Chromatography Forum

Afternoon all,

I am interested in analysing whether an insect species one of my colleagues is working with deposits a trail pheromone when walking. Having scanned the literature it seems that a number of authors have simply let their insects walk across a piece of filter paper for a number of hours before solvent extracting the filter paper.

I feel that there must be a better way than that. I am considering using aluminium foil as it would be possible to clean it before exposing the insects to it.

I'm essentially posting to gain some insight from the community as you have all been so helpful in the past.

Take care,

Joe

You research is always very interesting Joe. I'm with you on the aluminum foil idea. Does the insect always secrete the pheromone so you're sure you'll get it? Is there something about the filter paper the stimulates the insect to secrete the pheromone? Do you know the identity of the pheromone or are you trying to determine that?

This might be a classic application for your Markes TD system. If you can preclean the aluminum foil in an empty sample tube, then pull it out and expose it to the insects. Put it back in the tube and desorb it into your GCMS system. You could do it with filter paper as well but I'm betting there's a lot more extra "stuff" in filter paper that isn't on aluminum foil.

I think the filter paper is not an issue, if you make sure to also do an extraction of a blank filter paper so you can identify possible background signals.

How will you distinguish between pheromones and other, uh, traces of the insect? Do you know the molecules you're after or you plan to database search?

You could do a sort of quantification with in the x-axis "hours walked on the paper" and the y-axis the area of pheromone make sure to calculate the linearity!

I was intimating that you could do a direct thermal desorption/extraction of the filter paper as well. The aluminum foil will likely be cleaner than the filter paper.

X axis - temperature of desorption
y axis - pheremone A, pheremone B, urine, saliva, poop...
A complete library!

Hi Joe

Fascinating query from you as usual

Your idea and RB6 comments about the use of foil make sense.

Some random thoughts in no particular order

The paper may adsorb more but you will lose out on sensitivity on dilution with solvent extraction


see this interesting presentation
http://www.sussex.ac.uk/lasi/documents/ ... _6_120.pdf

You will only get a single track across your substrate?

Decay time appears to be from minutes to days for ants

Is there also a "no food" pheromone track?

As an off the wall idea - get them to march back and forwards through a "tunnel" of an empty glass open injection port liner to a food source to concentrate the trail. That liner could be just dropped into the injection port to be desorbed. You could also use that approach to establish a useful control of a "no food" trail. Alternatively line the liner tube with foil to be removed followed by TD. The no food control (also needed) could be made using this approach


Also see

http://mute-net.sourceforge.net/howAnts.shtml

What is the insect?

Cheers

Ralph

You research is always very interesting Joe. I'm with you on the aluminum foil idea. Does the insect always secrete the pheromone so you're sure you'll get it? Is there something about the filter paper the stimulates the insect to secrete the pheromone? Do you know the identity of the pheromone or are you trying to determine that?

This might be a classic application for your Markes TD system. If you can preclean the aluminum foil in an empty sample tube, then pull it out and expose it to the insects. Put it back in the tube and desorb it into your GCMS system. You could do it with filter paper as well but I'm betting there's a lot more extra "stuff" in filter paper that isn't on aluminum foil.

Thank you, i'm very lucky to be able to work in the field that I do. The behavioural assays indicate that the insects always secrete the trail pheromone, so I'm fairly certain I will collect at least some of it. Having looked through the literature it would appear that the filter paper doesn't serve any purpose and I have placed the insects on both filter paper and aluminium foil to determine whether they show a preference - they don't! I'm afaid that I have no idea on the identification of the pheromone, so it's a bit of a shot in the dark unfortunately.

I am currently awaiting some new O-rings for my TD system (I hope these will be delivered tomorrow), so as soon as they arrive I will try this method out. I think it could work very well!

I think the filter paper is not an issue, if you make sure to also do an extraction of a blank filter paper so you can identify possible background signals.

How will you distinguish between pheromones and other, uh, traces of the insect? Do you know the molecules you're after or you plan to database search?

You could do a sort of quantification with in the x-axis "hours walked on the paper" and the y-axis the area of pheromone make sure to calculate the linearity!

In order to minimise contamination from other bodily fluids (i.e. faeces and vomit), the insects have been starved for 48 hours before being introduced onto the filter paper. I like the quantification idea!

Hi Joe

Fascinating query from you as usual

Your idea and RB6 comments about the use of foil make sense.

Some random thoughts in no particular order

The paper may adsorb more but you will lose out on sensitivity on dilution with solvent extraction


see this interesting presentation
http://www.sussex.ac.uk/lasi/documents/ ... _6_120.pdf

You will only get a single track across your substrate?

