Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
JoVE Journal Biology
Click here for the English version

Biology

マッドクラブの卵巣成熟を促進するためのアイトークアブレーション

Published: March 31, 2023 doi: 10.3791/65039
Automatic Translation
1, 1, 1,2,3, 2,4, 5, 1,2,3
1Higher Institution Centre of Excellence (HICoE), Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 2Centre for Chemical Biology, Universiti Sains Malaysia, 3Department of Aquaculture, Faculty of Fisheries, Kasetsart University, 4School of Biological Sciences, Universiti Sains Malaysia, 5International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University

ERRATUM NOTICE

Summary

2つの眼球アブレーションプロトコル(すなわち、焼灼および 手術アプローチ)が、麻酔をかけられた雌のカニに対して実施された。マッドクラブのアイトークアブレーションは、生存率を低下させることなく卵巣の成熟を早めました。

Abstract

マッドクラブ(Scylla 属)は、インド西太平洋地域全体で見られる商業的に重要な甲殻類種です。培養中、卵巣成熟の誘導は、成熟したマッドクラブに対する消費者の需要を満たし、種子生産を早めるために重要です。アイトークアブレーションは、マッドクラブの卵巣成熟を促進するための効果的なツールです。ただし、マッドクラブのアイトークアブレーションに関する標準的なプロトコルはありません。この研究では、焼灼(麻酔をかけたカニの眼球を切除するための溶銑の使用)と手術(外科用ハサミを使用した眼球の除去)の2つの眼球アブレーション技術について説明します。眼球アブレーションの前に、性的に成熟した女性(CW > 86 mm)を海水の入ったアイスバッグ(-20°C)を使用して麻酔しました。水温が4°Cに達した時点で、アイスバッグを水から取り出した。眼球焼灼直後の麻酔からの回復には流す海水(周囲温度:28°C)を用いた。眼球切除術の過程または経過後に死亡率は発生しなかった。ここで紹介するアイトークアブレーションプロトコルは、マッドクラブの卵巣成熟を加速させました。

Introduction

Scylla属に属する4つのマッドクラブ種はすべて、水産養殖において商業的に重要な甲殻類種です1,2。マッドクラブを含む甲殻類の成長、および時期尚早(亜成体または思春期)段階から性的に成熟した(成体)段階へのそれらの変化は、古くてより小さな外骨格の定期的な脱落を含む脱皮プロセスを通じて起こります。甲羅の幅(CW)、頬骨、および腹部皮弁の形態は、Scyll a sppの性的成熟を決定するために広く使用されています。3,4,5。脱皮のプロセスは、さまざまなホルモンの作用によって調節されており、膨大な量のエネルギーを必要とします6。通常の脱皮プロセスに加えて、自発的にまたは外的要因によって誘発される四肢の喪失は、生存率に影響を与えることなくカニの脱皮を促進します7,8,9。したがって、四肢の解剖術は、ソフトシェルマッドクラブ養殖業における脱皮誘導に一般的に使用されています7,9。

片側または両側の眼茎アブレーションは、生殖腺の成熟と種子生産のために淡水エビと海産エビで主に人気があります10,11,12,13。甲殻類における一般的な眼球切除技術には、以下が含まれる:(i)紐14,15を用いた眼球基部での結紮;(ii)熱い鉗子または電気焼灼装置16を使用した眼茎の焼灼;(iii)開いた傷を残すために眼茎を除去または直接つまむこと12;(iv)かみそり17で眼の遠位部を切開して眼球内容物を除去する工程。アイトークX器官は、甲殻類の高血糖ホルモン(CHH)、脱皮阻害ホルモン(MIH)、および硝子体形成阻害ホルモン(VIH)を調節するため、甲殻類の重要な内分泌器官です6,18,19,20,21,22。アイトークX器官(または副鼻腔腺複合体)は、神経ペプチドホルモンファミリーに属する生殖腺阻害ホルモン(GIH)を合成して放出します6。片側または両側の眼球アブレーションはGIH合成を減少させ、刺激ホルモン(すなわち、生殖腺刺激ホルモン、GSH)の優勢をもたらし、甲殻類の卵巣成熟プロセスの加速をもたらします23,24,25,26。眼球切除後のGIHの影響がなければ、雌の甲殻類は卵巣の発達にエネルギーを捧げます27。甲殻類の卵巣成熟の誘導には片側眼茎切除で十分であり11、エビやカニの切除された眼茎は数回の脱皮後に再生できることがわかっています28。Scylla属に記録されている卵巣の発達段階は4つあります:i)未熟(ステージ1)、ii)早熟(ステージ2)、iii)前成熟(ステージ3)、およびiv)完全に成熟(ステージ4)29,30。未熟卵巣期は未熟女性に見られます。思春期の脱皮と交尾の後、未熟な卵巣は発達し始め、最終的に成熟し(ステージ4)、産卵します31。

