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Biology

Eyestalk ablation for å øke ovariemodning i mudderkrabber

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

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Summary

To øyestengelablasjonsprotokoller (dvs. cauterization og kirurgiske tilnærminger) ble utført på bedøvede hunnkrabber. Øyestengelablasjonen av mudderkrabber fremskyndet modningen av eggstokkene uten å redusere overlevelsesraten.

Abstract

Mudderkrabber (Scylla spp.) er kommersielt viktige krepsdyrarter som finnes i hele Indo-Vest-Stillehavsregionen. Under kultur er induksjon av eggstokkmodning viktig for å møte forbrukernes etterspørsel etter modne mudderkrabber og fremskynde frøproduksjonen. Eyestalk ablation er et effektivt verktøy for å forbedre ovariemodning i mudderkrabber. Det er imidlertid ingen standardprotokoll for øyestengelablasjon av mudderkrabber. I denne studien beskrives to øyestengelablasjonsteknikker: cauterization (bruk av varmt metall for å ablate øyestengelen til en bedøvet krabbe) og kirurgi (fjerning av øyestengelen ved hjelp av kirurgisk saks). Før øyestengelablasjon ble kjønnsmodne hunner (CW > 86 mm) bedøvet med ispose (−20 °C) med sjøvann. Når vanntemperaturen nådde 4 °C, ble isposen fjernet fra vannet. Rennende sjøvann (omgivelsestemperatur: 28 °C) ble brukt til gjenvinning fra anestesien umiddelbart etter øyestengelablasjon. Mortaliteten inntraff ikke under eller etter prosessen med øyestengelablasjon. Eyestalk-ablasjonsprotokollen som presenteres her, akselererte eggstokkmodningen til mudderkrabbene.

Introduction

Alle de fire mudderkrabbeartene i slekten Scylla er kommersielt viktige krepsdyrarter i akvakultur 1,2. Veksten av krepsdyr, inkludert mudderkrabber, og deres transformasjon fra pre-moden (sub-voksen eller pubertal) fase til seksuelt moden (voksen) fase skjer gjennom en smeltingsprosess som involverer periodisk shedding av eldre og mindre eksoskeletoner. Carapacebredde (CW), chelipeds og abdominal klaffmorfologier er mye brukt til å bestemme seksuell modenhet av Scylla spp. 3,4,5. Prosessen med smelting reguleres av virkningen av ulike hormoner og krever en stor mengde energi6. I tillegg til den normale smelteprosessen, fremskynder tap av lemmer, enten frivillig eller indusert av eksterne faktorer, smeltingen av krabber uten å påvirke overlevelsesraten 7,8,9. Derfor er lem autotomi ofte brukt for molt induksjon i soft-shell mud krabbe oppdrett industri 7,9.

Unilateral eller bilateral eyetalk ablation er mest populær i ferskvannsreke og marine reker for gonademodning og frøproduksjon10,11,12,13. Vanlige eyestalk-ablasjonsteknikker hos krepsdyr inkluderer følgende: (i) ligering ved foten av øyestengelen ved hjelp av en streng14,15; (ii) cauterization av eyestalk ved hjelp av varm tang eller elektrocautery enheter16; (iii) fjerning eller direkte klemming av øyestengelen for å etterlate et åpent sår12; og (iv) fjerning av innholdet i øyestengelen gjennom snitt etter å ha skåret den distale delen av øyet med en barberhøvel17. Eyestalk X-organene er viktige endokrine organer i krepsdyr da de regulerer krepsdyrhyperglykemiske hormoner (CHH), molthemmende hormoner (MIH) og vitellogenesehemmende hormoner (VIH)6,18,19,20,21,22. Eyestalk X-organer (eller sinuskjertelkomplekset) syntetiserer og frigjør gonadehemmende hormoner (GIH), også kjent som vitellogenesehemmende hormoner (VIH), som tilhører nevropeptidhormonfamilien6. Unilateral eller bilateral øyestengalablasjon reduserer GIH-syntese, noe som resulterer i dominans av stimulerende hormoner (dvs. gonadestimulerende hormoner, GSH) og akselerasjonen av ovariemodningsprosessen hos krepsdyr23,24,25,26. Uten påvirkning av GIH etter øyestengelablasjon, bruker kvinnelige krepsdyr sin energi til eggstokkutvikling27. Det er funnet at ensidig øyestengelablasjon er tilstrekkelig for induksjon av ovariemodning hos krepsdyr11 og at ablated eyetalk av reker og krabber kan regenerere etter flere moltings28. Det er fire ovarieutviklingsstadier registrert i Scylla spp.: i) umoden (stadium-1), ii) tidlig modning (stadium-2), iii) pre-modning (stadium-3) og iv) fullt moden (stadium-4) 29,30. Den umodne eggstokkstadiet finnes hos umodne kvinner. Etter pubertetsmelting og parring begynner den umodne eggstokken å utvikle seg og til slutt modnes (stadium 4) før gyting31.

