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How to collect beetles for DNA studies?

Preserving beetles for DNA studies is easy, but a few rules need to be followed.

You will first need to decide which specimens to preserve. It is ideal to have two or more specimens of a species preserved, so that the extra specimens can serve as backups in case the first specimen fails to yield good DNA. Also, because the specimen from which DNA sequences will be obtained serves as the voucher, it is important to choose the gender that contains the key morphological characters for distinguishing species. This will allow the DNA data to be properly associated with the morphological data, type specimens, etc. For Bembidion, males contain the most diagnostic characters, and so males should be preferentially preserved for DNA. The general rule of thumb is that whatever gender is best for a holotype is the gender you should choose for DNA preservation.

Exactly how a specimen should be preserved depends upon the materials you have available. I use one of two preservation methods: in 95-100% ethanol, or in silica gel. One of the most important things that both of these do is to remove water from the beetle’s tissue, which prevents nasty enzymes from destroying the DNA. Here are some procedures I use to ensure high-quality DNA.

Ethanol preservation
95-100% ethanol is ideal; lower concentration doesn’t work as well. Drop the live beetles into ethanol. Make sure there aren’t too many beetles in the vial; ideally there is at least four times as much ethanol as there is beetle mass. The vial on the left below will have well-preserved DNA; the vial on the right is too tightly packed with beetles, and the DNA won’t be as high quality website here.

Charge of ethanol
right charge of ethanol

Once the beetles have all died, it is ideal to pour off the ethanol and replace it with fresh ethanol. If you can’t do that right away, that’s OK; but you should change the ethanol in the next day or two. If you can change the ethanol again a few weeks later, that would be even better.

It can also help a great deal to open the body of the beetle up so that ethanol can penetrate, especially if the specimen is large or has a very thick cuticle. When I collect, I usually open up one or two specimens this way (and preserve a few other specimens whole, without opening them up), to ensure that at least those specimens will have excellent DNA. The simplest way to do this is to hold the specimen between the thumb and forefinger in one hand, such that the abdomen is exposed, and take forceps and gently pull the abdomen slightly off, as shown below. It is best if you don’t take the abdomen off completely; that way, the abdomen is kept associated with the forebody.

dissection of the beetle

The advantage of a dissection like this is the specimen can then have the soft tissue removed for DNA extraction, and the rest of the body is in great shape for a morphological specimen. If I want an even better morphological specimen (for example, for a holotype), after the dissection I pull off the soft tissue and put it into ethanol, and put the body into sawdust and ethyl acetate (with labels to associate the two pieces, of course).

Finally, the specimen should be kept as cool as possible; if you can keep it in a fridge, or a non-defrosting freezer, the DNA will be better preserved.

Thus, the ideal is to:

  •     use 95-100% ethanol
  •     don’t put too many beetles in each vial
  •     change the ethanol twice
  •     open up the specimen slightly to allow the ethanol to penetrate
    (optional – will improve DNA quality but if other steps are  followed the DNA will still be OK)
  •     keep the specimens cool

While this is the ideal approach, most specimens will be well preserved if you just do the first three of these. Certainly, if you can only manage the first three of these, that is much better than no specimen at all.

If you don’t have 95-100% ethanol, an alternative that I have used successfully is to boil the live beetle in water for 30 seconds, and preserve it in 80% ethanol. The DNA seems to be OK in 80% ethanol for at least a few weeks if it is boiled first.

Silica gel
Another approach is to preserve specimens with silica gel. This yields very good DNA, but very brittle specimens that break in pieces.Check this website.

Prepare each vial in advance by filling it half-full of dried silica gel. It is ideal to use indicating silica gel that changes color when it absorbs water; this allows you to see if the silica gel is still good, as it must be very dry to work well. (If it has changed color because it has absorbed water, you can dry it out by baking it in an oven.) Put a cotton plug on top of the silica gel to hold the silica gel in place in the vial. (Without the cotton plug, the silica gel will roll around and destroy the specimen.) Tighten the lid, and the vial is ready for use.

silica gel
right amount of silica gel

To preserve a specimen, open up the prepared vial, and put one to three live specimens in the top of the vial, above the cotton plug. Add whatever label you wish to use, and then close the lid tightly. The silica gel will dry out the beetle and preserve the DNA.