アイトークアブレーションプロトコルは、マッドクラブの繁殖資源の開発と種子の生産に不可欠です。世界の食品市場では、筋肉含有量の高いカニではなく、完全に成熟した卵巣(ステージ4)を持つ成熟したマッドクラブが消費者に好まれているため、大型のオスよりもさらに高い商品価値を持っています。マッドクラブのアイトークアブレーションのための完全なプロトコルはありません。この作業のアイトークアブレーションプロトコルは、完全に麻酔をかけられたカニを使用することでストレスを最小限に抑え、カニに刺されたことによる人員の身体的傷害を最小限に抑えます。このプロトコルは簡単で費用対効果に優れています。本稿では、生殖巣の成熟を誘導するス キュラ 属の眼茎切除のプロトコールを提示する。眼球焼灼術の2つの技術(焼灼と手術)をテストし、メスのマッドクラブの性腺発達率に基づいてそれらの効率を比較しました。

Subscription Required. Please recommend JoVE to your librarian.

Protocol

このプロトコルは、マレーシア実験動物科学協会によって概説された科学的目的のための動物の世話と使用のためのマレーシアの実施規範に従います。実験サンプルの犠牲は、実験動物の世話と使用のための国立衛生研究所ガイド(NIH出版物第8023号、1978年改訂)に従って行われました。性的に早産の雌の泥ガニ(オレンジ色の泥ガニ S.olivacea)は、マレーシアのセティウ湿地の地元の市場(5°66′62′N、102°72′33′′E)から収集されました。マッドクラブの種は、形態学的特徴に基づいて同定されました1。

1.サンプルの収集と消毒

  1. 健康で活動的で時期尚早のメスのマッドクラブを収集します(図1)。
    注:時期尚早の雌のカニは、三角形で明るい色の腹部フラップを持ち、CW範囲は80〜85mmです。
  2. カニを塩素化された水道水(淡水)で洗って、破片や浸透圧性寄生虫を取り除きます。
  3. カニを塩分濃度20pptの150ppmのホルムアルデヒドに30分間浸します。
  4. ホルムアルデヒド処理中は、エアストーンで継続的かつ穏やかな通気を維持します。曝気源は、中央曝気ラインまたは水槽曝気ポンプのいずれかからであり得る。
  5. 流れる海水でカニを洗い、残留ホルムアルデヒドを取り除きます。

Figure 1
図1:性的成熟段階を特定するために使用された雌のマッドクラブの腹部形態。 この図の拡大版を表示するには、ここをクリックしてください。

2.順応

  1. 消毒した各メスを別々の32 L円形タンクに移します。
  2. 雌を20pptの塩分で3日間飼育し、カニの体重の約4%〜5%で刻んだ海産魚を1日2回(朝09:00 amと夕方20:00 pm)与え続けます。
  3. 朝の餌の前に吸い上げて、余分な食べ残しの餌を取り除きます。
  4. カニ飼育海水の10%(20ppt)を毎日交換します。

