Beetles that keep their supercool

A few days ago, I wrote about antifreeze proteins in overwintering plants, during which I mentioned in passing that there is a similar system in some insects, and that I might write about that too. So, here goes...

Winter conditions in southern England

One of the key concepts here is 'thermal hysteresis' (TH), the difference that antifreeze chemicals (usually proteins, but there are exceptions) create between the melting and freezing points, thus inhibiting ice formation and crystal growth. In fish this effect can reduce the freezing point by up to 1.5°C, and in plants the effect is weaker, but in insects, it is much stronger, reflecting the colder temperatures experienced on land than in water (plants don't show this, but are very differently organised both morphologically and biochemically). For example, despite being intolerant to freezing, the Spruce Budworm moth Choristoneura fumiferana (family Tortricidae) can survive to around –30 °C due to the presence of antifreeze proteins (e.g. Doucet et al. 2002, Qin et al. 2007). More impressively, the Alaskan beetle Upis ceramboides (family Tenebrionidae) survives conditions as cold as –60 °C using a non-protein TH chemical called xylomannan (Walters et al. 2009) which is a combination of sugars (sacchardies) and fatty acids (Ishiwata et al. 2011) in the cell membrane where it appears to function by suppressing the freezing of water molecules within cells. Interestingly, xylomannan was already known to be present in the red seaweed Nothogenia fastigiata, and research on this has shown it to have anti-viral effects by inhibiting replication, including types of herpes, influenza and (to a lesser extent) HIV, among others (Damonte et al. 1994).

The Alaskan-Canadian 'red flat bark beetle' Cucujus clavipes puniceus (family Cucujidae) is another that can survive extremely low temperatures (some individuals 'supercooling' to -100 °C in the lab!), in this case due to the more typical TH proteins and also losing 60-70% of its water content in winter (presumably reducing the among that has to be prevented from freezing) (Sformo et al. 2010, Carrasco et al. 2012).

I could go on - there are plenty of other examples even if some of the mechanisms and biochemistry are not fullt understood - but the point is that (1) there are processes here which require more study (anti-virals anyone!) and (2) wherever we look, life is more resilient than we imagine, with tardigrades and bacteria able to survive in space and much work being done on 'extremophiles' in places such as hot springs and hydrothermal vents as well as the frozen Arctic. Maybe time for a bet at Ladbrokes on life being found in the liquid interior of Europa...

References

Carrasco, M.A., Buechler, S.A., Arnold, R.J., Sformo, T., Barnes, B.M. & Duman, J.G. (2012). Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics. Journal of Proteomics 75(4):1220-1234.
Damonte. E., Neyts, J., Pujol, C.A., Snoeck, R., Andrei, G., Ikeda, S., Witvrouw, M., Reymen, D., Haines, H. & Matulewicz, M.C. (1994). Antiviral activity of a sulphated polysaccharide from the red seaweed Nothogenia fastigiata. Biochemical Pharmacology 47(12): 2187-2192.
Doucet, D., Tyshenko, M.G., Davies, P.L. & Walker, V.K. (2002). A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferana. European Journal of Biochemistry 269(1): 38-46.
Ishiwata, A., Sakurai. A., Nishimiya, Y., Tsuda, S. & Ito, Y. (2011). Synthetic study and structural analysis of the antifreeze agent xylomannan from Upis ceramboides. Journal of the American Chemical Society
133(48): 19524-19535.
Qin, W, Doucet, D., Tyshenko, M.G. & Walker, V.K. (2007). Transcription of antifreeze protein genes in Choristoneura fumiferana. Insect Molecular Biology 16(4): 423-434.
Sformo, T., Walters, K., Jeannet, K., Wowk, B., Fahy, G.M., Barnes, B.M. & Duman, J.G. (2010). Deep supercooling, vitrification and limited survival to -100 °C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae. Journal of Experimental Biology 213(3): 502-509.
Walters, K.R., Serianni, A.S., Sformo, T., Barnes, B.M. & Duman, J.G. (2009). A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides. Proceedings of the National Academy of Sciences of the USA 106(48): 20210–20215.

Deadly crystals: keeping the ice at bay

Looking at our frozen-solid garden pond, I couldn't help notice how healthy the water plants looked - they might be encased in ice in sub-zero temperatures, but they clearly hadn't been destroyed (just think of the soggy green mush that's left if you freeze lettuce). This got me thinking - it isn't the temperature per se that causes damage, it's the formation of ice crystals in cells and tissues.

Water mint (Mentha aquatica) in our frozen pond.

