Anemones group project

July 31, 2010
Cattlepoint Field Collection:
Marie, Loni, and myself collected 15 Anthopleura elegantissima anemones from the lighthouse point at Cattlepoint intertidal for preliminary studies on anemone bacterial assemblages, exposure to V. tubiashii, and heat stress. We used a thin metal spatula to remove each anemone from high intertidal pools, trying not to disturb the pedal disc. The pools were continuously washed with tidal water and were not completely stagnant. The low tide was not low enough to collect anemones from low intertidal pools, but it should be perfect on Aug 8 and 9, 2010 with a -2.0 tide. Ten anemones were collected and placed into seawater in a zip-lock bag. I used gloved hands to collect 5 additional anemones and placed them in sterile 50 ml falcon tubes with water from the pools they were collected from. These 5 anemones were subjected to additional microbial sampling upon arrival at the lab at FHL.

  • Morgan and I decided to try several methods of plating for culture and swabbing for bacterial DNA. The first 2 anemones were placed on weigh boats and swabbed twice.
  • The first swab we placed into 300ul Qiagen Stool Kit lysing buffer and swished it around to remove the mucus, and then refridgerated O/N.
  • The second swab was swiped across ASWA (artificial seawater agar, DIFCO 2216), parafilmed, and left to grow O/N at RT.
  • The mucus + seawater remaining in the falcon tube was refrigerated O/N and will be centrifuged Sunday or Monday to concentrate mucus for plating purposes.

This procedure was repeated for the remaining 3 anemones with the addition of a sterile seawater wash of each anemone polyp prior to swabbing.


Aug 1, 2010
DNA Extraction:
Kathy used the Qiagen Stool Kit to extract DNA from the 5 A. elegantissima anemones Morgan and I swabbed yesterday. I included one negative extraction control. The initial incubation was between 85-90 degrees rather than 70 to lyse gram + bacteria. The only other amendments to the protocol were to use 1 ML lysis buffer and 1/3 of an inhibition removing tablet because we started with such a small quantity of DNA from the mucus swab. These samples are being stored at -20 degrees C (volume = 200 ul) in the left freezer.

Aug 2, 2010
PCR with universal bacterial primers on anemone mucus extracted yesterday with the Qiagen Stool Kit. Performed PCR reactions in duplicate including the negative extraction control. We also included 1 negative PCR control for contamination during PCR prep, or within PCR reagents (Go Taq, primers, water etc.). Total = 13 samples
25 ul rxn
  • 12.5 ul 2X Promega GoTaq
  • 1.5 ul 10X BSA
  • 1.0 ul 10uM 27F
  • 1.0 ul 10uM 1492R
  • 2 ul DNA template (hotstart @ 95C)
Thermal Profile:
  • Hotstart
  • 95 C 10 min
  • 95 C 30 sec
  • 55 C 30 sec
  • 72 C 50 sec
  • 40 Cycles
  • 72 C 8 min
  • 4 C HOLD

Product Check
  • Agarose gel (0.5 g agarose/ 50 ml buffer/ 5 ul Ethidium bromide)
  • Loaded 5 ul PCR product and 7 ul 1 Kb ladder
  • Run at 115 V for 30 minutes

Preparation for trial runs:
Marie and Loni made fresh Marine broth to be used for culturing VT


August 3, 2010

Matthew stressed a few anemone in distilled water and collected mucus. Method found in paper:
EFFECTS OF A TOXIN FROM THE MUCUS OF THE CARIBBEAN SEA ANEMONE (BUNODOSOMA GRANULIFERA)
ON THE IONIC CURRENTS OF SINGLE VENTRICULAR MAMMALIAN CARDIOMYOCYTES
EDUARDO M. SALINAS,’ JORGE CEBADA,’ ALBERT0 VALD&* ANOLAND GARATEIX,’ ABEL ANEIROS* and JULIO L. ALVAREZ3*
Toxicon, Vol. 35, No. 12, pp. 169!%1709, 1997
Centrifuged two tubes at 3000rpm for 10 minutes. A dark pellet formed but we weren't able to isolate mucus from the pellet or water.
Tried hanging an anemone in the air for a few minutes on 1mm x 1mm fine nylon mesh but did not see much mucus. Method found in paper:
Do anemonefishes use molecular mimicry to avoid being stung by host anemones?
J. K. Elliott*, R.N. Mariscal, K. H. Roux
J. Exp. Mar. Biol. Ecol. 179 (1994) 99-113

Preparation for trial runs:
The water bath was set up: cleaned and filled with dIH2O
We started an overnight culture of Vibrio tubiashii by adding 2 VT colonies (provided by Emma from 8/1/10) to 50 mLs of Marine broth, this tube was taken to lab 10 for O/N growth on the belly dancer.


PCR with universal bacterial primers (take 2) on anemone mucus extracted with the Qiagen Stool Kit. Because there was contamination in one of the two extraction controls and contamination in the PCR negative control, we decided to run another PCR and try to get clean negative controls. We used the same protocol as before (see below), however we UV irradiated the Master Mix (without primers and template) for 10 minutes prior to running the PCR. Our product check was completely blank, thus we are are going to run a 3rd PCR on Aug 4, 2010 without irradiation to see if that might have caused the PCR reaction to FAIL.

