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Online Resource
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Sea lamprey orient toward a source of a synthesized pheromone using odor-
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conditioned rheotaxis
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Nicholas S. Johnson,a,b Azizah Muhammad,c Henry Thompson,a Jongeun Choi,c*
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and Weiming Lia*
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a
Department of Fisheries and Wildlife, Michigan State University, Room 13 Natural
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Resources Building, East Lansing, MI 48824, USA
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b
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Road, Millersburg, MI 49759,USA
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c
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Building, East Lansing, MI 48824, USA
USGS, Great Lakes Science Center, Hammond Bay Biological Station, 11188 Ray
Department of Mechanical Engineering, Michigan State University, 2459 Engineering
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*Corresponding Authors: e-mail: liweim@anr.msu.edu, Phone: 517-432-6705, Fax: 517-
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432-1699 and e-mail: jchoi@egr.msu.edu, Phone: 517-432-3164, Fax: 517-353-1750
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Methods
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Synthesized 3kPZS, permits, and animals
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3kPZS was custom synthesized by Bridge Organics, Inc. (Vicksburg, MI, USA). 3kPZS
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was analyzed for chemical identity and purity using mass spectrometry and nuclear
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magnetic resonance at Michigan State University. Purity was greater than 97%.
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Application of synthesized 3kPZS to the Ocqueoc River (Millersburg, MI, USA) was
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approved by United States Environmental Protection Agency through experimental use
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permit 75437-EUP-2.
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Pre-ovulatory female sea lampreys (Petromyzon marinus) were captured by
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agents of the United States Fish and Wildlife Service, Marquette Biological Station, MI,
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USA, and Department of Fisheries and Ocean Canada, Sea Lamprey Control Centre, ON,
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Canada, in mechanical traps from tributaries to Lakes Huron and Michigan. Sea
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lampreys were transported to United States Geological Survey, Hammond Bay Biological
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Station, Millersburg, MI, USA, and maintained in 1000 L flow through tanks supplied
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with Lake Huron water at ambient temperatures (6 to 15 oC). Sea lampreys placed in
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holding cages to mature were checked at minimum every two days. When ovulated,
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females were returned to Hammond Bay Biological Station for tagging procedures.
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Visual observation of females, 3kPZS plume structure, water velocity mapping, and
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female movement track overlay
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To visually observe movement tracks of females, stream transects were physically
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marked with synthetic string every 5 m from 110 m downstream of the pheromone
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sources to within 10 m of pheromone sources. Transects were located every 2.5 m within
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10 m of the pheromone sources. Along each transect, electrical tape was attached to the
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string at 1 m intervals (Fig. S2). Transects and transect meter marks were transposed to
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stream maps drawn on graph paper where a square equaled 1 m2. During
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experimentation, a researcher would visually observe and describe the movement of a
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lamprey to another researcher that was recording the lamprey’s position on the stream
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map. The cross-sectional location of the female and experimental time was recorded
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whenever a female crossed a visually marked stream transect, where experimental time
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was defined as the time since the female was released. Therefore, the bifurcated stream
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channel contained 26 visual stream transects and at minimum, the location and time of
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each female was recorded during 26 events. These events occurred at different times
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depending on the upstream swimming speed of the female. Additional movement
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locations and times were recorded when the upstream swimming speed of the female
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permitted. Thus, sampling times were non-uniform among individuals.
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To trace 3kPZS distribution in the bifurcated test system, three dye tests were
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conducted: 1) dye was applied in both channels on 30May07, 2) dye was applied to the
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left channel on 31May07, and 3) dye was applied to the right channel on 31May07.
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Rhodamine was the 3kPZS surrogate and was applied to reach a final in-stream
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concentration of 1 x 10-8 M when mixed with the discharge from both channels using the
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same approaches and equipment as for 3kPZS application. After a 10 min dye
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introduction period, water samples were collected in 5 ml glass vials in transects across
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the stream at every 0.5 m. Stream transects were located in the same position as transects
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used for behavioral observation. The florescence intensity of each sample measured at
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556 nm was determined in a luminescence spectrometer (Perkin Elmer LSS55, Downers
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Grove, IL, USA) and rhodamine concentration was estimated using a standard curve (R2
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= 0.9998).