En øyestengelablasjonsprotokoll er essensiell for utvikling av stamfisk av mudderkrabbe og frøproduksjon. I det globale matmarkedet foretrekkes modne gjørmekrabber med fullt modne eggstokker (stadium-4) i stedet for krabber med høyere muskelinnhold av forbrukerne og har dermed en høyere kommersiell verdi, enda høyere enn store hanner. Det finnes ingen komplett protokoll for øyestengelablasjon av mudderkrabber. Eyestalk-ablasjonsprotokollen i dette arbeidet minimerer stress ved å bruke fullt bedøvede krabber og minimerer fysisk skade på personell fra krabbebitt. Denne protokollen er enkel og kostnadseffektiv. Her presenterer vi en protokoll for øyestengelablasjon av Scylla spp. som kan indusere modningen av gonaden. To teknikker for øyestengelablasjon (cauterization og kirurgi) ble testet og deres effektivitet sammenlignet basert på den gonadale utviklingshastigheten til kvinnelige mudderkrabber.

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Protocol

Denne protokollen følger den malaysiske anbefalingen for omsorg og bruk av dyr til vitenskapelige formål som er skissert av Laboratory Animal Science Association of Malaysia. Ofringen av de eksperimentelle prøvene ble gjort i henhold til National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Publications No. 8023, revidert 1978). Seksuelt pre-modne kvinnelige mudderkrabber (oransje mudderkrabbe S. olivacea) ble samlet inn fra det lokale markedet (5 ° 66′62 ′ ′ N, 102 ° 72′ 33 ′ Ø) på Setiu våtmarker i Malaysia. Mudderkrabbearten ble identifisert basert på morfologiske egenskaper1.

1. Prøveinnsamling og desinfeksjon

  1. Samle sunne, aktive og pre-modne kvinnelige mudderkrabber (figur 1).
    MERK: Førmodne hunnkrabber har trekantede og lyse bukklaffer sammen med et CW-område på 80-85 mm.
  2. Vask krabber med klorert vann fra springen (ferskvann) for å fjerne rusk og osmofile parasitter.
  3. Bløtlegg krabbene i 150 ppm formaldehyd med 20 ppt saltholdighet i 30 min.
  4. Oppretthold kontinuerlig og skånsom lufting med luftstein under formaldehydbehandlingen. Luftingskilden kan være fra enten en sentral luftingslinje eller en akvariumluftpumpe.
  5. Vask krabbene med rennende sjøvann for å fjerne eventuelle gjenværende formaldehyd.

Figure 1
Figur 1: Abdominal morfologi hos hunnlige mudderkrabber brukt for å identifisere kjønnsmodningsstadiene. Klikk her for å se en større versjon av denne figuren.

2. Akklimatisering

  1. Overfør hver desinfiserte kvinne til en separat 32 L sirkulær tank.
  2. Hunnene oppfostres i 3 dager i 20 ppt saltholdighet og fortsett å mate to ganger om dagen (morgen 09:00 og kveld 20:00) med hakket marin fisk på ca 4% -5% av krabbens kroppsvekt.
  3. Fjern overflødig og uoppladet fôr ved siphoning før morgenfôring.
  4. Utveksle 10% av krabben oppdrett sjøvann (20 ppt) daglig.

