DNA Sampling

[armata]

This summer a student from Bangor Uni UK is coming to SA - sampling of widespread snake species will go up a gear.

The student will make himself known on the forum soon.
Some of you have already provided samples, thanks for this.
What we require is scale ventral clip from live snakes, and a tissue sample from DORs (DORs must be fresh).
IMPORTANT - location, GPS prefered.
We will provide vials and ethanol.
Just to be clear these are what we consider to be the widespread species.

Bitis arietans
Bitis atropos
Causus rhombeatus
Naja nivea
Naja mossambica
Naja annulifera
Hemachatus haemachatus
Dendroaspis polylepis
Dispholidus typus
Aspedilaps lubricus (Aspidelaps? BV)
Psammophis crucifer
Psammophis notostictus
Psammophis mossambicus
Psammophis subtaeniatus
Crotaphopeltis hotamboeia
Psammophylax rhombeatus
Pseudaspis cana
Lamprophis capensis
Dasypeltis scabra
Lyconomorphus rufulus
Philothamnus semivariagatus
Duberria lutrix
Lycophidion capensis

Remember without the location the sample is US.

Thanks

[BushSnake]

Out of curiosity, why doesn't Psammophis brevirostris, and Bitis caudalis make it onto the list?

[armata]

Your right Andre, I must have missed a few, and both those you mention have a wide range. Also should have mentioned highlighted priority species, Psammophylax rhombeatus should be highlighted, my mistake.

[atropos]

Great will keep my eyes open. Busy looking for a 2nd hand gps...

[Sico]

erm i just caught a little L rufulus in my garden, but i am flying overseas tomorrow, so Armata tell me what you need in a PM and i shall try and provide.

[Bushviper]

When you say fresh is that opposed to frozen, or opposed to decomposed? What do we do with freshly run over DOR if we dont have vials and alcohol?

[armata]

Frozen is OK, so stick fresh DORs in freezer ASAP. I carry coolbag with ice packs, you know, just in case.

[Axel]

I think it's about time I made myself known on the forum (I have been 'lurking' for a while!). As armata has already mentioned, I am a PhD student at Bangor Uni in the UK currently researching the genetics of widespread South African snakes. The project will investigate the genetic diversity which occurs in these species across South Africa, in order to examine how historical climatic changes affected populations in different regions of the country. The research could also provide important information for the future conservation of these species, by identifying genetically distinct populations which may warrant special conservation status.

Anyway, the success of the project depends on having large numbers of samples from as many different locations as possible - so any samples which forum members can provide will be greatly appreciated and really contribute to the data we are collecting. A lot of people have already provided some really useful samples, so a big thanks to all who have.

As armata explained, we'd like ventral scale clippings, or tissue samples if snakes are DOR. Blood samples are also OK, but probably less practical to take in the field and give a lot less DNA than tissues. Shed skins are also good, as long as species ID is definite. Samples can be frozen if no alcohol tubes are available, and then thawed and put in alcohol later. Skins should be kept dry, in sealed plastic bags. I will provide alcohol tubes to everyone who is interested in taking samples - pm me or armata, and I will get this sorted next week. I am also interested in looking at colour variation across South Africa, particularly in puff adders and mole snakes. If you have pictures of snakes from which samples are taken it would be great to have these too.

thanks again, Axel.

[Bushviper]

Could you provide some pictures on what a ventral scale clipping looks like for the people who have never seen it done and dont want to mulilate the snake. What do you use to cut the scale and what should you treat the snake with before release?

Can the pictures just have a reference number which corresponds with the sample and then presented in digital format? What type of reference system would work for you? I would have thought something along the lines of AN Smith 0001/08 can be used for all specimens collected by Smith and then you can rename them on the photograph as well as the sample with your unique research number.

Which parts of the DOR specimen would give the best DNA? Would the heart, liver or just a muscle sliver be the best? How much do you need?

What about Python natalensis? I know technically you need permission to even have DNA but they all also fairly wide spread.

