Obervations
on the
Ultrastructure
of
Coelomocytes and Hemocytes
found in
Lumbricus terrestris
and
Placobdella parasitica
by Morgann Reilly
Student of Biology at Smith College
Abstract
In this study, an investigation was made into the ultrastructure of the coelomocytes and hemocytes of two species of the phylum annelida: Lumbricus terrestris, of the class oligochaeta, and Placobdella parasitica, of the class hirundinea. Samples of blood drawn from each species were dried by either the hexamethyldisilazane evaporative technique or critical point drying and then sputter coated before examination under a scanning electron microscope. Similar trends found in cell ultrastructures of both Lumbricus terrestris and Placobdella parasitica suggest the possibility of cellular association. However, further study is needed, particularly with reference to cellular infrastructure, before such a relationship can be established as fact.
Introduction
During the 1970s the ultrastructure of the oligochaeta coelomocytes and heomocytes, including Lumbricus terrestris (the common earthworm) was a prevailing topic of research in the biological community. Using both the traditional light microscope (LM) and the newly invented transmission electron microscope (TEM) the components (both shape and content) of cells found in the earthworm's blood were meticulously studied on a two dimensional level and classified by such visible characteristics as the presence and shape of organelles. As a direct result of the intervention of the TEM, the categories into which cells had previously been placed using characteristics visible with the LM were revised to take into account new characteristics observed using the more invasive TEM. By the early 1980s the cells had been re-categorized as follows (TEM classivications appear first with LM classifications following in parentheses): Type I lymphocytic coelomocytes (basophils), Type II lymphocytic coelomocytes (basophils), Type I granulocytes (neutrophils), Type II granulocytes (granulocytes), inclusion-containing coelomocytes (acidophils), and eleocytes (chloragogen cells). (Ratcliff 1981, Stein 1977, and Vetvicka 1994) Since then advances in the field of microscopy have resulted in the invention of the scanning electron microscope (SEM) making it possible to observe the ultrastructure of cells in three dimensions. Originally it was decided to examine the differences and similarites of two annelida classes, oligochaeta (the segmented worms) and hirundinea (the leeches) using Lumbricus terrestris and Placobdella parasitica (a species of turtle leech). Unfortunately, work with annelid blood has not kept pace with the technical developments in the field of biology, and few contemporary studies on the subject exist. Therefore, little information is available to draw on for reference on this topic except those written at a time when two-dimensional images were the extent of a microscope's capabilities. So because it is difficult to predict a three-dimensional structure from a two-dimensional image, the following is a comparative analysis of earthworm and leech blood cells rather than a definitive identification of said cells. Additionally, in hopes of engaging in some kind of comparison between the two species of annelids, a sample of each was subjected to either hexamethyldisilazane evaporative technique or critical point drying.
Materials and Methods
The first step of the experiment involved obtaining blood samples from both Lumbricus terrestris (the common earthworm) and Placobdella parasitica (the turtle leech). An earthworm was procured from a local bait shop. The worm was first rinsed with distilled water (dH2O) to remove the majority of dirt particles clinging to its skin, and then placed in a small beaker containing 5% ethanol for 10 min to anesthetize it. (Ownby 1993) While the worm was being anesthetized, a few drops of L-15 medium were placed on a glass coverslip (enough to cover the surface) labelled E1 (for "Earthworm blood, sample 1") resting in a small, plastic petri dish.
After 10 min, the worm was removed from the ethanol and carefully pinned to a sheet of dental wax. An incision was then made on its dorsal side, beginning a few segments back from the mouth, cutting towards the anus, but stopping before piercing the clitellum. A few drops of L-15 medium were deposited directly into the area of the incision and allowed to mix with the freely flowing blood of the worm. This mixture of L-15 medium and blood was then pipetted up and transferred to the L-15 medium on the previously prepared coverslip. Using the same worm, the procedure was repeated to acquire a sample for a second coverslip (labelled E2 for "Earthworm blood, sample 2"). The presence of blood cells in the E1 and E2 cultures was then verified using a dissecting microscope before proceeding to collect a similar blood sample from a leech.
In preparation for the leech sample (leeches obtained from the Connecticut Valley Biological Supply Co. in Southampton, MA), a few drops of the L-15 medium were placed on a glass coverslip (again, enough to cover its surface area) labelled L1 (for "Leech blood, sample 1) in a small, plactic petri dish. To obtain a leech's blood, the tip of its posterior sucker was severed using a razor blade. Then, holding the leech over the coverslip, the leech was bled directly into the medium. The procedure was repeated for a second coverslip (labelled L2 for "Leech blood, sample 2)). (Note: Due to a poor yield of blood from individual leeches, three separate leeches were bled: two for the L1 sample and one for the L2 sample.) A dissecting microscope was again used to ascertain the presence of blood cells in the L1 and L2 samples before continuing with the experiment.