Decay time appears to be from minutes to days for ants

Is there also a "no food" pheromone track?

As an odd idea - get them to march through a tunnel of an empty glass open injection port liner. That liner could be just dropped into the injection port to be desorbed. You could also use that approach to establish a useful control of a "no food" trail. Alternatively line the liner tube with foil to be removed followed by TD. The no food control is also needed and could be made using this approach


Also see

http://mute-net.sourceforge.net/howAnts.shtml

What is the insect?

Cheers

Ralph

I have placed 10 of my insects into a single petri dish:



The insect species I am working on is the soft fruit pest: vine weevil (Otiorhynchus sulcatus) (https://www.rhs.org.uk/advice/profile?PID=234). It's bigger than the mites I was previously working on (~ 2 cm), so I don't think i'll be able to get them to walk inside an inlet liner, although this is a nice idea for my mites!

I'm unsure as to whether there is a no food trail. I am mainly trying to figure out why they like to aggregate together. I have undertaken an analysis of the headspace to determine whether there is a volatile aggregation pheromone, but it appears there isn't. I used both TD and SPME to do this.

Cheers,

Joe

Hi Joe

Get your colleague to do the biology before you do the chemistry - if he/she can show that other individuals of the insect's species respond in some way to where it walked then there may be a pheromone involved. If there is, then is the time for some chemistry.

I don't doubt that with a sensitive enough analysis you can find an insects footprints, but that does not mean that it is laying a trail pheromone.

Trail pheromones can be very challenging - they are nearly always ephemeral, may only be laid under specific circumstances, and are the paradigm case of tiny quantities having big effects - there is some or other ant pheromone - from Atta texana I just googled it - that needs only 1 mg to make a trail three times round the world.

Hi Joe

Get your colleague to do the biology before you do the chemistry - if he/she can show that other individuals of the insect's species respond in some way to where it walked then there may be a pheromone involved. If there is, then is the time for some chemistry.

I don't doubt that with a sensitive enough analysis you can find an insects footprints, but that does not mean that it is laying a trail pheromone.

Trail pheromones can be very challenging - they are nearly always ephemeral, may only be laid under specific circumstances, and are the paradigm case of tiny quantities having big effects - there is some or other ant pheromone - from Atta texana I just googled it - that needs only 1 mg to make a trail three times round the world.

Hi Peter,

Sorry I didn't make it clear that the biology has already been done. Using a PTFE walkway they have shown that the vine weevil will follow the path laid down by the previous individual.

You do make a very good point about the difficulties analysing trail pheromones, I have tried and failed with my mites.

All the best,

Joe

Hi Joe

Do you know whether the trail substance is volatile ? And are thee any specific behaviours associated with depositing or sensing it ? That might give you some clies as to what kind of thing you are looking for.

Peter

Hi Joe

Do you know whether the trail substance is volatile ? And are thee any specific behaviours associated with depositing or sensing it ? That might give you some clies as to what kind of thing you are looking for.

Peter

Hi Joe

Do you know whether the trail substance is volatile ? And are thee any specific behaviours associated with depositing or sensing it ? That might give you some clies as to what kind of thing you are looking for.

Peter

Hi Peter,

I am unsure as to whether the trail substance is volatile or non-volatile at this time.

The vine weevil tend to aggregate in dark, confined spaces. Many people have previously attempted to identify a volatile aggregation pheromone without success.

Cheers,

Joe

Hi Joe

Aggregating beetles reminds me of something that I worked on years and years ago - some kind of beetle from the Kalahari that also aggregates. Like you I was helping out a biologist colleague. I cannot remember the name of the beetle but I do recall they were stinky that devils that crudded up my MS source something wicked ! Google Scholar Anne Rasa (she of dwarf mongoose fame) and you might find some papers.

Peter

Hi Peter,

Thanks for pointing me in the direction of Anne Rasa. I have read a few of her papers on the tenebrionid beetle this morning, although I could only find one that specifically mentions aggregation (http://onlinelibrary.wiley.com/doi/10.1 ... 161.x/full) and no chemical analysis was undertaken.

I'll try and dig a little deeper!

Cheers,

Joe

Joe, I honestly don't recall whether any of my chemistry got into print. I suspect not because I'm not a co-author on any of the papers. I was doing the analyses as a moonlighting project, so I couldn't do as much as I wanted.

No chance at all of getting my hands on any of the data either I'm afraid.

Peter