The second international conference on "Diapause in the Crustacea" was held August 24-29, 1997, in the ancient city of Ghent, Belgium, in a very old and lovingly restored monastery close to the heart of the city. Not surprisingly it attracted about 90 participants for a lively debate. Despite, or perhaps because of the conference having built on the successful previous international conference on the same topic held three years earlier in St. Petersburg, Russia, the definition of diapause surfaced repeatedly. What exactly is diapause? Precisely how do we define the word?

In general terms, many crustaceans (and numerous other taxa) can enter a state of suspended animation at a small embryonic or juvenile stage that can last for months or years. On the basis that you recognize it when you see it, that's usually diapause. The phenomenon can be of considerable economic significance. Understanding diapause is a potential route to controlling reservoir populations of major agricultural pests of cotton, grain, and fruit crops.

A favorite experimental model is the so-called "egg," truly an embryo at about the 12th cell division or 4,000-cell stage, of the brine shrimp Artemia. The embryo is encased in an extremely tough and impermeable cyst wall that can withstand all kinds of indignities and remain virtually dehydrated for decades until conditions are right for so-called hatching, again a loose word for release from diapause. In other genera the diapause stage is often less advanced, such as Daphnia, another favorite model.

The problem is where to draw the line between diapause and a lesser state of suspension known as quiescence. Nancy Marcus (Florida State University at Tallahassee) confronted the problem early in the conference. The key ingredient, if your reviewer as an interloping biochemist in a world of ecologists and developmental biologists has picked it up correctly, is in a cueing mechanism. True diapause is a part of the life cycle of the organism, perhaps optional, induced (and terminated) by one or more specific cues. On cue, the organism enters a state of suspension that can be relatively long, and it becomes resistant to environmental factors that may be severe such as drought or toxicity. Quiescence, by contrast, is a more immediate and short-term response to environmental fluctuations, leaving the organism less resistant and more vulnerable compared with diapause. The definition of quiescence seems to fade out rather than having sharp boundaries. Can mammalian hibernation and sleep be compared with states of quiescence?

Perhaps a further way of expressing what remains a fluid definition is this: in quiescence, the organism enters a state of suspense in direct response to an environmental factor such as low temperature or low dissolved oxygen, whereas in diapause the environmental factor is a trigger that pre-warns of alternative or associated forms of stress. For instance, increasing salinity as a lake evaporates may signal an impending reduction in food availability, or changes in day length may signal falling temperatures. Such triggers appear to include changes in photoperiod, salinity, temperature, pH, dissolved oxygen, and dehydration, according to the organism.

Conversely, release from diapause may require the opposite - water and oxygen, for example. But it is difficult to unravel whether these are true triggers or are merely truisms, because of course an embryo dependent on aerobic metabolism is not going to do anything in the absence of either water of oxygen. More subtle triggers are hard to find. Hatching of Artemia from diapause appears to require an environmental concentration of carbon dioxide/bicarbonate ions. On finer dissection of the mechanism, it is clear that this is not a trigger, but is instead an essential requirement for hatching to proceed once the process has reached a critical stage, probably by ionic exchange in the generation of a high osmotic potential coupled with water influx to expel the prenauplius mechanically.

James Clegg (Bodega Marine Laboratory) asked penetrating questions about another of the characteristics of deep diapause, namely the suspension of metabolic activity. Have desiccated diapause cysts totally ceased all biochemical action? Would this be theoretically plausible? Would it defy simple thermodynamic principles? Have we not learned that the macromolecules of life are inherently unstable? Don't proteins and nucleic acids require continual maintenance or protection to counter degradation? Which costs energy and requires metabolism? Long-term measurements of viability have been made on Artemia embryos on the boundary between diapause and quiescence, i.e., rehydrated but deprived of oxygen. They appear to violate the theoretical requirement to metabolize in order to retain viability.

The work of Tom MacRae's laboratory (Dalhousie University, Halifax, Canada) may throw some light on this mystery. An abundant protein (p26) found in the cysts has been shown to possess features of the small heat shock/alpha-crystallin family, and the molecular chaperone role of this protein may provide crucial protection throughout long-term diapause. So might another abundant molecule found in cysts, the glucose polysaccharide trehalose much studied by Clegg.

The diapause process is often geared to changes of a seasonal nature or may last many years, with obvious advantages for survival of the individual. Several participants including Carla Caceres (Illinois Natural History Survey, Champaign), Stephen Ellner (North Carolina State University at Raleigh), and Nelson Hairston (Cornell University, Ithaca, New York) discussed the more subtle consequences of a mixing of populations of different antiquities, sometimes centuries apart. The tendency for populations in the water phase to change over a period of years owing to competition between species may be slowed or confounded by the storage effect of the diapausing populations preserved in the sediments.

Diapause may be relatively long-lasting - decades or possibly centuries - but neither in theory nor in reality does it last forever. Which is a pity, because if diapause stages did have it both ways, staying alive while not really living, they would be obvious candidates for studies of ancient life and ancient DNA. Nevertheless, they are a step in the right direction of probing the comparatively recent history of life because, as Clegg has pointed out many times, the key point is that they entered into preservation as living stages, providing a distinct experimental advantage over the majority of sources of ancient DNA that were dead and degrading before preservation began. Diapause stages are adapted for the preservation of DNA.

The paper by Petra Limburg (Max-Planck-Institut, Germany) and colleagues discussed Daphnia "egg" traces dating to more than 3,000 and possibly 8,000 years ago. DNA has been amplified by PCR back to about 2,000 years, which would be far too recent to expect significant evolutionary changes at the exon level. However, by focusing on the 12S ribosomal RNA gene (which is mitochondrial and therefore originally present in multiple copies), and by focusing further on the short repetitive microsatellites within the introns of the gene (which are highly polymorphic and thereby diagnostic of strains and species), it is becoming possible to trace fluctuating population histories over hundreds if not thousands of years.

"With selected contributions on non-crustacean taxa," the definition of the conference helpfully allowed, with papers on jellyfish, seeds and desert plants, algae, insects, sponges, and with comment extending to all divisions of life. The dramatic flowering of the desert in quick response to rain is well known even to those who have never been near a desert. Lawrence Venable (University of Arizona at Tucson) brought the phenomenon to life with brilliant illustrations. The questions are easy to appreciate: what are the effects of seed diapause or quiescence on the long-term survival, competition, and territorial advancement of plants under desert conditions? There may be advantages in only a fraction of the seeds germinating when conditions are favorable. The long-term data-gathering necessary to answer these questions is well underway, but will extend over many decades.

A characteristic of the sponges (Porifera) is the ability to form a "reduced" state, in which the sponge may exist as a dormant mass of cells for many months. Sponges have a high degree of ability to repair themselves, to the extent that after dismemberment by passing through a mesh, the separated cells can reassociate into a new sponge. The encapsulated diapause state in sponges, known as the gemmule, can survive desiccation for a period of years, as in the crustacea.

The conference was superbly chaired by Luc Brendonck of the Royal Belgian Institute of Natural Sciences, Brussels, Belgium; upon request he will supply further information about contributors and abstracts. After a memorable afternoon touring the treasures of nearby Bruges, participants underwent training at a special extramural workshop on the finer distinctions of Belgisch Bier, verified by a splendid personal certificate of qualification signed by the Bieracademie. An unidentified ingredient triggered quiescence for the bus journey back to Ghent. The magnificent terminal banquet provided the cue for diapause.