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  • Erratum Notice
  • Özet
  • Özet
  • Giriş
  • Protokol
  • Sonuçlar
  • Tartışmalar
  • Açıklamalar
  • Teşekkürler
  • Malzemeler
  • Referanslar
  • Erratum
  • Yeniden Basımlar ve İzinler

Erratum Notice

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

Özet

Anestezi uygulanan dişi yengeçler üzerinde iki göz konuşması ablasyon protokolü (yani koterizasyon ve cerrahi yaklaşımlar) uygulandı. Çamur yengeçlerinin göz konuşması ablasyonu, hayatta kalma oranını düşürmeden yumurtalıkların olgunlaşmasını hızlandırdı.

Özet

Çamur yengeçleri (Scylla spp.), Hint-Batı Pasifik bölgesinde bulunabilen ticari olarak önemli kabuklu türleridir. Kültür sırasında, yumurtalık olgunlaşmasının indüksiyonu, olgun çamur yengeçlerine olan tüketici talebini karşılamak ve tohum üretimini hızlandırmak için önemlidir. Göz konuşması ablasyonu, çamur yengeçlerinde yumurtalık olgunlaşmasını arttırmak için etkili bir araçtır. Bununla birlikte, çamur yengeçlerinin göz konuşması ablasyonu için standart bir protokol yoktur. Bu çalışmada, iki göz konuşması ablasyon tekniği tanımlanmıştır: koterizasyon (anestezi uygulanmış bir yengecin göz konuşmasını ablate etmek için sıcak metal kullanımı) ve cerrahi (cerrahi makas kullanılarak göz konuşmasının çıkarılması). Göz konuşması ablasyonundan önce, cinsel olarak olgun dişiler (CW > 86 mm), deniz suyu ile bir buz torbası (-20 ° C) kullanılarak uyuşturuldu. Su sıcaklığı 4 ° C'ye ulaştığında, buz torbası sudan çıkarıldı. Akan deniz suyu (ortam sıcaklığı: 28 °C), göz sapı ablasyonundan hemen sonra anesteziden kurtulmak için kullanıldı. Mortalite, göz konuşması ablasyonu işlemi sırasında veya sonrasında ortaya çıkmadı. Burada sunulan göz konuşması ablasyon protokolü, çamur yengeçlerinin yumurtalık olgunlaşmasını hızlandırdı.

Giriş

Scylla cinsine ait dört çamur yengeç türünün tümü, su ürünleri yetiştiriciliğinde ticari açıdan önemli kabuklu türleridir 1,2. Çamur yengeçleri de dahil olmak üzere kabukluların büyümesi ve bunların pre-olgun (alt yetişkin veya ergen) fazdan cinsel olarak olgun (yetişkin) faza dönüşümü, yaşlı ve daha küçük dış iskeletlerin periyodik olarak dökülmesini içeren bir kalıplama işlemi ile gerçekleşir. Carapace genişliği (CW), şelayedler ve abdominal flep morfolojileri, Scylla spp'nin cinsel olgunluğunu belirlemek için yaygın olarak kullanılmaktadır. 3,4,5. Kalıplama işlemi çeşitli hormonların etkisiyle düzenlenir ve çok miktarda enerji gerektirir6. Normal kalıplama işlemine ek olarak, gönüllü olarak veya dış faktörler tarafından indüklenen uzuvların kaybı, hayatta kalma oranlarını etkilemeden yengeçlerin erimesini hızlandırır 7,8,9. Bu nedenle, uzuv ototomisi, yumuşak kabuklu çamur yengeç yetiştiriciliği endüstrisinde erime indüksiyonu için yaygın olarak kullanılmaktadır 7,9.

