dispersal of ticks and tick borne diseases by birds - Lista fuglestasjon

MATERIALS AND METHODS
Tick genetics: Making new
microsatellites (Paper I)
Twenty adult ticks were collected by
flagging in localities in southern
Norway. DNA was extracted by the spin
column technique using a DNeasy tissue
kit (Qiagen). Procedure: the specimens
were crushed/grinded (in this case, using
the tip of a Pasteur pipette to severe the
ticks’ cuticula). After lysis with a
protease, the specimens were transferred
to a spin column with a membrane that
binds to the DNA. The membrane was
washed twice by a buffer in a centrifuge.
After washing, the DNA was detached
from the membrane by an elution buffer
and spinned down into vials. The DNA
from all the ticks was pooled together.
Microsatellites were isolated by the
method described by Hamilton (1999).
After digesting the DNA into fragments
of 200 to 1000 base pairs, the DNA was
ligated to double-stranded SNX linkers
for “tagging” the fragments. To select
fragments that contained repeated
sequences, the DNA-fragments were
hybridised with a biotinylated
oligonucleotide probe (in this case only
one probe: (GT)15, not the whole range
of different probes that were used by
Hamilton) and captured on streptavidincoated
magnetic beads, as biotin strongly
binds to streptavidin. DNA that was not
hybridised was washed away using a
magnet. The enriched fragments were
ligated into a plasmid vector (XbaI
digested pBluescriptIIKS(+) vector,
Stratagene), which also carry the
property of ampicillin-resistance, and the
vector was transformed into E. coli cells
(XL1-Blue MRF'cells, Stratagene). The
cells were cultivated on LA plates
(Luria-Bertani medium supplemented
with 1.5% agar and 50 µg of ampicillin
per ml), the ampicillin was added to the
agarose medium to select the plasmidcontaining
cells. As not all of the cells
with plasmids would contain inserts of
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the DNA fragment, the cells had to be
screened for inserts. This was done by X-gal
and IPTG, which is included in the medium:
X-gal is a galactose analogue which is
broken down to a blue-coloured substance by
- galactosidase
with a carboxy-terminal, which
is inactive, and the plasmid contain the socalled
Lasz gene which in the presence of
IPTG produces itgalactosidases.
When the two inactive
enzymes are combined, they become active.
Plasmids with DNA-inserts do not produce
- galactosidase gene. The result is that
the colonies with inserts appear white, while
the remaining colonies appear blue. Positive
clones were picked directly from the agarose
plates denaturated at 95°C for 10 minutes and
amplified by using T7 and M13 reverse
primers. Polymerase chain reaction (PCR)
products of 300-800 bp were sequenced (see
Box IV). Primers were designed from the
sequences using Oligo software.
Tick genetics: Study of tick
family groups (Paper II)
Three adult female, fully engorged I. ricinus,
each with an attached, copulating male were
collected from cows on the island Hille, near
Mandal, Norway. They were allowed to
complete copulation and were incubated at
room temperature in a humid chamber
through oviposition and hatching of larvae.
DNA was extracted from the males, females
and 20 larvae from each female by using the
spin column technique, and PCR by using the
new microsatellites was performed. Paternity
of the attached male and the minimum
number of fathers involved in each family
was determined by Mendelian laws.
Collection of ticks from birds
(Paper III)
Northward migrating passerine birds were
caught with mist nets at four bird
observatories along the southern Norwegian
coast: Lista, Jomfruland, Store Færder and
Akerøya (Figure 1, Paper III). The birds were
examined for ticks around the beak, eyes and
ear-openings by using head-mounted
magnifying glasses (Figure 8). The ticks