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Neste Artigo

  • Erratum Notice
  • Resumo
  • Resumo
  • Introdução
  • Protocolo
  • Resultados
  • Discussão
  • Divulgações
  • Agradecimentos
  • Materiais
  • Referências
  • Erratum
  • Reimpressões e Permissões

Erratum Notice

Important: There has been an erratum issued for this article. Read More ...

Resumo

Dois protocolos de ablação de pedúnculo ocular (isto é, cauterização e abordagens cirúrgicas) foram realizados em caranguejos fêmeas anestesiados. A ablação ocular de caranguejos de lama acelerou o amadurecimento dos ovários sem diminuir a taxa de sobrevivência.

Resumo

Caranguejos da lama (Scylla spp.) são espécies de crustáceos comercialmente importantes que podem ser encontrados em toda a região do Pacífico Indo-Ocidental. Durante o cultivo, a indução da maturação ovariana é importante para atender à demanda dos consumidores por caranguejos maduros e acelerar a produção de sementes. A ablação do pedúnculo ocular é uma ferramenta eficaz para melhorar a maturação ovariana em caranguejos da lama. No entanto, não existe um protocolo padrão para a ablação do pedúnculo ocular de caranguejos da lama. Neste estudo, duas técnicas de ablação do pedúnculo ocular são descritas: cauterização (uso de metal quente para abater o pedúnculo ocular de um caranguejo anestesiado) e cirurgia (remoção do pedúnculo ocular com tesoura cirúrgica). Antes da ablação do pedúnculo ocular, fêmeas sexualmente maduras (LC > 86 mm) foram anestesiadas com uma bolsa de gelo (−20 °C) com água do mar. Quando a temperatura da água atingiu 4 °C, a bolsa de gelo foi removida da água. Água do mar corrente (temperatura ambiente: 28 °C) foi usada para recuperação da anestesia imediatamente após a ablação do pedúnculo ocular. A mortalidade não ocorreu durante ou após o processo de ablação do pedúnculo ocular. O protocolo de ablação do pedúnculo ocular aqui apresentado acelerou a maturação ovariana dos caranguejos da lama.

Introdução

Todas as quatro espécies de caranguejos pertencentes ao gênero Scylla são espécies de crustáceos comercialmente importantes na aquicultura 1,2. O crescimento de crustáceos, incluindo caranguejos da lama, e sua transformação da fase pré-madura (sub-adulta ou púbere) para a fase sexualmente madura (adulta) ocorrem através de um processo de muda que envolve a eliminação periódica de exoesqueletos mais velhos e menores. A largura da carapaça (LC), as morfologias dos quelípodos e dos retalhos abdominais são amplamente utilizadas para determinar a maturidade sexual de Scylla spp. 3,4,5. O processo de muda é regulado pela ação de vários hormônios e requer uma enorme quantidade de energia6. Além do processo normal de muda, a perda de membros, voluntária ou induzida por fatores externos, agiliza a muda dos caranguejos sem afetar sua taxa de sobrevivência 7,8,9. Portanto, a autotomia de membros é comumente utilizada para indução de muda na indústria de criação de caranguejos de casca mole 7,9.

A ablação unilateral ou bilateral do pedúnculo ocular é mais popular em camarões de água doce e camarões marinhos para maturação de gônadas e produção de sementes10,11,12,13. Técnicas comuns de ablação de pedúnculo ocular em crustáceos incluem: (i) ligadura na base do pedúnculo ocular com corda14,15; (ii) cauterização do pedúnculo ocular com pinça quente ou eletrocautério16; (iii) remoção ou pinçamento direto do pedúnculo ocular para deixar a ferida aberta12; e (iv) remoção do conteúdo do pedúnculo ocular por incisão após corte da porção distal do olho com navalha17. Os órgãos X do pedúnculo ocular são importantes órgãos endócrinos em crustáceos, pois regulam os hormônios hiperglicêmicos (CHH) dos crustáceos, os hormônios inibidores da muda (MIH) e os hormônios inibidores da vitelogênese (VIH)6,18,19,20,21,22. Os órgãos X do pedúnculo ocular (ou complexo da glândula sinusal) sintetizam e liberam hormônios inibidores das gônadas (GIH), também conhecidos como hormônios inibidores da vitelogênese (VIH), pertencentes à família dos hormônios neuropeptídeos6. A ablação unilateral ou bilateral do pedúnculo ocular reduz a síntese de GIH, resultando na dominância de hormônios estimulantes (i.e., hormônios estimulantes das gônadas, GSH) e na aceleração do processo de maturação ovariana em crustáceos23,24,25,26. Sem a influência do GIH após a ablação do pedúnculo ocular, crustáceos fêmeas dedicam sua energia ao desenvolvimento ovariano27. Verificou-se que a ablação unilateral do pedúnculo ocular é suficiente para a indução da maturação ovariana em crustáceos11 e que o pedúnculo ocular ablado de camarões e caranguejos pode se regenerar após várias mudas28. Há quatro estágios de desenvolvimento ovariano registrados em Scylla spp.: i) imaturo (estágio-1), ii) maturação precoce (estágio-2), iii) pré-maturação (estágio-3) e iv) totalmente maduro (estágio-4)29,30. O estágio ovariano imaturo é encontrado em fêmeas imaturas. Após a muda puberal e o acasalamento, o ovário imaturo começa a se desenvolver e finalmente amadurece (estágio 4) antes da desova31.