I use clear plastic vials with screw-top lids that contain an O-ring for a tighter seal. Here’s what they look like:

different sizes of vials

The larger vial is about 65 mm long (with the lid on) by 15 mm wide, and is available from Sarstedt (their catalogue number 60.542.007). The smaller vial is 46 mm long by 11 mm wide, and is available from USA Scientific (their catalogue number 1420-9700).

UPDATE: Kip Will notes that he uses a slightly different procedures for the much larger beetles he works on. He twists them at the prothorax-mesothorax junction and break them open there so the ethanol can penetrate. For the larger specimens he changes the ethanol three times. If they are >25mm then he puts a leg or two separately into ethanol.

Written by David Maddison

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Why beetles have “hard” elytra?

One of the most important features of Coleoptera is their ‘elytra’, the hard exoskeletal which covers their wings. The ‘elytra’ helps to protect the beetle but also has many other functions, too. Some beetles trap moisture in their wings and the elytra protects it from drying in heat and wind, this means the beetles can travel across arid deserts without dehydrating. Other Coleoptera can live under water because they can store air in their wings, which is again protected by the elytra. Coleoptera (beetles) are most probably the most versatile creatures on earth. The beetles exoskeleton is made up of numerous plates called sclerites (a hardened body part), separated by thin sutures. This design creates the armoured defenses of the beetle while maintaining flexibility check.

The morphology of a fiddler beetle

Scientists interested in cuticle structure[1] have examined cuticle from the elytra of the red flour beetle, Tribolium castaneum. The elytra have two proteins in large amounts that are not present in the membranous hindwings. These proteins are associated with hard cuticle both in the elytra and elsewhere on the beetle. The cuticle of the elytra becomes hard and rigid from extensive crosslinking, or chemical connections between the protein strands. This evidence from red flour beetle suggests that in the evolutionary past, an ancestor of modern beetles had a mutation caused proteins for cuticle crosslinking to be expressed in the forewings. The up regulation of two cuticle genes in the forewings may be a key to the evolution of elytra withえろたれす/.

The elytra are not used for flight, but tend to cover the hind part of the body and protect the second pair of wings. The elytra must be raised in order to move the hind flight wings. A beetles flight wings are crossed with veins and are folded after landing, often along these veins, and are stored below the elytra.

Cerymbycid beetle ready for takeoff [3]

In some beetles, the ability to fly has been lost. These include the ground beetles (family Carabidae) and some ‘true weevils’ (family Curculionidae), but also some desert and cave-dwelling species of other families. Many of these species have the two elytra fused together, forming a solid shield over the abdomen. In a few beetle families, both the ability to fly and the elytra have been lost, with the best known example being the glow-worms of the family Phengodidae, in which the females are larviform (where the females in the adult stage of metamorphosis resemble the larvae to various degrees) throughout their lives.

“Nature is replete with examples of layered-structure materials that are evolved through billions of years to provide high performance. Insect elytra (a portion of the exoskeleton) have evoked worldwide research attention and are believed to serve as fuselages and wings of natural aircraft. This work focuses on the relationship between structure, mechanical behavior, and failure mechanisms of the elytra. We report a failure-mode-optimization (FMO) mechanism that can explain elytra’s mechanical behaviors. We show initial evidence that this mechanism makes bio-structures of low-strength materials strong and ductile that can effectively resist shear forces and crack growth. A bio-inspired design of a joint by using the FMO mechanism has been proved by experiments to have a potential to increase the interface shear strength as high as about 2.5 times. The FMO mechanism, which is based on the new concept of property-structure synergetic coupling proposed in this work, offer some thoughts to deal with the notoriously difficult problem of interface strength and to reduce catastrophic failure events.” [2]


[1] Mi Young Noh, Karl J. Kramer, Subbaratnam Muthukrishnan, Michael R. Kanost, Richard W. Beeman, Yasuyuki Arakane. Two major cuticular proteins are required for assembly of horizontal laminae and vertical pore canals in rigid cuticle of Tribolium castaneum. Insect Biochemistry and Molecular Biology. 53: 22-29.

[2] Fan, J.; Chen, B.; Gao, Z.; Xiang, C. 2005. Mechanisms in Failure Prevention of Bio-Materials and Bio-Structures. Mechanics of Advanced Materials and Structures. 12(3): 229-237.

[3] Species 2000 & ITIS Catalogue of Life