[Terry]

Over the past couple of years I have been experimenting on the best technique for sterilising Yellow Admiral eggs. The main reason being my ongoing struggle with a disease that has been devastating my Yellow Admiral stock here in the UK. There is no doubt that the main problem is caused by very high densities of insects in a small confined area (10ft x 6ft x 7ft Greenhouse structure). The reasons for trying to maintain such high densities of insects is that Yellow Admirals are not native to the UK and therefore safety in numbers is a reasonable way of making sure I have plenty of time to pull the stock back should any generation fail to produce another. Over the years (15 to date) this has worked very well but recently I have encountered a disease known as wilt which has made the project very difficult to maintain with huge losses of larvae, mainly in the last instar but also at any of the earlier instars. I used to control the health of my stock by sterilising eggs in Formaldehyde solution but this is becoming more difficult to obtain due to it being very toxic if used without precautions, so I embarked on finding a suitable replacement.

I now use a common household cleaner/steriliser, Tradename, Domestos Extended germ kill. When I first tried this on my Yellow Admiral eggs I was convinced it would kill them even at dilute strengths or that at high dilution it would be rendered useless, however I was wrong and this product has proved to be an adequate replacement for formaldehyde.

One of the major differences of using this product instead of formaldehyde is that formaldehyde does not effect the glue like substance the female butterfly uses to stick the eggs to the leaf of the food-plant. Domestos solution dissolves this glue and therefore the eggs will drop to the bottom of the container used to sterilise the eggs in. I found this very frustrating to begin with but it has now been recognised as a significant advantage in achieving maximum hatch rate of the sterilised eggs.

I have found that Domestos is best used at a 10% solution to 90% water as any lower and it may not kill all the disease pathogens and any higher it can dissolve the egg shell and kill the developing larvae. It should also never be used on eggs laid within the previous 24 hours as the shells have not hardened off sufficiently. I normally soak the eggs for 15 minutes in this solution although I have on occasion left them for 20 minutes with few problems.

The major discovery/benefit from using this new sterilisation method is that I can now keep the eggs separate from the leaf they were laid on in a sterile plastic box completely dry and with no risk of mould destroying them before the larvae emerge. After the allotted time in the solution and after removing the leaves that the eggs were originally attached to, (still check as a few eggs still remain attached), I carefully drain away most of the solution by careful pouring, the eggs sink to the bottom so there is little chance if cautious of tipping them away as well. Then after filling this container up with the eggs still inside to full, with fresh water to dilute the domestos I get another sterile container, place a sheet of absorbent kitchen paper over this and after carefully draining the container with the eggs in to 1/4 full I pour the eggs onto the Kitchen paper and let the water drain through. When drained I carefully remove the paper with the eggs on it and place it on several other dry sheets of Kitchen paper to absorb more moisture and leave to dry for 20 minutes. When sufficiently dry I get the sterile plastic box I will store the eggs in and picking up the sheet with the eggs on, using an artists paint brush "carefully" using the tip of the brush sweep the eggs into the box. The eggs are quite hard and bounce like rubber when they hit the bottom of the container. Using this method the vast majority of the eggs will hatch and losses are minimal. One important thing to remember is that you must keep a lid on the box and check at least twice a day for emerging larvae which must be carefully moved using a paint brush to another container with the food-plants as they will starve quite quickly if forgotten. The plastic boxes I use for this sterilisation technique are 73mm x 73mm x 73mm Square stacking boxes as shown in this picture http://www.watdon.co.uk/the-naturalists/acatalog/Clear_plastic_boxes.html

http://www.domestos.co.uk/products/multiuse-bleach.php

If any of the monarch trust members get problems with diseased stock I hope this will help towards a quick and successful solution. I have never tried this on Monarch eggs so be very careful as this species eggs may not be as tough as the Admiral eggs. You may have to vary the experiment yourselves to get it just right for the species you are breeding.

[NormTwigge]

Pepetuna – As the human flu virus has different strains, so does NPV have numerous strains which can be insect specific ie – coeloptera, hymenoptera, lepidoptera etc. but not necessarily species specific within lepidoptera. In other words the same NPV that infects admirals can also infect monarchs. The disease must be ingested by the caterpillar to incubate the infection, so the fact that the two species have completely different hostplants will prevent that to some extent. However the virus can be trasmitted between totes or containers by handling a “clean” container after an infected one whether gloves are worn or not, and similarly the virions can be transmitted from one plant to another in the same way, so sterilisation is a big factor in controlling any disease. Your hygiene procedure must have at least been sufficient to prevent cross contamination to the monarchs, but obviously had a good foothold with the admirals. The butterfly farmers suggest destroying all plants and stock when it gains a foothold and start afresh again with everything. However that is not something that Terry can do with his Yellow Admirals and hence the ongoing battle.
Monarch Watch (USA) reports that NPV is the pathogen that affects monarch the most, but realise that USA may be different to NZ.

[Darren]

last one for the night, this paper shows that monarchs are affected by at least 5 different NPVs: AcMNPV-HPP, PxMNPV, AfMNPV, AgMNPV, and HzSNPV.

Establishment of a monarch butterfly (Danaus plexippus, Lepidoptera: Danaidae) cell line and its susceptibility to insect viruses. Arthur H. MCINTOSH and James J. GRASELA. Appl. Entomol. Zool. 44 (2): 331?336 (2009) retrieved from https://www.jstage.jst.go.jp/article/aez/44/2/44_2_331/_article

[Darren]

Excellent introduction to Baculoviruses (which include the NPVs):

http://www.microbiologybytes.com/virology/kalmakoff/baculo/baculo.html

It includes a great illustration of the polyhedra dissolving in the caterpillars midgut.
http://www.microbiologybytes.com/virology/kalmakoff/baculo/pics/NPVinfection.jpg

[Terry]

Very interesting research indeed!
I was going to suggest that maybe the Monarchs were not susceptible to this specific NPV virus that attacked Pepetuna’s Red Admirals. But before I could post, Darren reported this research, which makes perfect sense as in my butterfly house the species are all Nymphalidae.

[Darren]

NPVs are a group of viruses, which now seem to consist of a number of sub-groups. Some insects are infected by a number of NPVs from different sub-groups. NPVs can be host specific to varying degrees, so for example an NPV collected from a cabbage moth, Mamestra brassicae was tested on 66 lepidopterous species and found to infect 32 species from the Noctuidae, Geometridae, Yponomeutidae, and Nymphalidae families. However, 91% of the susceptible species were in the Noctuidae.

[Pepetuna]

When all of you have been researching NPV, have you found anything that suggests that the form of the virus is species-specific? I.e. if your Admirals get it can they or do they pass it on to other species such as Monarchs? The reason I ask, is that a) Terry seems to have suffered with it attacking both Admirals and Peacocks b) I had it in my Yellow Admirals, but there were never any signs of it in the Monarchs I raised last season. I did not raise the larvae together in the same containers, but I have serious doubts that my hygiene procedures were THAT good that there would have been no infection on my person or gear. I did use gloves (as Anna suggested a couple of posts ago) and I was careful with the order I did my “chores” but still couldn’t get rid of it in the Admirals and never got it with the Monarchs. Any ideas/comments?

[NormTwigge]

Darren I’m sure you are right when you say “our current discoveries have only scratched the surface”. With the rate technology and discovery is progressing, new information replaces or accompanies the old in a short space of time.
Imagine in years to come a treatment for disease infected farmed caterpillars. Perhaps a little far fetched, although I’m sure Terry, and myself, would welcome that with open arms if we were still around.

[Darren]

Norm, Altizer’s comment was published in 2008 and the book you reference was published in 2009. I don’t take her comment to mean no research has been done, afterall she has spent 15 years herself researching the subject, and she started as a student in Karen Oberhauser’s lab. In the mid 70s it was estimated that over a thousand entomopathogens had already been discovered. My interpretation of her comment is that our current discoveries may have only scratched the surface. They may be many more entomopathogens out there still awaiting discovery.

However since Terry is certain he has NPV virus it seems this isn’t the situation here.

[Terry]

There is no doubt about it! Wilt is the disease I have in my stock. Norm has described it in perfect detail. This also accounts for my sterilisation efforts partial success. I had commented on previous posts that I feared my stock may be becoming inbred only for them to miraculously bounce back but after careful consideration I am sure it is because of this disease and the way it spreads. Also as stated the virus is inside the egg and so I must try to control this by keeping the inside of the Butterfly House as clean as possible and keep the stock to a more reasonable density. I feel some new experiments coming on quite soon!

[NormTwigge]

Perhaps Professor Sonia Altizer made the statement before the
recent book “Principles and Procedures for Rearing High Quality Insects” was published. Edited by John C. Schneider, Department of Entomology and Plant Pathology of Mississippi State University, the content of the book deals exhaustively with technical research into problems encountered by commercial insect rearing facilities producing insects for biological control of crop pests such as caterpillars. A comprehensive chapter on diseases covers the subject in great detail, as disease is a major weapon in the war over crop pests.
Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide in New Zealand, and NPV (Nuclear polyhedrosis virus) which occurs naturally, is commercially produced for farmers in Australia to combat pests (caterpillars) in a variety of field crops, indicating much research has been done on diseases relating to lepidoptera. So it may appear the Professors comments could be outdated.

[Darren]

?For the vast majority of butterflies, we have no idea what fungi, what viruses, what bacteria are out there, and it?s mainly because nobody?s looked.? Professor Sonia Altizer

[NormTwigge]

Entomopathogens, and in particular Lepidoptera diseases is a subject that I find interesting and have sourced as much information as I can. The diseases comprise of virus,bacteria,fungi,protozoa and microsporidia, and nematodes. Wilt is recognised by the larva stopping feeding,climbing to a high point on the plant and eventually hanging by its claspers in an inverted V. The internal contents of the larva liquify which drips from the head, the skin can rupture releasing millions of virions which can infect the plant and containers.
However the virus can be killed by exposure to ultraviolet light in sunlight, high temperatures and of course sterilisation. In a typical butterfly house with shade cloth or similar covering the UV probably does not penetrate, and virus particles can remain active for some months.

[Terry]

Many thanks for all the feedback!
I have come to the conclusion that I will probably just have to soldier on the best I can with this disease and experiment along the way with different methods to try and control it. I really have been fortunate that over all the years when I could have had this particularly nasty disease in my Butterfly House I had somehow avoided it! Maybe I introduced it with the Small Tortoiseshell and Peacock collected from the wild 2 years ago, because other introductions over the preceding 15 years, vanessa atalanta, aglais milberti, cynthia cardui, Bassaris gonerilla, and additional species, produced no problems. I should have stuck with just the Vanessa Itea and maybe all would still be well? But that’s just guessing and what I have to do now is cope with the situation in hand. Maybe other enthusiasts have something to add to this thread that could broaden our knowledge further?

[Anna]

Yes Darren its something like this though the fungi that occurred on mine was white. (By the way this was on caterpillars that were on a nettle in the garden, so it wasn’t in cramped conditions or anything)
Isn’t the bug world interesting??

I googled the name, then clicked on images, and found all sorts of photos with the fungi and websites to go to.

[Pepetuna]

I’ve seen Monarch larvae with the fungal Metarhizium, and they look quite different to wilt. The infected larvae don’t liquefy, but get covered with a kind of pale greenish-beige fungus and seem quite stiff and hard when they die.

BTW, I don’t think Norm said “bleaching won’t stop the NPV virus”. I think Norm was saying sterilizing the eggs won’t stop the NPV virus, whatever sterilizing agent is used. The virus is not in spores on the outside of the egg (like Oe) but if it is passed on by an infected parent to the eggs, it is INSIDE the egg.

[Darren]

Is this it?
http://en.wikipedia.org/wiki/Metarhizium_anisopliae

[Anna]

Does anyone know about Metorhizium? I read that its a fungi that kills insects to supply plants with nitrogen. I think I got a bit of something like that at the end of last breeding season. I also had some with a kind of wilt like Terry mentioned. Fortunatley it didn’t get too many though.
I find disposible gloves great.

[Darren]

Well I’m certainly no expert, but reading the above something struck me.

1)Terry says bleaching is helping him fight “a disease known as wilt”.
2)Norm says bleaching won’t stop the NPV virus.

Might it be possible that Terry’s “wilt” is not being caused by the NPV virus, but by some other parasite (viruses, bacteria, fungi, protozoans, nematodes, or mites)?

[Pepetuna]

I have just realised that the previous post looks like I am trying to tell Terry what to do – and that wasn’t my intention at all. The “you” in the above post refers to anyone here in New Zealand who gets hit with NPV in Admirals, as I have done. Terry has been raising those Yellow Admirals successfully for 15 years, and is obviously an extremely competent breeder. However, I do stand by my suggestion to people here that they safely destroy infected larvae and follow preventative hygiene measures.

[Pepetuna]

Yes, I go along with Norm on this – to protect against “wilt” or NPV, it is the plant material, the containers, your hands etc etc (anything that comes in contact with the larvae) that you must sterilise, rather than the eggs. There are good notes on preventative treatment here http://butterflybreeders.org/public/health/disease_prevention_Polyhedrosis.html

I am not criticising Terry’s raising methods – he is obviously a very competent and successful breeder to have kept the Yellow Admirals going through so many generations over such a long period, and I avidly read his posts on the Yellow Admiral project. But here in New Zealand where we do have the option to destroy the whole infected batch, we should do so to prevent further infection. And we should use tried and true hygiene procedures to make sure we don’t keep subjecting subsequent batches of caterpillars to this very nasty virus.

In summary, if you get it in a batch of Admiral caterpillars, you should destroy them (sadly), and use bleach to sterilise the plant material, caterpillar castles or plastic containers, your hands, your overalls and gumboots etc for any subsequent batches of caterpillars. And you should take steps to prevent any moisture build-up in plastic containers if you are using them to rear caterpillars.

[Terry]

That is a very interesting post Norm! If the virus is inside the egg then I must make a guess that some virus must be on the outside as well because this sterilisation technique is definitely having an effect and my losses are much less than with non sterilised eggs. However you have given me an idea although very risky knowing how toxic this chemical can be at the wrong dose, that maybe it could be sprayed directly on to the nettles as they regenerate outside of the Butterfly House and thus when they are placed back inside the virus will be at a lower level. I once observed how my father had used domestos to sterilise his Pigeon drinkers (He keeps racing pigeons) and then emptied the contents against a fence where urtica dioica grows. I thought it would kill them outright but it had little effect, just a very slight discolouring of the leaves, but they recovered and soon looked normal again. Maybe the nettles can draw this chemical into the system and neutralise this virus. It’s just a thought, and as I have admitted I am no chemist so if this is a stupid suggestion I will be prepared for some criticism from the more knowledgeable members of Monarch Trust. Another problem of rearing Butterflies inside the Butterfly House is that the glass and netting filters out much of the ultra violet range from the sunlight and this of course is one of natures way of controlling these types of diseases in the wild. The best way for me to go of course would be to have 2 Butterfly Houses and let one remain dormant for a year at a time. Not forgetting in an ideal situation I could keep the stock at more normal less overcrowded densities but other lepidopterists who do this find that if something goes wrong the stock is lost completely with no chance of saving it apart from getting fresh wild stock and this is why I opted for the safety in numbers approach. I do feel I have been very fortunate to have managed to keep this project going for so long with so few major disease outbreaks up until recently, so I have no reason to feel aggrieved by the current situation, it’s more of an interesting challenge which ever way it turns out in the end.

[NormTwigge]

Bleach (Sodium hypochlorite) as a diluted solution is the standard sterilising agent used by commercial butterfly farmers to kill Oe spores (a protozoan parasite) on monarch butterfly eggs, which an infected female butterfly transfers to the surface of the egg when she oviposits. An emerging caterpillar eats its way out of the shell and by doing so ingests the Oe spores which then propagates the disease, hence the sterilising process.

Wilt is the common name for NPV (Nuclear polyhedrosis virus) which is a disease that occurs occasionally in the wild, but more prominently in crowded conditions where many caterpillars are bred. Entomological researchers have found that stress created by crowded conditions can start the disease, which can spread rapidly through a colony.

The frazz (droppings) of an infected caterpillar can contaminate the leaf with the virus, and then ingested by another caterpillar, thus passing on the disease. The virus can remain on a plant for a long period and spread by contact.
Rarely does an infected caterpillar make it through to pupation, but if it does the emerging adult harbours the disease and a female can deposit infected eggs. However the virus is contained INSIDE the egg and therefore sterilising the eggs will not eliminate it.

A google search on ?Nuclear Polyhedrosis Virus? will access lots of information.

[Darren]

My guess is that it is the Sodium Dichloro Isocyanurate Dihydrate(NaDCC)which is giving you the germ killing you wanted, and the surfactant Sodium Lauryl Sulphate which is dissolving the “glue”, as Sodium Lauryl Sulphate (SLS) aka Sodium dodecyl sulfate (SDS) is used in laboratories for unravelling protein molecules.

The perfumes, fillers, “Aqua” (like water only much more expensive! ;-> ) and so forth probably don’t do much, so you may be able to get a similar but cheaper effect with swimming pool disinfectant (NaDCC) and shampoo (SDS). But if you find domestos good value for money its probably easier to just stick with that.

[Terry]

Wow that’s a lot of different chemicals! I am amazed it doesn’t kill the eggs but even as I write this I have many Yellow Admiral larvae hatching from eggs in the plastic boxes behind me on the table. And using the new technique of dry eggs loose in the clean dry plastic boxes I am getting approx 99% emergence.

[Darren]

From information on the unilever website:
Domestos contains:

Sodium Dodecylbenzenesulfonate Surfactant
Sodium sulfate Builder
Calcium carbonate Bulking Agent
Sodium Dichloro Isocyanurate Dihydrate Oxidising Agent
Sodium Lauryl Sulfate Surfactant
Parfum Fragrance
Ethylenediamine/Stearyl Dimer Dilinoleate Copolymer Binder
Pentasodium triphosphate pH Adjuster
Aqua Process by-product
Sodium citrate pH Adjuster
Non-Detergent Organic Matter Process by-product
Calcium Sodium EDTMP Sequestrant
Lauryl Alcohol Process by-product
Mica Pearlescer
Butylphenyl Methylpropional Fragrance
Coumarin Fragrance
CI 77266 Colourant

[Author]

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