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Dye concentration and water velocity throughout the experimental test systems
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were modeled by a Monotonic Piecewise Cubic Hermite Interpolation Polynomial
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(Fritsch and Carlson 1980). Stream maps, dye distribution maps, and water velocity
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maps were produced in Python (Version 2.4, http://www.python.org/ Copyright © 1990-
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2006, Python Software Foundation) and ovulated female movement tracks were overlaid
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in Python Imaging Library (Version 1.1.6 http://www.pythonware.com/ products/pil/
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Copyright © 1997-2006 by Secret Labs AB & Copyright © 1995-2006 by Fredrik Lundh,
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Publisher: Secret Labs AB) and exported to Photoshop (Version CS2) for final display.
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Dye map color contours were exponentially scaled to match the back calculated
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concentration of 3kPZS in the stream.
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Algorithm pseudo-code
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The following symbols and parameters were used in the control algorithms:
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
3kPZS. z0  10
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
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z 0 was the minimum concentration value at which sea lampreys exhibit a response to
14
M (Johnson et al. 2009).
In odor-conditioned rheotaxis, zth was the threshold concentration value for the sea
lamprey to determine if it was within the odor plume.

In klinotaxis, z max (k ) was a memory function based on the last measured maximum
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concentration. It was used to determine if the sea lamprey was directed toward
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increasing concentration.
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
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zt arg et was the threshold concentration value for the sea lamprey to determine if the
current position was the source of the pheromone.

0    1 was the threshold for the sea lamprey to determine if it was directed
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toward increasing concentration due to klinotaxis. The value was related to the
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resolution of the chemo-sensory input and was chosen as   1014 (Johnson et al.
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2009).
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
In klinotaxis, the forgetting factor 0    0.5  1 was used for the sea lamprey to
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discount z max (k ) such that the half of the maximum value in the previous iteration
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was remembered when the sea lamprey did not find a good direction.
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
v was the forward velocity of the sea lamprey. This quantity was calibrated for each
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algorithm to yield results most similar to observed movements using least squares
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optimization.
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
vmag (k ) was the flow velocity magnitude when the sea lamprey is at ( x(k ), y(k )) .
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
 flow(k ) was used in rheotaxis and odor-conditioned rheotaxis to determine the
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direction of the odor source. It is parallel to the flow but in the upstream direction, i.e.
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180o plus the flow direction.
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
 d (k ) was the desired heading angle of control effort of the algorithms.
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
In odor-conditioned rheotaxis, 1 and  2 defined the degree by which the sea
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lamprey was allowed to deviate from  flow(k ) in two cases: before the first
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detection of the odor plume ( 1 ) and when it was within the plume (  2 ). These
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parameters were chosen such that the sea lamprey was headed in an upstream
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direction in both of these cases; during plume reacquisition, the sea lamprey is
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allowed to orient itself downstream when need to re-enter the plume. 1   2 so
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that the sea lamprey is able to explore a wider area before the first detection of the
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plume than when it is within it.
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
 1 and  2 were the gains for the obstacle avoidance (all algorithms) and odor-
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conditioned rheotaxis strategy.  (k ) was the number of degrees the sea lamprey
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rotated to avoid an obstacle or reacquire the odor plume and these gains determined
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by how much.  1 and  2 were calibrated for each algorithm for the best results.
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
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T  0.5 sec was the sampling time determined by the period of the sea lamprey’s
inhalation (Kleerekoper and Sibakin 1956).
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wx (k ) ~ N (0, x2 ) , w y (k ) ~ N (0, 2y ) , and w (k ) ~ N (0, 2 ) were random
variables from the environment and control effort modeled by the Gaussian white
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noise. These parameters were chosen as  x  0.1 m,  y  0.1 m, and    0.1 m
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(Chang et al. 1998).