3. Indusert smelting for seksuell modenhet

  1. Klipp alle bena unntatt svømmebena ved hjelp av sterilisert saks.
    1. Fang krabben med et øsenett, og hold krabben forsiktig. Klipp begge chelipeds først og deretter gangbena på det andre leddet ved hjelp av saks. Krabben vil automatisk tømme de skadede vedleggene. Anestesi er ikke nødvendig ved autotomi i ekstremiteter.
  2. Vask krabben i ferskvann umiddelbart etter lem autotomi.
  3. Overfør de lem-autotomiserte krabbene individuelt i perforerte plastkurver (28 cm L x 22 cm B x 7 cm H), og legg dem i en glassfibertank (305 cm L x 120 cm B x 60 cm H).
    MERK: To kurver kan bindes og klippes sammen. Den øverste kurven brukes som et deksel slik at krabben ikke kan rømme fra kurven.
  4. Bruk et resirkulerende akvakultursystem (RAS) med 20 ppt saltholdighet og en vanndybde på minst 10 cm for å sikre at hele plastkurven er nedsenket.
  5. Fortsett å mate den lem-autotomiserte kvinnelige krabben med hakket marin fisk to ganger per dag ved 5% -7% av krabbens kroppsvekt.
  6. Bak krabbene til de er kjønnsmodne gjennom smelting (35 dager).
    MERK: Indusert smelting kan hoppes over for kommersiell ovariemodning og frøproduksjon med vilt modne kvinnelige mudderkrabber. Høstede modne kvinner fra naturen må akklimatiseres og direkte utsatt for kaldsjokkbedøvelse og påfølgende øyestengelablasjon.

4. Anestesi

  1. Velg kjønnsmodne hunner med en mørkfarget ovalformet bukklaff med CW >86 mm (figur 1).
  2. Fang krabber med et scoop nett, og hold dem individuelt i små akvarier for anestesi.
  3. Etter 5 min akklimatiseringsperiode, tilsett 2-fenoksyetanol (2-PE) ved 2 ml / l i hvert akvarium og la 15 minutters anestesibehandling.
  4. Sørg for at krabber er fullt bedøvet av mangel på spontan bevegelse.