Many animals have antifreeze chemicals in their blood and tissues e.g. cold-water fish such as the largely Antarctic notothenioids and the unrelated Arctic cod (Boreogadus saida). These have various types of glycoproteins that bind to small ice crystals, preventing them from growing and/or recrystallising (and in some cases there are systems allowing resistance to the damage caused by ice crystal formation). The precise mechanisms of the various classes of antifreeze protein aren't fully understood, but research has continued (e.g. Wierzbicki et al. 2007), with the shapes and orientations of the proteins being clearly important (as in enzymes) with effects seen at the ice-water interface where ice has many different faces for potential binding. So, what's the situation in plants? In broad terms it's quite similar - many overwintering plants produce antifreeze proteins that work in a similar way to those of fish, but there are some differences.

The first of these relates to the concept of 'thermal hysteresis'. This is the difference that antifreeze proteins create between the melting and freezing points, thus inhibiting ice formation and crystal growth. In fish this effect can reduce the freezing point by up to 1.5°C, but in plants the effect is weaker. Insect antifreeze proteins, by the way, have a much stronger hysteresis effect and are not all proteins (e.g. Walters et al. 2009 who report one comprised of saccharides and fatty acids, found in an Alaskan beetle) - maybe something for a future post...

Secondly, their function appears to be more to do with inhibiting the recrystallization of ice rather than  preventing its formation (Griffith & Yaish 2004). They also have a different evolutionary origin, mostly having developed from proteins involved in tackling pathogens - indeed, some retain antifungal properties. In hindsight, maybe this isn't so surprising as a protein that can bind to a pathogen might plausably have a straightforward evolutionary path to be able to bind to somthing else.

Lastly, unlike the equivalent proteins found in fish and insects, plant antifreeze proteins have multiple ice-binding points (Griffith & Yaish 2004), though it is unknown (as far as I am aware) whether this is directly adaptive or simply a left-over from its pathogen-fighting evolutionary history.

So, next time you see a healthy looking plant in wintry conditions, these are the sorts of biochemical shenanigans going on inside - and a subject where a bit of extra biophysical amd molecular research could yield genuine breakthroughs.

References

Griffith, M. & Yaish, M.W. (2004). Antifreeze proteins in overwintering plants: a tale of two activities. Trends in Plant Science 9(8): 399–405.
Walters, K.R., Serianni, A.S., Sformo, T., Barnes, B.M. & Duman, J.G. (2009). A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides. Proceedings of the National Academy of Sciences of the USA 106(48): 20210–20215.

Wierzbicki, A., Dalal, P., Cheatham, T.E., Knickelbein, J.E., Haymet, A.D.J. & Madura, J.D. (2007). Antifreeze proteins at the ice/water interface: three calculated discriminating properties for orientation of Type I proteins. Biophysical Journal 93(5): 1442–1451.

Leaf-beetle Larval Challenge

A few years ago I took on the role of organiser of the UK's Chrysomelidae Recording Scheme. At that time there was no up-to-date identification guide to this family of beetles - to become familiar with them meant spending a lot of time and/or money accumulating papers from a variety of sources, some rather obscure. Fortunately the landmark publication of 'the Atlas' (Cox 2007) listed many of these so it was at least feasible to find them. Then I decided that there should be a new and affordable key - everyone knew this to be honest, but I had both the time (sort of) and stubborn-ness/drive (plenty of that) to write it myself, and so a couple of years later, Hubble (2012) was born! Of course, this isn't the end of the story - keys are after all artificial things and different approaches can be taken. For example, Andrew Duff's 'Beetles of Britain & Ireland' will take a different approach when the Chrysomelid volume comes out (vol. I is here) and that's most welcome. Some genera are inherently tricky (Longitarsus springs to mind...) so having alternative ways of separating them is likely to be helpful, not to mention covering a range of budgets.

However, there is another way in which the story continues. These and almost any other guides you can find cover adults - eggs, larvae and pupae are rarely mentioned. Of course, it is adults that are usually found and which form the core of any biological collection. However, it does mean much of the group's life history is neglected. There are good reasons for this - juvenile stages tend to be small, hidden away (not always the case for larvae though - see below) and tricky to identify, the latter because in many cases they've never knowingly been found. A quick skim through 'the Atlas' shows just how often the words 'Larva - undescribed' are used plus of course larvae come in various stages/instars and these may vary considerably within a single species. A difficulty for sure, but also an opportunity for research and publication, and one that I'm becoming increasingly tempted to work on. To do so will not be easy and those gaps in research may mean a truly comprehensive guide is not yet possible, but I think that there is enough knowledge out there - as specimens, existing publications and the contents of entomologists' brains - to produce something genuinely interesting and useful (e.g. for conservation purposes). First job - collect existing literature, then work through the better known species and lastly fill in as many of the trickier gaps as I can. So, you read it here first  - a guide to the juvenile forms of leaf beetles, but don't hold your breath! And to show that not everything is hidden away and hard to find, I'll leave you with a cluster of larvae of the common dock beetle Gastrophysa viridula doing what it does best - eating dock leaves!