Tek taraflı veya iki taraflı göz konuşması ablasyonu çoğunlukla gonad olgunlaşması ve tohum üretimi için tatlı su karidesi ve deniz karidesinde popülerdir10,11,12,13. Kabuklularda yaygın göz konuşması ablasyon teknikleri şunları içerir: (i)14,15 ipi kullanarak göz konuşmasının tabanında ligasyon; (ii) sıcak forseps veya elektrokoter cihazları kullanılarak göz konuşmasının koterizasyonu16; (iii) açık bir yara bırakmak için göz konuşmasının çıkarılması veya doğrudan sıkıştırılması12; ve (iv) gözün distal kısmını jiletle kestikten sonra kesi yoluyla göz konuşması içeriğinin çıkarılması17. Göz sapı X-organları, kabukluların hiperglisemik hormonlarını (CHH), molt inhibe edici hormonları (MIH) ve vitellogenezi inhibe eden hormonları (VIH) düzenledikleri için kabuklularda önemli endokrin organlardır 6,18,19,20,21,22. Göz konuşması X-organları (veya sinüs bezi kompleksi), nöropeptit hormon ailesi6'ya ait vitellogenezi inhibe eden hormonlar (VIH) olarak da bilinen gonad inhibe edici hormonları (GIH) sentezler ve serbest bırakır. Tek taraflı veya iki taraflı göz konuşması ablasyonu, GIH sentezini azaltır, bu da uyarıcı hormonların (yani, gonad uyarıcı hormonlar, GSH) baskınlığına ve kabuklularda yumurtalık olgunlaşma sürecinin hızlanmasına neden olur23,24,25,26. Göz konuşması ablasyonundan sonra GYH'nin etkisi olmadan, dişi kabuklular enerjilerini yumurtalık gelişimine adamışlardır27. Tek taraflı göz konuşması ablasyonunun, kabuklularda yumurtalık olgunlaşmasının indüksiyonu için yeterli olduğu bulunmuştur11 ve karides ve yengeçlerin ablatlı göz konuşmasının birkaç kalıplamadan sonra yenilenebileceği bulunmuştur28. Scylla spp.'de kaydedilen dört yumurtalık gelişim aşaması vardır: i) olgunlaşmamış (evre-1), ii) erken olgunlaşma (evre-2), iii) olgunlaşma öncesi (evre-3) ve iv) tamamen olgun (evre-4)29,30. Olgunlaşmamış yumurtalık evresi olgunlaşmamış kadınlarda bulunur. Pubertal erime ve çiftleşmeden sonra, olgunlaşmamış yumurtalık gelişmeye başlar ve nihayet yumurtlamadan önce olgunlaşır (aşama-4)31.

Bir göz sapı ablasyon protokolü, çamur yengeç yavrularının gelişimi ve tohum üretimi için gereklidir. Küresel gıda pazarında, daha yüksek kas içeriğine sahip yengeçler yerine tam olgun yumurtalıklara (evre-4) sahip olgun çamur yengeçleri tüketiciler tarafından tercih edilmektedir ve bu nedenle büyük erkeklerden bile daha yüksek bir ticari değere sahiptir. Çamur yengeçlerinin göz konuşması ablasyonu için tam bir protokol yoktur. Bu çalışmadaki göz konuşması ablasyon protokolü, tamamen anestezi uygulanmış yengeçler kullanarak stresi en aza indirir ve yengeç ısırıklarından personelin fiziksel yaralanmasını en aza indirir. Bu protokol kolay ve uygun maliyetlidir. Burada, gonadın olgunlaşmasını tetikleyebilecek Scylla spp.'nin göz konuşması ablasyonu için bir protokol sunuyoruz. İki göz konuşması ablasyon tekniği (koterizasyon ve cerrahi) test edildi ve dişi çamur yengeçlerinin gonadal gelişim hızına göre etkinlikleri karşılaştırıldı.

