The genetic variation of a local subpopulation (South Hadley, MA) of the freshwater crayfish Orconectes rusticus was surveyed using random amplified polymorphic DNA (RAPD) analysis. By comparing the similarity of the bands produced by RAPDs, it was found that there was variation within this population. Four primary genotypes (A,B,C, and D) were found within a sampling of 25 individuals. Within the study, more than half of the individuals were found to have 31.29% dissimilarity (variability). Although this data suggests a noticeable level of diversity, a continuation of this study using mitochondrial DNA (mDNA) analysis, RFLP fingerprinting, or the sequencing of hypervariable regions of the mitochondrial genome may increase the viability of this conclusion.

Within the last decade, technological advancement has increasingly supported the use of genetics in determining population diversity (Wolf 1993). Many molecular techniques are now available which allow ecologists and evolutionary biologists to determine the genetic architecture of a wide variety of closely related individuals (West). DNA markers that are known to be genetically linked to a trait of interest can be used for gene cloning, medical diagnostics, and for trait introgression in plant and animal breeding programs.

Several different methods for documenting genetic information are used. These methods include isozyme analysis, restriction fragment length polymorphisms (RFLP), and random amplified polymorphic DNA (RAPD) (Mulcahy et al. 1993). Although isozyme analysis and RFLPs are a source of readily obtainable genetic information which is easily reproduced, they often do not show polymorphisms which are necessary to determine variation within a group of genetically similar individuals.

The RAPD technique employs 10 base pair random primers to locate random segments of genomic DNA to reveal polymorphisms. These primers adhere to a specific nucleotide segment of the genomic DNA. The DNA is cut into many segments of a specific length which can be measured using gel electrophoresis. For a mutation to change the RAPD pattern, it must occur in the priming region or must change the length of the DNA between priming regions. In this way the RAPD analysis can provide a simple and reliable method for measuring genomic variation. Because it is a relatively straight-forward technique to apply, and the number of loci that can be examined is unlimited, RAPD analysis is viewed as having a number of advantages over RFLP's and other techniques (Lynch and Milligan 1994).

The RAPD technique has further advantages over other systems of genetic documentation because it has a universal set of primers, no preliminary work such as probe isolation, filter preparation, or nucleotide sequencing is necessary (Williams et al. 1990). The ease and simplicity of the RAPD technique make it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics. In many instance, only a small number of primers are necessary to identify polymorphism within species (Williams et al. 1990). Indeed, as Mulcahy et al. (1995) report, a single primer may often be sufficient to distinguish all of the sampled varieties. Williams et al. (1990) state that the ease of the RAPD technique could lead to the automation of genetic mapping and to the extension of genetic analysis to cover organisms which lack an ample number of phenotypic markers to completely describe their genome.

The RAPD technique was first employed by Williams et al. (1990) to examine human DNA samples. While they found that the information returned for an individual RAPD analysis was quite low, studies using multiple markers to define a genome yielded the best results. This technique was also used by Mulcahy et al. (1993) in an examination of apple cultivars. Using the RAPD technique, they were able to distinguish eight distinct apple cultivars. The RAPD results were fairly consistent within each group which indicates that RAPD are likely to provide reliable identifications.

Vicario et al.(1995) used the RAPD technique in their analysis of the genetic relationship among a unique population of Abies nebrodensis and Abies alba populations along a north-south geographic gradient. While allozyme analysis was also employed in this study, they report that genetic differences in the populations were easily detectable using the RAPD analysis with single-primer DNA amplifications. Their RAPD analyses showed high levels of genetic difference between the populations which confirms their hypothesis that the two groups are taxonomically distinct.

For any population a selective process can produce change only if there are some variations to select among. No amount of reproduction can affect a population's genetic composition if all individuals are identical. From an evolutionary standpoint the progressive accumulation of genetic variation is thought to have given rise, beginning with common ancestors, to the diversity of life. The process of continued evolution is critically dependent on renewed variation. Thus, genetic variation can be thought of as the "fuel" for evolution.

