; Alife - Project Report ; Ken Webb ; (C) Copyright 2004 Ken Webb ; All rights reserved ; January 9, 2004 ; ProSort_MinimalModel_2.nlogo ; There are three types of objects in this system (as in all NetLogo programs): ; - a single observer object, ; - a large number of patch objects, ; - a large number of protein objects (a breed of turtle). ; Using standard NetLogo practice, each procedure in this program is identified ; as being an observer procedure, a patches procedure, or a protein procedure. ; PROGRAM DECLARATIONS ; ==================== ; Proteins ; -------- ; this is a built-in NetLogo command that declares protein as a breed of turtle breeds [ proteins ] ; Lipid variables ; --------------- ; this is a built-in NetLogo command that declares instance variables that belong to all patches patches-own [ lipidType lipidThickness ] ; Protein variables ; ----------------- ; this is a built-in NetLogo command that declares instance variables that belong to all proteins proteins-own [ proteinL ; length of protein hydrophobic length (Lundbaek p.2081, 2082) ] ; Global Constants and Variables ; ------------------------------ ; this is a built-in NetLogo command that declares global constants and variables globals [ ; Types of lipids sopcType ; 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC) (100%) cholSopcType ; Cholesterol and Sopc together (1:1 50% each) ; Thicknesses of the lipid types in nanometers (nm) sopcThickness ; Sopc lipids cholSopcThickness ; CholSopc lipids meanThickness ; Mean thickness of bilayer when Sopc:CholSopc is 1:1 meanL ; mean protein TMD length ; Colors used to display the lipid types sopcColor ; Sopc cholSopcColor ; CholSopc proteinColor ; Base color to use when coloring proteins ; Indices into list used in swap routine LTypeIx LThickIx LColIx ; Misc. globalValList ; used for swap generationCount ; shows passage of time (time steps) budInterval ; try budding every budInterval time steps bud_Radius ; radius of the bud region budTotalLipidsInRadius ; total number of lipids within random radius budCompareVal ; number if n% are cholSopcType chromosome ; list of lists of possible Protein & Lipid movement probability values ; Probability matrix of protein (P_) movement ; see seek-optimal-site function ; Gt = greater than ; Eq = equal ; Lt = less than P_ProbGtGt P_ProbGtEq P_ProbGtLt P_ProbEqGt P_ProbEqEq P_ProbEqLt P_ProbLtGt P_ProbLtEq P_ProbLtLt ; Probability matrix of lipid (L_) movement L_ProbIf0 L_ProbIf1 L_ProbIf2 L_ProbIf3 L_ProbIf4 L_ProbIf5 L_ProbIf6 L_ProbIf7 L_ProbIf8 ] ; SETUP ; ===== ; Setup - invoked when user presses the setup button in the GUI ; ----- to setup ; observer procedure ca ; clear all clear-output ; clear the output window set-globals set-probabilities ChromosomeNum chromosome set budTotalLipidsInRadius value-from (patch 0 0) [count patches in-radius bud_Radius] set budCompareVal budTotalLipidsInRadius * 0.5 ; bud when 50% are cholSopcType initialize-lipidBilayer ask patches [ color-lipid-type ] report-colors initialize-proteins plot-sorting ; plot an initial value on the graph end ; Give values to all the global variables ; --------------------------------------- to set-globals ; observer procedure ; Constants set sopcType 1 ; type set cholSopcType 2 set sopcThickness 3.0 ; thickness in nm set cholSopcThickness 3.3 set sopcColor green ; color set cholSopcColor red set proteinColor blue ; Bud set budInterval 1 ; try budding every budInterval time steps set bud_Radius 7 ; default ; List indices used in swap routine set LTypeIx 0 set LThickIx 1 set LColIx 2 ; Variables set meanThickness mean list sopcThickness cholSopcThickness set globalValList [] set generationCount 0 ; Chromosome ; ---------- ; ChromosomeNum is the first value in each sub-list, 1, 2, ... ; Probability matrix for Protein (P_) movement (values in range from 1 to n). ; prob = 1 is 100% certain ; prob = 100000 is highly unlikely ; default 8 3 2 12 3 4 2 3 4 ; perfect sorting 100000 3 2 100000 3 100000 2 3 100000 ; Probability matrix for Lipid (L_) swap (values in range from 1 to n). ; default 2 16 64 160 