Um protocolo de ablação ocular é essencial para o desenvolvimento de matrizes de caranguejo e produção de sementes. No mercado global de alimentos, caranguejos maduros com ovários totalmente maduros (estágio 4) em vez de caranguejos com maior teor muscular são preferidos pelos consumidores e, portanto, têm um valor comercial mais alto, até mesmo maior do que os machos grandes. Não há um protocolo completo para a ablação ocular de caranguejos da lama. O protocolo de ablação do pedúnculo ocular neste trabalho minimiza o estresse com o uso de caranguejos totalmente anestesiados e minimiza os danos físicos causados às mordidas de caranguejos. Este protocolo é fácil e econômico. Apresentamos um protocolo para a ablação do pedúnculo ocular de Scylla spp., que pode induzir a maturação da gônada. Duas técnicas de ablação do pedúnculo ocular (cauterização e cirurgia) foram testadas e suas eficiências comparadas com base na taxa de desenvolvimento gonadal de fêmeas de caranguejos da lama.

Protocolo

Este protocolo segue o Código de Prática da Malásia para o Cuidado e Uso de Animais para Fins Científicos delineado pela Associação de Ciência Animal de Laboratório da Malásia. O sacrifício das amostras experimentais foi feito de acordo com o National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revisado em 1978). Caranguejos de lama fêmeas sexualmente pré-maduros ( S. olivacea) foram coletados no mercado local (5°66′62′′N, 102°72′33′′E) nas Zonas Húmidas de Setiu, na Malásia. As espécies de caranguejos lamacentos foram identificadas com base em característicasmorfológicas1.

1. Coleta e desinfecção de amostras

  1. Coletar caranguejos fêmeas saudáveis, ativos e pré-maduros (Figura 1).
    NOTA: Caranguejos fêmeas pré-maduros têm abdômen triangular e de cor clara, juntamente com uma faixa de LC de 80 a 85 mm.
  2. Lave os caranguejos com água da torneira clorada (água doce) para remover detritos e parasitas osmofílicos.
  3. Mergulhe os caranguejos em formol de 150 ppm com salinidade de 20 ppt por 30 min.
  4. Manter a aeração contínua e suave com airstones durante o tratamento com formaldeído. A fonte de aeração pode ser de uma linha de aeração central ou de uma bomba de aerador de aquário.
  5. Lave os caranguejos com água do mar corrente para remover qualquer formaldeído residual.

figure-protocol-1591
Figura 1: Morfologia abdominal de fêmeas de caranguejos de lama utilizados para identificar os estágios de maturação sexual. Clique aqui para ver uma versão maior desta figura.

2. Aclimatação

  1. Transfira cada fêmea desinfetada para um tanque circular separado de 32 L.
  2. Crie as fêmeas por 3 dias em 20 ppt de salinidade e continue se alimentando duas vezes por dia (manhã 09:00 am e noite 20:00 pm) com peixes marinhos picados a cerca de 4%-5% do peso corporal do caranguejo.
  3. Retire o excesso e a ração não consumida por meio de sifões antes da alimentação matinal.
  4. Troque 10% da água do mar de criação de caranguejo (20 ppt) diariamente.