In preparing the specimens for drying, each was fixed with McDowell-Trump fixative solution. (McDowell 1976) To do this, the coverslips were left in their individual petri dishes, covered with the fixative solution, the lid put on the dish to prevent contamination, and allowed to sit for 20 min. The specimens were then dehydrated by cycling them through the following series of ethanol washes, leaving the dishes covered during each interval to prevent evaporation of the wash:
30% ethanol x 1 for 5 min
50% ethanol x 1 for 5 min
70% ethanol x 1 for 5 min
80% ethanol x 1 for 5 min
95% ethanol x 1 for 5 min
100% ethanol x 3 for 10 min
100% ethanol x 1 for 40 min
In the running of these cycles, after removing the previous proof of ethanol and replacing it with the next one in succession, the coverslips were gently lifted from the bottom of the petri dish to allow the new proof of ethanol to circulate underneath them as well as above before covering the petri dish again.
The first drying method performed was the hexamethyldisilazane evaporative technique. To do this, the E2 and L2 coverslips were each placed into their own small glass petri dish, immediately covered with 100% ethanol to prevent the specimen from drying out, the coverslip lifted to allow circulation of the new solution, the dish covered, and allowed to sit for 20 min. The 100% ethanol was then removed from the petri dishes and replaced with a mixture of approximately one-half 100% ethanol and one-half hexamethyldisilazane; the coverslip was lifted and the dish covered, then left alone for another 20 min. This solution was then replaced with one of pure hexamethyldisilazane; the coverslip was lifted and the dish covered, then allowed to sit for 20 min. A second cycle of pure hexamethyldisilazane repeated the same steps as the first for the same duration of 20 min. After this last 20 min cycle was completed, the petri dishes were placed in a well-ventilated area and their lids propped open to allow the pure hexamethyldisilazane solution to evaporate.
The two remaining samples, E1 and L1, were dried using a critical point drier. The holder for the critical point drier was loaded, from bottom to top while submerged in a beaker of 100% ethanol with: a metal ring, the E1 coverslip, a second metal ring, the L1 coverslip, a third metal ring, and a nickel plate. The holder was then transferred to the chamber of the critical point drier which was also filled with 100% ethanol. The chamber was then sealed and flooded with liquid carbon dioxide (CO2) and cooled to 5oC. By opening the exhaust valve for 30 sec at 3 min intervals over the next 15 min, the chamber was gradually purged of all the 100% ethanol until it contained only pure liquid CO2. At this point, the inlet valve for the CO2 was closed and the temperature in the chamber gradually raised to 40oC. Concurrently, the pressure in the chamber was maintained at approximately 1400 millitorr. Once the temperature reached 40oC, the exhaust valve of the critical point drier was opened, allowing the pressure in the chamber to slowly return to zero before the specimens were removed.
After all four samples had completed their respective drying methods they were prepared for the sputter coater. Each coverslip was placed onto its own cylindrical aluminum mount with a portion of double-sided sticky tape. To secure the coverslip to the mount, the underside of each slide was painted with a few drops of isopropanol carbon paint which was then allowed to dry for 15 min.
The specimens were then put through the sputter coater to give them a thin coating of gold polladium, thereby enabling the specimen to produce an image while under the SEM. The specimens were secured to the pedestal in the chamber of the sputter coater with double-sided sticky tape to prevent them from moving and the coverslips from overlapping during the coating. An initial vacuum of 45 millitorr was achieved and was then increased to approximately 50 millitorr; the voltage was then set to 9 volts and the current to 10 milliamps. The specimens were allowed to coat for 3 min before the voltage setting was returned to zero and the vacuum released, allowing the pressure in the chamber to return to zero. The specimens were then removed from the sputter coater and placed in a lint-free box until they could be examined under the SEM.
Results
When looked at under the SEM, nearly all of the earthworm cells from the critical point drier (sample E1) appear to be ruptured. Some look as though they were in the process of extending pseudopodia and beginning to flatten out, similar in appearance to a fried egg, with a mound of membrane encased cytoplasm (the "yolk") surrounded by extending pseudopodia. But rather than having a smooth appearance, the "yolk" of the E1 cells are severely pocked with a number of deep craters; Figure 1: E1-1 is an example of such a cell. Other cells seem to have simply burst, leaving behind a ruptured and empty membranous shell (see Figure 2: E1-2).
Figure 1: E1-1
Lumbricus terrestris cell dried by critical point drying with extending pseudopodia and cratered membrane. Bar represents 10 um. X2000.
Figure 2: E1-2
Empty membranous shell of ruptured Lumbricus terrestris cell dried by critical point drying. Bar represents 10 um. X2700.