320 640 10000 100000 1000000 ; BudRadius ; 99 = get value from GUI rather than from this chromosome set chromosome [ [1 [ 8 3 2 12 3 4 2 3 4 2 16 64 160 320 640 10000 100000 1000000 99]] ; default [2 [100000 3 2 100000 3 100000 2 3 100000 1 2 4 5 10000 10000 10000 100000 1000000 99]] ; perfect sorting ] end ; Set the probability variables from values in chromosome list ; ------------------------------------------------------------ ; this is a standard lisp-like recursive function to set-probabilities [probID pList] ; observer procedure ifelse empty? pList [print "Can't find pList"] [ifelse first first pList = probID [set-prob-detail 1 first but-first first pList] [set-probabilities probID but-first pList] ] end ; Set value for one probability variable (for use only by set-probabities) ; -------------------------------------- to set-prob-detail [itemID pList] ; observer procedure ; Protein movement (P_) probabilities if itemID = 1 [set P_ProbGtGt first pList] if itemID = 2 [set P_ProbGtEq first pList] if itemID = 3 [set P_ProbGtLt first pList] if itemID = 4 [set P_ProbEqGt first pList] if itemID = 5 [set P_ProbEqEq first pList] if itemID = 6 [set P_ProbEqLt first pList] if itemID = 7 [set P_ProbLtGt first pList] if itemID = 8 [set P_ProbLtEq first pList] if itemID = 9 [set P_ProbLtLt first pList] ; Lipid movement (L_) probabilities if itemID = 10 [set L_ProbIf0 first pList] if itemID = 11 [set L_ProbIf1 first pList] if itemID = 12 [set L_ProbIf2 first pList] if itemID = 13 [set L_ProbIf3 first pList] if itemID = 14 [set L_ProbIf4 first pList] if itemID = 15 [set L_ProbIf5 first pList] if itemID = 16 [set L_ProbIf6 first pList] if itemID = 17 [set L_ProbIf7 first pList] if itemID = 18 [set L_ProbIf8 first pList] ; Bud Radius if itemID = 19 [ set bud_Radius first pList if bud_Radius = 99 [set bud_Radius BudRadius] ; allow it to be set from GUI ] ; recursively handle next value in the list if itemID < 19 [set-prob-detail itemID + 1 but-first pList] end ; Initialize the lipid bilayer ; ---------------------------- to initialize-lipidBilayer ; observer procedure ask patches [set-lipid-type-Random] ask patches [set-lipid-thickness] end ; Initialize proteins ; ------------------- to initialize-proteins ; observer procedure set-default-shape proteins "circle" create-custom-proteins NumberOfProteins [ setxy (random screen-size-x) (random screen-size-y) ; only 2 possible values in this minimal model ifelse (random 2 = 1) [ set proteinL 2.85] [ set proteinL 3.60] ] set meanL mean values-from proteins [proteinL] ask proteins [set color proteinColor - ((proteinL - meanL) * 10)] end ; Set lipid type ; -------------- to set-lipid-type-Random ; patches procedure ifelse random 100 < PercentCholesterolSopc [set lipidType cholSopcType] ; CholSopc [set lipidType sopcType] ; SOPC end ; Set default lipid thickness ; --------------------------- to set-lipid-thickness ; patches procedure ifelse lipidType = sopcType [ set lipidThickness sopcThickness ] [ set lipidThickness cholSopcThickness ] end ; Color lipids based on their type only ; ------------------------------------- to color-lipid-type ; patches procedure ifelse lipidType = sopcType [ set pcolor sopcColor ] [ set pcolor cholSopcColor ] end ; Report the meaning of the different colors in the model ; ------------------------------------------------------- to report-colors ; observer procedure type "Sopc lipid is " report-color sopcColor type ". CholSopc combination is " report-color cholSopcColor print "." print "For protein TMD colors, see legend in graph at left." end ; for use only by report-colors ; ----------------------------- to report-color [lColor] ; observer procedure locals [strColor] set strColor lColor if shade-of? red lColor [set strColor "red"] if shade-of? green lColor [set strColor "green"] if shade-of? blue lColor [set strColor "blue"] type strColor end ; PROCESS ; ======= ; Go - invoked when user presses the go button in the GUI ; -- to go ; observer procedure ask proteins [seek-optimal-site] ask patches [adjust-domain-size-continuous] if generationCount mod budInterval = 0 [bud-and-dock] plot-sorting set generationCount generationCount + 1 end ; Seek optimal site depending on TMD length ; ----------------------------------------- ; The protein has two questions: ; 1. What state am I currently in? (compare myVal with 0) ; 2. What state would I be in if I moved? (compare bnVal with myVal) ; to seek-optimal-site ; protein procedure locals [pL bestNeighbor bnVal myVal x y] set pL proteinL set bestNeighbor random-one-of neighbors set bnVal value-from bestNeighbor [pL - lipidThickness] set myVal pL - lipidThickness ; value at current location set x value-from bestNeighbor [pxcor] set y value-from bestNeighbor [pycor] if myVal > 0 ; TMD partly exposed to water [ if bnVal > myVal and random P_ProbGtGt = 1 [setxy x y] ; get worse if bnVal = myVal and random P_ProbGtEq = 1 [setxy x y] ; stay the same if bnVal < myVal and random P_ProbGtLt = 1 [setxy x y] ; get better ] if MyVal = 0 ; TMD optimal [ if bnVal > myVal and random P_ProbEqGt = 1 [setxy x y] ; get much worse if bnVal = myVal and random P_ProbEqEq = 1 [setxy x y] ; stay optimal if bnVal < myVal and random P_ProbEqLt = 1 [setxy x y] ; get somewhat worse ] if myVal < 0 ; TMD embedded in lipid with room to spare [ if bnVal > myVal and random P_ProbLtGt = 1 [setxy x y] ; get better if bnVal = myVal and random P_ProbLtEq = 1 [setxy x y] ; stay the same if bnVal < myVal and random P_ProbLtLt = 1 [setxy x y] ; get somewhat worse ] end ; Adjust domain size (continuously) ; --------------------------------- to adjust-domain-size-continuous ; patches procedure locals [neighCount] ; Sopc lipids swap their position if surrounded by CholSopc lipids if lipidType = sopcType [ if count neighbors with [lipidType = sopcType] < 2 [swap-lipids cholSopcType] ] ; CholSopc lipids may swap their positions based on number of neighbors of ; their own type, and on a randomly generated value within a range ; determined by a probability value. if lipidType = cholSopcType [ set neighCount count neighbors with [lipidType = cholSopcType] if neighCount = 0 and random L_ProbIf0 = 0 [swap-lipids sopcType] if neighCount = 1 and random L_ProbIf1 = 0 [swap-lipids sopcType] if neighCount = 2 and random L_ProbIf2 = 0 [swap-lipids sopcType] if neighCount = 3 and random L_ProbIf3 = 0 [swap-lipids sopcType] if neighCount = 4 and random L_ProbIf4 = 0 [swap-lipids sopcType] if neighCount = 5 and random L_ProbIf5 = 0 [swap-lipids sopcType] if neighCount = 6 and random L_ProbIf6 = 0 [swap-lipids sopcType] if neighCount = 7 and random L_ProbIf7 = 0 [swap-lipids sopcType] if neighCount = 8 and random L_ProbIf8 = 0 [swap-lipids sopcType] ] end ; Bud and Dock ; ------------ to bud-and-dock ; patches procedure locals [x y r lCount pCount] ; create an invisible circular region at a random location set x (random screen-size-x) - screen-edge-x set y (random screen-size-y) - screen-edge-y set r bud_Radius ; count the number of CholSopc lipids within that circular region set lCount value-from (patch x y) [count (patches in-radius r) with [lipidType = cholSopcType]] ; check if this count is bigger than the threshold if lCount > budCompareVal [ ; Bud lipids ask (patch x y) [ask (patches in-radius r) [set-all-lipid-variables sopcType sopcThickness lime]] ; Bud and Dock proteins - by random repositioning, rather than die and recreate ask (patch x y) [ask (proteins in-radius r) [setxy ((random screen-size-x) - screen-edge-x) ((random screen-size-y) - screen-edge-y)]] ; Dock lipids - fill in same number of random locations while [lCount > 0] [ set x (random screen-size-x) - screen-edge-x set y (random screen-size-y) - screen-edge-y if value-from (patch x y) [lipidType] = sopcType [ ask (patch x y) [set-all-lipid-variables cholSopcType cholSopcThickness yellow] ] set lCount lCount - 1 ] ; end while ] ; end if end ; Plot Sorting - plot one set of values on the graph ; ------------ to plot-sorting ; observer procedure locals [tmdShort tmdLong] if count proteins < 100 [stop] ; prevent a divide-by-zero error set-current-plot "Proteins in CholSopc Lipid" ; TMD Short amino acids (len 15 16 