This study attempts to identify and estimate genetic variation within a lacustrine crayfish population. There are over 350 species of crayfish in North America. Sixty-five of these species belong to the genus Orconectes (Gunderson 1995). Orconectes rusticus inhabits freshwater environments similar to Mount Holyoke's Stony Brook. Generally, they can be identified by their robust claws and by the dark rusty spots on either side of the carapace (Holdrich 1988). Like lobsters, freshwater crayfish walk on their four back legs, or periopods. The first pair of periopods has adapted as a mechanism for both fighting and defense. Other legs extend from the abdomen of the crayfish, but they cannot support the weight of the body. Instead, they seem to be specifically adapted for other functions, including feeding or egg-bearing (Banister and Campbell 1985).

O. rusticus lay its eggs in the spring (late April or May) as water temperatures increase. Each females lays from 80 to 575 eggs (the actual number is directly proportional to the size of the female), which hatch after approximately three weeks, depending on water temperature (Thorp 1991). Crayfish reach sexual maturity in 16 to 17 months at which point (the following June or July) the male molts. He will molt once again in August or September. Most males die in the late spring or early summer at the age of 36 to 37 months. Female have approximately the same life span, but they molt only once after they lay their first eggs. All crayfish burrow in October, but the females do not leave their burrows until April, when the young are able to survive on their own (Smart 1962). O. rusticus eats a variety of other invertebrates (worms, leeches, clams, aquatic insects, and amphipods), aquatic plants, detritus, fish eggs, and small fish (Pekarsky 1990). Their main predators are grackles, fish, people, and other crayfish (crayfish are extremely aggressive particularly the males) (Holdrish 1988).

In our research we employed the RAPD technique to look for genetic variation in crayfish individuals for Stony Brook on the Mount Holyoke College campus, South Hadley, Massachusetts. We hypothesize that the crayfish in Stony Brook represent multiple genotypes. Information on the number of genotypes evident in the brook is important as it can give us further information as to the vulnerability of the population as a whole. Human and environmental changes such as dam construction and temperature change will have a much greater effect on a population which is comprised of a single genotype when compared to one with a number of different variations. Setting up a genetic fingerprint for the crayfish in Stony Brook will also enable us and other researchers to identify the range of genetic variation within the species by providing a key to which other crayfish individuals and populations from other locations can be compared.

To obtain DNA from O. rusticus, it was first necessary to procure several specimens. The specimens were collected from Stony Brook in South Hadley, Massachusetts, just below the Upper Lake dam on the Mount Holyoke College campus on October 12, 1996. To capture the O. rusticus, members of the study waded into the stream and visually located a specimen. It was slowly approached and when the crustacean was within reach, a 20 oz plastic cup was either placed in front of it a and a ()12 inch stick was used to prod the O. rusticus into the container, or the cup was held behind the specimen and the stick used to frighten it into swimming backwards into the cup. It was observed that the method of frightening the specimen and getting it to swim backwards into the cup worked best, given the relative speed of movement of the specimen in comparison to that of the members of the study. Once caught, the cups containing the O. rusticus were gathered together on the shore. Approximately 30 individuals were collected; only those at least 2.5cm long were deemed acceptable for this experiment, as the larger the specimen, the more easily tissue could be extracted.

In the lab, the O. rusticus were placed into plastic bags and placed alive into a -80oC freezer. The freezing both killed the specimens and preserved them. It was imperative that they be kept at -80oC to arrest the degeneration of DNA due to the decomposition of the cells upon the death of the organism. The specimens were stored in the -80oC freezer until their dissection.

Since the observation of the expression of polymorphisms in O. rusticus DNA was the primary objective in this study, a RAPD analysis of the DNA was selected as the most suitable procedure. Random amplified polymorphic DNA, or RAPD, analysis is a widely used technique for detecting genomic polymorphisms at a number of different loci using minimal quantities of genomic DNA. Using a oligonucleotide primer of random sequence in a PCR (polymerase chain reaction), a reproducible array of strain-specific products are generated that can be analyzed using gel electrophoresis. RAPD analysis can be carried out on organism for which there is little or no information concerning genomic sequences...thus making it possible to analyze polymorphism for virtually any organism from which relatively pure genomic DNA can be isolated (Pharmicia Biotech 5).

