How to obtain temperature shocked Papilio machaon?

[corradocancemi]

Hi, i'd be very curious to know how somebody creates some temperature shocked forms of Papilio machaon, as the very dark specimens (often with black markings fusing), and the very pale forms as the evittatus. Do you know wich is the method to create this kind of trasformations?
24 hours before the emergence of an adult of machaon, i have put the pupa either in the fridge and under a very hot air conditioning. But nothing happened when adult emerged

[Ascalaphus]

I haven't tried it yet, but I know that the 24 hours prior to pupation are a much more important period for creating aberrations than the hours before emergence of the adult.

I wonder if anyone would want to discuss their secret with us, because creating P. machaon aberrations can be a lucrative business.

[bobw]

I don't know how easy it is with machaon; Nymphalids are far more prone to producing variations with temperature shock than anything else.

The shock needs to be done to freshly formed pupae, before they've hardened. It also needs to be a fairly extreme shock, e.g. over 40 degrees Celsius. The more extreme the temparature the more you will kill, but the more extreme the survivors will be.

Bob

[irisscientist]

Corradocancemi,

I could not agree more with Bobw. It was proposed with Nymphalids that extreme temperature variation, at critical times in pupal development were the most likely cause for the induction of species aberrations. Ultimately wing patterns are genetic traits, but somehow exposure to extremes of temperatures during critical stages in pupal development effect the genetic transcription (or translation) machinery to produce the observed aberrations. Like Bobw also states, normally the more the extreme the shock, the more extreme the observed aberration. My own thoughts on this subject matter include the putative involvement of temperature induced hydration levels on the developing pupae and their potential effect on the transcription/translation machinery?

As with many butterfly species the duration of the pupal incubation period of Papilio machaon is extremely variable, but it is generally around 4 weeks. If I were to conduct the experiments myself to test this hypothesis, I would acquire some scientific grade temperature controlled incubators (ideally multi-chambered) and first incubate the pupae at various medium to high temperatures and observe which temperatures produced any wing pattern variations. As consistent high (or low) temperatures are likely to cause death, it would be advisable to initially incubate the pupae at various shorter duration developmental stages. Once you had obtained the results from these initial, test experiments, you should then be able to pin point more exact time frames (and durations) for required aberrations as well as also fine tune exact temperature shock conditions in order to induce the degree of aberration required.

Although this certainly not something I would condone (outside of the pure scientific interest), as Ascalaphus clearly states, tayloring species aberrations could potentially be a very lucrative business for anyone wishing to explore it enough!


Mark

[bluemoth]

On the old forum I asked how to shock pupa to make aberrations. I tryed putting pupa of Painted Lady Butterflies in freezer for veryed amounts of time. All died at a few minuts past 30 minutes in the freezer. All servived at befor or at exactly 30 minutes in the freezer. Most hatched with messed up wings. Abberration occured on frount wings only on some of the butterflies left in the freezer for 15 minutes or longer. Pupa were put in freezer just after shell was fully expanded but not fully hardened. We definatly need an expert to share their secrits with us here in shocking pupa. They should also not worry about sharing because I do not think they will loose income from selling abberants buy sharing their secrits. It will take all us begeners a long time to get good abberants to sell.

[irisscientist]

Dear Bluemoth,

The information relating to temperature induced butterfly aberrations is far from secret and is ‘mostly’ freely available online. It can easily be self researched if you simply know how and where to look (google scholar should always be a first port of call) and as with any scientific endeavor, thorough research (before starting any actual experiments) is the key to success. I have only spent a few minutes on this subject matter, but am able to summarize what I have read as follows:

A lot of the early successful work relating to this subject matter was confirmed by Sir Cyril Clarke and was reported in articles such as “Abnormalities of wing pattern in the Eastern Tiger Swallowtail butterfly, Papilio glaucus” (1983), the abstract of which can be found here:

onlinelibrary.wiley.com/doi/10.1111/j.1365-3113.1983.tb00465.x/abstract

Clarke concluded that the melanic background patterns of butterflies develop prior of adult eclosion. Concurrent studies performed by Ritland (1983), also discovered that pattern development in butterflies was susceptible to temperature induced shock during their pupal stages. This work was however unfortunately performed as part of his PhD thesis and consequently was only published therein. Some of this work however is summarized in some his later work, such as “The Effect of Temperature on Expression of the Dark Phenotype in Female Papilio glaucus (Papilionidae)” (1986) the pdf of which and can be found here:

lepidopteraresearchfoundation.org/journals/25/PDF25/25-179.pdf

The importance of this work is however jumping the gun a little, and so I will return back to it below.

Prior to the publication of the results by Clarke (and Ritland), observations on seasonal variations within Nymphalidae species lead to the conduction of cold shock experiments, such as those reported by Shapiro (1981) “Phenotypic plasticity in temperate and subarctic Nymphalis antiopa (Nymphalidae): Evidence for adaptive canalization”, of which the pdf can be found here:

peabody.research.yale.edu//jls/pdfs/1980s/1981/1981-35(2)124-Shapiro.pdf

In summary, 8hour old pupae were incubated for 2 weeks at 2C, prior to being returned back to normal (25C) incubation temperatures until eclosion. Please note that nearly all cold-shock literature available suggests 2C shock temperatures, not -20C (household freezer temperature) as you had previously reported and tested.