17) set-current-plot-pen "TmdShort" set tmdShort count proteins with [proteinL = 2.85 and lipidType = cholSopcType] / count proteins with [proteinL = 2.85] * 100 plot tmdShort ; TMD Long amino acids (len 18 19 20) set-current-plot-pen "TmdLong" set tmdLong count proteins with [proteinL = 3.6 and lipidType = cholSopcType] / count proteins with [proteinL = 3.6] * 100 plot tmdLong end ; Swap lipids ; ----------- to swap-lipids [swapeeType] ; patches procedure locals [lType lThick lCol valList somebody] set lType lipidType set lThick lipidThickness set lCol pcolor ; the entire swap operation is atomic and must not be interrupted without-interruption [ set somebody random-one-of neighbors with [lipidType = swapeeType] if somebody != nobody [ask somebody [swap-all-lipid-variables lType lThick lCol]] set valList globalValList ] set lipidType item LTypeIx valList set lipidThickness item LThickIx valList set pcolor item LColIx valList end ; Swap all lipid variables ; ------------------------ to swap-all-lipid-variables [lType lThick lCol] ; patches procedure locals [oldVals] set oldVals (list lipidType lipidThickness pcolor) set-all-lipid-variables lType lThick lCol set globalValList oldVals end ; Set all lipid variables ; ----------------------- to set-all-lipid-variables [lType lThick lCol] ; patches procedure set lipidType lType set lipidThickness lThick set pcolor lCol end @#$#@#$#@ GRAPHICS-WINDOW 321 10 733 443 100 100 2.0 0 10 0 0 CC-WINDOW 321 447 734 566 Command Center BUTTON 231 62 314 95 NIL setup NIL 1 T OBSERVER SLIDER 7 10 205 43 PercentCholesterolSopc PercentCholesterolSopc 0 100 20 1 1 NIL MONITOR 7 472 179 521 AverageBilayerThickness sum values-from patches [lipidThickness] / count patches 3 1 SLIDER 7 98 205 131 NumberOfProteins NumberOfProteins 10 5000 2000 10 1 NIL BUTTON 231 99 314 132 NIL go T 1 T OBSERVER SLIDER 7 199 145 232 ChromosomeNum ChromosomeNum 1 2 2 1 1 NIL MONITOR 231 10 314 59 Time Step generationCount 0 1 PLOT 7 244 293 467 Proteins in CholSopc Lipid Time Step Concentration (% in rafts) 0.0 100.0 0.0 100.0 true true PENS "TmdShort" 1.0 0 -4210689 true "TmdLong" 1.0 0 -16777152 true SLIDER 150 199 314 232 BudRadius BudRadius 1 20 7 1 1 NIL @#$#@#$#@ WHAT IS IT? ----------- Eukaryotic cells (those found in all higher organisms) contain multiple compartments that each perform distinct functions. The endoplasmic reticulum (ER) is a major site of protein production. These proteins are transported in small spherical membrane-bounded vesicles to the Golgi complex for sorting, a process that determines the proteins’ final destinations. Some of the fine details of sorting are not yet known, such as the exact mechanism by which proteins become selectively attached to specific domains in the Golgi membrane bilayer. These regions subsequently bud off to become vesicles destined for the cell’s outer plasma membrane. The goal of this project is to produce a minimal model that demonstrates the emergence of protein sorting in the Golgi, given a set of simpler lipid and protein movement behaviors that depend on relative size. The current individual-based model is an early prototype that shows one way to achieve protein sorting. The model exhibits: 1. The self-organization of lipids into domains, including thicker cholesterol-rich lipid rafts. 2. The selective association of protein trans-membrane domains (TMDs) with different lipid domains based on thickness, resulting in protein sorting. 3. The continuous budding off of vesicles from regions containing lipid rafts with associated sorted proteins as cargo, and replacement of this material through the docking of less self-organized vesicles. 