5. Eyestalk ablasjon

  1. Cauterization teknikk
    1. Utfør alle prosedyrer på toppen av et bord og i et åpent område.
    2. Ta en nikkel-stålstang med flatt hode (f.eks. en skrutrekker) med et tre- eller plasthåndtak, og dekk håndtaket med et vått bomullshåndkle.
    3. Steriliser to rustfrie kirurgiske tang i en autoklav.
    4. Forbered 70% etanol i en sprayflaske og hold den borte fra brannrelaterte kilder, for eksempel blåsebrenner og rødglødende skrutrekker. Ha silkepapir klar til bruk.
      MERK: Etanol er svært brannfarlig. Hold sikker avstand fra brannkilder.
    5. Koble en blowtorch til en gassflaske (butan) sikkert.
      FORSIKTIG: Følg instruksjonene på blowtorch og gassflaske. Forsikre deg om at blowtorch er slått av når du kobler til gassflasken. Les og følg alle brannsikkerhetsforanstaltninger som er nevnt på gassflasken.
    6. Bruk tykke bomullshansker for å unngå skade fra varme gjenstander.
    7. Utsett spissen av metallstangen for blåselampens brann til metallstangen er lys rød.
    8. Dekk den bedøvede krabben med et vått bomullshåndkle.
      MERK: Dekk antennene på krabben for å unngå unødvendig skade.
    9. Hold det ene øyet på krabben med sterilisert tang.
      MERK: Steriliser tangen i en autoklav for første gangs bruk, og desinfiser med 70% etanol for senere bruk på andre krabber.
    10. Hold den rødglødende metallspissen på krabbens øye og trykk litt i ca. 10-15 s til øyestengelen får en oransje eller rødoransje farge. Vær forsiktig når du utfører dette trinnet for å unngå skade på tilstøtende strukturer.
      MERK: To personer er nødvendig for å utføre øyestengelablasjon etter cauterization-metoden: en for å holde krabben og en annen for å utføre ablasjonsprosedyren.
    11. Desinfiser tangen med 70% etanolspray for å sikre at krysskontaminering mellom krabber.
      MERK: Utfør bare dette trinnet minst 5 minutter etter øyestengelablasjonsprosedyren for å sikre at tangen avkjøles før desinfeksjon ved hjelp av 70% etanol for å forhindre potensielle brannfarer.
    12. Etter å ha utført øyestengelablasjonen på alle krabber, dypp den varme nikkelstålmetallstangen (skrutrekkeren) i vann fra springen.
    13. Desinfiser håndkleet før gjenbruk. Flere håndklær kan brukes for å spare tid.
      NOTAT: Vask håndkleet med vann fra springen, og dypp det i 30 ppm klorert vann i 5 minutter. Vask deretter håndkleet med vann fra springen igjen, og dypp det i en 1 g/l natriumtiosulfatløsning.
    14. Oppbevar blåselampen på et trygt sted etter at du har slått den av, og vent til den vender tilbake til omgivelsestemperatur (ca. 30 min) før du kobler fra.
  2. Kirurgi teknikk
    1. Utfør prosedyren i et godt ventilert område.
    2. Steriliser to kirurgiske saks og tang i en autoklav.
    3. Hell 50 ml 70% etanol i et 100 ml glassbeger.
    4. Bruk tykke bomullshansker.
    5. Hold den bedøvede krabben, og dekk den med et vått bomullshåndkle.
    6. Hold det ene øyet på krabben med sterilisert tang.
    7. Klipp raskt av øyestengelen ved hjelp av sterilisert kirurgisk saks.
      MERK: Hemolymfe kan gå tapt fra den sårede delen av krabben.
    8. Dypp saks og tang i 70% etanol etter hver bruk, og tørk dem med silkepapir før gjenbruk.

6. Post-anestesi omsorg

  1. Forbered 20 ppt filtrert sjøvann, og hold i en overliggende tank med kontinuerlig lufting.
  2. Koble et fleksibelt rør til lufttanken for gravitasjonsvannstrøm.
  3. Umiddelbart etter øyestengelablasjon legger du krabben i kurven, og utsetter krabben for rennende sjøvann (omgivelsestemperatur: 28 °C) fra lufttanken.
  4. Hold sjøvannet strømmende, og overvåk krabben til den kan bevege seg spontant, noe som indikerer utvinning fra anestesien.
    MERK: Sjøvann kan tilberedes i en bakketank, og en nedsenkbar vannpumpe kan brukes til vannstrømmen.
  5. Hold krabbene individuelt i 20 ppt sjøvann med lufting i et akvarium i 30 minutter for videre observasjon.
    MERK: De gjenvunnede krabbene vil bli dyrket individuelt i den påfølgende stamfiskkulturprosessen.

7. Observasjon av modning av eggstokkene

  1. Stamfisk oppdrett
    1. Overfør de modne krabbene til individuelle 32 L sirkulære tanker.
    2. Fortsett fôringen med hakket marin fisk (frossen ved −20 °C) to ganger per dag (morgen kl. 09.00 og kveld kl. 20.00), og fjern uspist fôr før morgenfôringen.
    3. Bak stamfisken individuelt i 30 dager i 20 ppt saltholdighet.
    4. Fjern avføring, og bytt 10% av sjøvannet (20 ppt) daglig.
  2. Disseksjon
    1. Rengjør et dissekerbrett, saks og tang med 70% etanol.
    2. Bedøv hunnene individuelt med 2-PE nedsenking anestesimetoden.
    3. Velg tilfeldig nylig modne kvinner (etter smelting av pre-modne kvinner) som ikke har gått gjennom øyestengablasjon for å bekrefte deres gonadale stadier.
    4. Ofre alle øynene, eksperimentelle hunner individuelt, og identifiser gonademodningsstadiene. Ødelegg krabbens thoraxganglia ved hjelp av en skarp steril øl. Fjern den øverste karapellen først og deretter hepatopancreas for å gjøre eggstokken synlig. Observer eggstokkfargen, og identifiser ovariemodningsstadiet (figur 2).
  3. Identifikasjon av modningsstadier i eggstokkene
    1. Vær oppmerksom på eggstokkfargen med det blotte øye eller under et stereomikroskop.
    2. Identifiser modningsstadiene for eggstokkene basert på fargestoffer30: den umodne (stadium-1) viser en gjennomsiktig eller kremaktig hvit farge; den tidlige modningen (stadium 2) viser en blek til lys gulaktig farge; (iii) pre-modning (stadium-3) viser en gul til lys oransje farge; og (iv) den fullt modne (trinn 4) viser en mørk oransje til rødlig farge.