3.性的成熟のための誘発脱皮

  1. 滅菌ハサミを使用して水泳脚を除くすべての脚を切ります。
    1. スクープネットでカニを捕まえ、カニを慎重に持ちます。最初に両方の頬骨を切り、次にハサミを使用して第2関節の歩行脚を切ります。カニは損傷した付属物を自動的に自動トマイズします。四肢の解剖術には麻酔は必要ありません。
  2. 四肢の解剖直後に淡水でカニを洗います。
  3. 手足の自動トーミズされたカニを個別に穴あきプラスチックバスケット(長さ28 cm x 幅22 cm x 高さ7 cm )に移し、グラスファイバータンク(長さ305 cm x 幅120 cm x 高さ60 cm )に入れます。
    注意: 2つのバスケットを結んでクリップで留めることができます。トップバスケットは、カニがバスケットから逃げないようにカバーとして使用されます。
  4. 塩分濃度20ppt、水深10cm以上の再循環養殖システム(RAS)を使用して、プラスチックバスケット全体が水没するようにします。
  5. 四肢オートトミズされたメスのカニに、カニの体重の5%〜7%で1日2回、刻んだ海産魚を与え続けます。
  6. 脱皮によって性的に成熟するまで(35日)カニを育てます。
    注:誘発された脱皮は、野生の成熟した雌のマッドクラブによる商業的な卵巣の成熟と種子の生産のためにスキップすることができます。野生から収穫された成熟した雌は順応し、直接冷震麻酔とそれに続く眼球切除を受けなければなりません。

4.麻酔

  1. CW >86 mmの暗い楕円形の腹部皮弁を持つ性的に成熟した女性を選択します(図1)。
  2. スクープネットでカニを捕まえ、麻酔のために小さな水槽に個別に保管してください。
  3. 5分間の順応期間の後、2-フェノキシエタノール(2-PE)を2 mL / Lで各水槽に加え、15分間の麻酔治療を行います。
  4. 自発的な動きの欠如によってカニが完全に麻酔されていることを確認してください。

5.アイトークアブレーション

  1. 焼灼技術
    1. すべての手順は、テーブルの上と空きスペースで実行します。
    2. 木製またはプラスチック製のハンドルが付いた平らな頭のニッケル鋼の金属棒(ドライバーなど)を取り、濡れた綿タオルでハンドルを覆います。
    3. オートクレーブで2つのステンレス製外科用鉗子を滅菌します。
    4. スプレーボトルに70%エタノールを準備し、ブロートーチや赤いホットドライバーなどの火災関連の発生源から遠ざけてください。ティッシュペーパーを使用する準備をしてください。
      注:エタノールは非常に可燃性です。火源から安全な距離を保ってください。
    5. ブロートーチをガスボンベ(ブタン)にしっかりと接続します。
      注意: ブロートーチとガスボンベの指示に従ってください。ガスボンベに接続するときは、ブロートーチのスイッチがオフになっていることを確認してください。ガスボンベに記載されているすべての火災安全上の注意を読み、それに従ってください。
    6. 高温物による怪我を防ぐために、厚い綿の手袋を着用してください。
    7. 金属棒が真っ赤になるまで、金属棒の先端をトーチの火にさらします。
    8. 麻酔をかけたカニを濡れた綿タオルで覆います。
      注意: 不必要な損傷を避けるために、カニのアンテナを覆います。
    9. 滅菌した鉗子でカニの片目を持ちます。
      注:初めて使用する場合はオートクレーブで鉗子を滅菌し、その後他のカニに使用する場合は70%エタノールを使用して消毒します。
    10. 赤熱した金属の平らな先端をカニの目に持ち、目茎がオレンジ色または赤みがかったオレンジ色に変わるまで約10〜15秒間わずかに押します。隣接する構造物への損傷を避けるために、この手順を実行するときは注意してください。
      注:焼灼法に従って眼茎アブレーションを実行するには、カニを保持する人とアブレーション手順を実行する人の2人が必要です。
    11. カニ間の相互汚染がないことを確認するために、70%エタノールスプレーで鉗子を消毒します。
      注意: このステップは、眼球アブレーション手順の後、少なくとも5分間待ってから、潜在的な火災の危険を防ぐために70%エタノールを使用して消毒する前に鉗子が冷却されていることを確認するために実行してください。
    12. すべてのカニにアイトークアブレーションを行った後、熱いニッケル鋼の金属棒(ドライバー)を水道水に浸します。
    13. 再利用する前にタオルを消毒してください。時間を節約するために複数のタオルを使用できます。
      注意: タオルを水道水で洗い、30ppmの塩素水に5分間浸します。その後、タオルを再度水道水で洗い、1 g / Lのチオ硫酸ナトリウム溶液に浸します。
    14. 電源を切った後、トーチを安全な場所に保管し、環境温度(約30分)に戻るまで待ってから切断してください。
  2. 手術技術
    1. 換気の良い場所で手順を実行してください。
    2. オートクレーブで2つの外科用ハサミと鉗子を滅菌します。
    3. 50 mLの70%エタノールを100 mLのガラスビーカーに注ぎます。
    4. 厚い綿の手袋を着用してください。
    5. 麻酔をかけたカニを持ち、濡れた綿タオルで覆います。
    6. 滅菌した鉗子でカニの片目を持ちます。
    7. 滅菌した手術用ハサミを使用して眼球をすばやく切断します。
      注:カニの負傷部分から血リンパが失われる可能性があります。
    8. 使用するたびにハサミと鉗子を70%エタノールに浸し、ティッシュペーパーを使用して乾燥させてから再利用してください。