4. Achievement of a steady-state in which the pathways of lipid self-organization, protein sorting, and vesicle budding and docking, collectively maintain a homeostatic organization. HOW IT WORKS ------------ Lipids are modeled as NetLogo patches. There are two types of lipids, 1-stearoyl-2-oleoyl-phosphatidylcholine (sopcType), and a 1 to 1 combination of cholesterol and Sopc (cholSopcType). Each lipid type is a different thickness and is displayed in a different color: lipidType lipidThickness lipidColor --------- -------------- ---------- sopcType 3.0 nm green cholSopcType 3.3 nm red The only possible operation for a lipid is to swap itself with an immediate neighbor of a different type. In a real cell, a fluid lateral diffusion occurs as lipids of all types constantly randomly swap places with other lipids in their local neighborhood. Here is the algorithm used by each lipid patch at each time step (adjust-domain-size-continuous): If I am a CholSopc lipid Then If 0 of my 8 neighbors is a CholSopc lipid Then With probability ProbIf0, swap with a random one of my Sopc neighbors If 1 of my 8 neighbors is a CholSopc lipid Then With probability ProbIf1, swap with a random one of my Sopc neighbors … (same idea for 2, 3, 4, 5, and 6) … If 7 of my 8 neighbors is a CholSopc lipid Then With probability ProbIf7, swap with a random one of my Sopc neighbors If all 8 of my 8 neighbors are CholSopc lipids Then do nothing If I am a Sopc lipid Then If fewer than 2 Of my 8 neighbors are Sopc lipids Then Swap with a random one of my CholSopc neighbors Proteins are modeled as a breed of NetLogo turtles, that interact over time with the lipid patches. A protein’s only attribute is a hydrophobic trans-membrane domain (TMD), which can be either short (light blue) or long (very dark blue). The TMD seeks to embed itself within a region of lipid bilayer whose thickness is compatible with its TMD length. A long TMD-protein seeks the thicker cholesterol-rich CholSopc lipid rafts. A short TMD-protein seeks the thinner cholesterol-free Sopc lipid regions. At each time step, all proteins follow the same set of rules in deciding whether to stay where they are or possibly move to a neighboring patch that may lead to a region of optimal lipid thickness. Here is the algorithm (seek-optimal-site): If my TMD is partly exposed to water (i.e. my hydrophobic length > the thickness of the lipid in which I am embedded) Then If a random one of my lipid neighbors is even thinner Then With very low probability P_ProbGtGt, move to that patch If a random one of my lipid neighbors is the same thickness Then With some probability P_ProbGtEq, move to that patch If a random one of my lipid neighbors is thicker Then With relatively high probability P_ProbGtLt, move to that patch If my TMD is already optimal (i.e. my hydrophobic length = the thickness of the lipid in which I am embedded) Then If a random one of my lipid neighbors is thinner Then With very low probability P_ProbEqGt, move to that patch If a random one of my lipid neighbors is the same thickness Then With some probability P_ProbEqEq, move to that patch If a random one of my lipid neighbors is thicker Then With relatively low probability P_ProbEqLt, move to that patch If my TMD has room to spare ((i.e. my hydrophobic length < the thickness of the lipid in which I am embedded) Then If a random one of my lipid neighbors is thinner Then With relatively high probability P_ProbLtGt, move to that patch If a random one of my lipid neighbors is the same thickness Then With some probability P_ProbLtEq, move to that patch If a random one of my lipid neighbors is even thicker Then With relatively low probability P_ProbLtLt, move to that patch Lipid rafts gradually emerge out of an initial random configuration and through the local actions of individual lipid patches, as described above. Lipid rafts are regions consisting largely of patches containing lipids of cholSopcType. They are high in cholesterol (Chol). These constructs emerge without being explicitly mentioned anywhere in the rules. In the model, as a lipid raft grows in size, there is an increasing probability that a circular region of the membrane containing a raft will bud off to become a separate vesicle. The material that buds off is replaced by lipids in vesicles that dock with the membrane. A similar mechanism takes place in a real biological cell. Vesicles from the endoplasmic reticulum (ER) dock with the Golgi. Vesicles that are higher in cholesterol content later bud off from the Golgi and find their way to the plasma membrane on the outside of the cell. An extended neighborhood is specified using the NetLogo in-radius primitive. The number of patches, including the current patch, that are within one radius unit, is 5. 