3. Muda induzida para a maturidade sexual

  1. Corte todas as pernas, exceto as pernas de natação usando tesoura esterilizada.
    1. Pegue o caranguejo com uma rede de coleta e segure o caranguejo com cuidado. Corte primeiro os dois quelípodos e depois as pernas de caminhada na segunda articulação usando uma tesoura. O caranguejo irá autotomizar os apêndices danificados automaticamente. A anestesia não é necessária para a autotomia do membro.
  2. Lave o caranguejo em água doce imediatamente após a autotomia dos membros.
  3. Transfira individualmente os caranguejos autotomizados com membros para cestos plásticos perfurados (28 cm L x 22 cm W x 7 cm H) e coloque-os em um tanque de fibra de vidro (305 cm L x 120 cm W x 60 cm H).
    NOTA: Dois cestos podem ser amarrados e cortados juntos. A cesta superior é usada como cobertura para que o caranguejo não possa escapar da cesta.
  4. Use um sistema de aquicultura recirculante (RAS) com 20 ppt de salinidade e uma profundidade de água de pelo menos 10 cm para garantir que todo o cesto de plástico fique submerso.
  5. Continue alimentando a fêmea de caranguejo autotomizado com peixes marinhos picados duas vezes por dia a 5% a 7% do peso corporal do caranguejo.
  6. Criar os caranguejos até amadurecer sexualmente através de muda (35 dias).
    NOTA: A muda induzida pode ser ignorada para maturação ovariana comercial e produção de sementes com caranguejos de lama fêmeas maduras selvagens. Fêmeas maduras colhidas da natureza devem ser aclimatadas e submetidas diretamente à anestesia por choque frio e subsequente ablação do pedúnculo ocular.

4. Anestesia

  1. Selecionar fêmeas sexualmente maduras com retalho abdominal ovalado de coloração escura com LC >86 mm (Figura 1).
  2. Pegue os caranguejos com uma rede de coleta e mantenha-os individualmente em pequenos aquários para anestesia.
  3. Após 5 min do período de aclimatização, adicionar 2-fenoxietanol (2-PE) a 2 mL/L em cada aquário e permitir 15 min de tratamento anestésico.
  4. Certifique-se de que os caranguejos estejam totalmente anestesiados pela falta de movimento espontâneo.

5. Ablação do pedúnculo ocular

  1. Técnica de cauterização
    1. Execute todos os procedimentos em cima de uma mesa e em uma área aberta.
    2. Pegue uma haste de metal de níquel-aço de cabeça plana (por exemplo, uma chave de fenda) com uma alça de madeira ou plástico e cubra a alça com uma toalha de algodão molhada.
    3. Esterilizar duas pinças cirúrgicas inoxidáveis em autoclave.
    4. Prepare etanol 70% em um borrifador e mantenha-o longe de quaisquer fontes relacionadas ao fogo, como maçarico e chave de fenda vermelha quente. Tenha papel higiênico pronto para uso.
      NOTA: O etanol é altamente inflamável. Mantenha uma distância segura das fontes de incêndio.
    5. Conecte um maçarico a um botijão de gás (butano) com segurança.
      CUIDADO: Siga as instruções sobre o maçarico e o cilindro de gás. Certifique-se de que o maçarico está desligado ao conectar com o cilindro de gás. Leia e siga todas as precauções de segurança contra incêndio mencionadas no botijão de gás.
    6. Use luvas grossas de algodão para evitar ferimentos causados por objetos quentes.
    7. Sujeite a ponta da haste metálica ao fogo do maçarico até que a haste metálica fique vermelha brilhante.
    8. Cubra o caranguejo anestesiado com uma toalha de algodão molhada.
      OBS: Cubra as antenas do caranguejo para evitar danos desnecessários.
    9. Segure um olho do caranguejo com pinça esterilizada.
      OBS: Esterilizar a pinça em autoclave para primeiro uso e desinfetar com etanol 70% para posterior uso em outros caranguejos.
    10. Segure a ponta plana de metal quente no olho do caranguejo e pressione levemente por cerca de 10 a 15 s até que o talo do olho fique com uma cor laranja ou laranja-avermelhada. Tenha cuidado ao realizar esta etapa para evitar danos às estruturas adjacentes.
      OBS: Duas pessoas são necessárias para realizar a ablação do pedúnculo ocular seguindo o método de cauterização: uma para segurar o caranguejo e outra para realizar o procedimento de ablação.
    11. Desinfete a pinça com spray de etanol 70% para garantir que não haja contaminação cruzada entre caranguejos.
      NOTA: Só execute esta etapa pelo menos aguardar 5 minutos após o procedimento de ablação do pedúnculo ocular para garantir que a pinça seja resfriada antes da desinfecção usando etanol 70% para evitar possíveis riscos de incêndio.
    12. Depois de realizar a ablação do pedúnculo ocular em todos os caranguejos, mergulhe a haste de metal de aço níquel quente (chave de fenda) em água da torneira.
    13. Desinfete a toalha antes de reutilizar. Várias toalhas podem ser usadas para economizar tempo.
      NOTA: Lave a toalha com água da torneira e mergulhe-a em água clorada de 30 ppm por 5 min. Em seguida, lave a toalha com água da torneira novamente e mergulhe-a em uma solução de tiossulfato de sódio a 1 g/L.
    14. Mantenha o maçarico em um local seguro depois de desligá-lo e aguarde até que ele retorne à temperatura ambiente (cerca de 30 min) antes de desconectar.
  2. Técnica cirúrgica
    1. Realize o procedimento em uma área bem ventilada.
    2. Esterilizar duas tesouras cirúrgicas e pinças em autoclave.
    3. Despeje 50 mL de etanol a 70% em um copo de vidro de 100 mL.
    4. Use luvas grossas de algodão.
    5. Segure o caranguejo anestesiado e cubra-o com uma toalha de algodão molhada.
    6. Segure um olho do caranguejo com pinça esterilizada.
    7. Corte rapidamente o pedúnculo ocular usando tesoura cirúrgica esterilizada.
      NOTA: A hemolinfa pode ser perdida da parte ferida do caranguejo.
    8. Mergulhe a tesoura e a pinça em etanol 70% após cada uso e seque-as com papel higiênico antes de reutilizá-las.

