r.d.j.butcher at dundee.ac.uk
Mon Jul 19 07:26:16 EST 1999
i have had a number of requests for info on our crude IVFsystem in
drosophila, and it has been suggested i reply on the newsgroup, so
here goes. Sorry for this delay, but i really have been busy on our
field course and the inevitable preparing for and then dealing with
all the data and students afterwards.
These methods were developed on Ichneumonoidea (Hymenoptera),
modified with some succes for Pyralid moths (Lepidoptera) but have
only been roughly tested on Drosophila and thus do NOT represent
optimised systems. Rather this is a very preliminary system. I am
hoping to optimise them later this year (I am not working with them
at present) and will report the results and any new protocols on the
Note that these preliminary experiments were carried out using males
and females from the two lines:-
Cyo P(2,3)/Bc1 Egfr1; y*w*/+
which although far from ideal, were available at hand at the time
and allowed the biparental nature of any eclosing imagoes to be
1. Virgin females (1-3 days old) were allowed to mate with males
(more than one copulation was allowed) and held away from
oviposition sites (i.e. on yeast paste only) over a 5 hours
period prior to dissection of the female and removal of the
sperm or spermataphore  into modified drosophila ringers (130mM
NaCl, 5mM KCl, 1.5mM CaCl2, 2mM Na2HPO4, 5% (w/v) BSA, 10% w/v
filtered homogenate from the spermatheca ). Typically the
free sperm / spilled spermatophore contents of ten females
spermatheca were microdissected into 20-30ul of ringers, but it
appears imperative to reduce dessication so both speed and use of a
locally saturated atmosphere is important .
2. Microdissect out the mature eggs from virgin females  and add
(bath for up to 1 minute) to the sperm mixture obtained above.
3. Load into a microinjector (standard inert oil as per embryo
microinjections) and "oviposit" via the microinjector  into
media. maintain as per normal.
>From the limited preliminary trials this results in some 20-27%
(range) of embryos hatching, and 10-14% developing as imagoes, but
the system has not yet been optimised.
. The importance of the number of matings and time in spermatheca
prior to dissection is not known, and needs optimising, but appears
that sperm removed from the spermatophore shortly after copulation
may be less suitable for this IVF sytem.
. I havent tried the less time consuming extraction of sperm by
microdissection from males. If it worked it would be a mjour tisaver
and is worthy of checking. This would however avoid the male
accessory gland secretions (importance in an IVF system, as opposed
to sperm competition and egg maturation / oviposition stimulants, is
unknown to me and can be evaluated), and maternal secretions
into the spermatheca (same points), would also add immature (sterile)
. relavance of female spermatheca homogenate in this system is
unknown, its left over from Leps.
. keeping the microdissected sperm at cooler temperatures(e.g.
0-4C) to allow larger bulk procedures has not been evaluated yet. If
they remained viable this would improve yields per day etc.
. This seems important. I have tried this procedure on embryos
laid by older virgin females, or those laid by females mated to XO
males (derived from the gynF9 (2,3) stock following mating to
ms(3)K81 males) and they failed to develop. Removal of the chorionic
and vitelline membranes, prior to IVFfertilissation, and maintaining
under oil afterwards (standard embryo microinjection procedures) all
failed in this preliminary system.
. This is left over from parasitic wasps... Passage through an
orafice (microinjection needle) of a slightly narrower dimension than
the egg is essential in the Hymenoptera case. Its importance to
drosophila has not been tested yet.
. not important, the IVF embryo could be hatched on a glass slide
etc, it is just morconvenient as the first instar can burrow in and
Lastly, the ability of this IVF system to work with cryopreserved
sperm, or embryos, obtained by these methods has not been
checked yet and i will look at that later on.
Any more queries please ask. Hope this helps and sorry for all the
space to thiose who have no interest in this
Evolutionary and Ecological Entomology Unit,
Department of Biological Sciences,
Dundee, DD1 4HN,
Work Phone:- 01382-344291 (Office), 01382-344756 (Lab).
e-mail:- r.d.j.butcher at dundee.ac.uk
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