13 patches are within a radius of 2, and 149 patches are within a radius of 7. The model uses a default radius of 7 (BudRadius) to specify a region large enough to, with some probability, bud off. Every n time steps (default: 1), a random patch is sampled to see if the number of lipids with lipidType = cholSopcType within this extended neighborhood exceeds some threshold (BudRadius). If so, a vesicle buds off. In the model this means that CholSopc lipids change their state to Sopc, and all proteins that were within this region cease to exist. The “lost” CholSopc lipids and proteins are recreated at random locations in the membrane, simulating the docking of vesicles from the ER. The result is a reduction in order that counteracts the fine-grained order-increasing behavior of individual lipids described previously. HOW TO USE IT ------------- Set the PercentCholesterolSopc slider to a value between 0% and 100% cholesterol. The default for the Golgi is 20%. Set the NumberOfProteins slider to a value between 100 and 5000. The default is 2000. Set the ChromosomeNum to 1 or 2. This determines the probabilities of position-swapping and movement for lipids and proteins. It is called a chromosome because it’s a list of values that could be optimized using a genetic algorithm. With a value of 1, behavior is somewhat more exploratory. With a value of 2 (the default), the lipids and proteins tend to maintain current good locations. Set the BudRadius to a value from 1 to 10, but typically between 5 and 8. The default is 7. Press the setup button. You will see a random initial configuration. Look at the Command Center window for information on the colors used. Press the Go button. THINGS TO NOTICE ---------------- The thicker CholSopc lipids will gradually cluster together to form lipid rafts, red domains within the green Sopc lipid. The long-TMD proteins (darker color) will mostly sort themselves into the thicker lipid rafts, while the short-TMD proteins (lighter color) will be retained in the Sopc. Watch the graph. It shows the percentage of each type of protein (short-TMD and long-TMD) embedded within CholSopc lipids, a rough measure of protein sorting. Note that this measure does not discriminate between solitary CholSopc patches and CholSopc found in lipid raft regions. After about 800-900 time steps, some 90% of long-TMD proteins (the darker curve) will be embedded in CholSopc, while only about 4% of short-TMD proteins (the lighter curve) will be embedded in CholSopc. Also at around 800-900 time steps, budding will start to occur. Sufficiently large lipid rafts will bud off as vesicles carrying whatever lipids and proteins were within a BudRadius radius of the approximate center of the lipid raft. The budding will appear as a circular region in a different shade of green. The same number of CholSopc lipid and protein units will be randomly distributed over the entire NetLogo grid, representing docking of new vesicles from the ER. The new CholSopc lipids will appear yellow, but are otherwise the same as the red ones. By some 4000 time steps, the long-TMD protein curve will be trending downward. Because of the randomness introduced by many cycles of budding and docking, more and more of the long TMD proteins become transiently associated with individual CholSopc lipid patches that are not part of a cluster. These CholSopc are constantly moving so a high proportion of the long TMD are constantly being moved back into Sopc patches. The slow downward trend will continue for many thousands of time steps. The overall pattern on the graph for the default starting conditions, is hyperbolic growth, followed by exponential decay. THINGS TO TRY ------------- (1) Change the ChromosomeNum to 1, while keeping the other values at their defaults. Press setup and go. The behavior is quite