6.麻酔後のケア

  1. 20pptのろ過海水を準備し、連続通気のあるオーバーヘッドタンクに保管します。
  2. 重力水流のためにオーバーヘッドタンクにフレキシブルパイプを接続します。
  3. アイトークアブレーションの直後にカニをバスケットに入れ、頭上のタンクから流れる海水(周囲水温:28°C)にカニをさらします。
  4. 海水の流れを保ち、麻酔からの回復を示す自発的に動くことができるまでカニを監視します。
    注:海水は地上タンクで準備でき、水中ポンプを使用して水流を行うことができます。
  5. カニを個別に20pptの海水に入れ、水槽で30分間通気して観察します。
    注:回収されたカニは、その後の家畜養殖プロセスで個別に培養されます。

7.卵巣成熟の観察

  1. 家畜飼育
    1. 成熟したカニを個々の32 L円形タンクに移します。
    2. 刻んだ海産魚(-20°Cで冷凍)を1日2回(朝09:00 amと夕方20:00 pm)給餌を続け、朝の給餌の前に食べ残しを取り除きます。
    3. 20pptの塩分で30日間、個別に飼育します。
    4. 糞を取り除き、毎日海水の10%(20 ppt)を交換します。
  2. 解剖
    1. 解剖トレイ、はさみ、鉗子を70%エタノールで清掃します。
    2. 2-PE浸漬麻酔法で女性を個別に麻酔します。
    3. 性腺の段階を確認するために、眼球アブレーションを経ていない新しく成熟した女性(未熟な女性の脱皮後)をランダムに選択します。
    4. すべての眼茎切除実験雌を個別に犠牲にし、生殖腺の成熟段階を特定します。鋭い滅菌千枚通しを使用してカニの胸部神経節を破壊します。最初に上部の甲羅を取り除き、次に肝膵臓を取り除き、卵巣を見えるようにします。卵巣の色を観察し、卵巣の成熟段階を特定します(図2)。
  3. 卵巣成熟段階の識別
    1. 肉眼または実体顕微鏡で卵巣の色を観察します。
    2. 着色30に基づいて卵巣の成熟段階を特定します:未熟(ステージ1)は半透明またはクリーミーな白色を示します。早熟(ステージ2)は淡い黄色がかった色を示します。(iii)前成熟(ステージ3)は黄色から淡橙色を示す。(iv)完全に成熟した(ステージ4)は、濃いオレンジ色から赤みがかった色を示します。