The "Ready-To-Go" RAPD Analysis Kit selected for use contained six different random primers, two samples of control Escherichia coli DNA (C1A and BL21), and 100 reaction tubes. Each tube contains a bead which holds the reagents for the RAPD reaction. Within each bead is contained the correct measurements of thermostable polymerase (AmpliTaq® and Stoffel fragment), BSA, dNTPs (dATP, dCTP, dGTP, dTTP), and buffer (MgCl2, KCl, and Tris). The only other reagents that need to be added to the tube for reaction besides the bead are genomic DNA and the primer of choice (Pharmicia Biotech 6). The beads reduce the risk of measurement error of the reagents and of contamination in the reaction.

There are six primers in a Ready-To-Go RAPDs kit, and each amplifies the same random section of DNA within an organism each time. Some of the primers may amplify a section of DNA that may show few or no polymorphisms. Finding a primer that consistently amplifies polymorphisms involves testing each one of the primers on several individuals.

The primers in the kit were initially stored at room temperature because their contents are lyophilized. They each were reconstituted with 500µL of distilled water as needed and were then stored in the -20oC freezer. Specific primers were defrosted as needed for each reaction.

PCR reactions were set up as following using the RAPDs kit: in each tube containing a Ready-To-Go bead, 2µL genomic O. rusticus DNA, 18µL dH2O, and 5µL primer. A positive control was made with 2µL E. coli DNA instead of O. rusticus DNA, a negative control with the 2µL of DNA replaced by 2µL of dH2O. Each tube contained a total volume of 25µL. A different tube was prepared for each O. rusticus individual and run in a thermocyler. The thermocyler was programmed to run the following cycle: 1 cycle at 95oC for 5 minutes, followed by 45 cycles at 95oC for 1 minute, 36oC for 1 minute, and 72oC for 2 minutes (Pharmicia Biotech 14). After the completion of the cycles, the thermocycler kept the samples at 4oC until they could be used or stored. Storage of PCR products was in a 4oC refrigerator.

An initial test of the RAPDs kit was performed using the control E. coli DNA C1A and BL21 and primer 2. Once PCR was performed on the controls, these samples needed to be analyzed. The method used to do this is Gel Electrophoresis. The first step in Electrophoresis is to make argarose gels. In this experiment we used two sizes of gels, a mini one to test samples and the success of the PCR and a larger gel for clear resolution and separation of polymorphic bands.

To make a mini gel combine 0.4g of argarose (type V, obtained through pfs), 40µL of 1X tris-acetate buffer (TAE) which is diluted from a 10X stock stored at 4o, and 8µL of ethidium bromide in a beaker with a cross stir-bar. For a large gel the components are: 1.5g of argarose, 150µL of TAE, and 60µL of ethidium bromide also placed in a beaker as above. Place the beaker on a heating plate at temperature 7 and stir 3. As the mixture begins to boil, remove the stir bar and place the beaker in the microwave to complete heating. Allow the mixture to boil until it is clear, and then allow it to cool until it can be comfortably handled (about 50oC). Pour the mixture into a mold with the gel tray already in it and place a well comb into the gel. It will need to cool for about 20 minutes or until it is somewhat firm and opaque.

The gel is transferred into the gel box after it is set. Fill the box with 1X TAE buffer slightly over the top edge of the gel so that the wells are covered. At the positive terminal 12µL of ethidium bromide is added. Wells are loaded with the following: for the ladder only: 1µL of ladder, 2µL of 10X dye, and 3µL of dH2O; all other samples: 2µL of 10X dye and 5µL of PCR product. Loading totals are 6µL and 7µL, respectively. Mini gels are run at 100 volts for an hour, and large gels at 100V for 2 and a half to 3 hours, or until dye has traveled to about an inch from the bottom of the gel.

Check completed gels first on a ultraviolet light box for bands and adequate separation. Secondly take a photograph. Photos are taken with a Pharmicia Biotech camera system, using photo preset #1. Finally gels are disposed of in an ethidium bromide waste bottle.

A large gel was prepared and loaded with the products from the test PCR reaction as well as a sample of 100bp ladder. Ladder, negative control, BL21 E. coli, and C1A E. coli were loaded into wells 2 through 5. All other wells were empty. The gel was run, checked on the UV light box, and a photo was taken. Bands appeared in all wells including the negative control. The bands in the negative control are not the result of contamination, because bands and/or smears in a negative control are normal in RAPD analysis (Pharmicia Biotech 21). The bands that appear are the result of small amounts of DNA contamination in the polymerases (Pharmicia Biotech 21). Differences in banding patterns were observed in the two species of E. coli, and those bands were compared to pictures provided by the RAPDs instruction manual. The bands matched, and therefore, it was concluded that the reaction was done properly and there was no contamination of samples.