6. Cuidados pós-anestésicos

  1. Prepare 20 ppt de água do mar filtrada e mantenha em um tanque aéreo com aeração contínua.
  2. Conecte um tubo flexível com o tanque superior para o fluxo gravitacional de água.
  3. Imediatamente após a ablação do talo ocular, coloque o caranguejo no cesto e submeta o caranguejo à água do mar corrente (temperatura da água ambiente: 28 °C) do tanque superior.
  4. Mantenha a água do mar fluindo e monitore o caranguejo até que ele possa se mover espontaneamente, o que indica recuperação da anestesia.
    NOTA: A água do mar pode ser preparada em um tanque de solo, e uma bomba de água submersível pode ser usada para o fluxo de água.
  5. Mantenha os caranguejos individualmente em água do mar de 20 ppt com aeração em um aquário por 30 min para observação adicional.
    OBS: Os caranguejos recuperados serão cultivados individualmente no processo de cultivo subsequente de matrizes.

7. Observação da maturação ovariana

  1. Criação de matrizes
    1. Transfira os caranguejos maduros para tanques circulares individuais de 32 L.
    2. Continue a alimentação com peixes marinhos picados (congelados a -20 °C) duas vezes por dia (manhã 09:00 am e noite 20:00 pm), e remova a ração não consumida antes da alimentação matinal.
    3. Criar o reprodutor individualmente por 30 dias em salinidade de 20 ppt.
    4. Retire as fezes e troque 10% da água do mar (20 ppt) diariamente.
  2. Dissecção
    1. Limpe uma bandeja dissecante, tesoura e pinça com etanol 70%.
    2. Anestesiar as fêmeas individualmente com o método de anestesia de imersão em 2-PE.
    3. Selecionar aleatoriamente fêmeas recém-maduras (após a muda de fêmeas pré-maduras) que não passaram pela ablação do pedúnculo ocular para confirmar seus estágios gonadais.
    4. Sacrificar individualmente todas as fêmeas experimentais ablativas com o olhículo e identificar os estágios de maturação das gônadas. Destrua os gânglios torácicos do caranguejo usando uma ave estéril afiada. Remova a carapaça superior primeiro e, em seguida, o hepatopâncreas para tornar o ovário visível. Observe a coloração dos ovários e identifique o estágio de maturação ovariana (Figura 2).
  3. Identificação dos estágios de maturação ovariana
    1. Observe a cor do ovário a olho nu ou sob um estereomicroscópio.
    2. Identificar os estágios de maturação ovariana com base na coloração30: o imaturo (estágio-1) apresenta uma cor branca translúcida ou cremosa; o início da maturação (estágio-2) apresenta coloração pálida a amarelada clara; (iii) o pré-maturação (estágio 3) apresenta coloração amarela a laranja claro; e (iv) o totalmente amadurecido (estágio-4) apresenta coloração laranja-escura a avermelhada.

Resultados

Maturação gonadal
Tecidos ovarianos brancos cremosos (ovários imaturos, estágio 1) foram encontrados em 100% das fêmeas dissecadas (n = 6) antes da realização da ablação do pedúnculo ocular (Figura 2). A taxa de maturação gonadal das fêmeas de caranguejos com haste ocular (n = 63; 31 fêmeas com a técnica de cauterização e 32 fêmeas com a técnica cirúrgica) foi maior em comparação com as fêmeas que não foram submetidas à ablação do pedúnculo ocu...