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

生殖腺の成熟
クリーム状の白い卵巣組織(未熟卵巣、ステージ1)は、眼茎アブレーションを行う前に、解剖された女性(n = 6)の100%に見られました(図2)。眼茎切除雌ガニ(n=63、焼灼術31頭、手術法32頭)の生殖巣成熟率は,眼茎切除を行わなかった雌ガニ(n=31)と比較して30日飼育後に高かった(図3)。成熟前の卵巣の割合が最も高かった(ステージ3)は、眼茎切除された雌のカニで見られ(図3;焼灼と手術の両方の技術)、一元配置分散分析(ANOVA)では、実験的な雌のカニの卵巣成熟段階間で有意差(p < 0.05)が示されました(表1)。対照群は、焼灼および手術治療群と比較して、未熟な雌のカニの有病率が高かった(Tukey's HSDテスト、p < 0.001)。焼灼と手術の治療は、すべての成熟段階で雌のカニの割合に関して有意差を示さなかった(TukeyのHSDテスト、すべてp > 0.1)。焼灼(テューキーのHSD試験、p = 0.004)と手術(テューキーのHSD試験、p = 0.006)の両方の処理は、対照処理よりも成熟前のステージ3の雌のカニの割合が有意に高く、焼灼と手術の治療のみが、治療後30日以内に未熟な段階からステージ4の雌のカニを生産することができました(表2)。

Figure 2
図2:メスのマッドクラブの4つの卵巣成熟段階。 ステージ間の卵巣の色と体積の違いは、黒い矢印ではっきりと指摘されています。 この図の拡大版を表示するには、ここをクリックしてください。

Figure 3
図3:30日間の飼育期間(n=94)後の眼球アブレーション(手術および焼灼)および対照を受けた雌カニの卵巣成熟段階。エラーバーは標準偏差を表します。上付き文字は、p < 0.05の各成熟段階での処理間の有意差を示しています。この図の拡大版を表示するには、ここをクリックしてください。

成熟段階 平方和 ティッカー 平均二乗 F P
未熟(ステージ1) グループ間 3755.556 2 1877.778 169 <0.001
グループ内 66.667 6 11.111
トータル 3822.222 8
早熟(ステージ2) グループ間 1355.556 2 677.778 8.714 0.017
グループ内 466.667 6 77.778
トータル 1822.222 8
熟成前(ステージ3) グループ間 4688.889 2 2344.444 17.58 0.003
グループ内 800 6 133.333
トータル 5488.889 8
完全に成熟(ステージ4) グループ間 822.222 2 411.111 9.25 0.015
グループ内 266.667 6 44.444
トータル 1088.889 8
注: 平均差はp = 0.05水準で有意です。

表1:一元配置分散分析試験後の眼球切除(焼灼および手術)と対照雌性腺の性腺成熟段階の比較。 平均差は p = 0.05で有意であった。

成熟段階 処遇 比較治療 P
未熟(ステージ1) 焼灼 手術 1
焼灼 コントロール <0.001
手術 コントロール <0.001
早熟(ステージ2) 焼灼 手術 0.129
焼灼 コントロール 0.014
手術 コントロール 0.232
熟成前(ステージ3) 焼灼 手術 0.934
焼灼 コントロール 0.004
手術 コントロール 0.006
完全に成熟(ステージ4) 焼灼 手術 0.109
焼灼 コントロール 0.012
手術 コントロール 0.237
注: 平均差はp = 0.05水準で有意です。

表2:眼球切除(焼灼および外科)と対照雌ガニの性腺成熟段階の違いに関する事後テューキーのHSDテスト。 平均差は p = 0.05で有意であった。

生存率
眼茎切除雌ガニの平均生存率は、30日間の飼育期間で95.45%±4.98%(平均±標準偏差)であった。眼球アブレーションおよび取り扱い後の最初の7日以内に死亡は発生しなかった。眼球アブレーション後の30日間の飼育期間中、死亡率は治療間で有意差はなかった(クラスカル-ウォリス検定、 p = 0.67)。四肢自動トミートマイズガニの脱皮成功率は80%±2.86%(n=115)であった。

Subscription Required. Please recommend JoVE to your librarian.