RAPD analysis is very sensitive to changing any of the conditions of the reaction and contamination (West). Therefore experimental conditions must be kept constant and all steps to avoid contamination must be taken. To avoid any changes in the procedure, each member of the study performed the same tasks continually with few substitutions. Also, every attempt was made to maintain a sterile environment. Gloves and goggles were worn and reactions were done in the most sterile environment available.

Once PCR and electrophoresis procedures have been understood and practiced, the next step is to begin the analysis of O. rusticus' DNA. The first step in this process involves the extraction of DNA from the specimens collected earlier. A sample of heart tissue and claw tissue was isolated from one of the O. rusticus. To extract DNA from the O. rusticus tissue GeneReleaserTM was used.

A 1mm3 section of tissue is isolated from the O. rusticus specimen. The tissue is placed in the bottom of a 1.5µL tube, and 25µL of 1X TE (pH 7.4) is added. Next the tissue is minced by thrusting a pestle against the tissue and compressing the tissue against the tube. Ten thrusts are sufficient. If more than one sample is being prepared at a time, each homogenate should be stored at 4oC as each additional homogenate is being prepared.

After the homogenate(s) have been prepared, transfer 1µL into a standard PCR amplification tube. Resuspend the GeneReleaserTM mixture by vortexing for 2-3 seconds and then add 20µL to the 1µL of homogenate in the PCR tube. Vortex the tube for 2-3 seconds. Then place the tubes in a microwaveable 96 well rack, and place the rack in the center of the microwave oven. Microwave for 6 minutes, and remove. Transfer tubes to a heating plate preheated to 80oC and equilibrate for 5 minutes. Samples are then stored at -20oC.

GeneReleaserTM was used on samples on heart and claw tissue dissected earlier. The products were then run on a mini gel to determine first whether or not DNA isolation had been successful, and secondly, which tissue and concentration would be optimal for the actual comparison of individuals. Three samples of each heart and claw tissue in concentrations of 3, 5, and 7µL in addition to 2µL of 10X dye were loaded into wells 1-3 (heart) and 5-7 (claw). This gel was run only for a half and hour and was also run at 150V.

The gel was checked and a photo was taken. The Genomic DNA was visible in all samples and concentrations. The DNA isolated from the heart tissue showed the brightest bands, but the bands from the claw tissue were also quite bright. Deciding to follow the less is more principle of PCR we chose to dilute the least concentrated sample of claw DNA for our actual study.

We began the search for the appropriate concentration of claw tissue by performing serial dilutions of genomic DNA. One µL of genomic DNA and subsequent serial dilutions to 10-4 were run on a mini gel using primer 2 to compare the clarity and brightness of the bands. The dilution of 10-3 was chosen because of its clarity and band definition. As stated before, the less DNA used in the RAPD reactions the more amplification that takes place and the clearer the bands are for analysis. The next step was the dissection and preparation with GeneReleaserTM on the first 8 individuals.

At this point a primer that would amplify polymorphic DNA strands in different individuals needed to be identified. We began by testing Primers #1 and #2. We ran PCR reactions with two samples each per primer of individuals 1 and 2. After PCR the samples were run on a large gel with a sample of ladder and the positive and negative controls. The gel was run for 2 and a half hours at 150V, in addition no ethidium was put into the gel. The gel was checked and a photo was taken. We observed no polymorphic bands between individuals 1 and 2 with neither primers #1 or #2. The bands on the gel were faint and the resolution of the photo was not very good, a problem that was addressed later.

The same procedure was repeated as above except with individuals 2 and 4, primers #3 and #4, and no positive controls were run. Once again no polymorphic bands were observed between individuals 2 and 4 with neither primers #3 and #4. The gel was again very faint and did not have good resolution. Finally the procedure was repeated again with no changes, but with individuals 8 and 9 and primers #5 and #6.In checking the gel this time, however, we did observe polymorphic bands between individuals 8 and 9 with primer #5, but not with primer #6. Once again the gels run were not of good quality. Based upon the observed presence of polymorphic bands using primer #5, we choose to use this primer in the remainder of our PCR reactions.