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In klinotaxis, N 0 and N1 were the number of iterations the sea lamprey was
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allowed to keep its current direction before exploring a new one. When the odor was not
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present, the lamprey would use N 0 . When the odor was present, except when the
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concentration was increasing, the lamprey would use N1 . This sequence was inspired by
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the “run” and “tumble” observed in bacterial klinotaxis (Adler 1966; Block et al. 1982).
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Control algorithm 1: Rheotaxis (Fig. S3)
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INITIALIZE
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I1. Let the initial position and heading angle be ( x(1), y(1)) and  (1) respectively.
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I2. Measure vmag (1) .
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I3. if vmag (1)  0 then: sea lamprey selects a random direction for  flow(1)
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else:  flow(1) is measured at ( x(1), y(1)) end if
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I4. Let  (0)  0 and  d (0)   flow(1) .
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I5. Let k  1 .
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FIND ODOR SOURCE
S1. if obstacle is encountered then:  d (k ) is chosen such that the obstacle is avoided
end if
S2. (State update) x(k  1)  x(k )  Tv cos (k )  wx (k )
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y(k  1)  y(k )  Tv sin  (k )  wy (k )
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 (k  1)   d (k )  w (k )
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S3. Measure vmag (k  1) .
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S4. if vmag (k  1)  0 then:  flow(k  1)   flow(k )
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else:  flow(k  1) is measured at ( x(k  1), y(k  1)) end if
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S5. Increment k by 1
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S6. Perform steps S1-S5.
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Control algorithm 2: Odor-conditioned rheotaxis (Fig. S4)
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INITIALIZE
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I1. Let the initial position and heading angle be ( x(1), y(1)) and  (1) respectively.
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I2. Measure z (1) and vmag (1) .
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I3. if vmag (1)  0 then:  flow(1) is random direction
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else:  flow(1) is measured at ( x(1), y(1)) end if
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I4. Let  (0)  0 .
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I5. if z (1)  zth then:  d (0) is random direction in
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[ flow(1)  1, flow(1)  1 ]
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else:  d (0) is random direction in [ flow(1)   2 , flow(1)   2 ]
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I6. Let k  1 .
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FIND ODOR PLUME
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P1. while sea lamprey is not in the odor plume ( z (k )  zth ) do:
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P2. if obstacle is encountered then:  d (k ) is chosen such that the obstacle is avoided
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end if
P3. if  (k )   flow(k )  1 AND  (k )   flow(k )  1 then:
 d (k )   flow(k ) end if
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P4. (State update) x(k  1)  x(k )  Tv cos (k )  wx (k )
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y(k  1)  y(k )  Tv sin  (k )  w y (k )
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 (k  1)   d (k )  w (k )
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P5. Measure z (k  1) and vmag (k  1) .
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P6. if vmag (k  1)  0 then:  flow(k  1)   flow(k )
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else:  flow(k  1) is measured at ( x(k  1), y(k  1)) end if
P7. Increment k by 1 end while
TRACK PLUME
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T1. while sea lamprey has not located the odor source ( z (k )  zt arg et ) do:
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T2. if sea lamprey exits odor plume ( z (k )  zth ) then: go to step R1
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else: go to step S3 end if
T3. if obstacle is encountered then:  d (k ) is chosen such that the obstacle is avoided
else:  d (k )   d (k  1) and  (k )  0 end if
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T4. if  d (k )   flow(k )   2 OR  d (k )   flow(k )   2 then:
 d (k )   flow(k ) end if
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T5. Perform steps P4-P6.
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T6. Increment k by 1 end while
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REACQUIRE PLUME
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R1. while sea lamprey is not in odor plume ( z (k )  zth ) do:
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R2. if obstacle is encountered then:  d (k ) is chosen such that the obstacle is
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avoided
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else: go to step R3 end if
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R3. if sea lamprey exited odor plume on right side then:
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 d (k )   d (k  1)   (k  1) and  (k )   1013 z (k )  zth
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else:  d (k )   d (k  1)   (k  1) and  (k )   1013 z (k )  zth
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end if
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R4. Perform steps P4-P6.
