DOI: 10.5061/dryad.5hqbzkhgt
title: Juvenile and adult sea Lamprey behaviour (twitch and movement)

description: Sea lamprey are invasive fish species in the Laurentian Great Lakes and
 parasitically feed on many commercially important fishes. Sound has been
 used as a deterrent for invasive species, but its potential for
 manipulating sea lamprey behaviour in natural stream conditions is
 under-tested. Both the behavioural acoustic threshold and differential
 responses at different life stages of sea lamprey have also yet to be
 established. To fill in some of these literature gaps, low-frequency tones
 of 70 or 90 Hz were used in a laboratory setting to determine the
 behavioural responses of adult and juvenile sea lamprey. Both stages of
 sea lamprey exhibited a change in swimming behaviour and a twitch
 (startle) in response to both frequencies. Adults had a lower threshold to
 70 Hz than 90 Hz tone bursts with juveniles showing no difference between
 frequencies. These thresholds and behaviours can be used in natural
 settings to control or impact the behaviour of sea lamprey to aid in
 directing lamprey movement either toward existing traps or away from fish
 bypass structures.
description: Data was collected using experimental tones, of 70 or 90 Hz,
 played at varying amplitude levels (level 0-15 on the MP3 player, 129 –
 169 dB re 1 µPa, -29.95 to -34.75 dB re 1 m/s<sup>2</sup>). To
 determine behavioural reactions and thresholds, sea lamprey would need to
 be consistently still for 1 minute; sound would then be played for 1
 minute, and behavioural reactions (i.e. movement/swimming) were recorded
 during the sound treatment; this was repeated with multiple sessions (1
 minute of no movement followed by 1 minute of sound exposure) within one
 trial. The behavioural reaction of the sea lamprey was also recorded
 directly after (1-10 seconds) the sound was played for 1 minute (swimming
 needed to start within 1-10 seconds but would be recorded for 1 minute to
 look at proportion of the time spent swimming). A stepwise method was used
 for decibel changes between sessions within a trial, where sea lamprey
 were started at a low decibel level sound and no sea lamprey movement
 (i.e. swimming) within the minute of sound exposure, would lead to a
 higher decibel level for the next session. Sea lamprey movement response
 (i.e. swimming) would lead to a lower decibel levels or a repeated decibel
 level (if there was a question of small movement and needed to be
 re-checked), similar to methods used and modified in other studies
 (Tavolga and Wodinsky 1965; Lu et al. 1996; Hawkins and Chapman 2020).
 Sessions within a trial would continue until sea lamprey stopped
 behaviourally responding (swimming), potentially resulting from
 habituation or exhaustion or until the highest decibel level was
 reached.  The immediate response to sound application
 was also used to compare the behaviour of adult and juvenile sea lamprey.
 This “twitch” response, defined by a sudden, short-term tail or body
 movement that occurred within the first second of sound presentation, and
 was quantified as present or not present for the first presentation of any
 decibel level for each individual. The twitch response was compared to the
 individual sea lamprey’s baseline behaviour during the one-minute period
 immediately preceding the sound. Analysis of all trials examined the
 proportion of time spent swimming, prior (no treatment), during (onset of
 sound) and after (offset of sound) noise treatments. In
 total, 54 sea lamprey were used in this experiment, 22 juvenile sea
 lamprey and 32 adult sea lamprey. 32 trials were performed on adult sea
 lamprey with 17 trials with 70 Hz and 15 trials with 90 Hz. 22 trials were
 performed on juvenile sea lamprey, with 10 trials with 70 and 11 trials
 with 90 Hz tones. Five trials included both frequencies, three trials
 started with 90 Hz, followed by high amplitude 70Hz tones (level 12-15;
 161-169 dB re 1 µPa), and two trials started with 70 Hz followed by high
 amplitude 90 Hz tones (level 12-15; 160-167 dB re 1 µPa) to compare high
 amplitude frequencies. Behavioural threshold levels for
 movement during sound exposure were determined by quantifying the
 proportion of time spent swimming; this was compared to the sea lamprey
 baseline taken from unmoving behaviour needed for a full 1 minute before
 playing sound. Since the trials started with lower decibel levels which
 were followed by increases (if no movement was detected), the behavioural
 threshold was quantified as the first decibel level of either frequency
 used (70 or 90 Hz) that elicited a “positive response” behaviour during
 sound. To determine the threshold of movement during sound playing, we
 examined the time spent swimming during the one minute of sound for a
 “positive response” if a sea lamprey was moving more than or equal to 50%
 of the time (30 sec of 60 sec or 0.50 proportionally) then it was
 characterized as a “positive response”. To determine the behavioural
 threshold of movement directly after sound had stopped playing, we
 similarly examined the proportion of time spent swimming after the sound
 for a “positive response,” defined as a sea lamprey that moved within the
 first 10 seconds after the offset. A behavioural threshold for movement
 after sound has ceased is harder to determine and therefore, we used more
 resilient standards for “positive response”; movement after sound must
 have been more than or equal to 80% of the one minute period (48 sec of 60
 or 0.80 proportionally) and the sea lamprey must not have made any