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Representative Results

Gonade modning
Kremhvitt ovarievev (umodne eggstokker, stadium 1) ble funnet hos 100 % av de dissekerte hunnene (n = 6) før øyestengelablasjonen ble utført (figur 2). Modningsraten for gonaden hos øyestengelableblerte hunnkrabber (n = 63; 31 hunner med cauteriseringsteknikk og 32 hunner med operasjonsteknikk) var høyere sammenlignet med hunnkrabber som ikke ble utsatt for øyestengelablasjon (n = 31) etter 30 dagers individuell oppdrett (figur 3). De høyeste andelene av førmodne eggstokker (stadium-3) ble funnet i øyestengelableblerte hunnkrabber (figur 3; både cauterization og surgery techniques), og en enveisvariansanalyse (ANOVA) viste signifikante forskjeller (p < 0,05) mellom de eksperimentelle hunnkrabbenes ovariemodningsstadier (tab 1). Kontrollgruppen hadde høyere forekomst av umodne hunnkrabber sammenlignet med gruppene med cauterization og kirurgisk behandling (Tukeys HSD-test, p < 0,001). Cauterization og kirurgiske behandlinger viste ingen signifikante forskjeller når det gjaldt andel hunnkrabber på tvers av alle modningsstadier (Tukeys HSD-test, alle p > 0,1). Både cauterization (Tukeys HSD-test, p = 0,004) og kirurgi (Tukeys HSD-test, p = 0,006) behandlinger hadde signifikant høyere prosentandeler av pre-modne stadium 3 kvinnelige krabber enn kontrollbehandlingen, og bare cauterization og kirurgiske behandlinger var i stand til å produsere stadium-4 kvinnelige krabber fra et umodent stadium innen 30 dager etter behandling (tabell 2).

Figure 2
Figur 2: Fire ovariemodningsstadier hos hunngjørmekrabber. Forskjeller i fargen og volumet av eggstokkene mellom stadiene er tydelig påpekt av de svarte pilene. Klikk her for å se en større versjon av denne figuren.

Figure 3
Figur 3 Ovariemodningsstadier hos hunnkrabber utsatt for øyestengelablasjon (kirurgi og cauterization) og kontroll etter oppdrettsperioden på 30 dager (n = 94). Feillinjen representerer standardavviket. De hevede bokstavene indikerer signifikante forskjeller mellom behandlingene ved hvert modningsstadium på p < 0,05. Klikk her for å se en større versjon av denne figuren.

Modningsstadium Summen av kvadrater Df Gjennomsnittlig firkant F P
Umoden (stadium-1) Mellom grupper 3755.556 2 1877.778 169 <0.001
Innenfor grupper 66.667 6 11.111
Total 3822.222 8
Tidlig modning (stadium-2) Mellom grupper 1355.556 2 677.778 8.714 0.017
Innenfor grupper 466.667 6 77.778
Total 1822.222 8
Pre-modning (stadium-3) Mellom grupper 4688.889 2 2344.444 17.58 0.003
Innenfor grupper 800 6 133.333
Total 5488.889 8
Fullt modnet (stadium-4) Mellom grupper 822.222 2 411.111 9.25 0.015
Innenfor grupper 266.667 6 44.444
Total 1088.889 8
Merk: Den gjennomsnittlige forskjellen er signifikant på p = 0,05 nivå.