We then completed the dissection and isolation of genomic DNA of the remaining O. rusticus individuals (9-25). Each individual sample of O. rusticus DNA was diluted and PCR reactions with the RAPDs kit were performed using primer 5.

The problems that we had noticed with our gels, bands being faint and overall resolution being poor, were getting progressively worse as our experimentation continued. To attempt to alleviate the problem we made sure to return to the gel procedure used initially and outlined above. We also changed the stock of all of our reagents. We obtained new argarose, made fresh stocks of 1X TAE, this time diluted from a 50X stock, used new 10X dye and dH2O, allowed the gel mixture to thoroughly boil, placed ethidium bromide in the gels, and ran the gels again at 100V.

We first ran two test mini gels prepared this way with ladder and PCR products. The first gel was run with PCR products from individuals 13, 16, and 15, and the second with products from individuals 23, 24, and 25. Both gels were run at 100V for one hour. Each of the gels showed good brightness and resolution of the bands, and this method of running gels was adopted for running our final PCR products.

The final two gels were run containing ladder, positive control (E. coli BL21), negative control, and the PCR products from individuals 1-25. Two gels were run, both with ladder and negative control. Gel #1 was run with the positive control and individuals 1-12. Gel #2 was run with individuals 13-25. The gels were run at 80V for a total of three hours, they were then checked and photos were taken. The final gels were then analyzed.

Tissue used in this experiment was extracted from the claws of O. rusticus specimens. Claw tissue was chosen over that of the heart for three reasons. First, maintaining an experimental environment without contamination is essential in RAPD reactions, therefore, tissue from the heart was not chosen because of the possibility of it being contaminated by the contents of the stomach which is in close proximity to the heart. Secondly, it is easier to extract a sufficient amount of tissue through dissection from the claw than from internal organs. Finally, PCR reactions produce more effective results when lesser amounts of DNA are utilized.

Wells 1-3 contain genomic heart DNA, in Figure 1, and wells 5-7 contain genomic claw DNA . Significant amounts of genomic DNA appeared in each of the tested samples. Genomic DNA from heart tissue was more abundant than that from the claw tissue. Due to the PCR principle that less DNA yields more clear results, claw tissue was chosen.

In testing primers for their ability to amplify polymorphic bands of DNA, the following test reaction was run on individuals 8 and 9. Each individual was tested twice with both primers 5 and 6. Figure 2 shows the results of this test. Lanes 3 and 4, which contained two samples of DNA from individual #8 treated with primer 5, show no differences, indicating that primer 5 is able to consistently amplify a single locus. Lanes 5 and 6, which contained samples of DNA from individual #9 treated with primer 5, were identical, but different from the bands present in lanes 3 and 4. Being as primer 5 was able to consistently amplify identical bands within one individual, but the bands amplified between two individuals were not the same, it was selected for further use in this study. The sequence for primer 5 is (5'-d[AACGCGAAC]-3') which means that at every point in the genomic DNA that this sequence appears, the primer will bind. After binding, the sections between priming regions are replicated and amplified during PCR. It is these sections that are viewed on the gel.

Final PCR reactions were performed on individuals 1 through 25 using primer 5. The resulting gels are pictured in Figure 3. The bands were scored in comparison to the 100 bp ladder, which is found in lane one of both gels. The results of the scoring are exhibited in Figure 4. Bands ranged in length from 90 bp to 1400 bp, with the majority of individuals exhibiting bands in the 500 bp region (Band G) and the 1000 bp region (Band D). Based upon the information in Figure 4, a phenogram was constructed (Figure 5) to compare the similarity of the individuals. Figure 5 also shows the relationship between the dissimilarities of each genotype. It was observed that there was 63.25% dissimilarity, or variability, between genotype A and B,C, and D. Within genotype A, there was 50% dissimilarity between individuals. Between genotypes B,C, and D 45% dissimilarity. Within both genotypes B and C, 31.29% dissimilarity was noted. Individuals 4 and 15, 11 and 25, and 16 and 22 showed 0% dissimilarity.