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R5. Increment k by 1 end while
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As can be seen in the description of odor-conditioned rheotaxis, the sampling time
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was chosen as a period of inhalation, which is simplistic and might limit the scope of the
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model set. Particularly, with evidence that sea lamprey make decisions based on averaged
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measurements over multiple periods of inhalations, the model needs to be extended
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appropriately. The scope of the model set can be extended by selecting a sampling time
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as a multiple periods of inhalations and replacing the single measurement with the
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average of measurements over the multiple periods. This will produce another discrete-
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time system with a larger sampling time and new measurement noise with a smaller
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variance due to averaging. With this new model, the exact same performance evaluation
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procedure as described in this paper can be performed.
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Control algorithm 3: Klinotaxis (Fig. S5)
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INITIALIZE
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I1. Let the initial position and heading angle be ( x(1), y(1)) and  (1) respectively.
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I2. Measure z (1) .
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I3. Let z max (1)  0 ,  (0)  0 and  d (0) is random direction.
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I4. Let k  1 .
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DETECT ODOR
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O1. while the sea lamprey does not detect any odor ( z (k )  z0 ) do:
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O2. if obstacle is encountered then:  d (k ) is chosen such that the obstacle is avoided
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end if
O3. if  d has been the same direction for the last N 0 iterations then:
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 d (k ) is random direction and  (k )  0
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else:  d (k )   d (k  1) and  (k )  0 end if
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O4. (State update) x(k  1)  x(k )  Tv cos (k )  wx (k )
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y(k  1)  y(k )  Tv sin  (k )  wy (k )
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 (k  1)   d (k )  w (k )
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O5. z max (k  1)  max{ z (k ), z max (k )}
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O6. Measure z(k  1) .
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O7. Increment k by 1 end while
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FIND ODOR SOURCE
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S1. while sea lamprey has not located the odor source ( z (k )  zt arg et ) do:
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S2. if obstacle is encountered then:  d (k ) is chosen such that the obstacle is avoided
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end if
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else: go to step S2 end if
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S3. if 3kPZS concentration is not increasing ( z (k )  z max (k )   ) then:
if sea lamprey does not detect odor ( z (k )  z0 ) then:
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if  d has been the same direction for the last N 0 iterations then:
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 d (k ) is random direction and  (k )  0
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else:  d (k )   d (k  1) and  (k )  0 end if
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else:
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if  d has been the same direction for the last N1 iterations then:
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 d (k ) is random direction and  (k )  0
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else:  d (k )   d (k  1) and  (k )  0 end if
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end if
else:  d (k )   (k  1) and  (k )  0 end if
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S4. Perform steps O3-O5.
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S5. if z (k )  z max   then: z max (k  1)  z max (k )
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else: z max (k  1)  z (k ) end if
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S6. Measure z(k  1) .
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S7. Increment k by 1 end while
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In the control algorithms the obstacle avoidance algorithm was written as
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if obstacle is encountered then:  d (k ) is chosen such that the obstacle is avoided end if.
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The details of the obstacle avoidance algorithm is given as follows.
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1) while obstacle is encountered do:
2) if obstacle was encountered at (k-1)th iteration then: go to step 3
else: go to step 4 end if
3)  (k  1)   (k  2)
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4) if  (k )   flow (k ) then:  (k  1)   1
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else:  (k  1)   1 end if
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5)  d (k )   d (k  1)   (k  1)
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6) Increment k by 1 end while
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When the obstacle avoidance algorithm is activated, the first step is to decide which
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direction the lamprey must turn to swim away from the obstacle. To do this, we first
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determine whether or not obstacle avoidance was active in the previous iteration. We do
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this because the direction in which the lamprey must turn to avoid the obstacle has
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already been decided, and we want to avoid the possibility of turning back into the
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obstacle. In this case, we use the same turning direction until the lamprey is clear of the
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obstacle. In the case that the obstacle avoidance was not previously active, we use the
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lamprey’s heading direction relative to the flow direction to determine the turning
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direction. Obstacle avoidance overrides all other algorithms, because a higher priority is
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placed on clearing obstacles. The obstacle avoidance algorithm is deactivated when the
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sea lamprey has cleared the obstacle.