Discussão

Este protocolo foi desenvolvido para a ablação do pedúnculo ocular do caranguejo-da-lama, Scylla spp., e pode ser aplicado como um método eficiente para induzir a maturação gonadal. Este protocolo pode ser facilmente replicado para a maturação comercial dos ovários de caranguejos lama e pode ser implementado para reduzir o período latente (tempo de uma desova para outra) na produção de sementes de caranguejo lama.

A ablação do pedúnculo ocular de crustáceos (i.e., cama...

Divulgações

Nenhum dos autores tem conflitos de interesse.

Agradecimentos

Este estudo foi apoiado pelo Ministério da Educação, Malásia, no âmbito do programa Higher Institution Centre of Excellence (HICoE), Malásia, acreditado no Instituto de Aquicultura Tropical e Pesca, Universiti Malaysia Terengganu (Vot No. 63933 & Vot No. 56048). Agradecemos o apoio da Universiti Malaysia Terengganu e Sayap Jaya Sdn. Bhd. através da Bolsa de Pesquisa de Parceria Privada (Vot. No. 55377). Uma posição de bolsista acadêmico adjunto da Universiti Sains Malaysia para Khor Waiho e Hanafiah Fazhan também é reconhecida.

Materiais

NameCompanyCatalog NumberComments
Aeration tube Ming Yu ThreeN/Aaquarium and pet shop
AirstoneMing Yu ThreeN/Aaquarium and pet shop
Autoclave machineHIRAYAMA MANUFACTURING CORPORATIONN/AMADE IN JAPAN
Bleaching powder (Hi-Chlon 70%)Nippon Soda Co.Ltd,JapanN/AN/A
Blow torch MR D.I.Y. Group BerhadN/AN/A
Circular tank (32L)BEST PLASTIC INDUSTRY SDN. BHD. N/AN/A
Cotton hand gloves (thick) MR D.I.Y. Group BerhadN/AN/A
Cotton towelMR D.I.Y. Group BerhadN/AN/A
Digital thermometerHanna InstrumentHI9814Hanna Instruments GroLine Hydroponics Waterproof pH / EC / TDS / Temp. Portable Meter HI9814
Digital Vernier CaliperINSIZE Co., Ltd.N/A
Dissecting trayHatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu
Dropper bottle/Plastic Pipettes DropperShopee MalaysiaN/AN/A
Ethanol 70%Thermo Scientific Chemicals033361.M1Diluted to 70% using double distilled water
Fiberglass tank (1 ton)Hatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu
Fine sandN/AN/Acollected from Sea beach of Universiti Malaysia Terengganu
First Aid KitsWatsons MalaysiaN/AN/A
Flat head nickel steel metal rod (Screw driver)MR D.I.Y. Group BerhadN/AN/A
FormaldehydeThermo Scientific Chemicals119690010
Gas cylinder (butane gas) for blow  torchMR D.I.Y. Group BerhadN/AN/A
Gas lighter gun (long head)MR D.I.Y. Group BerhadN/AN/A
Glass beaker (100 mL))Corning Life Sciences1000-100
Ice bag Watsons MalaysiaN/AN/A
Perforated plastic baskets Eco-Shop Marketing Sdn. Bhd.N/AN/A
PVC pipe 15mmBina Plastic Industries Sdn Bhd (HQ)N/AN/A
RefractometerATAGO CO.,LTD.
RefrigeratorSharp Corporation JapanN/AChest Freezer SHARP 110L - SJC 118
Scoop netMR D.I.Y. Group BerhadN/A
SeawaterHatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu
Siphoning pipeMR D.I.Y. Group BerhadN/AN/A
Spray bottleMr. DIY Sdn BhdN/AN/A
Stainless surgical forceps N/AN/AN/A
Stainless surgical scissors N/AN/AN/A
Submersible water pump ASN/Amodel: Astro 4000
Tincture of iodine solution  (Povidone Iodine)Farmasi Fajr Sdn BhdN/AN/A
Tissue paper N/AN/A
Transparent plastic aquariumMing Yu ThreeN/Aaquarium and pet shop
Waterproof tableHatcheri AKUATROP N/AResearch Center of Universiti Malaysia Terengganu