Discussion

本プロトコールはマッドクラブ Scylla 属の眼茎切除術のために開発されたもので、生殖腺の成熟を誘導する効率的な方法として応用できる。このプロトコルは、マッドクラブの商業的卵巣成熟のために容易に複製することができ、マッドクラブ種子生産における潜伏期間(ある産卵から別の産卵までの時間)を短縮するために実施することができる。

甲殻類(すなわち、淡水エビ、海産エビ)の眼茎切除は、通常、生殖腺の成熟と季節外れの産卵を誘発するために行われます11,12,13。ブラチュランクラブのアイトークアブレーションは、脱皮25,32,33、ホルモン調節18、生殖腺成熟34、および誘導された繁殖および繁殖能力の研究にも行われています35,36,37,38,39.2-フェノキシエタノールへの浸漬による麻酔は、節足動物におけるメタンスルホン酸トリカイン(MS-222)の使用に匹敵するが、より安価であり、追加の緩衝液の使用を必要としないため、使用した40。片側または両側の眼茎切除は甲殻類の生理機能に影響を与えます。この研究で述べられているプロトコルに従った眼球アブレーションは、マッドクラブの卵巣成熟率にも影響を及ぼします。対照治療(眼球アブレーションなし)では、雌のカニの43.33%±5.77%が未熟な卵巣を持っていました(ステージ1)。しかし、同じ飼育期間(30日)に、眼茎切除された雌ガニは成熟前の卵巣を有し(ステージ3;焼灼術および手術技術でそれぞれ56.67%±11.55%および53.33%±15.28%)、眼茎切除がマッドクラブの生殖腺成熟を増加させる可能性があることを示しています。以前の研究では、無傷のカニ(眼球切除なし)の卵巣発達は、眼茎切除されたカニの卵巣発達よりも遅いことも報告されています25,31。無傷の甲殻類では性腺の発達が遅いため、アイトークアブレーションは市販のエビやエビの孵化場で広く行われています。このプロトコルでは、眼球切除を受けた雌ガニは、眼球切除治療を受けていない雌ガニと比較して、より高い卵巣成熟率を達成しました(図3)。

マッドクラブの生殖腺の成熟はホルモン21,41,42によって調節されています。アイトークには、マッドクラブの性腺成熟プロセスで重要な役割を果たす重要な内分泌腺(すなわち、X-organ-副鼻腔腺複合体)が含まれています18,21。焼灼または手術による片側の眼球切除は、阻害ホルモン(VIHなど)の合成と放出に関与する主要な内分泌腺の1つを損傷し、それによって生殖腺刺激ホルモン(すなわちVSH)のレベルが高くなります。

Scylla属の卵巣成熟段階は、卵巣組織の着色を肉眼で観察することで区別できます29,30,43。半透明またはクリーミーな白い卵巣組織は、未熟卵巣29,30,43,44の兆候です。この研究では、卵巣の成熟プロセスが遅いため、眼茎アブレーションのない雌のカニのグループで未熟卵巣(ステージ1)がまだ見つかりました。しかし、眼球切除群のカニ(焼灼と手術の両方による)は、ほとんどが成熟前の卵巣を示し(ステージ3)、一部の個体は完全に成熟した卵巣を示しました(ステージ4)。したがって、ここで説明するアイトークアブレーションのプロトコルは、メスのマッドクラブの卵巣成熟を促進するために使用できます。このプロトコルは、野生で収集された成熟したメスのマッドクラブに直接適用して、種子の生産を早めることもできます。マッドクラブの生殖腺成熟に対する焼灼と手術法の有効性を評価し、脱皮期間の正確な推定を確実にするために、性的に早すぎるカニを使用しました。性的に早産の雌のカニの(誘発された)脱皮後、それらの卵巣はまだ未熟または初期の発達段階にあることに気づきました29,45。新たに成熟した雌のカニ(眼球切除または眼葉切除なし)を30日間飼育した後、卵巣組織の色によって卵巣の発達段階(ステージ1からステージ4)を決定しました。このプロトコルは、血リンパの喪失を回避し、切除部位での感染を防ぐために、マッドクラブでアイトークアブレーションを実行するための焼灼技術の使用を奨励しています。焼灼はすぐに傷を密封しますが、手術技術は傷が治癒するのに時間がかかり、これは感染の可能性を可能にします。商業目的のために、より大きな成熟したカニは、できれば卵巣成熟の後期段階で、眼球アブレーションのために選択され、その後の商業またはひなのストック培養のために完全に成熟した卵巣段階に到達するまでの時間を短縮する必要があります。眼茎アブレーションに加えて、砂基質による個体飼育と十分な給餌、できれば生きた飼料による飼育は、飼育下のマッドクラブの生殖腺成熟率を高めることができます30,35,46,47。

