Million-Year-Old Cells

George Poinar and Roberta Poinar

FROM: The Quest for Life in Amber: The Discovery of Fossil DNA. (Excerpted from pp. ix-x; 63-69), 1994 by George and Roberta Poinar. Addison-Wesley Publishing.

Millions of years ago, trees from now vanished forests produced deposits of resin that transformed into what we know today as one of nature's most beautiful gems, amber. The forces that caused the sticky resin to slowly harden remain a mystery. While still viscous, the resin acted like flypaper and tenaciously held insects, plant parts, and even small vertebrates that touched it. Eventually the partially hardened yellow clumps, complete with their assemblage of enclosed organisms, fell from the trees to the earth. There, first leaves and debris, and later soil and rock, covered them, and eventually they were buried in layers of sandstone, limestone, or even coal. All this time, the original resin was developing the hardness, density, and melting point characteristic of amber. This durable organic gemstone outlasted the parent trees, the forests they formed, and the ecosystem of which they were a part. In some places the rock layers containing fossilized resin settled beneath the sea, and, over eons, the currents slowly loosened the amber from its grave. On the sea bottom, the amber was exposed to marine creatures that would even grow on the surface of larger pieces. Eventually the loosened amber was washed up on beaches, where humans eagerly competed with each other to collect this gold from the sea. In other regions, the amber-bearing rock layers were shoved up into mountain ranges, where release of the gem depended on the forces of erosion. Where amber is locked up in layers of the earth, humans have dug mines to locate the "veins" and follow them as far as they can. Whether because of its color, its feel, the mystery surrounding its formation, or the fascinating insect or plant remains it contains, amber is, and has been for centuries, valued by people. Any substance that can weather the forces of time must have magical powers. Just behold the sunlight as it reverberates off the internal fracture planes of raw amber and you will understand why many people consider it mystical. Few things rival the beauty of fossils in amber. When examining insects entombed in this gem of the sun, you can hardly be unmoved by the wonder of seeing marvelously preserved invertebrates frozen forever in their everyday tasks worker ants carrying food, bees with outstretched wings carrying pollen, flies mating. But it is even more miraculous that under the dissecting microscope one can easily see in some specimens intact tissues, remnants of their internal organs. This alone makes amber-embedded fossils unique, because few other fossils have been found in which soft tissues have been so well preserved. It was therefore only a matter of time before the right circumstances presented themselves a well-preserved fossil with tissue, an experienced microscopist with an electron microscope, people with a love of amber and the willingness to look-and the idea that the fine structure of these internal tissues might also be preserved would be born and ultimately pursued, especially when our previous studies had so

strongly suggested it to be true. That someone as involved in the study of amber and amber fossils as I am would share their magic with his or her family is only natural, and amber really is a family affair in our case. One day in 1980, Roberta and I were taking turns peering down the oculars of a dissecting scope at a particularly well preserved set of Baltic amber fossils. The pile glittered in the sunlight, really fossil gold. Typically, when we are sharing some very special amber fossils, we utter many of the same exclamations of delight and wonderment that you would hear at some awe-inspiring event such as an especially vivid fireworks show. When that soon-to-be-famous female fly appeared under the spotlight of the dissecting scope, we cried, "Oh my God, look at this!" There was so much tissue, so well preserved. That fly looked like a freshly embedded specimen ready for a microscopist's knife. When we looked up at each other, the same thought was written across our faces surely this fly's cell structure must be intact too! From then on, that mycetophilid fly became the center of our research project. The day finally arrived when Roberta could begin work on the fly. We hoped that all the bugs excuse the pun had been worked out of the system in the mid-1970s, when we had worked on the fossil nematodes. I had already photographed the intact specimen, and a small piece of the amber had been chipped off and sent to Dr. Curt Beck for infrared spectroscopy analysis and confirmation that our fly was indeed in Baltic amber. We had decided to break the fly into two pieces and save one half for future studies. The piece for safekeeping was stored in an airtight container in the dark. The specimen had broken through the abdomen, and Roberta started to work on her piece. The tissue was obvious as a dark strip adjacent to the cuticle. The center of the fly, however, was an open cavity, and this proved to be the first hurdle in obtaining ultrafine sections. After a frustrating day attempting to obtain any usable sections, we decided that we would have to fill the cavity with embedding plastic before we could go any further just another delay. It was several days later that Roberta was finally able to look at our tissue in the electron microscope. When I returned to my office after a trip to the library a note was hanging on the door: "Success!" I went immediately to the microscope room and breathlessly looked down at the lighted screen, straight onto tissue that was 40 million years old. Tissue with nuclei and organelles mitochondria, lipid droplets and ribosomes was there before my eyes, as well as entire muscle bands with easily identifiable components such as fibrils and mitochondria. Tracheoles, the breathing apparatus of insects, had intact linings, recognizable tubercles, and even possibly remnants of the plasma membrane. There is something almost spiritual in a discovery such as this. It certainly makes one feel humble to look on intact cells that have been around for 40 million years. Long before human beings were even considered in the evolutionary time scale, cells existed with the same structures and organelles that they have today. How insignificant and mundane we really are. The structure of the cells seen in our amber has a different appearance than that of cells standardly prepared for electron microscopy today. This is because scientists strive to obtain what they regard as the best fixation with cell structures having clarity and definition. Some say that