Referências

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  14. Diarte-Plata, G., et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
  15. Vargas-Téllez, I., et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
  16. Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
  17. Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
  18. Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493 (2022).
  19. Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
  20. Kornthong, N., et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
  21. Kornthong, N., et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122 (2019).
  22. Liu, C., et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
  23. Chen, H. -. Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552 (2019).
  24. Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
  25. Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
  26. Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
  27. Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. Wu, Q., et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
  30. Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
  31. Farhadi, A., et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8, (2021).
  32. Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
  33. Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
  34. Jang, I. K., et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus. Crustaceana. 83 (2), 129-141 (2010).
  35. Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
  36. Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
  37. Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
  38. Yi, S. -. K., Lee, S. -. G., Lee, J. -. M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
  39. Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
  40. Archibald, K. E., Scott, G. N., Bailey, K. M., Harms, C. A. 2-phenoxyethanol (2-PE) and tricaine methanesulfonate (MS-222) immersion anesthesia of American horseshoe crabs (Limulus polyphemus). Journal of Zoo and Wildlife Medicine. 50 (1), 96-106 (2019).
  41. Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
  42. Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
  43. Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
  44. Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
  45. Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity. Journal of King Saud University - Science. 31 (4), 827-835 (2019).
  46. Wu, X., et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
  47. Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
  48. Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
  49. Zhang, C., et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
  50. Zhang, C., et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
  51. Diarte-Plata, G., Sainz-Hernandez, J. C., Aguiñaga-Cruz, J. A., Fierro-Coronado, J. A., Polanco-Torres, A., Puente-Palazuelos, C. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 130 (3-4), 172-178 (2012).
  52. Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
  53. Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
  54. Fazhan, H., et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).

Erratum


Formal Correction: Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs
Posted by JoVE Editors on 5/26/2023. Citeable Link.

An erratum was issued for: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs. The Introduction, Protocol, Discussion and References were updated.

The forth sentence in the third paragraph of the Introduction has been updated from:

The eyestalk ablation protocol in this work minimizes stress by using fully sedated crabs and minimizes physical injury to personnel from crab bites. 

to:

The eyestalk ablation protocol in this work minimizes stress by using fully anesthetized crabs and minimizes physical injury to personnel from crab bites. 

The start of the Protocol has been updated from:

This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia. The sacrifice of the experimental samples was done according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Sexually pre-mature female mud crabs (orange mud crab S. olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics1.

to:

This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia and was approved by the Universiti Malaysia Terengganu's Research Ethics Committee (Animal ethics approval number: UMT/JKEPHMK/2023/96). The sacrifice of the experimental samples was done according to the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. Sexually pre-mature female mud crabs (orange mud crab Scylla olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics1.

Section 4 of the Protocol has been updated from:

4. Cold-shock anesthesia

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for cold shock anesthesia.
  3. Prepare 2 L of 4 °C to 1 °C seawater (20 ppt) in a transparent plastic aquarium. Maintain the temperature using (−20 °C) ice bags for cold shock anesthesia.
    NOTE: Check the temperature with a digital thermometer.
  4. Immerse the crab in the 4 °C seawater until sedated (about 3−5 min).
  5. Ensure the crabs are fully anesthetized by the lack of spontaneous movement. The legs and chelipeds joints will still show minor movements when touched with forceps.

to:

4. Anesthesia

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for anesthesia.
  3. After 5 min of acclimatization period, add 2-phenoxyethanol (2-PE) at 2 mL/L into each aquarium and allow 15 min of anesthesia treatment.
  4. Ensure the crabs are fully anesthetized by the lack of spontaneous movement.

Section 5 of the Protocol has been updated from:

5. Eyestalk ablation

  1. Cauterization technique
    1. Perform all procedures on top of a table and in an open area.
    2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
    3. Sterilize two stainless surgical forceps in an autoclave.
    4. Prepare 70% ethanol in a spray bottle. Have tissue paper ready for use.
      NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
    5. Connect a blowtorch to a gas cylinder (butane) securely.
      CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
    6. Wear thick cotton gloves to avoid injury from hot objects.
    7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
    8. Cover the anesthetized (sedated) crab with a wet cotton towel.
      NOTE: Cover all the tentacles of the crab to avoid unnecessary damage.
    9. Hold one eye of the crab with sterilized forceps.
      NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
    10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color.
      NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
    11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
    12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
    13. Disinfect the towel before reuse. Multiple towels can be used to save time.
      NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
    14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
  2. Surgery technique
    1. Perform the procedure in a well-ventilated area.
    2. Sterilize two surgical scissors and forceps in an autoclave.
    3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
    4. Prepare the tincture of iodine solution in a dropper bottle.
      NOTE: Tincture of iodine (iodine tincture or weak iodine solution) is made up of 2%-7% elemental iodine and potassium iodide, or sodium iodide, dissolved in ethanol and water.
    5. Wear thick cotton gloves.
    6. Hold the sedated crab, and cover it with a wet cotton towel.
    7. Hold one eye of the crab with sterilized forceps.
    8. Swiftly cut off the eyestalk using sterilized surgical scissors.
      NOTE: Hemolymph may be lost from the wounded part of the crab.
    9. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.
    10. Apply two to three drops of iodine tincture to the wounded part of the eyestalk immediately after cutting it off.
      NOTE: Tincture of iodine is used for healing and to prevent infection.