 movement during the 1 minute of sound exposure. The two separate
 thresholds (movement during sound and movement after sound) were then
 determined by examining all positive responses and finding the first and
 the lowest sound level leading to, maximum, one threshold per sea lamprey
 for during sound and after sound. Level 0 was not included as a threshold
 even if the behavioural response was classified as “positive response” due
 to no sound change from background noise, this was assumed to be random
 behaviour (one level 3 (133 re 1 µPa, -30.43 dB re 1
 m/s<sup>2</sup>) was not included because it was first level
 played of the session and behavioural response was 0.5 and we were unable
 to determine if it was a behavioural threshold or random behaviour). The
 sound levels (determined for each responding sea lamprey) were classified
 as the dependent variable, while frequency (70 and 90 Hz) and life stage
 (adult and juvenile) were the independent variables, using an ANOVA in
 SPSS (IBM). The twitch response of lamprey to sound was
 also used to determine the behavioural thresholds. The proportion of
 twitches were calculated for each level (0-15, 129-169 dB re 1 µPa, -29.95
 to -34.75 dB re 1 m/s<sup>2</sup>) for each life stage (adults
 and juveniles) and frequency (70 and 90 Hz) by the proportion of
 individual sea lamprey that presented the twitch behaviour to the total
 number of individuals exposed to the sound decibel within that life stage
 and frequency. The proportion of twitches was classified as the dependent
 variable in adults and juveniles, while frequency (70 and 90 Hz), life
 stage, and differences between the different sound levels (0-15, 129-169
 dB re 1 µPa, -29.95 to -34.75 dB re 1 m/s<sup>2</sup>) were
 classified as independent variables, which were evaluated using a
 Kruskal-Wallis test as this data did not follow a normal distribution. To
 test the difference between levels, arbitrary numbers (0-15, based on
 volume levels of the Mp3 player) were used in statistics, which could then
 be translated to decibel level using Table 1. Sound levels that were
 presented to all sea lamprey less than three times in total during all
 trials, were not considered within the life stage and frequency tested
 (adult and juvenile, 70 or 90 Hz) since all provided insufficient data for
 statistical analysis. Further insight into the data was needed, so a
 separate test specifically comparing each level proportion (1-15; 129-169
 dB re 1 µPa, -29.95 to -34.75 dB re 1 m/s<sup>2</sup>) with
 level 0 (no sound/129 dB re 1 µPa, -29.95 dB re 1
 m/s<sup>2</sup>), was used to statistically determine which
 levels were significantly different from level 0 or no sound. Comparisons
 of levels to level 0 (no movement) were evaluated with simple contrasts
 relative to level 0, with a statistical difference of
 <em>p</em>&lt;0.05 showing a difference from zero for each
 level tested against.
description: # Juvenile and adult sea Lamprey behaviour (twitch and movement)
 [https://doi.org/10.5061/dryad.5hqbzkhgt](https://doi.org/10.5061/dryad.5hqbzkhgt) ## Description of the data and file structure ### Files and variables #### File: Proportion of Twitches.xlsx **Description:**  In total, 54 sea lamprey were used in this experiment, 22 juvenile sea lamprey and 32 adult sea lamprey. 32 trials were performed on adult sea lamprey with 17 trials with 70 Hz and 15 trials with 90 Hz. 22 trials were performed on juvenile sea lamprey, with 11 trials with 70 and 11 trials with 90 Hz tones. Sea lamprey behaviour of immediate response to sound application as a “twitch” behaviour was quantified by tail or body movement that occurred in the first second of sound presented. Adult and juvenile sea lamprey twitches were found to be significant for 70 Hz and a follow up comparison of each Level/Sound Pressure Level/Particle Acceleration to 0 was conducted with the results in this excel, along with the proportion of twitches for each level and the data corresponding to proportion (amount of twitches and no twitches). ##### Variables * "Life Stage" - Life stage of the lamprey tested. Either "Juvenile" meaning before spawning age or "Adult" meaning around spawning age. * "Frequency" - Frequency, two frequencies were used on different lamprey 70 Hz or 90 Hz but only 70 Hz was significant and is the only one depicted here. * "Level" - using experimental tones played at varying levels on the MP3 player, (range from 0-15). Each level played corresponded to a specific sound pressure level (129 – 169 dB re 1 µPa) and particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). * "Pressure" - Each level played corresponded to a specific sound pressure level ( range from 129 – 169 dB re 1 µPa). * "Acceleration" - Each level played corresponded to a specific particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). * "Proportion of Twitches" - Proportion of reactions to tone with twitch. A mean of all adult or juvenile sea lamprey that either did not did not twitch at the different sound levels (0.00-1.00)  * "Twitch" - Amount of sea lamprey that displayed a twitch at the first instance of the sound level of that row * "No Twitch" - Amount of sea lamprey that did not display a twitch at the first instance of the sound level of that row #### File: Juvenile\_Twitch\_Proportion.xlsx **Description:** 22 trials were performed on juvenile sea lamprey, 11 trials using 70 Hz and 11 trials using 90 Hz tones. Five of those trials included both frequencies (three 90 Hz using high amplitude 70 Hz and two vice versa these are marked with red lettering). Juvenile