Multiple genotypes were found within the Stony Brook population of O. rusticus. This statement is based upon the discovery of 63.25% dissimilarity, or variability, between the 25 individuals studied. This degree of variability is high enough to suggest that there is gene flow within this population. In this situation gene flow is defined as the exchange of genetic traits between populations through movement of individuals (Stiling 1996). Since heterozygosity is reduced in inbred or local populations, the large variability of this population must be representative of a population that is exhibiting gene flow (Stiling 1996). In addition as time goes on, populations that are isolated diverge from each other, each losing heterozygosity. Original variation that was exhibited within populations now appears as variation among populations. This principle is genetic drift. Given the amount of variation within this population, the principle of genetic drift can also be discarded in this situation (Griffiths, et al 1993).

From our knowledge of O. rusticus and other species of crayfish we can determine that the evolutionary fitness of this organism is fairly high, based upon its ability to adapt itself to its environment. It does this by changing its body shape to accommodate flow rate, adapting its fecundity in accordance with its environment's ability to support the population, the female's alteration of the amount of eggs she lays in relation to the water temperature, and its ability to out compete other species of crayfish (Holdich 1988).

Further study can be continued by analyzing genetic variability within different populations of this species. Because allele frequencies differ at loci more from one population than they do between populations, genetic diversity in a species consists of diversity within a population and among-population diversity. An equation that models this genetic diversity is

where Ht = total genetic variation in the species, Hp = average diversity within populations, and Dpt = average diversity among populations across the total species range (Stiling 1996). Methods that could be used to determine genetic variability between populations and support the RAPD analysis are: restriction fragment length polymorphic (RFLP) fingerprinting, sequencing of hypervariable regions in the mitochondrial genome, isozyme analysis using microsatellite markers, simple sequence repeats, and coupled PCR and single strand conformational polymorphism (SSCP) assay of the intergenic transcribed spacer (ITS) region.

In addition if this study was to be continued or replicated, there are few things that should be altered. First, there should be no deviations from the original procedure put forward in the materials and methods section. Specific deviations which caused difficulty in gathering experimental data were due to procedural problems with the gels. Specifically, voltage should not exceed 100V and gels should contain ethidium bromide. Figure 2 is an example of these two specific problems in interpreting experimental data. A second alteration that should be made, would be to measure the specimens to determine what stage of development they're in. This observation would be valuable in making generational determinations between individuals and their genotypes. Figure 3 shows the genetic variability within the local population of crayfish. It would probably have been useful to measure the length of the crayfish because size and age are directly correlated. If they are approximately the same length the crayfish in any of the genotypes that we designated could be members of the same cohort.

This study gives evidence that supports the use of RAPD analysis in determining variability within a population. This method of fingerprinting is valuable in that it is relatively easy to obtain valuable data. This study allows for a more introspective interpretation of diversity within a population. This study can also serve as a reference point for future examinations of genetic variation within populations of O. rusticus. Futhermore, it can be a model for other studies relating to genetic diversity within a deme.

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The list of individuals that assisted us with advice, encouragement, knowledge, and general help is highly significant to the successful completion of this experiment. Without the support of certain individuals and the Department of Biological Sciences at Mount Holyoke College, this project would never have been possible. Therefore, first we need to thank the Department of Biological Sciences for making all of the necessary materials for this study available to us, and also thanks to the entire department for their overall support and encouragement.

The first individual we need to acknowledge is our laboratory instructor, Oona West. Oona was integral in helping us to design and conduct this experiment. Without her knowledge of these technologies and her time spent instructing us about these procedures we would not have been able to begin this project.

Next we need to offer our thanks to Professor Craig Woodard of the Department of Biological Sciences for his invaluable assistance and unlimited access to any equipment in his lab that we needed. His devotion of time to our questions and assistance in trouble-shooting and in general procedures was essential to the successful completion of this study.

We would also like to give our thanks to Linda Young of the Department of Biological Sciences for helping us gather supplies by ordering what we needed for this study. We would also like to thank our Ecology and Evolution Professor Aaron Ellison for requiring all of his students to engage in an extended research project, and for approving this study as an acceptable project for the class.

Finally, we must thank the group that made this all possible in the first place- the noble population of O. rusticus in Stony Brook at Mount Holyoke College. This simple crayfish has been overlooked too long by the Mount Holyoke community, and we thank the valiant martyrs whose participation in this study will ensure an interest in the O. rusticus population in Stony Brook for years to come.