Protokol

Bu protokol, Malezya Laboratuvar Hayvanları Bilimi Derneği tarafından özetlenen Bilimsel Amaçlar için Hayvanların Bakımı ve Kullanımı için Malezya Uygulama Kurallarına uygundur. Deneysel örneklerin kurban edilmesi, Ulusal Sağlık Enstitüleri Laboratuvar Hayvanlarının Bakımı ve Kullanımı Kılavuzu'na göre yapılmıştır (NIH Yayınları No. 8023, gözden geçirilmiş 1978). Cinsel olarak olgunlaşmamış dişi çamur yengeçleri (turuncu çamur yengeci S. olivacea), Malezya'daki Setiu Sulak Alanları'ndaki yerel pazardan (5 ° 66 ′ 62 ′ ′ N, 102 ° 72 ′ 33 ′ ′ E) toplandı. Çamur yengeç türleri morfolojik özelliklere göre tanımlanmıştır1.

1. Numune toplama ve dezenfeksiyon

  1. Sağlıklı, aktif ve olgunlaşmamış dişi çamur yengeçlerini toplayın (Şekil 1).
    NOT: Prematüre dişi yengeçler, 80-85 mm'lik bir CW aralığı ile birlikte üçgen ve açık renkli karın fleplerine sahiptir.
  2. Kalıntıları ve ozmofilik parazitleri gidermek için yengeçleri klorlu musluk suyuyla (tatlı su) yıkayın.
  3. Yengeçleri 30 dakika boyunca 20 ppt tuzluluk ile 150 ppm formaldehit içinde bekletin.
  4. Formaldehit tedavisi sırasında hava taşlarıyla sürekli ve nazik havalandırma sağlayın. Havalandırma kaynağı, merkezi bir havalandırma hattından veya bir akvaryum havalandırma pompasından olabilir.
  5. Artık formaldehitleri gidermek için yengeçleri akan deniz suyuyla yıkayın.

figure-protocol-1582
Şekil 1: Cinsel olgunlaşma aşamalarını tanımlamak için kullanılan dişi çamur yengeçlerinin karın morfolojisi. Bu şeklin daha büyük bir versiyonunu görmek için lütfen buraya tıklayın.

2. İklimlendirme

  1. Dezenfekte edilen her dişi ayrı bir 32 L dairesel tanka aktarın.
  2. Dişileri 20 ppt tuzlulukta 3 gün boyunca destekleyin ve yengeç vücut ağırlığının yaklaşık% 4 -% 5'inde doğranmış deniz balıklarıyla günde iki kez (sabah 09: 00 ve akşam 20: 00 pm) beslenmeye devam edin.
  3. Sabah beslenmeden önce sifonlayarak fazla ve yenmemiş yemleri çıkarın.
  4. Yengeç yetiştiriciliği deniz suyunun% 10'unu (20 ppt) günlük olarak değiştirin.

3. Cinsel olgunluk için indüklenmiş kalıplama

  1. Sterilize edilmiş makas kullanarak yüzme bacakları hariç tüm bacakları kesin.
    1. Yengeci bir kepçe ağı ile yakalayın ve yengeci dikkatlice tutun. Önce her iki şelayeti ve ardından ikinci eklemdeki yürüme bacaklarını makas kullanarak kesin. Yengeç, hasarlı uzantıları otomatik olarak otonize edecektir. Ekstremite ototomisi için anestezi gerekli değildir.
  2. Ekstremite ototomisinden hemen sonra yengeci tatlı suda yıkayın.
  3. Ekstremite ototomize yengeçleri ayrı ayrı delikli plastik sepetlere (28 cm L x 22 cm G x 7 cm Y) aktarın ve bir fiberglas tanka (305 cm L x 120 cm G x 60 cm Y) yerleştirin.
    NOT: İki sepet birbirine bağlanabilir ve kırpılabilir. Üst sepet kapak olarak kullanılır, böylece yengeç sepetten kaçamaz.
  4. Tüm plastik sepetin suya batırıldığından emin olmak için 20 ppt tuzluluğa ve en az 10 cm su derinliğine sahip bir devridaim su ürünleri yetiştiriciliği sistemi (RAS) kullanın.
  5. Ekstremite ototomize dişi yengeci, yengeç vücut ağırlığının% 5-7'sinde günde iki kez doğranmış deniz balıklarıyla beslemeye devam edin.
  6. Yengeçleri kalıplama yoluyla cinsel olarak olgunlaşana kadar destekleyin (35 gün).
    NOT: Ticari yumurtalık olgunlaşması ve yabani olgun dişi çamur yengeçleri ile tohum üretimi için indüklenmiş küflenme atlanabilir. Vahşi doğadan hasat edilen olgun dişiler iklimlendirilmeli ve doğrudan soğuk şok anestezisine ve ardından göz konuşması ablasyonuna tabi tutulmalıdır.