Similar effects to those induced by cold shock can also be induced via Tungstate injections, as reported by Otaki (2004), “Species-specific color-pattern modifications of butterfly Wings”. The pdf can be found here:

onlinelibrary.wiley.com/doi/10.1111/j.1440-169X.2004.00721.x/pdf

In opposition to the cold-shock (forward) method, heat-shock (reverse) treatment (as well as Thapsigasrgin injections) has also been shown to induce similar wing pattern aberrations in certain species. Data for this was also reported by Otaki (2007), “Reversed type of color-pattern modifications of butterfly wings: A physiological mechanism of wing-wide color-pattern determination”. The pdf can be found here:

www.sciencedirect.com/science?_ob=MiamiImageURL&_cid=271905&_user=776054&_pii=S0022191007000364&_check=y&_origin=&_coverDate=30-Jun-2007&view=c&wchp=dGLzVlk-zSkzV&md5=aa45f415acbd3e8db9d3c2243d4b6355/1-s2.0-S0022191007000364-main.pdf

This paper is exceptionally good as it reports on the effects of cold-shock, heat-shock, Tungstate injections, Thapsigasrgin injections, as well as other forms of treatments. Other equally good papers covering various forms of treatments (including UV) were also published by Kolyer (1970), “VARIATIONS IN THE MARKINGS OF PIERIS RAPAE (PIERIDAE) INDUCED DURING THE PUPAL STAGE”. The pdf can be found here:

peabody.research.yale.edu//jls/pdfs/1970s/1970/1970-24(2)125-Kolyer.pdf

Slightly different from Shapiro, Otaki chose to use 12hour old pupae for his experiments and cold shocked his pupae for 24 hours (at 2C), as well as heat shocking them for up to 3 days at varying (38C to 42C) temperatures.

As you had reported with your own experiments, cold and heat shock treatments of pupae has proved to induce very high morbidity rates and those that do hatch are often crippled. As a consequence of these results, Ritland’s (1986) paper (as mentioned above) demonstrated that aberrations could also be induced by raising the constant rearing temperature of species such as Papilio glaucus.

All in all, some very interesting reading. As you can see for yourself, all of this information is freely available online and very easily accessible. You just need to know where to look! Although I have tried to summarize some of the work relevant to this subject matter, I have actually only just scratched its surface. There is a much more out there to read on this (as well as every other subject), you just need to willingness to go and look for it!

Mark

[bluemoth]

Thank you so much irissicientist! I did not know ware to look to find this info. It will be a lot of interesting reading indead.

[corradocancemi]

Many thanks for your posts, guys! Irisscientist, your contribution is great ;D
This topic has become very interesting. If i undertand correctly, is almost impossibile to do this experiments with normal instruments we have at home, like a normal fridge or the air conditioning

[irisscientist]

Corrado Cancemi,

As with all forms of common, main-stream fields of science, Entomology is another example of science which at one time or another started at home. As a consequence, ingenious focused individuals have built/engineered equipment, which has enabled them to perform the functions they required. This I doubt is an exception to these rules, it just depends on the length you are willing to go to in order to achieve your own objectives? Modern science insists that data is reproducible and in order to comply with this, common variables (such as time and temperature) are nowadays tightly controlled. Ultimately however, where there is a will, you will find a way! In this particular instance, many modern fridges/freezers are nowadays fitted with digital, thermostatically controlled units and with internet sites like ebay, I assume these can be purchased (second hand) rather cheaply. How accurate they are however remains to be tested? Outside of this however there are always options to build/engineer your own, or alternatively pay the price and purchase one of the many which has already been specifically designed and engineered for that particular purposes. Like I said, it all depends on the level of commitment you are willing to go to in order to achieve your goals!


Mark

[Chris Grinter]

These devices are perfect for temperature controlled rearing (or brewing beer!). spyderrobotics.com/products/herpstat.html

[irisscientist]

Like I said, where there is a will!

It would appear however this is just a thermostat control until, which electronically controls the powder output to external devises which themselves heat or cool. I have one for my greenhouse temperature control (a Bio-Green thermo 2 thermostat). This one however has a minimum setting of 10C, and would therefore not reach temperatues as low as 2C. Many home beer brewing technologies however would be ideal for the kind of use we are talking about.


Mark

[papiliotheona]

In my opinion, experimenting like this, with the exception of extremely common stuff like V. cardui, is a waste of valuable pupae. Putting stuff in the freezer is a big nono as the temp inside a fridge is around -12 with is much more than just about any lab-reared lepidopteran can withstand. Some wild Papilio pupae can take this but only after being hardened off and acclimated to natural wild temperatures for many months, which doesn't occur in the lab.

If any of you want to try this, I'd suggest sticking a J'd or slung larva or fresh pupa in the fridge for a few hours.