Tabell 1: Sammenligning av øyestengelabler (cauterization og kirurgi) og kontroll av hunnkrabbens gonadale modningsstadier etter enveis ANOVA-test. Gjennomsnittlig forskjell var signifikant ved p = 0,05.

Modningsstadium Behandling Sammenligning behandling P
Umoden (stadium-1) Cauterisering Kirurgi 1
Cauterisering Kontroll <0.001
Kirurgi Kontroll <0.001
Tidlig modning (stadium-2) Cauterisering Kirurgi 0.129
Cauterisering Kontroll 0.014
Kirurgi Kontroll 0.232
Pre-modning (stadium-3) Cauterisering Kirurgi 0.934
Cauterisering Kontroll 0.004
Kirurgi Kontroll 0.006
Fullt modnet (stadium-4) Cauterisering Kirurgi 0.109
Cauterisering Kontroll 0.012
Kirurgi Kontroll 0.237
Merk: Den gjennomsnittlige forskjellen er signifikant på p = 0,05 nivå.

Tabell 2: Post-hoc Tukeys HSD-test av forskjellene mellom øyestengelablede (cauterization og kirurgiske) og kontrollhunnkrabbens gonademodningsstadier. Gjennomsnittlig forskjell var signifikant ved p = 0,05.

Overlevelse
Gjennomsnittlig overlevelse for øynene, ablerte hunnkrabber var 95,45 % ± 4,98 % (gjennomsnittlig ± standardavvik) i oppdrettsperioden på 30 dager. Ingen mortalitet inntraff i løpet av de første 7 dagene etter øyestengelablasjon og håndtering. I løpet av oppvekstperioden på 30 dager etter øyestengelablasjon var mortaliteten ikke signifikant forskjellig (Kruskal-Wallis' test, p = 0,67) mellom behandlingene. Den smeltende suksessraten for de lem-autotomiserte hunnkrabbene var 80 % ± 2,86 % (n = 115).

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Discussion

Denne protokollen ble utviklet for øyestengelablasjon av mudderkrabben, Scylla spp., og kan brukes som en effektiv metode for å indusere gonademodning. Denne protokollen kan enkelt replikeres for kommersiell eggstokkmodning av mudderkrabber og kan implementeres for å redusere den latente perioden (tid fra en gyting til en annen) i mudderkrabbefrøproduksjon.

Øyestengelablasjonen av krepsdyr (dvs. ferskvannsreker, marine reker) gjøres vanligvis for å indusere gonademodning og gyting utenfor sesongen11,12,13. Eyetalk ablasjon i brachyuran krabber har også blitt gjort for å studere smelting 25,32,33, hormonell regulering 18, gonademodning 34, og indusert avl og reproduktiv ytelse 35,36,37,38,39 . Anestesi via nedsenking i 2-fenoksyetanol ble brukt da det er sammenlignbart med bruk av trikain metansulfonat (MS-222) i leddyr, men billigere og krever ikke bruk av ekstra buffer40. Unilateral eller bilateral øyestengelablasjon påvirker krepsdyrets fysiologi. Eyetalk-ablasjon etter protokollen oppgitt i denne studien påvirker også ovariemodningshastigheten til mudderkrabber. I kontrollbehandlingen (uten øyestengelablasjon) hadde 43,33 % ± 5,77 % av hunnkrabbene en umoden eggstokk (stadium 1). I samme oppdrettsperiode (30 dager) hadde imidlertid øyestengelablede hunnkrabber eggstokkene før modningen (stadium 3; 56,67 % ± 11,55 % og 53,33 % ± 15,28 % med henholdsvis cauterization og kirurgiske teknikker), noe som viser at øyestengelablasjon kan øke gonademodningen hos mudderkrabber. Tidligere studier har også rapportert at ovarieutviklingen av intakte krabber (uten eyestalk-ablasjon) er langsommere enn for eyestalk-ablated krabber 25,31. På grunn av den langsommere gonadeutviklingen hos intakte krepsdyr, er øyestengelablasjon mye gjort i kommersielle reke- og rekeklekkerier. I denne protokollen oppnådde øyestengelablede hunnkrabber høyere prosentandel ovariemodning sammenlignet med hunnkrabbene uten øyestengelablasjonsbehandling (figur 3).