[armata]

Re scale clipping - it is easier to tube the snake, even non-venomous. The ventral scale can be cut quite deep, but not enough to draw blood, and in a neat rectangle. Two such scales will suffice. Fine sharp scissors are best, strangely perhaps but i find fine needlework scissors work best. The area can be wiped with an antiseptic swab. With dead snakes a section of skin with tissue attached is fine, but so is a sliver of heart or liver. BTW, if you are studying snakes the ventral clips also provide identification for that individual. If you do this then you must clip to a code for each snake.

Axel and I will probably devise a form for data. But main thing is to give the sample a ref no which will appear on the vial and form. If you don't have GPS then nearest town, nature reserve etc. But remember back in Bangor they may not be familiar with just small town names, so add province. The form will probably be on excel, most have access to that.
Thanks all in advance.
BV, Python natalensis, you are right of course, and these would be welcome.
One more thing, be aware of legalities when sampling live snakes. Thos of you doing catch & release should have no probs..

[BushSnake]

Why use ventral scales vs dorsal scales? Is it only because of the ease of clipping? The other sources that I provide DNA data for (mainly lizzards) recommended the tip of the tail for all reptiles. This is far easier to clip, and doesn't hurt the snake, but would that be sufficient?

The other thing that I forgot when I started sampling for Armata was that large samples do not absorb the alcohol as well and become rotten, even in the vials. This is true for any sample (large samples of insects, moths, beetles, etc) so I though I'll share my mishaps to avoid more. The other sources recommended a sample about the size of a pin head. Armata / Axel, can you comment on the size of the sample you require?

[armata]

Two times half a ventral scale is the norm.

re clipping elsewhere - for one my permit applications clipping the tip of the tail of snakes was regarded as 'mutilation', but, wait for it, was OK for lizards! I even had trouble convincing this particular application that ventral clipping was standard scientific practice; had to describe as same as clipping your toe nails.
We will confine samples to scale clipping and tissue; caudal puncture for blood is fiddly, especially with adders, and needs experience and extra equipment and special buffer.
It should be mentioned that those of you who have WC snakes and are 100% sure of location then this would also be fine; as long as you don't mind 'mutilating' your snakes!!

[armata]

One more thing, and I should have mentioned this earlier. If you are NOT experienced with handling venomous snakes then please limit your samples to dead specimens. I am sorry that we cannot supply handling tubes, but maybe most of you have got anyway.
Be safe.

[Axel]

Why use ventral scales vs dorsal scales? Is it only because of the ease of clipping? The other sources that I provide DNA data for (mainly lizzards) recommended the tip of the tail for all reptiles. This is far easier to clip, and doesn't hurt the snake, but would that be sufficient?

The other sources recommended a sample about the size of a pin head. Armata / Axel, can you comment on the size of the sample you require?

Most of the DNA is located around the scale base, rather than at the edge, so with ventral scales being larger it is easier to get a clipping which includes some of the appropriate tissue. I also believe people have had problems here in the UK with getting permission to take DNA samples from wild snakes. However, ventral clipping to mark individuals is OK, and if you then want to use the tissue for DNA anaysis that's fine (strange, I know).

Re. sizes of sample: I typically use an approximately 6mmX4mm sized piece of scale for a DNA extraction, which gives a good yeild. However, it's always good to have extra material so subsequent extractions can be done in the future. It's also surprising how little material can be used and still provide sufficient DNA, albeit at lower concentrations. I have successfully done extractions on dwarf Bitis samples from armata that were around 2mmX0.5mm - so sampling should not be limited to just adult/large snakes.

Which parts of the DOR specimen would give the best DNA? Would the heart, liver or just a muscle sliver be the best?

muscle tissue actually gives fairly low yeilds, and in liver samples the DNA can be degraded due to the powerful enzymes contained in the liver. The ideal would be a small section of skin, including the scales and underlying tissues. Tail tips are also fine if this is more convenient.

I will compile all this information into an instruction sheet, so everyone is clear on how to take samples. Oh, and if people have captive WC snakes of known locality, and don't fancy clipping scales, a shed skin would be fine.

Thanks.

[Mongoose]

This might be a silly question - but if I collect DNA, may I send various people tissue from the same specimen?