Refined Histological Staining of Carcınoscorpıus Rotundıcauda (Mangrove Horseshoe Crab) Eggs Encapsulated by Chitinous Proteins

Abstract

Haematoxylin and Eosin staining is less practical for Carcinoscorpius rotundicauda eggs because of chitinous proteins H, B1, B2, and Rest. A modified protocol with alcohol and haematoxylin washing, soak adjustments for Sudan IV and brief dips with Toluidine-O was introduced. Clearly, cellular protein like peripheral amoebocytes were stained blue using Toluidine-O and was consistent with first embryonic moult on day 8. Sudan IV distinguished lipid structures during limb budding, body segmentation, and extraembryonic shell formation because xylene washing made lipid structures colourless. Additionally, gradual yolk reduction, including yolk platelets, granules, and pits, was witnessed as eggs developed across days 8-15. This showed protein- and lipid-specific stains can detail events such as yolk lipid and protein breakdown as nutrients are supplied to the embryo. Stains like Sudan IV and Toluidine-O also indicate development and structural integrity, suggesting the embryo was alive and still progressing. The combination of conventional and specific stains was crucial to observe developmental progression that coincide with detection of delayed development or death. This refined protocol therefore enhances clarity, supports reliable staging, and improves interpretation of embryonic condition across time, making it a valuable approach for detailed histological assessment of C. rotundicauda eggs.

Keywords

• embryo
• yolk
• microscopy
• lipid
• colorimetry

Project Number

OT-01-2021

Ethical Statement

Ethical approval was not required for this study, as the investigated arthropod species is not listed under any annexes of local regulations, including the Fisheries Act 1985 and the Wildlife Protection Act 2023.

Thanks

The authors gratefully acknowledge the support of their respective institutions for providing laboratory facilities, workspace, and essential equipment necessary for this research. This study was supported by the Ocean Park Conservation Fund, Hong Kong (Project Code: OT-01 2021).

Kitinli proteinlerle kapsüllenmiş Carcinoscorpius rotundicauda yumurtalarının histolojik boyanmasında rafine yöntem

Abstract

Hematoksilen ve Eozin boyası, Carcinoscorpius rotundicauda yumurtalarında H, B1, B2 ve Rest gibi kitinli proteinler nedeniyle daha az uygulanabilir bulunmuştur. Bu nedenle alkol ve hematoksilen yıkama, Sudan IV için bekletme ayarlamaları ve Toluidine‑O ile kısa daldırmalar içeren değiştirilmiş bir protokol geliştirilmiştir. Toluidine‑O kullanımıyla çevresel amebositler maviye boyanmış ve bu durum 8. günde gerçekleşen ilk embriyonik deri değişimiyle tutarlı bulunmuştur. Sudan IV ise lipid yapıları ayırt etmiş; uzuv tomurcuklanması, vücut segmentasyonu ve ekstraembriyonik kabuk oluşumu sırasında xilen yıkaması lipid yapılarını renksiz hale getirmiştir. Ayrıca 8–15. günler arasında yumurta gelişimi boyunca yumurta plakaları, granüller ve çukurlar dahil olmak üzere kademeli yumurta sarısı azalması gözlenmiştir. Bu durum, protein ve lipitlere özgü boyaların embriyonun beslenmesi için yumurta sarısındaki protein ve lipitlerin parçalanmasını ayrıntılı biçimde gösterebildiğini ortaya koymuştur. Sudan IV ve Toluidine‑O gibi boyalar aynı zamanda gelişim ve yapısal bütünlüğü işaret ederek embriyonun canlı ve ilerlemekte olduğunu göstermiştir. Geleneksel ve özgül boyaların kombinasyonu, gecikmiş gelişim veya ölümün tespit edilmesiyle eşzamanlı olarak gelişimsel ilerlemenin gözlenmesinde kritik rol oynamıştır. Bu rafine edilmiş protokol, açıklığı artırmakta, güvenilir evrelemeyi desteklemekte ve embriyonik durumun zaman içindeki yorumunu geliştirmekte olup, C. rotundicauda yumurtalarının ayrıntılı histolojik değerlendirmesi için değerli bir yaklaşım sunmaktadır.

Keywords

• Embriyo
• yumurta sarısı
• mikroskopi
• lipit
• kolorimetri

Project Number

OT-01-2021

Ethical Statement

Bu çalışma için etik onay gerekli görülmemiştir; çünkü incelenen eklembacaklı türü, 1985 Balıkçılık Yasası ve 2023 Yaban Hayatı Koruma Yasası da dahil olmak üzere yerel düzenlemelerin herhangi bir ekinde listelenmemektedir

Thanks

Yazarlar, bu araştırma için gerekli laboratuvar olanaklarını, çalışma alanını ve temel ekipmanları sağlayan kurumlarına verdikleri destekten dolayı teşekkür ederler. Bu çalışma, Ocean Park Conservation Fund, Hong Kong (Proje Kodu: OT-01 2021) tarafından desteklenmiştir.

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