electron microscopy is a study of artifacts that tissues are frozen in time with harsh chemical fixatives, and cells are viewed when caught in a millisecond of life, with their whole life cycle extrapolated from these images. There are obviously many parallels between studying cells with the electron microscope and studying insects entombed in amber. But in the case of amber, nature used its own methods of fixation and dehydration. We feel that the fossil fly best resembles modern-day tissues that have been processed by inert dehydration using ethylene glycol. Sugars, terpenes, and other compounds in the tree sap may have combined with water in the cells to dehydrate and preserve the tissue of entrapped insects almost a mummification process. In the past, humankind was aware of the preservative qualities of tree resins. The Egyptians used resins to embalm their nobility and wealthy citizens. Resins have antibiotic qualities that destroy fungi and bacteria and retard decaying, plus they have components that preserve tissue. Myrrh, a common embalming agent, is a mixture of the resin, gum, and essential oils from the Commiphora plant. This was poured into the cranial, chest, abdominal, and pelvic cavities of ancient Egyptians, and the bandages that wrapped mummies were soaked in it. Resins have also been used topically on wounds as an antiseptic and in wines to prevent spoilage. Greek Retsina wines still use resin for flavoring. Our study of the fossil fly continued over many weeks and followed the routine cycle of ups and downs associated with electron microscope research. Although some areas of the fly were well preserved especially a strip of hypodermal tissue immediately adjacent to the undersurface of the fly's cuticle other tissues, further into the body cavity, lacked substructure and were composed of ghostly cell outlines. Muscle cells were the best preserved, and that has been observed by researchers in other studies of ancient tissues, such as mummies or woolly mammoths. Preliminary results of our studies were first sent off in July 1981 to the journal IRCS Medical Science, where they were published later that year. More conclusive results after months more of study were published in Science in March 1982. This paper represented a pivotal point in our research plans. If tissues could be discovered so well preserved in amber-embedded insects 40 million years old, what else could be found? If the nuclei contained chromatin, a darkly staining structural component known to include genetic material, could DNA (deoxyribonucleic acid) actually be there too, just waiting to be discovered? If there were ribosomal structures, why then not ribonucleic acid (RNA)? We wondered if insects in older amber also had well-preserved tissue remains. On March 5, 1982, we wrote and asked Frank Carpenter at Harvard if he could send us an insect fossil in Canadian amber. On May 12 he sent us a braconid wasp from 70- to 80millionyear-old Cedar Lake amber. Roberta treated the specimen in exactly the same manner as she had treated the Baltic amber fly. The Canadian amber also sectioned with the glass knives and under the electron microscope, the sections showed well-defined tissue in the wasp's abdomen. Although the few sections we examined did not appear to pass through any nuclei, we did observe distinct sections of trachea and surrounding laminated membranous structures in partially vacuolated cytoplasm. We now had evidence that amber from different ages and plant sources could preserve insect tissues, cells, and cell organelles. Due to our work on fossil bacteria, we had unprovable evidence, but evidence nonetheless, that even life forms might lie dormant entombed in amber. What about nucleic acids? As we were

asking ourselves these questions, unknown forces were at work in other places, fitting together the pieces of a complex pattern of techniques, knowledge, and expertise that would ultimately enable us to find answers to these questions.