25 ul rxn
  • 12.5 ul 2X Promega GoTaq
  • 1.5 ul 10X BSA
  • 1.0 ul 10uM 27F
  • 1.0 ul 10uM 1492R
  • 2 ul DNA template (hotstart @ 95C)
Thermal Profile
  • 95 C 10 min
  • 95 C 30 sec
  • 55 C 30 sec
  • 72 C 50 sec
  • 40 Cycles
  • 72 C 10 min
  • 4 C HOLD


Product Check
  • Agarose gel (0.5 g agarose/ 50 ml buffer/ 5 ul Ethidium bromide)
  • Loaded 5 ul PCR product and 7 ul 1 Kb ladder
  • Run at 115 V for 30 minutes




August 4, 2010

Matthew suspended a large anemone on the 1mm x 1mm fine mesh over a custard glass for 5 minutes and scraped the underside of the mesh. 0.25mL of mucus was collected. 50uL of the mucus was pipetted onto each marine agar and TCBS plate. A metal loop was bent into an L, dipped in ethanol, flamed and used to spread the mucus over each plate. The plates and lids were exposed to UV light for 10 minutes to sterilize them. The plates were then allowed to sit at room temperature (lab 10) to see if we get any bacterial growth to confirm if the UV treatment worked.

Preparation for trial runs:
Loni and Marie separated anemones into individual (labeled) glass dishes in the sea table to acclimate
We made a map of the position of the dishes on the outside of the tank
The temperature of the water in the sea table is 12-15C
All 13 anemones were weighed and divided into test groups as follows:
Heat 35C
H1 4.57g
H2 3.12g
H3 4.37g
H4 4.42g
Vibrio tubiashii
V1 1.88g
V2 3.10g
V3 0.68g
V4 3.44g
Vibrio tubiashii and Heat 35C
VH1 4.22g
VH2 4.19g
VH3 3.29g
VH4 2.81g
Control (for our test runs we only have 1 animal per treatment)
C1 1.38g
C2 2.97g
C3 1.38g

We set up VT serial dilutions: 10^1, 10^2, 10^3, 10^4, 10^5, 10^6, 10^7, 10^8, 10^9, 10^10
by adding 4.5 mLs of dIH2O to 10 falcon tubes and then adding 0.5 mLs of our VT culture to the first dilution, mixing well, and then adding 0.5 mLs from that tube to the next, mixing well and continuing on...

We plated these dilutions along with the pure VT culture and the dIH20 that was used for the dilutions

We set up a new culture today by adding 49 mLs of marine broth to a 50 mL tube and addind 1 mL of our inital VT culture to that tube, O/N on the belly dancer in lab 10
The growth on these plates will give us an idea of ~how many VT we grow over night
When we do our real experiment we will probably only plate the concentration that we use to expose the anemones to

The hot water bath was set for 35C

Heat stress trial run:
The hot water bath was ready to go in the afternoon at 35C
The experimental individual were photographed in the sea table in their happy state
Marie and Loni took the 4 selected individuals (H1-H4) out of the sea table and wiped down the outside of the glass dishes to not get any sea water into the heat bath
Start time= 1:45 pm



FISH (Fluorescent in situ hybridization):

Morgan and I cross referenced publications and probe queries on probebase (http://131.130.66.201/probebase/) to find probes specific to particular bacterial groups of interest. We hope to order these probes with a Cy5 fluorophore at 5' end. These primers will bind to the target bacteria within the anemone mucus and fluoresce under specific wavelengths of light so that we can both ennumerate and partially identify the bacteria in our samples. Another great FISH reference site: http://www.arb-silva.de/fish-probes/fish-protocols/


|| Bacterial Taxa || Sequence || Probe || Reference ||

Alphaproteo
GGT AAG GTT CTG CGC GTT
ALF968
Neef 1997
Betaproteo
GCC TTC CCA CTT CGT TT
BET42a
Manz et al. 1992
Gammaproteo
GCC TTC CCA CAT CGT TT
GAM42a
Manz et al. 1992
Vibrio
AGG CCA CAA CCT CCA AGT AG
GV
Eilers et al. 2000
Actinobacteria
TCC GGT CTC CCC TAC CAT
Actino-658
Kong et al. 2005
Acidobacteria
CTG AGA TGG TTC ACT TCC
LS_HOL189
Meisinger et al. 2007
Cyanobacteria
AGA GGC CTT CAT CCC TCA
405_Syn
West et al. 2001
PCR Take 3
Today Morgan ran another PCR this afternoon, without UV treating the master mix and without the hot start. We got product this time, but we also had bands in all of our negative controls. Boo. It should be noted that for most of our samples (4/5), the bands are at much greater intensity than for the negative controls, suggesting that we successfully extracted microbial DNA from the mucus using the Stool kit. Tomorrow we will repeat the PCR with new reagents and with new working solutions of our primers, as we realized the entire class dipped into these primers when we amplified microbial DNA from the snails. We will also use the sterile hood on the bottom level of lab 10.