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Orientation of female sea lampreys to a source of 3kPZS in a flow and reduced-flow
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environment
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Test system
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A PIT antenna system with multiplexer recorded the number of females that moved
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upstream during a trial. Cross channel PIT antennas were located 2.5 m upstream of the
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confluence of the major and minor channel and at the furthest upstream point of the
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minor channel (Fig. S6). A 0.5 m2 PIT antenna was placed around the 3kPZS application
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location which was located 14 m upstream of the female release cage.
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Procedures
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Flow and reduced-flow experiments were not conducted at random because of the
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difficulty of replicating fine-scale flow characteristics when switching the test stream.
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Flow experiments were conducted from 12Jun08 to 17Jun08, reduced-flow experiments
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were conducted from 18Jun08 to 26Jun08, and flow experiments were conducted again
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on 27Jun08 and 28Jun08. A coin was flipped to determine whether 3kPZS or control
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solvent was applied as the test odorant under each flow condition. Six trials were
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conducted with flow and 3kPZS application, 4 trials were conducted with flow and
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control solvent application, 8 trials were conducted with reduced-flow and 3kPZS
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application, and 4 trials were conducted with reduced-flow and control solvent
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application. Between 8 and 10 ovulated females were released per trial. Individual
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females were not exposed to more than one treatment because once ovulated, female
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lifespan is typically less than 7 days.
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Under flow and reduced-flow conditions 3kPZS was applied at a rate to achieve a
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fully mixed in-channel concentration of 5 x 10-13 M. It was assumed that under reduced-
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flow conditions that 25 L/sec of water passed through the channel due to leakage of the
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wing-dam and subsurface water flow. The amount of synthesized 3kPZS required for an
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experiment was dissolved in 1 ml of methanol, mixed with 25 L of river water, and
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applied to the stream at a rate of 167 ml/min via peristaltic pump through a 4.8 mm inside
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diameter tube over a 2.5 h application period. Control solvent consisted of 1 ml of
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methanol mixed with 25 L of river water applied to the stream at a rate of 167 ml/min for
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2.5 h.
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Ovulated females were pre-exposed to the test odorant 30 min prior to release and
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were observed for 2 h after release. Movement tracks of individual sea lampreys were
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manually observed and recorded on a stream map referencing a 0.5 m2 stream grid
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system as described above (Johnson et al. 2009). The number of females leaving the
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release cage, moving upstream through the downstream PIT antenna (move upstream),
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entering inside the 0.5 m2 PIT antenna around the odorant source (visit odor), and
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swimming through the upstream PIT antenna without visiting the odorant source (pass
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odor) was confirmed by the PIT tag system. Significant differences among treatments in
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the above listed metrics were determined with logistic regression models. Data were also
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analyzed with mixed effect logistic regression with a random effect of trial date. All
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statistical results from logistic regression models are robust to the inclusion of the
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random effect of trial date, supporting the assumption that a single ovulated female can
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be treated as an individual sample (Siefkes et al. 2005). Statistical results reported are
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from two-tailed analyses conducted in R (R Development Core Team 2009).
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3kPZS plume structure, water velocity mapping, and female movement track overlay
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In the minor channel the reduced-flow condition dye test was conducted on 26Jun08 and
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the flow condition dye test was conducted on 30Jun08. Under flow and reduced-flow
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conditions, rhodamine was applied to reach a final in-stream concentration of 5 x 10-8 M
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(assuming a discharge of 25 L/sec under no-flow conditions) using the same approaches
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and equipment as used for 3kPZS application. Rhodamine was applied to the
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experimental channel 30 min prior to sampling. Water samples were collected from the
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middle of the water column in 5 ml glass vials in transects across the channel at every 0.5
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m. Channel transects were located every 2.5 m starting at the furthest downstream
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portion of the channel extending to 5 m upstream of the odorant application location.