The earthworm cells dried by the hexamethyldisilazane evaporative technique do not, however, exhibit the same problematic characteristics as those which were critical point dried. Figure 3: E2-7 shows one of the occasional clumps of smaller cells that formed on the E2 slide. Measuring approximately 54 um across, it contains four of the varieties of cells (indicated with arrows and figure references) prominent in the culture of earthworm blood cells. One of these appears in Figure 4: E2-1; best described as having a cabbage-like appearance, it measures about 14 um in diameter. Contained within the mass of cells shown in Figure 5: E2-6 is another cell type, which appears to be composed of many spherical granules, in this case surrounded by strings of pseudopodia. Figure 6: E2-5 offers a magnified image of the granular portion (12 um in size) of the same multi-cellular mass while Figure 7: E2-8 shows what appears to be a free-standing version of the same granulated cell type (approximately 15 um in diameter) minus the pseudopodial strings. Though the granules are less defined, the cell in Figure 8: E2-3 (16 um in size) seems to be of the same variety. Just as Figure 7: E2-8 and Figure 8: E2-3 are similar in appearance, so too are Figure 9: E2-9 and Figure 10: E2-4 (whose cells measure 20 um and 24 um in magnitude, respectively). The surfaces of both are covered with balloon-like structures which have shrunk in on themselves. Figure 11: E2-2, measuring around 18 um, shows a cell with a complete absence of granules, but with a variety of concave depressions.
Figure 3: E2-7
Multi-cellular mass of Lumbricus terrestris cells dried by hexamethyldisilazane evaporative technique with cell types that appear later indicated with arrows and figure references. Bar represents 10 um. X1200.
Figure 4: E2-1
Lumbricus terrestris cell dried by hexamethyldisilazane evaporative technique displaying a cabbage-like surface structure. Bar represents 10 um. X3500.
Figure 5: E2-6
Multi-cellular mass of Lumbricus terrestris cells dried by hexamethyldisilazane evaporative technique with granulated cell indicated by arrow. Bar represents 10 um. X1300.
Figure 6: E2-5
Further magnification of granulated Lumbricus terrestris cell indicated in Figure 5: E2-6. Bar represents 10 um. X2200.
Figure 7: E2-8
Free-standing version of Lumbricus terrestris cell dried by hexamethyldisilazane evaporative technique with well-defined granules. Bar represents 10 um. X3700.
Figure 8: E2-3
Lumbricus terrestris cell dried by hexamethyldisilazane evaporative technique with less well-defined granules. Bar represents 10 um. X3700.
Figure 9: E2-9
Lumbricus terrestris cell dried by hexamethyldisilazane evaporative technique with collapsing granules. Bar represents 10 um. X4000.
Figure 10: E2-4
A second Lumbricus terrestris cell dried by hexamethyldisilazane evaporative technique with withering granules. Bar represents 10 um. X2700.
Figure 11: E2-2
Lumbricus terrestris cell dried by hexamethyldisilazane evaporative technique with concave depressions. Bar represents 10 um. X3300.
While the earthworm cells survived the hexamethyldisilazane evaporative technique without any apparent complications, the leech cells subjected to the same drying technique (L2) appeared featureless and very few in number when examined under the SEM. Instead, it was the cells subjected to the critical point drier (those on the L1 coverslip) that yielded the best results. Figure 12: L1-2 and Figure 13: L1-5 (each approximately 17 um in diameter), with their cabbage-like appearances, are similar to the earthworm cell shown in Figure 4: E2-1. Unlike the earthworm cells, however, the leech cells show the beginnings of pseudopodial formations at their base. The cells shown in Figure 14: L1-1 and Figure 15: L1-3 (11 um and 13 um, respectively), though their surface textures are slightly less cabbage-like, display an even greater number of pseudopodial extensions radiating from the central body of the cell. Figure 16: L1-4 (13 um in size), though slightly distorted due to the charging that occurred inside the SEM, is unique among the leech cells chronicled here in that its surface appears to be covered with a number of small, spherical granules, as opposed to the characteristic cabbage-like appearance present to some extent on all the other recorded leech figures.
Figure 12: L1-2
Placobdella parasitica cell dried by critical point drying with cabbage-like surface structures and spreading pseudopodia. Bar represents 10 um. X4500.
Figure 13: L1-5
A second Placobdella parasitica cell dried by critical point drying with cabbage-like surface structures and the beginning of pseudopodial extensions. Bar represents 10 um. X4500.
Figure 14: L1-1
Placobdella parasitica cell dried by critical point drying with remnants of cabbage-like surface structures and fibrous pseudopodial extensions. Bar represents 10 um. X2700.