4. Anestezi

  1. CW >86 mm ile koyu renkli oval şekilli karın flebi olan cinsel olarak olgun kadınları seçin (Şekil 1).
  2. Yengeçleri bir kepçe ağı ile yakalayın ve anestezi için ayrı ayrı küçük akvaryumlarda saklayın.
  3. 5 dakikalık iklimlendirme süresinden sonra, her akvaryuma 2 mL / L'de 2-fenoksietanol (2-PE) ekleyin ve 15 dakikalık anestezi tedavisine izin verin.
  4. Yengeçlerin kendiliğinden hareket eksikliği ile tamamen uyuşturulduğundan emin olun.

5. Göz konuşması ablasyonu

  1. Koterizasyon tekniği
    1. Tüm yordamları bir masanın üstünde ve açık bir alanda gerçekleştirin.
    2. Ahşap veya plastik saplı düz başlı nikel-çelik metal bir çubuk (örneğin bir tornavida) alın ve sapı ıslak pamuklu bir havluyla örtün.
    3. İki paslanmaz cerrahi forseps'i bir otoklavda sterilize edin.
    4. Bir sprey şişesinde% 70 etanol hazırlayın ve üfleme meşalesi ve kırmızı sıcak tornavida gibi yangınla ilgili kaynaklardan uzak tutun. Kağıt mendili kullanıma hazır bulundurun.
      NOT: Etanol oldukça yanıcıdır. Yangın kaynaklarından güvenli bir mesafeyi koruyun.
    5. Bir üfleme fenerini bir gaz tüpüne (bütan) güvenli bir şekilde bağlayın.
      DİKKAT: Üfleme feneri ve gaz tüpü üzerindeki talimatları izleyin. Gaz tüpüne bağlanırken üfleme fenerinin kapalı olduğundan emin olun. Gaz tüpünde belirtilen tüm yangın güvenliği önlemlerini okuyun ve takip edin.
    6. Sıcak nesnelerden yaralanmayı önlemek için kalın pamuklu eldivenler giyin.
    7. Metal çubuğun ucunu, metal çubuk parlak kırmızı olana kadar üfleme fenerinin ateşine maruz bırakın.
    8. Anestezi uygulanan yengeci ıslak pamuklu bir havluyla örtün.
      NOT: Gereksiz hasarları önlemek için yengeç antenlerini örtün.
    9. Yengecin bir gözünü sterilize forseps ile tutun.
      NOT: Forsepsleri ilk kez kullanım için bir otoklavda sterilize edin ve daha sonra diğer yengeçlerde kullanmak için% 70 etanol kullanarak dezenfekte edin.
    10. Kırmızı-sıcak metal düz ucu yengeç gözünün üzerine tutun ve göz konuşması turuncu veya kırmızımsı-turuncu bir renge dönene kadar yaklaşık 10-15 s hafifçe bastırın. Bitişik yapılara zarar vermemek için bu adımı uygularken dikkatli olun.
      NOT: Koterizasyon yöntemini takiben göz konuşması ablasyonunu uygulamak için iki kişiye ihtiyaç vardır: biri yengeci tutmak için, diğeri ablasyon prosedürünü gerçekleştirmek için.
    11. Yengeçler arasında çapraz kontaminasyon olmadığından emin olmak için forsepsleri% 70 etanol spreyi ile dezenfekte edin.
      NOT: Potansiyel yangın tehlikelerini önlemek için% 70 etanol kullanılarak dezenfeksiyondan önce forsepslerin soğutulmasını sağlamak için göz sapı ablasyon prosedüründen sonra bu adımı sadece en az 5 dakika bekleyerek gerçekleştirin.
    12. Tüm yengeçlerde göz sapı ablasyonunu gerçekleştirdikten sonra, sıcak nikel çelik metal çubuğu (tornavida) musluk suyuna batırın.
    13. Tekrar kullanmadan önce havluyu dezenfekte edin. Zamandan tasarruf etmek için birden fazla havlu kullanılabilir.
      NOT: Havluyu musluk suyuyla yıkayın ve 5 dakika boyunca 30 ppm klorlu suya batırın. Ardından, havluyu tekrar musluk suyuyla yıkayın ve 1 g / L sodyum tiyosülfat çözeltisine batırın.
    14. Blowtorch'u kapattıktan sonra güvenli bir yerde tutun ve bağlantıyı kesmeden önce ortam sıcaklığına (yaklaşık 30 dakika) dönene kadar bekleyin.
  2. Cerrahi teknik
    1. Prosedürü iyi havalandırılan bir alanda gerçekleştirin.
    2. İki cerrahi makas ve forsepsleri bir otoklavda sterilize edin.
    3. 100 mL'lik bir cam beherin içine 50 mL% 70 etanol dökün.
    4. Kalın pamuklu eldivenler giyin.
    5. Anestezi uygulanmış yengeci tutun ve ıslak pamuklu bir havluyla örtün.
    6. Yengecin bir gözünü sterilize forseps ile tutun.
    7. Sterilize cerrahi makas kullanarak göz konuşmasını hızla kesin.
      NOT: Hemolenf, yengecin yaralı kısmından kaybolabilir.
    8. Makas ve forsepsleri her kullanımdan sonra %70 etanol içine batırın ve tekrar kullanmadan önce kağıt mendil kullanarak kurulayın.

