Updates from July, 2012 Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 19:56 on 2012/07/30 Permalink
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    During the weekend I induced the spawning using light and it was quite effective. A good amount of gametes were shed (together with poop…).

    I washed a few times before adding a couple of drops of 0.5M EDTA solution. Eggs did not show any cleavage stage a few hours after activation. On the next day there was a small number o embryos at 2 or 4-cell stage. Not significant.

    On Sunday I made a 0.1mM EDTA solution and left the embryos for 1h at 10 °C and no shaking. No cleavage stages again, but on the next morning there were quite a few embryos in cleavage stages up to 64-cell, apparently.

    Today I left the embryos for 2h in 0.2mM EDTA (exchanging the solution after the first hour). 2-cell stage embryos were already present a couple of hours after spawning. More embryos seem to be cleaving. I washed the dish many times and put the dish in the cold room (5-7 °C).

    Sabrina helped to set a 4D recording with a 2-cell embryo that will go overnight.

     
  • Bruno Vellutini 19:08 on 2012/07/27 Permalink
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    Tried scraping Membranipora for eggs with new animals. I ripped zooids only from colonies with eggs and sperm in small bowls. Transferred the deposited material to dishes and washed many times. A drop of EDTA was added before the initial washes. During washes all the discoid eggs became round and I could see many spermatozeugma and spermatozoids around.

    Dishes were kept on ice and put in the cold room. I observed the dishes hourly and there was no sign of cleavage. Five hours after “fertilization” most eggs look healthy with its characteristic membrane and some are degrading; I found almost none disc-shaped oocytes. A few show very active cytoplasmic movements.

    What could be wrong?

     
  • Bruno Vellutini 16:03 on 2012/07/26 Permalink
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    Membranipora spawning, first try 

    24/07/12

    Aina brought Membranipora colonies in the morning for spawning. They stayed in the PlayMate box until 12h00 (project discussion) when I transferred the leafs to the 5 liter transparent bucket with clean sea water from the cold room (in the dark). I cut a part and verified that the zooids had eggs inside. With forceps I scraped the colonies destroying the zooids to release eggs and sperm in water. After waiting 10 min at the cold room I collected the deposited material and transferred to petri dishes for washing.

    Samples were still quite dirty after 3-5 washes. I kept the dishes in the cold room during lunch, washed a couple of times more, and put in the incubator at 18 °C. Looking under the scope I found many eggs with the membrane (fertilized) and some disc-shaped unfertilized oocytes.

    4h later I checked under the scope again and eggs looked the same (less oocytes).

    25/07/12

    Exchanged the water of the bucket once. Checked the embryos and most were still alive with the membrane, but did not cleave; changed the water 3 times.

    26/07/12

    Changed the water once more and checked the colonies under the stereoscope for spawning, but everything was dead. Embryo cultures also do not looked well anymore.

    Failure.

    Lessons:

    • Upon arrival, cut leaves with colonies in smaller pieces and wash out the jelly. Repeat washes after a while to get them really clean. Keep them in glass bowls in the cold room in the dark.
    • After scraping clean well and keep embryos in the cold room.
    • Maybe use EDTA to help fertilization? Check reference…
     
  • Bruno Vellutini 15:04 on 2012/07/26 Permalink
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    Juveniles do not seem much interested in Platynereis nectochaeta larvae, they seem to prefer the eggs/zygotes much more.

    It should be better to clean the eggs well before adding to the dish so that they can be kept for longer without making the water dirty.

     
  • Bruno Vellutini 12:29 on 2012/07/23 Permalink
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    PCR mostly stem/germ cell genes of Terebratalia 

    Setting up a pcr with gradient (63 +- 3 °C) at 12h:

    Gene Primers Tm mean
    Mm Six3/6 64.10
    Tt Dicer1b 61.60
    Tt Runx 63.15
    Tt Mael 65.15
    Tt Piwia 65.55
    Tt Tudor1 61.95
    Tt Ago 62.95
    Tt PL10 63.10
    Tt Pum 65.35

    Extraction and ligation overnight at 4°C, except for MAEL.

    24/07/12

    Transformation and plating of colonies.

    25/07/12

    PL10 and Pumilio had very few colonies on the plate (insert numbers) and together with Piwia they did not produce any positive colony.

    27/07/12

    Put positive colonies to grow for minipreps.

    BV162 Terebratalia transversa Dicer1b
    BV163 Terebratalia transversa Dicer1b
    BV164 Terebratalia transversa Runx2
    BV165 Terebratalia transversa Runx2
    BV166 Terebratalia transversa Runx2
    BV167 Terebratalia transversa Tudor1
    BV168 Terebratalia transversa Argonaute
    BV169 Terebratalia transversa Argonaute
    BV170 Terebratalia transversa Argonaute
    BV171 Terebratalia transversa NK6
    BV172 Terebratalia transversa NK6
    BV173 Terebratalia transversa NK6
    BV174 Terebratalia transversa Wnt7
    BV175 Terebratalia transversa Wnt7
    BV176 Terebratalia transversa Wnt7

     
  • Bruno Vellutini 18:37 on 2012/07/20 Permalink
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    Fed juveniles with Platynereis eggs and they managed to swallow a bunch of them. It seems that keeping the eggs/embryos in the dish for a while might be worthwhile.

     
  • Bruno Vellutini 16:20 on 2012/07/04 Permalink
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    Fed L. ruber with shrimp and changed the water. Also cleaned juvenile cultures and tried to feed them. Some did swallow small pieces of muscle. So they are starting to eat.

     
  • Bruno Vellutini 12:50 on 2012/07/04 Permalink
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    Sent poster to print for the EuroEvoDevo 2012 Lisbon meeting. Here is the final art:

    EED12 poster

     
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