different. Watch the effect of this change on clustering, and watch the graph. (2) For a much faster moving simulation, but one in which it will be hard to see the clustering, set values as follows: ChromosomeNum = 1, BudRadius = 5, NumberOfProteins = 500. Also you will need to change the size of the NetLogo grid, to the NetLogo default of 35 by 35. Press the “more ...” button just above the grid. Set “Screen Edge X” to 17, “Screen Edge Y” to 17, and “Patch Size” (pixels) to 9.0. Press OK. This configuration produces clustering into lipid rafts, budding, and a flat curve. EXTENDING THE MODEL ------------------- Add additional intermediate protein TMD lengths, and try to get the longer sizes sorted into the lipid rafts. NETLOGO FEATURES ---------------- This model does not yet work with 2.x versions of NetLogo. It has only been tested with NetLogo 1.3.1. RELATED MODELS -------------- There are no other models I am aware of that deal with protein sorting. CREDITS AND REFERENCES ---------------------- Copyright (C) 2004 by Ken Webb. All rights reserved. @#$#@#$#@ default true 0 Polygon -7566196 true true 150 5 40 250 150 205 260 250 ant true 0 Polygon -7566196 true true 136 61 129 46 144 30 119 45 124 60 114 82 97 37 132 10 93 36 111 84 127 105 172 105 189 84 208 35 171 11 202 35 204 37 186 82 177 60 180 44 159 32 170 44 165 60 Polygon -7566196 true true 150 95 135 103 139 117 125 149 137 180 135 196 150 204 166 195 161 180 174 150 158 116 164 102 Polygon -7566196 true true 149 186 128 197 114 232 134 270 149 282 166 270 185 232 171 195 149 186 149 186 Polygon -7566196 true true 225 66 230 107 159 122 161 127 234 111 236 106 Polygon -7566196 true true 78 58 99 116 139 123 137 128 95 119 Polygon -7566196 true true 48 103 90 147 129 147 130 151 86 151 Polygon -7566196 true true 65 224 92 171 134 160 135 164 95 175 Polygon -7566196 true true 235 222 210 170 163 162 161 166 208 174 Polygon -7566196 true true 249 107 211 147 168 147 168 150 213 150 arrow true 0 Polygon -7566196 true true 150 0 0 150 105 150 105 293 195 293 195 150 300 150 bee true 0 Polygon -256 true false 151 152 137 77 105 67 89 67 66 74 48 85 36 100 24 116 14 134 0 151 15 167 22 182 40 206 58 220 82 226 105 226 134 222 Polygon -16777216 true false 151 150 149 128 149 114 155 98 178 80 197 80 217 81 233 95 242 117 246 141 247 151 245 177 234 195 218 207 206 211 184 211 161 204 151 189 148 171 Polygon -7566196 true true 246 151 241 119 240 96 250 81 261 78 275 87 282 103 277 115 287 121 299 150 286 180 277 189 283 197 281 210 270 222 256 222 243 212 242 192 Polygon -16777216 true false 115 70 129 74 128 223 114 224 Polygon -16777216 true false 89 67 74 71 74 224 89 225 89 67 Polygon -16777216 true false 43 91 31 106 31 195 45 211 Line -1 false 200 144 213 70 Line -1 false 213 70 213 45 Line -1 false 214 45 203 26 Line -1 false 204 26 185 22 Line -1 false 185 22 170 25 Line -1 false 169 26 159 37 Line -1 false 159 37 156 55 Line -1 false 157 55 199 143 Line -1 false 200 141 162 227 Line -1 false 162 227 163 241 Line -1 false 163 241 171 249 Line -1 false 171 249 190 254 Line -1 false 192 253 203 248 Line -1 false 205 249 218 235 Line -1 false 218 235 200 144 bird1 false 0 Polygon -7566196 true true 2 6 2 39 270 298 297 298 299 271 187 160 279 75 276 22 100 67 31 0 bird2 false 0 Polygon -7566196 true true 2 4 33 4 298 270 298 298 272 298 155 184 117 289 61 295 61 105 0 43 boat1 false 0 Polygon -1 true false 63 162 90 207 223 207 290 162 Rectangle -6524078 true false 150 32 157 162 Polygon -16776961 true false 150 34 131 49 145 47 147 48 149 49 Polygon -7566196 true true 158 33 230 157 182 150 169 151 157 156 Polygon -7566196 true true 149 55 88 143 103 139 111 136 117 139 126 145 130 147 139 147 146 146 149 55 boat2 false 0 Polygon -1 true false 63 162 90 207 223 207 290 162 Rectangle -6524078 true false 150 32 157 162 Polygon -16776961 true false 150 34 131 49 145 47 147 48 149 49 Polygon -7566196 true true 157 54 175 79 174 96 185 102 178 112 194 124 196 131 190 139 192 146 211 151 216 154 157 154 Polygon 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