6. Anestezi sonrası bakım

  1. 20 ppt filtrelenmiş deniz suyu hazırlayın ve sürekli havalandırmalı bir baş üstü tankında tutun.
  2. Yerçekimi suyu akışı için havai tanka esnek bir boru bağlayın.
  3. Göz konuşması ablasyonundan hemen sonra, yengeci sepete yerleştirin ve yengeci baş üstü tanktan akan deniz suyuna (ortam suyu sıcaklığı: 28 ° C) maruz bırakın.
  4. Deniz suyunun akmasını sağlayın ve yengeci kendiliğinden hareket edene kadar izleyin, bu da anesteziden iyileşmeyi gösterir.
    NOT: Deniz suyu bir toprak tankında hazırlanabilir ve su akışı için bir dalgıç su pompası kullanılabilir.
  5. Daha fazla gözlem için yengeçleri ayrı ayrı 20 ppt deniz suyunda 30 dakika boyunca bir akvaryumda havalandırma ile saklayın.
    NOT: Geri kazanılan yengeçler, sonraki yavru kültür sürecinde ayrı ayrı kültürlenecektir.

7. Yumurtalık olgunlaşmasının gözlenmesi

  1. Damızlık yetiştiriciliği
    1. Olgun yengeçleri bireysel 32 L dairesel tanklara aktarın.
    2. Günde iki kez doğranmış deniz balıklarıyla (-20 ° C'de dondurulmuş) beslenmeye devam edin (sabah 09:00 ve akşam 20:00) ve sabah beslenmeden önce yenmemiş yemleri çıkarın.
    3. Yavruları 20 ppt tuzlulukta 30 gün boyunca ayrı ayrı destekleyin.
    4. Dışkıyı çıkarın ve günlük deniz suyunun% 10'unu (20 ppt) değiştirin.
  2. Diseksiyonu
    1. Diseksiyon tepsisini, makası ve forsepsleri %70 etanol ile temizleyin.
    2. 2-PE daldırma anestezi yöntemi ile kadınları ayrı ayrı uyuşturun.
    3. Gonadal aşamalarını doğrulamak için göz konuşması ablasyonundan geçmemiş yeni olgun kadınları (pre-matür dişilerin erimesinden sonra) rastgele seçin.
    4. Tüm göz ağrısı ablatlı deney kadınlarını ayrı ayrı feda edin ve gonad olgunlaşma aşamalarını tanımlayın. Keskin bir steril baykuş kullanarak yengeç torasik ganglionlarını yok edin. Yumurtalıkları görünür kılmak için önce üst kabuğu ve ardından hepatopankreası çıkarın. Yumurtalık rengini gözlemleyin ve yumurtalık olgunlaşma aşamasını tanımlayın (Şekil 2).
  3. Yumurtalık olgunlaşma aşamalarının tanımlanması
    1. Yumurtalık rengini çıplak gözle veya stereomikroskop altında gözlemleyin.