Larvae of Gastrophysa viridula on a dock (Rumex) leaf

References

Cox, M.L. (2007). Atlas of the Seed and Leaf Beetles of Britain and Ireland. Pisces, Newbury.
Hubble, D. (2012). Keys to the Adults of Seed and Leaf Beetles of Britain and Ireland. FSC, Telford.

Return to the Tomb of Zootoca

One of my recent posts took a close look at the mummified corpse of a juvenile common lizard (Zootoca vivipara). Apart from noting that the fungus on it suggested it hadn't mummified too well, I also made the point (aided by a friend who knows about those strange tetrapod thingies) that there was surprisingly little written about lizard teeth/dentition. While mammalian dentition commands many pages of diagrams showing the different types of teeth, lizards - at least in the books and websites I could find - tend to simply be described as 'homodont' i.e. having all teeth of the same form. While the variation isn't as striking as in mammals (canines, incisors, molars etc), this is an oversimplification, so I decided to stay well outside my invertebrate comfort zone and delve further. First of all, an overview of the upper and lower jaws.

Fig. 1. Dorsal view of the lower jaw.

Fig. 2. Ventral view of the upper jaw.

Fig. 3. Side view of the upper jaw.

Fig. 4. Side view of the lower jaw (the red line parallels the curve of the jaw).

Comparing Figs. 1 & 2, the outline shapes of the jaws are clear and it can be seen that they match well, widening behind the snout - unsurprising as they wouldn't work if they didn't fit together. In Fig. 1, the dark central structure is taken to be the tongue which has mummified and appears to match the shape of the upper palate in Fig. 2. Looking at Figs. 3 & 4, the upper jawline is approximately straight in side view, while there is a slight curve in the lower jawline. However, the front section of the lower jaw (where the majority of teeth can be seen) is quite straight and so still fits well with the upper jaw, and I imagine that there could be some flexing when biting, especially as this is a juvenile with small bones. This is somewhat speculative however, and I'm more than happy for anyone who knows more about such things to send me info on errors or omissions! Now, zooming in on the jaws...

Fig. 5. Lower jaw - curved tooth arrowed.

Fig. 6. Lower jaw - 'appendiculate' teeth arrowed.

Fig. 7. Lower jaw - apparently serrated tooth arrowed.

Fig. 8. Upper jaw - three apparently fused teeth indicated by the position of the scale bar.

The homodont description of lizard teeth where they are all considered to be 'conical' is clearly not the whole story when looking at Fig. 5 which shows backwards-curving teeth at the front of the lower jaw - presumably for holding on to prey when it is initially captured. While some of the cheek-teeth do have simple conical points on a cylindrical shaft, others are again more complex as in Fig. 6 (you may need to enlarge it) which shows that some are 'appendiculate' or at least asymmetrical with a protrusion on the forward side. I don't know what the function of this might be - maybe a closer fit is ensured with the upper jaw, and/or there could be some element of shearing'cutting which a simple conical point would not achieve. To me, the latter idea is possibly supported by Fig. 7 which (as well as the asymmetrical shape) shows what appears to be a serrated edge. Given that Z. vivipara feeds on prey such as invertebrates with tough exoskeletons, such dentition may be useful.

Lastly, Fig. 8 shows a small section of the upper jaw where three teeth appeared to be fused. Again, the function is not clear (to me, anyway), but I can imagine the 'inter-tooth' sections acting like blades and/or providing a close fit against teeth in the lower jaw. As you can see from the scale bar, the structures here are small and were distinctly difficult to dissect but I hope that the key features are clear. As I say, I particularly welcome comments on this topic and fully expect this to get updated as I learn more.

Get some nyger inside ya

It's time for the first post of the new year, and my attention was grabbed this morning by the activity on our garden nyger seed feeder. It sees a lot of goldfinch activity most days, but among the colourful flits and flights were a couple of less brightly plumaged finches - redpolls.

Goldfinches on the nyger seed feeder, plus a redpoll (arrowed) nearby.

Redpoll taxonomy has changed over the last few years, but this is a common redpoll (Carduelis flammea) and not a species I've seen in our garden before. Redpolls tend to move around a lot, rarely staying in one place for long, so I was lucky to spot them (there were two but they were never close enough to appear in the same photo). This is also true of another recent 'garden first', a brambling seen a few weeks ago.

A brambling on nyger seed.

These are all species known to favour small seed such as nyger (or without kindly humans, teasels and the like) - however redpoll and brambling are not common visitors to urban gardens, so I have to wonder if the poor, very wet summer conditions have led to a low yield of seed in their native foodplants. If so, they may rely on feeders more than usual and be back, and I'll also keep an eye out for another potential visitor - the siskin.