Gonademodningen til mudderkrabben reguleres av hormonene21,41,42. Øyestengelen inneholder viktige endokrine kjertler (dvs. X-organ-sinuskjertelkomplekset) som spiller viktige roller i den gonadale modningsprosessen til mudderkrabber 18,21. Ensidig øyestenglablasjon, enten ved cauterization eller kirurgi, skader en av de viktigste endokrine kjertlene som er involvert i syntese og frigjøring av hemmende hormoner (f.eks. VIH), og resulterer dermed i et høyere nivå av gonadestimulerende hormoner (dvs. VSH).

Ovariemodningsstadiene av Scylla spp. kan differensieres ved å observere eggstokkvevsfargen med det blotte øye 29,30,43. Gjennomsiktig eller kremaktig hvitt eggstokkvev er indikasjoner på umodne eggstokkene 29,30,43,44. I denne studien ble umodne eggstokker (stadium-1) fortsatt funnet i gruppen av hunnkrabber uten øyestengelablasjon på grunn av den langsommere modningsprosessen for eggstokkene. Krabbene i øyestengelgruppene (både ved cauterization og kirurgiske teknikker) viste imidlertid for det meste pre-modne eggstokker (stadium-3), med noen individer som viste fullt modne eggstokker (stadium-4). Derfor kan protokollen for øyestengelablasjon beskrevet her brukes til å øke ovariemodningen hos hunngjørmekrabber. Denne protokollen kan også brukes direkte på viltinnsamlede modne kvinnelige mudderkrabber for å fremskynde frøproduksjonen. For å evaluere effektiviteten av cauterization og kirurgiske metoder på mud krabbe gonad modning og for å sikre nøyaktig estimering av smeltevarighet, ble seksuelt pre-modne krabber brukt. Etter (indusert) smelting av seksuelt pre-modne kvinnelige krabber, la vi merke til at eggstokkene deres fortsatt var i de umodne eller tidlig utviklende stadiene29,45. Etter 30 dager med oppdrett av de nylig modne hunnkrabbene (enten øyestengelblated eller uten eyestalk-ablation), ble eggstokkutviklingsstadiene (stadium-1 til stadium-4) bestemt av fargen på eggstokkvevet. Denne protokollen oppfordrer til bruk av cauterization-teknikken for å utføre øyestengelablasjon i mudderkrabber for å unngå hemolymfetap og forhindre infeksjon på de ablerte stedene. Cauterization forsegler umiddelbart såret, mens operasjonsteknikken tar tid for såret å helbrede, og dette vil tillate sjanse for infeksjon. For kommersielle formål bør større modne krabber, fortrinnsvis på et senere stadium av ovariemodning, velges for øyestengelablasjon for å forkorte tiden for å nå det fullt modne eggstokkstadiet for senere handel eller stamfiskkultur. I tillegg til øyestengelablasjon kan individuell oppdrett med sandsubstrat og tilstrekkelig fôring, gjerne med levendefôr, øke gonademodningshastigheten hos mudderkrabber i fangenskap 30,35,46,47.

Krepsdyrblod kalles hemolymfe og kan gå tapt under øyestengelablasjon. Et overdreven tap av hemolymf kan føre til død av øyestengel-ablated krabber, spesielt når du utfører kirurgi for å fjerne øyestengelen. Hemolymfen kan koagulere i den sårede delen for å forhindre tap. Imidlertid, i forhold til operasjonsteknikken, forsegler cauterization-teknikken den sårede delen umiddelbart, og forhindrer dermed tap av hemolymf og mulig infeksjon.

Dødelighet av mudderkrabbe etter ensidig øyeablasjon med enten cauterization eller kirurgi ble ikke funnet i løpet av de første 7 dagene. Dermed kan eyestalk-ablasjon gjøres med høyere overlevelsesrate. Ensidig øyestengelablasjon hemmer ikke overlevelsesraten til krabben33.