    2. Yumurtalık olgunlaşma aşamalarını renklendirme30'a göre tanımlayın: olgunlaşmamış (aşama-1) yarı saydam veya kremsi beyaz bir renk gösterir; erken olgunlaşma (aşama-2) soluk ila açık sarımsı bir renk gösterir; (iii) ön olgunlaşma (aşama-3) sarı ila açık turuncu renk gösterir; ve (iv) tamamen olgunlaşmış (aşama-4) koyu turuncu ila kırmızımsı bir renk gösterir.

Sonuçlar

Gonad olgunlaşması
Diseke edilen dişilerin %100'ünde (n=6) göz konuşması ablasyonu yapılmadan önce kremsi beyaz over dokuları (olgunlaşmamış yumurtalıklar, evre-1) bulundu (Şekil 2). Göz sapı ablatlı dişi yengeçlerin gonad olgunlaşma oranı (n = 63; koterizasyon tekniği ile 31 dişi ve cerrahi teknik ile 32 dişi), 30 günlük bireysel yetiştirmeden sonra göz sapı ablasyonuna (n = 31) tabi tutulmayan dişi yengeçlere göre daha yüksekti (

Tartışmalar

Bu protokol, çamur yengeci Scylla spp.'nin göz konuşması ablasyonu için geliştirilmiştir ve gonad olgunlaşmasını indüklemek için etkili bir yöntem olarak uygulanabilir. Bu protokol, çamur yengeçlerinin ticari yumurtalık olgunlaşması için kolayca çoğaltılabilir ve çamur yengeç tohumu üretiminde gizli süreyi (bir yumurtlamadan diğerine geçen süre) azaltmak için uygulanabilir.

Kabukluların (yani, tatlı su karidesi, deniz karidesi) göz konuşması ablasyon...

Açıklamalar

Yazarların hiçbirinde çıkar çatışması yoktur.

Teşekkürler

Bu çalışma, Malezya Eğitim Bakanlığı tarafından, Malezya Terengganu Üniversitesi Tropikal Su Ürünleri Yetiştiriciliği ve Balıkçılık Enstitüsü'ne akredite edilmiş Yüksek Kurum Mükemmeliyet Merkezi (HICoE) programı kapsamında Malezya Eğitim Bakanlığı tarafından desteklenmiştir (Vot No. 63933 & Vot No. 56048). Universiti Malaysia Terengganu ve Sayap Jaya Sdn. Bhd.'nin Özel Ortaklık Araştırma Bursu (Vot. No. 55377) aracılığıyla desteğini kabul ediyoruz. Universiti Sains Malaysia'dan Khor Waiho ve Hanafiah Fazhan'a kadar ek bir Akademik Araştırmacı pozisyonu da kabul edilmektedir.

Malzemeler

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

Referanslar

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  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).
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  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).
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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).
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