甲殻類の血液は血リンパと呼ばれ、眼球アブレーション中に失われる可能性があります。血リンパの過剰な喪失は、特に眼球を除去する手術を行う場合、眼茎切除カニの死につながる可能性があります。血リンパは、喪失を防ぐために負傷した部分で凝固することができます。しかし、手術技術と比較して、焼灼技術は負傷した部分を即座に密封し、それによって血リンパの喪失および起こりうる感染を防ぐ。

焼灼または手術のいずれかによる片側の眼球アブレーション後のマッドクラブの死亡率は、最初の7日以内には見つかりませんでした。したがって、眼球アブレーションはより高い生存率で行うことができます。片側の眼茎切除は、カニ33の生存率を妨げない。

カニの取り扱いやアイトークアブレーション中のストレスは、カニの死亡率に寄与する可能性があります。眼球アブレーション中の取り扱いストレスを最小限に抑えるには、適切な麻酔が必要です。甲殻類の眼茎アブレーションでは、化学麻酔薬(すなわち、キシロカイン、リドカイン)が眼茎アブレーションの前に眼葉の基部で使用されます14,15,17,48。しかし、マッドクラブは攻撃的な性質とサイズが大きいため、眼茎の付け根でのみ麻酔を使用するだけでは不十分であり、注射中に動物に追加のストレスを与える可能性があります。一方、それらをより低い水温にさらすことによる麻酔はより経済的で安全です。マッドクラブの麻酔に冷水を使用することは一般的であり、その効率、単純さ、および回復と生存への影響が最小限であるため、他の研究で使用されています37,49,50。さらに、淡水エビMacrobrachium americanum51で明らかなように、泥ガニの眼茎アブレーション後の痛みの評価に関する将来の研究が推奨されています。

焼灼法と手術法の両方を使用した眼球アブレーションは、カニの生存への影響を最小限に抑え、卵巣の成熟を促進しますが、眼球アブレーションを実行するには、技術の専門的な習得が必要です。プロトコル間の遅延はカニに追加のストレスを追加するため、ステップ間のタイミングは重要です。手術技術とは異なり、焼灼技術は可燃性機器(すなわち、ブロートーチおよびブタンガス)の使用を伴うため危険である。したがって、焼灼技術を実行する際には特別な注意が必要です。

カニは本質的に共食いであり、脱皮を終えたばかりでまだソフトシェルの状態にある他のカニを捕食することが知られています7,52,53。したがって、カニを個別に飼育することで、共食いによる不必要な死亡を避けることができます。マッドクラブ養殖における個体飼育の使用は、肥育およびソフトシェルクラブ養殖の目的で、高密度培養と池養殖の両方で一般的に行われています8,53。このプロトコルは、個別の飼育と維持も利用しました。飼育や商業のためのカニの輸送中、カニの頬は、戦闘、不必要な怪我、および手足の喪失を防ぐためにしっかりと縛られます(または自動調整さえされます)34。

眼球アブレーションのための記述されたプロトコルは、複数の人で実行されるべきです。アイトークアブレーションが完了したら、使い捨てではない機器(水槽、トレイ、タオルなど)を30ppmの塩素で消毒する必要があります。カニは少なくとも1日に2回監視する必要があります。死んだカニ、食べられない飼料、切除された手足、または脱皮したカニの殻は、病気の蔓延の可能性を防ぐために、迅速に処分する必要があります(つまり、漂白剤を含む土壌に埋める必要があります)。

