
amazonicum
.
7. As taxas atômicas O:N mostraram que durante todos os estágios de desenvolvimento de
M. amazonicum
predomina o catabolismo de proteínas para a produção de energia.
8. Na despesca de tanques de larvicultura, as pós-larvas, precisam de um maior tempo de
aclimatação, ao serem transferidas da água de salinidade 10‰ para água doce.
10
9. As relações entre as taxas metabólicas e o peso seco, indicam que o consumo de oxigênio
aumentou em taxa menor que a taxa de crescimento no período estudado e que a excreção de
amônia é proporcional à biomassa do corpo.
10. A sobrevivência, a produtividade, o ganho de peso e o IEL decresceu linearmente com o
aumento da concentração de nitrito no ambiente. Entretanto, não houve diferença
15
significativa entre as concentrações 0-0,8mg.L
-1
NO
2
-N nos parâmetros avaliados. Assim, a
faixa de 0–0,8mg.L
-1
NO
2
-N pode ser considerada segura para o cultivo.
11. A concentração 1,6mg.L
-1
NO
2
-N retardou o desenvolvimento larval em todos os
parâmetros avaliados e afetou as taxas metabólicas das larvas de
M. amazonicum.
O nitrito
aparentemente causa aumento no metabolismo protéico na fase lecitotrófica e no uso de
20
lipídeos como fonte de energia na fase de alimentação exógena.
12. Estudos precisam ser realizados para avaliar o efeito do nitrito no desenvolvimento
larval de
M. amazonicum
no intervalo 0,8–1,6mg.L
-1
NO
2
-N.
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