to:

5. Eyestalk ablation

  1. Cauterization technique
    1. Perform all procedures on top of a table and in an open area.
    2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
    3. Sterilize two stainless surgical forceps in an autoclave.
    4. Prepare 70% ethanol in a spray bottle and keep it away from any fire-related sources, such as blow torch and red hot screwdriver. Have tissue paper ready for use.
      NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
    5. Connect a blowtorch to a gas cylinder (butane) securely.
      CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
    6. Wear thick cotton gloves to avoid injury from hot objects.
    7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
    8. Cover the anesthetized crab with a wet cotton towel.
      NOTE: Cover the antennae of the crab to avoid unnecessary damage.
    9. Hold one eye of the crab with sterilized forceps.
      NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
    10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color. Be careful when conducting this step to avoid damage to adjacent structures. 
      NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
    11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
      NOTE: Only perform this step at least waiting for 5 min after the eyestalk ablation procedure to ensure the forceps are cooled down before disinfection using 70% ethanol to prevent potential fire hazards.
    12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
    13. Disinfect the towel before reuse. Multiple towels can be used to save time.
      NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
    14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
  2. Surgery technique
    1. Perform the procedure in a well-ventilated area.
    2. Sterilize two surgical scissors and forceps in an autoclave.
    3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
    4. Wear thick cotton gloves.
    5. Hold the anesthetized crab, and cover it with a wet cotton towel.
    6. Hold one eye of the crab with sterilized forceps.
    7. Swiftly cut off the eyestalk using sterilized surgical scissors.
      NOTE: Hemolymph may be lost from the wounded part of the crab.
    8. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.

Step 7.2.2 of the Protocol has been updated from:

Sedate the females individually with the cold shock anesthesia method.

to:

Anesthetize the females individually with the 2-PE immersion anesthesia method.

The Discussion has been updated from:

This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.

The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning11,12,13. Eyestalk ablation in brachyuran crabs has also been done to study molting25,32,33, hormonal regulation18, gonad maturation34, and induced breeding and reproductive performance35,36,37,38,39. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs25,31. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones21,40,41. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs18,21. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye29,30,42. Translucent or creamy white ovarian tissues are indications of immature ovaries29,30,42,43. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages29,44. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique requires an additional step of disinfection using iodine. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity30,35,45,46.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. The application of a tincture of iodine can prevent infection of the wounded part. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab33.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation14,15,17,47. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival37,48,49.

Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions7,50,51. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes8,52. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss34.

The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.

to:

This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.

The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning11,12,13. Eyestalk ablation in brachyuran crabs has also been done to study molting25,32,33, hormonal regulation18, gonad maturation34, and induced breeding and reproductive performance35,36,37,38,39. Anesthesia via immersion in 2-phenoxyethanol was used as it is comparable to the use of tricaine methanesulfonate (MS-222) in arthopods but cheaper and does not require the use of additional buffer40. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs25,31. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones21,41,42. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs18,21. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye29,30,43. Translucent or creamy white ovarian tissues are indications of immature ovaries29,30,43,44. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages29,45. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique takes time for the wound to heal and this would allow for chance of infection. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity30,35,46,47.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab33.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation14,15,17,48. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival37,49,50. In addition, future research on pain assessment following eyestalk ablation on mud crabs is recommended to highlight the change in behaviours associated with pain and stress, as evident in freshwater prawn Macrobrachium americanum51.

Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions7,52,53. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes8,53. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss34.

The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.

The References have been updated from:

  1. Keenan, C. P., Davie, P. J. F., Mann, D. L. A revision of the genus Scylla de Haan, 1833 (Crustacea: Decapoda: Brachyura: Portunidae). Raffles Bulletin of Zoology. 46 (1), 217-245 (1998).
  2. Fazhan, H. et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066 (2020).
  3. Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., Zakaria, M. Z. Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. Journal of Sustainability Science and Management. 5 (2), 97-109 (2010).
  4. Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
  5. Waiho, K. et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
  6. Mykles, D. L., Chang, E. S. Hormonal control of the crustacean molting gland: Insights from transcriptomics and proteomics. General and Comparative Endocrinology. 294, 113493 (2020).
  7. Fujaya, Y. et al. Is limb autotomy really efficient compared to traditional rearing in soft-shell crab (Scylla olivacea) production? Aquaculture Reports. 18, 100432 (2020).
  8. Waiho, K. et al. Moult induction methods in soft-shell crab production. Aquaculture Research. 52 (9), 4026-4042 (2021).
  9. Rahman, M. R. et al. Evaluation of limb autotomy as a promising strategy to improve production performances of mud crab (Scylla olivacea) in the soft-shell farming system. Aquaculture Research. 51 (6), 2555-2572 (2020).
  10. Okumura, T. et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
  11. Pervaiz, P. A., Jhon, S. M., Sikdar-bar, M. Studies on the effect of unilateral eyestalk ablation in maturation of gonads of a freshwater prawn Macrobrachium dayanum. World Journal of Zoology. 6 (2), 159-163 (2011).
  12. Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
  13. Shyne Anand, P. S. et al. Reproductive performance of wild brooders of Indian white shrimp, Penaeus indicus: Potential and challenges for selective breeding program. Journal of Coastal Research. 86 (sp1), 65 (2019).
  14. Diarte-Plata, G. et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
  15. Vargas-Téllez, I. et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
  16. Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis (Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
  17. Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
  18. Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493 (2022).
  19. Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
  20. Kornthong, N. et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
  21. Kornthong, N. et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122 (2019).
  22. Liu, C. et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
  23. Chen, H.-Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552 (2019).
  24. Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
  25. Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
  26. Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
  27. Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. Wu, Q. et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
  30. Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
  31. Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
  32. Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
  33. Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
  34. Jang, I. K. et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus.Crustaceana. 83 (2), 129-141 (2010).
  35. Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
  36. Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
  37. Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
  38. Yi, S.-K., Lee, S.-G., Lee, J.-M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
  39. Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
  40. Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
  41. Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
  42. Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
  43. Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
  44. Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity? Journal of King Saud University - Science. 31 (4), 827-835 (2019).
  45. Wu, X. et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
  46. Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
  47. Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
  48. Zhang, C. et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
  49. Zhang, C. et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
  50. Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
  51. Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
  52. Fazhan, H. et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).

to:

  1. Keenan, C. P., Davie, P. J. F., Mann, D. L. A revision of the genus Scylla de Haan, 1833 (Crustacea: Decapoda: Brachyura: Portunidae). Raffles Bulletin of Zoology. 46 (1), 217-245 (1998).
  2. Fazhan, H. et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066 (2020).
  3. Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., Zakaria, M. Z. Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. Journal of Sustainability Science and Management. 5 (2), 97-109 (2010).
  4. Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
  5. Waiho, K. et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
  6. Mykles, D. L., Chang, E. S. Hormonal control of the crustacean molting gland: Insights from transcriptomics and proteomics. General and Comparative Endocrinology. 294, 113493 (2020).
  7. Fujaya, Y. et al. Is limb autotomy really efficient compared to traditional rearing in soft-shell crab (Scylla olivacea) production? Aquaculture Reports. 18, 100432 (2020).
  8. Waiho, K. et al. Moult induction methods in soft-shell crab production. Aquaculture Research. 52 (9), 4026-4042 (2021).
  9. Rahman, M. R. et al. Evaluation of limb autotomy as a promising strategy to improve production performances of mud crab (Scylla olivacea) in the soft-shell farming system. Aquaculture Research. 51 (6), 2555-2572 (2020).
  10. Okumura, T. et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
  11. Pervaiz, P. A., Jhon, S. M., Sikdar-bar, M. Studies on the effect of unilateral eyestalk ablation in maturation of gonads of a freshwater prawn Macrobrachium dayanum. World Journal of Zoology. 6 (2), 159-163 (2011).
  12. Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
  13. Shyne Anand, P. S. et al. Reproductive performance of wild brooders of Indian white shrimp, Penaeus indicus: Potential and challenges for selective breeding program. Journal of Coastal Research. 86 (sp1), 65 (2019).
  14. Diarte-Plata, G. et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
  15. Vargas-Téllez, I. et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
  16. Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis (Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
  17. Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
  18. Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493 (2022).
  19. Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
  20. Kornthong, N. et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
  21. Kornthong, N. et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122 (2019).
  22. Liu, C. et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
  23. Chen, H.-Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552 (2019).
  24. Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
  25. Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
  26. Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
  27. Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. Wu, Q. et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
  30. Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
  31. Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
  32. Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
  33. Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
  34. Jang, I. K. et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus.Crustaceana. 83 (2), 129-141 (2010).
  35. Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
  36. Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
  37. Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
  38. Yi, S.-K., Lee, S.-G., Lee, J.-M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
  39. Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
  40.  Archibald, K. E., Scott, G. N., Bailey, K. M., Harms, C. A. 2-phenoxyethanol (2-PE) and tricaine methanesulfonate (MS-222) immersion anesthesia of American horseshoe crabs (Limulus polyphemus). Journal of Zoo and Wildlife Medicine. 50 (1), 96-106 (2019).
  41. Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
  42. Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
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