Figure 15: L1-3
A second Placobdella parasitica cell dried by critical point drying with remnants of cabbage-like surface structures as well as numerous fibrous pseudopodial extensions. Bar represents 10 um. X3000.
Figure 16: L1-4
Granulated Placobdella parasitica cell dried by critical point drying. Bar represents 10 um. X7000.
Discussion
The results obtained through the separate preparative techniques were interesting when compared both by method and by specimen. Although both E1 and L1 were processed in the critical point drier at the same time, the cells of the two samples experienced very different levels of success. If the two slides had been processed at different times, it could have been possible that the pressure was released too quickly while E1 was in the chamber, resulting in such a sudden and massive change in pressure that caused the cells to explode. But since E1 and L1 were prepared at the same time this cannot be the case. The results of the hexamethyldisilazane evaporation technique are equally as puzzling since the E2 cells do not appear abnormal when looked at under the SEM, yet the L2 cells are barely present and those that are visible are almost featureless. Since the conflicting reactions of the two species to the different drying techniques cannot be easily dismissed by the possibile explanation of human error, it is most likely species-specific cellular preference that resulted in such dissimilar responses to identical methods.
Looking at the cells both within and between species, it is possible to infer a number of similarities and possible relationships. For example, the cells in Figure 4: E2-1, Figure 12: L1-2, and Figure 13: L1-5 all exhibit the same cabbage-like exterior. Because the earthworm and leech are from the same phylum, annelida, it is not improbable that similarities would present themselves between classes. But without any knowledge the infrastructure of the cells, it would be presumptuous to declare the existence of any inter-class relationships barring further study.
Nor is it difficult to see relationships in the ultrastructure of cells within the same species. Figure 7: E2-8 has well-defined granules while those in Figure 8: E2-3 are slightly less well-defined, becoming less distinct as they seem to sink into themselves. Conceivably, Figure 8: E2-3 could be of the same cell type as Figure 7: E2-8, only at a later stage in its development. Perhaps this particular variety of cell was degenerating into that which is shown in Figure 11: E2-2, a cell whose surface is covered with a series of concave depressions. It is possible that these impressions are the result of dozens of granules collapsing in upon themselves. Continuing to broaden this same theory of granular shrinkage, Figure 9: E2-9 and Figure 10: E2-4 show what could be considered an intermediate level in the degeneration from the well-defined granules to the depressionary surfaces. The withered extensions seen in these figures could be deflating granules on their way to complete collapse. Though it has been suggested by other researchers that all types of Lumbricus terrestris cells are the same cell type at different developmental stages, the theory has remained unproven. (Burke 1974) Therefore, all of these observations remain mere speculations based upon the ultrastructure of a few commonly recurring cells found in the blood of Lumbricus terrestris.
The similar surface structures of the leech cells also yield cause for conjecture. For instance, given time, rounded, blunt pseudopodia have been known to extend into finer, more filamentous structures. Figure 14: L1-1 and Figure 5: L1-3 could be the result of the continued extension of pseudopodia present in Figure 12: L1-2 and Figure 13: L1-5. This, again, is drawing on the theory that many of the cells present in the sample are, in fact, of the same cell type at different stages in its development. Looking at Figure 16: L1-4, with its beaded surface appearance, it could be deduced that, should the granules somehow burst, they could give way to the cabbage-like surface structures seen in Figure 12: L2-2 and Figure 13: L2-5. The ruffle-like protrusions of such cells could then eventually degenerate into the strings of pseudopodia seen in Figure 14: L1-1 and Figure 15: L1-3. Again, however, these are purely speculative observations as cellular relationships cannot be established solely through the examination of their ultrastructure. More comprehensive research of these same cell types, especially with regards to their infrastructure, is needed before such observations on the Placobdella parasitica as those made here can be considered conclusions.
References
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McDowell, Elizabeth M. and Trump, Benjamin F. "Histological Fixatives Suitable for Diagnostic Light and Electron Microscopy." Archives of Pathology Laboratory Medicine. 100 (1976) 405-414.
Ownby, David W. et al. "The Extracellular Hemoglobin of the Earthworm, Lumbricus terrestris: Determination of subunit stoichiometry." The Journal of Biological Chemistry. 268 (1993): 13539 - 13540.
Ratcliffe, NA and Rowley, AF (eds). Invertebrate Blood Cells, Volume 1. New York: Academic Press Inc, 1981. Pp. 135 - 165, 203 - 243.
Stein, Elizabeth et al. "The Coelomocytes of the Earthworm Lumbricus terrestris: Morphology and phagocytic properties." Journal of Morphology. 153 (1977): 467 - 478.
Vetvicka, Vaclav et al. Immunology of Annelids. Boca Raton, FL: CRC Press Inc, 1994. Pp. 81 - 140, 151 - 159.