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

どの著者も利益相反を持っていません。

Acknowledgments

この研究は、マレーシア教育省の支援を受け、マレーシアの高等教育センターオブエクセレンス(HICoE)プログラムの下で、マレーシアトレンガヌ大学熱帯水産養殖水産研究所に認定されました(Vot No. 63933およびVot No. 56048)。我々は、マレーシア・トレンガヌ大学及びサヤップ・ジャヤSdn. Bhd.のプライベート・パートナーシップ研究助成金(Vot. No. 55377) を通じた 支援に感謝する。マレーシア科学大学からコール・ワイホ、ハナフィア・ファザンの非常勤アカデミックフェローの地位も認められています。

Materials

Name Company Catalog Number Comments
Aeration tube  Ming Yu Three N/A aquarium and pet shop
Airstone Ming Yu Three N/A aquarium and pet shop
Autoclave machine HIRAYAMA MANUFACTURING CORPORATION N/A MADE IN JAPAN
Bleaching powder (Hi-Chlon 70%) Nippon Soda Co.Ltd,Japan N/A N/A
Blow torch  MR D.I.Y. Group Berhad N/A N/A
Circular tank (32L) BEST PLASTIC INDUSTRY SDN. BHD.  N/A N/A
Cotton hand gloves (thick)  MR D.I.Y. Group Berhad N/A N/A
Cotton towel MR D.I.Y. Group Berhad N/A N/A
Digital thermometer Hanna Instrument HI9814 Hanna Instruments GroLine Hydroponics Waterproof pH / EC / TDS / Temp. Portable Meter HI9814
Digital Vernier Caliper INSIZE Co., Ltd. N/A
Dissecting tray Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Dropper bottle/Plastic Pipettes Dropper Shopee Malaysia N/A N/A
Ethanol 70% Thermo Scientific Chemicals 033361.M1 Diluted to 70% using double distilled water
Fiberglass tank (1 ton) Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Fine sand N/A N/A collected from Sea beach of Universiti Malaysia Terengganu
First Aid Kits Watsons Malaysia N/A N/A
Flat head nickel steel metal rod (Screw driver) MR D.I.Y. Group Berhad N/A N/A
Formaldehyde Thermo Scientific Chemicals 119690010
Gas cylinder (butane gas) for blow  torch MR D.I.Y. Group Berhad N/A N/A
Gas lighter gun (long head) MR D.I.Y. Group Berhad N/A N/A
Glass beaker (100 mL)) Corning Life Sciences 1000-100
Ice bag  Watsons Malaysia N/A N/A
Perforated plastic baskets  Eco-Shop Marketing Sdn. Bhd. N/A N/A
PVC pipe 15mm Bina Plastic Industries Sdn Bhd (HQ) N/A N/A
Refractometer ATAGO CO.,LTD.
Refrigerator Sharp Corporation Japan N/A Chest Freezer SHARP 110L - SJC 118
Scoop net MR D.I.Y. Group Berhad N/A
Seawater Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Siphoning pipe MR D.I.Y. Group Berhad N/A N/A
Spray bottle Mr. DIY Sdn Bhd N/A N/A
Stainless surgical forceps  N/A N/A N/A
Stainless surgical scissors  N/A N/A N/A
Submersible water pump  AS N/A model: Astro 4000
Tincture of iodine solution  (Povidone Iodine) Farmasi Fajr Sdn Bhd N/A N/A
Tissue paper  N/A N/A
Transparent plastic aquarium Ming Yu Three N/A aquarium and pet shop
Waterproof table Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu

DOWNLOAD MATERIALS LIST

References

  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).
  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).

Tags

今月のJoVE、第193号、ス キュラオリバセア、冷ショック麻酔、焼灼、性腺発達、誘発繁殖

Erratum

Formal Correction: Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs
Posted by JoVE Editors on 05/26/2023. 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).
  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. 140 (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).
マッドクラブの卵巣成熟を促進するためのアイトークアブレーション
Play Video
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Asmat-Ullah, M., Rozaimi, R.,… More

Asmat-Ullah, M., Rozaimi, R., Fazhan, H., Shu-Chien, A. C., Wang, Y., Waiho, K. Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs. J. Vis. Exp. (193), e65039, doi:10.3791/65039 (2023).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter