The selection of a certain preservation method is dependant on whether one is on expedition or at home and the final purpose of the specimens. Is it meant to be for exhibitional purpose with the aim of a good colour preservation, or for scientific study which requires conservation of the original body shape and other important structures ?
In the following, different preservation techniques which were tested by us are summarized with their advantages and disadvantages discussed.
1. Immersion in acetone
In this method the set phasmids are immersed head foremost in acetone. Airtight plastic-boxes are suitable containers to store the acetone and are filled with a layer of at least 5 cm. Depending on their size, the phasmids should be left in the acetone for 3-10 days and need to be dried for 2-3 days afterwards. Drying after the immersion in acetone can be achieved by wrapping the setting board with newspaper and placing it in the sun, putting it on a heater or in a drying cupboard. When drying in the sun, the newspaper is sufficient to prevent bleaching by the sunlight. Always ensure that there is enough air circulation as this speeds up the drying process and never dry the setting boards and phasmids in a closed room !
Immersion in acetone works very well with brown phasmids of all sizes and the natural body shape is well preserved. The disadvantage of this method is clearly that the acetone will discolour green or very colourful phasmids. Adverse effects on the insects are not known, but most certainly the acetone removes lipids from the cuticle. Due to the space, weight and quantity of acetone required,this is not a suitable method for expeditions.
2. Immersion in a Bohric solution: isopropanol (98%) and formaldehyde (35%)
This technique is similar to method No. 1 and once again involves the use of large airtight plastic containers which are filled with at least 5 cm chemical solution. The set phasmids are placed head foremost in a solution of 1000 parts isopropanol (98 %) and 30 parts formaldehyde (35 %). Independant from their size, the phasmids need to remain in the solution for 10-14 days and are dried in the sun or on a heater for 2-3 days afterwards. The setting boards and phasmids should never be dried in closed rooms, and caution is needed due to the toxic nature of formaldehyde. It has proven best to wrap the complete setting board in newspaper and place it in the sun or to put it on a heater. Always ensure that there is plenty of fresh air when the insects are dried indoors (e.g. by opening a window) !
Fresh solutions should only be used for brown phasmids, as the isopropanol will discolour green or other colourful species. Older solutions will discolour the insects less seriously and it is recommended to use a separate solution for green phasmids only.
This method is almost perfect for brown phasmids of all sizes but the disadvantages are that formaldehyde is highly toxic and that the solution seriously discolours green phasmids. Often green insects will discolour even more after dehydration or change colour to pale brown. Better results concerning the preservation of green colour may be achieved by including cane-sugar in the solution. Apart from the mentioned disadvantages, the method is very satisfactory for preserving the original body shape and the dried phasmids can be easily relaxed by placing them in hot water steam. Due to the space, weight, airline safety regulations and quantity of solution required, thi is is not a suitable method for field conditions.
3. Drying with silica-gel
Drying phasmids in a box with a large quantity of silica gel is an effective method and is preferably used by many entomologist to preserve Orthoptera, dragonflies (Odonata) or various Rhynchota. Silica gel are small, spherical crystals which have innumerable hollow spaces in their interior. The crystals of this silicate- connection are able to deprive water from their surrounding and can take up as much water as 1/3 of their own mass. Heat (up to 130 °C) will make the crystals loose the water again. Thus, the silica gel can be easily regenerated in ones oven and may be used several hundred times. If applicated to often, the silica gel will loose its water depriving effect due to giving off “dust”. This means, that because of movement, tiny fractions of the crystals brake off and stuff the hollow spaces, so that no water can be sucked up anymore. This is seen by a white dusky layer on the surface of the crystals. Silica gel can be obtained in different colours (blue, orange, red) and will change its colour as soon as it takes up water; the more water, the stronger colour is changed. Blue silica gel will change its colour to red or pink and orange silica gel will become slightly bluish or transparent.This colour indicator allows to see how much water has already been taken up and makes it easier to decide wheter more fresh silica gel is required to dry the insects properly.
The use of silica gel is rather easy and the insects simly need to be stored in an airtight plastic container with a large amount of silica gel. On expeditions some silica gel can be included in each paper- or butterfly-bag depening on the size and body mass of the phasmids. The phasmids themselves are best placed on a soft layer of material which also sucks up water, like e.g. cotton-wool or tissue. Then the paper packets (à see „Packing & Transport“) filled with phasmids and silca gel are stored in an airtight container and further silica gel may be added in the container to ensure proper drying, in particular if larger species are inculded. When the silica-gel has completely changed colour, it can be poured away and regenerated. Back home, the dried and papered phasmids are relaxed by placing them in hot water steam (à see “Setting & Repair”) and can be set in the usual manner. However, care must be taken that no limbes are broken off when removing the phasmids from the paper packets.
4. Immersion in ethanol (70%)
Immersion in 70% ethanol is the traditional method to preserve animals, plants or organs in most subjects of biology and medicine. Followingly, most of the historic material from museum collections was originally preserved in ethanol and pinned and dried subsequently. The method works very well with organs and most animals like fishes and small mammals but is rather unsatisfactory with phasmids.
This method will effectively preserve the phasmids but will seriously discolour it, turning reen, red or blue to pale yellow or brown. The phasmids will remain flexible for many years, but the joints of the antennae and legs become stiff which makes subsequent setting of the insects difficult, or almost impossible as the legs can not be unfolded. If more than one specimen is put into a container the legs will become tangled and damaged and very soft bodied species will almost completely disintegrate after some years. For expeditions it is unsuitable due to the space, weight, quantity of ethanol required and airline safety regulations which prohibit the transport of inflammable liquids.
Suitable containers are airtight plastic- or glass-bottles or tubes. The collecting data can be included on a small paper label and need to be written with pencil, as this is resistant to ethanol.
5. Drying in heat or an air-conditioned environment
Brown and very thin phasmids (e.g. males of Ramulus or Gratidia) can be easily dried without the use of chemical solutions or silica gel. Possibilities are to place the insects in an air-conditioned room, which have a very cool and dry atmosphere, placing them on a heater or drying them in the sun. All three methods may allow drying before rotting occurs, but are best combined with other methods (e.g. injection of 98% ethanol), especially for larger species. Drying untreated phasmids in direct sunlight is quick and effective for most smaller species, but the intense sunlight destroys almost all the colour. To prevent the insects from bleaching by the sunlight, the setting board should be wrapped in newspaper.
6. Injecting with ethanol (98%) or formalin
A fine hypodermic needle or drain tube and a small syringe can be used to inject 98% ethanol or formalin. The needle is best introduced between the meso- and metathorax and liquid is injected until it emerges from the mouth and anus of the phasmid. If the ethanol or formalin does not emerge from both body ends, a second injection is required at the appropriate end to ensure the body cavity is completely irrigated. Both chemicals preserve the phasmid well but will cause some loss of colour; especially ethanol will make some green pigmentation become yellow, but generally less than which occurs if the insect is dried in direct sunlight or completele immersed in ethanol. Formalin will even have less effect on green pigmentation but caution is needed, due to the highly toxic nature of this chemical.
Both chemicals will effectively preserve the phasmid but no drying process is achieved by injection only. The insects need then to be left in either in sunlight (wrapped in newspaper), on a heater, stored in a drying cupboard or placed in an airtight container with plenty of silica gel. The method is useful for expeditions but only works well with slender phasmids up to 10 cm (not larger than e.g. Lonchodes, Aretaon, Dares)
7. Evisceration and stuffing with cotton-wool
This method has been suggested by several authors for large, soft-bodied Orthoptera like e.g. large Ensifera (long-horned grasshoppers) or crickets and is often recommended for large phasmids as well. The colour of the phasmids is well preserved but the method also shows clear disadvantages, why it is only recommended for specimens meant to be for exhibitional purpose or when numerous dublicates are present in one´s scientific collection. Evisceration destroys most of the internal morphological features, some of which might have to be examined at a later date. Furthermore, it is very difficult to retain the original body shape of very thin or soft-bodied species and much care must be taken, that the internal pigmentation is not accidentally rubbed off. Nevertheless, if evisceration and stuffing with cotton-wool is well done , the insects dry very quickly and the original colours are almost perfectly retained.
There are numerous possibilities to place the cuts on the ventral body surface of a phasmid to eviscerate. In order to make only short cuts it is best to use a scalpel or fine scissors to make either a transverse cut between the metasternum and the 2nd abdominal sternite (1st sternite fused with metasternum): For larger and very long phasmids it is recommended to make a longitudinal cut along the lateral margin of the metasternum and a further cut along the lateral margins of the 4th and 5th abdominal sternites. Then the viscera is removed by using different pairs of forceps, depending on the size of the phasmid, but care must be taken as the pigmentation (colour) is easily rubbed off the inside of the cuticle. Afterwards, the insect is stuffed with white cotton-wool which is a time-consuming process. Although other colours (e.g. green cotton-wool for green phasmids) may give better looking, the use of white cotton-wool is preferable as it is less likely to be mistaken for the natural colour in places where the internal pigmentation was accidentially rubbed off. Before stuffing, large species can be placed in a solution of isopropanol and formaldehyde (à see method No. 2) or Bohric solution for a few minutes, which gives good results with green species (e.g. Phyllium, Diapherodes, Eurycnema) and speeds up the drying process. Finally the stuffed phasmids are pinned, set and placed on a heater or in the sunlight. In the latter case, the setting board needs to wrapped in newspaper, as the direct sunlight would seriously bleach the colours.
8. Freeze drying
Freeze drying is undoubtly one of the best and satisfying methods to preserve animals or plants and gives almost perfect results with phasmids. The preservation of colour and body shape is stunning and even very colourful species will remain in perfect condition almost appearing as being still alive !. However, the method is obviously not a possibility when on expedition, and is also unsuitable when more than a few specimens are wished to be preserved. Furthermore, it is connected with high financial expenses and mostly used by large institutions for preserving larger animals like fishes, amphibians or small mammals. As a special, very expensive freeze-drying apparatus is needed, only a brief explanation is given below.
The process of freeze drying is quite simple but can only be achieved with the necessary equipment. Freeze-dryers are produced in small numbers in the USA and can be ordered for private use but are very expensive. Mostly the apparatus is based on a modified freezer and works as follows: The specimens are set and afterwards frozen in a vacuum-chamber. From time to time a thermostat allows the specimens to thaw. When slightly thawed a small pump, which is attached to the outer wall of the vacuum-chamber, deprives all free water molecules. This process is repeated until the specimen is completely dehydrate.