Stress under krabbehåndtering og øyestengelablasjon kan bidra til krabbedødelighet. Riktig anestesi er nødvendig for å minimere håndteringsstress under øyestengelablasjon. I krepsdyr øyestengelablasjon brukes kjemiske bedøvelsesmidler (dvs. xylokain, lidokain) i bunnen av øyestengelen før øyestengelablasjon14,15,17,48. På grunn av den aggressive naturen og den store størrelsen på gjørmekrabber, er imidlertid bruk av anestesi bare ved bunnen av øyestengelen ikke tilstrekkelig, og kan føre til ekstra stress for dyrene under injeksjonen. På den annen side er anestesi ved å utsette dem for en lavere vanntemperatur mer økonomisk og tryggere. Bruk av kaldt vann til anestesi i mudderkrabber er vanlig og har blitt brukt i andre studier på grunn av effektiviteten, enkelheten og minimal innvirkning på utvinning og overlevelse 37,49,50. I tillegg anbefales fremtidig forskning på smertevurdering etter øyestengelablasjon på mudderkrabber for å belyse endringen i atferd forbundet med smerte og stress, som tydelig i ferskvannsreker Macrobrachium americanum51.

Selv om eyestalk-ablasjon ved hjelp av både cauterization og kirurgiske metoder har minimal effekt på krabbeoverlevelse og forbedrer ovariemodning, krever det å utføre øyestengelablasjon profesjonell mestring av teknikkene. Tidspunktet mellom trinnene er kritisk, da enhver forsinkelse mellom protokollene gir ekstra stress for krabbene. I motsetning til operasjonsteknikken er cauterization-teknikken farlig fordi den innebærer bruk av brennbart utstyr (dvs. en blåsebrenner og butangass). Dermed er det nødvendig med ekstra forsiktighet når du utfører cauterization-teknikken.

Krabber er kannibalistiske i naturen, og de er kjent for å bytte på andre som nettopp har fullført sin molt og fortsatt er i deres soft-shell forhold 7,52,53. Dermed kan oppdrett av krabber individuelt unngå unødvendig dødelighet på grunn av kannibalisme. Bruk av individuell oppdrett i gjørmekrabbekultur praktiseres vanlig, både i kultur med høy tetthet og damkultur, for fôring og soft-shell krabbeoppdrett formål 8,53. Denne protokollen benyttet også individuell oppdrett og vedlikehold. Under transporten av krabbene for oppdrett eller handel, blir krabben bundet fast (eller til og med autotomisert) for å forhindre kamp, unødvendig skade og tap av lemmer34.

Den beskrevne protokollen for øyestengellablasjon skal utføres med flere personer. Etter å ha fullført øyestengelablasjonen, skal ikke-engangsutstyr (f.eks. Akvariet, brettet, håndkleet, etc.) desinfiseres med 30 ppm klor. Krabbene må overvåkes minst to ganger per dag. Eventuelle døde krabber, uspist fôr, ablerte lemmer eller smeltede krabbeskjell skal kastes raskt (dvs. begravet i jord med blekepulver) for å forhindre potensial for sykdomsspredning.

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Disclosures

Ingen av forfatterne har interessekonflikter.

Acknowledgments

Denne studien ble støttet av utdanningsdepartementet, Malaysia, under Higher Institution Center of Excellence (HICoE) -programmet, Malaysia, akkreditert til Institutt for tropisk akvakultur og fiskeri, Universiti Malaysia Terengganu (Vot nr. 63933 &; Vot nr. 56048). Vi anerkjenner støtten fra Universiti Malaysia Terengganu og Sayap Jaya Sdn. Bhd. via Private Partnership Research Grant (Vot. No. 55377). En adjungerende akademisk stipendiatstilling fra Universiti Sains Malaysia til Khor Waiho og Hanafiah Fazhan er også anerkjent.

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).

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Denne måneden i JoVE Scylla olivacea kald-sjokk anestesi cauterization gonadal utvikling indusert avl

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).
Eyestalk ablation for å øke ovariemodning i mudderkrabber
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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).

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