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Dye concentration and water velocity throughout the experimental test systems was
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measured, modeled, and displayed as described earlier.
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Online Resource References
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Adler J (1966) Chemotaxis in bacteria. Science 153:708-716
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Block SM, Segall JE, Berg HC (1982) Impulse responses in bacterial chemotaxis. Cell
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Chang H-G, Freeman WJ, Burke BC (1998) Biologically modelled noise stabilizing
neurodynamics for pattern recognition. Int J Bifurcat Chaos 8:321-345
Fritsch FN, Carlson RE (1980) Monotone Piecewise Cubic Interpolation. SIAM J Numer
Anal 17:238–246
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Johnson NS, Yun S-S, Thompson HT, Brant CO, Li W (2009) A synthesized pheromone
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induces upstream movement in female sea lamprey and summons them into traps. P
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Natl Acad Sci USA 106:1021-1026. doi:10.1073/pnas.0808530106
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Kleerekoper H, Sibakin K(1956) Spike potentials produced by the sea lamprey
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(Petromyzon marinus) in the water surrounding the head region. Nature 178:490-
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491. doi:10.1038/178490b0
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R Development Core Team (2009) R: a language and environment for statistical
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computing. R Foundation for Statistical Computing. Vienna, Austria. ISBN: 3-
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900051-07-0. http://www.R-project.org. Accessed 25Aug09
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Siefkes MJ, Winterstein JS, Li W (2005). 3-keto petromyzonol sulfate specifically
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attracts ovulating female sea lamprey (Petromyzon marinus). Anim Behav 70:1037-
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Online Resource Figure Captions
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Fig. S1 Water velocity in bifurcated stream channel where sea lamprey orientation
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behaviors were observed. Color coding indicates the estimated water velocity through
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the stream channel. Passive integrated transponder (PIT) antennas were placed around
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the 3kPZS sources which were located 45 m upstream of the channel confluence and
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cross channel PIT antennas were placed 1 m upstream of the channel confluence (white
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lines). PIT antennas determined how many lampreys entered the left and right stream
357
channels and located the left and right source of 3kPZS
358
Fig. S2 Photograph of the downstream section of the bifurcated stream showing stream
359
transects used for behavioral observation and dye sampling
360
Fig. S3 Flowchart for control algorithm 1 (rheotaxis)
361
Fig. S4 Flowchart for control algorithm 2 (odor-conditioned rheotaxis)
362
Fig. S5 Flowchart for control algorithm 3 (klinotaxis)
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Fig. S6 Water velocity of novel experimental system under flow (a) and reduced-flow
364
conditions (b). Passive integrated transponder antennas located at 5 m, 20 m, and around
365
the odor application location at 15 m (O). Females were released from the cage at the 1
366
m mark (RC). Color coding indicates the estimated water velocity through the stream
367
channel
19
368
369
370
Fig. S1
20
371
372
Fig.S2
21
373
START
INITIALIZE
FIND ODOR
SOURCE
374
375
376
Fig. S3
22
START
INITIALIZE
z (k )  z th
NO
YES
FIND PLUME
NO
z (k )  zth
YES
TRACK
PLUME
z (k )  zth
YES
NO
NO
z (k )  zt arg et YES
REACQUIRE
PLUME
NO
END
377
378
379
Fig. S4
23
z (k )  zth
YES
START
INITIALIZE
NO
z (k )  z0
YES
DETECT
ODOR
NO
z (k )  z0
YES
FIND ODOR
SOURCE
YES
END
380
381
382
Fig. S5
24
z (k )  zt arg et
NO
383
(a)
(b)
384
385
Fig. S6
25