sea lamprey behaviour of immediate response to sound application. This “twitch” behaviour was quantified by tail or body movement that occurred in the first second of sound presented. The behaviour of the sea lamprey as the response during the first second of sound which could be described as a twitch. Each trial, for each lamprey, has a row for each Level/Sound Pressure Level/Particle Acceleration even if that level was not tested in that trial. A "0.00" or "1.00" indicates that level was presented with "1.00" being a positive response (movement) and "0.00" being a negative response (no movement) from the sea lamprey. Organized by "Level" largest to smallest. A column for date was added for the date of that trial conducted and corresponded with trial number in the first column. ##### Variables * "Trial #" - Trial number (#) was the order of trials conducted (1-22) and corresponds with the date of the trial. * "Level" - using experimental tones played at varying levels on the MP3 player, (range from 0-15). Each level played corresponded to a specific sound pressure level (129 – 169 dB re 1 µPa) and particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). * "Pressure" - Each level played corresponded to a specific sound pressure level ( range from 129 – 169 dB re 1 µPa) * "Acceleration" - Each level played corresponded to a specific particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). * "Frequency" - using experimental tones of either 70 Hz or 90 Hz * "Proportion" - Reaction to tone with twitch. A "0.00" or "1.00" indicates that level was tested with "1.00" being a positive response (movement) and "0.00" being a negative response (no movement). A "n/a" indicates that the level indicated was not used during one of the trials, therefore no data can be submitted.  * "Date" - Date of the trial conducted, year-month-day format (yyyy-mm-dd). #### File: Adult\_Twitch\_Proportion.xlsx **Description:**  32 trials were performed on adult sea lamprey with 17 trials with 70 Hz and 15 trials with 90 Hz. Adult sea lamprey behaviour of immediate response to sound application. This “twitch” behaviour was quantified by tail or body movement that occurred in the first second of sound presented. The behaviour of the sea lamprey as the response during the first second of sound which could be described as a twitch. Each trial, for each lamprey, has a row for each Level/Sound Pressure Level/Particle Acceleration even if that level was not tested in that trial. A "0.00" or "1.00" indicates that level was presented with "1.00" being a positive response (movement) and "0.00" being a negative response (no movement) from the sea lamprey. Organized by "Level" largest to smallest. A column for date was added for the date of that trial conducted and corresponded with trial number in the first column. ##### Variables * "Trial #" - Trial number (#) was the order of trials conducted (1-22) and corresponds with the date of the trial. * "Level" - using experimental tones played at varying levels on the MP3 player, (range from 0-15). Each level played corresponded to a specific sound pressure level (129 – 169 dB re 1 µPa) and particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). * "Pressure" - Each level played corresponded to a specific sound pressure level ( range from 129 – 169 dB re 1 µPa) * "Acceleration" - Each level played corresponded to a specific particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). * "Frequency" - using experimental tones of either 70 Hz or 90 Hz * "Proportion" - Reaction to tone with twitch. A "0.00" or "1.00" indicates that level was tested with "1.00" being a positive response (movement) and "0.00" being a negative response (no movement). A "n/a" indicates that the level indicated was not used during one of the trials, therefore no data can be submitted. * "Date" - Date of the trial conducted, year-month-day format (yyyy-mm-dd).   #### File: Adult and Juvenile Proportion Movement Sound Threshold Determination.xlsx **Description:** In total, 54 sea lamprey were used in this experiment, 22 juvenile sea lamprey and 32 adult sea lamprey. 32 trials were performed on adult sea lamprey with 17 trials with 70 Hz and 15 trials with 90 Hz. 22 trials were performed on juvenile sea lamprey, with 11 trials with 70 and 11 trials with 90 Hz tones. Five of the juvenile trials included both frequencies (three 90 Hz using high amplitude 70 Hz and two vice versa). Each trial contained sessions where the sea lamprey would have to remain still for one minute before being exposed to sound of a certain decibel level, followed by a minute after sound exposure. These sessions would be repeated and swimming behaviour during and after sound was recorded by proportion of time (1 min) spent swimming (0.00-1.00) up to 15 sessions in one trial. Behavioural sound thresholds were determined by either 0.50 or more time spent swimming during sound (behavioural threshold sound onset) or 0.80 or more time spent swimming after sound (behavioral threshold sound offset), these are "positive responses" and depicted in two sheets labeled "During" and "After". The behavioural threshold was quantified as the first decibel level of either frequency used (70 or 90 Hz) that elicited a positive response, these were identified with red lettering. Each trial is depicted within each sheet with all sessions labeled by sound level used and proportion of time spent swimming in that session. For some of the early adult sea lamprey trials, behaviour after sound exposure was not recorded and is therefore labeled as "N/A". Also Level 0 (no sound) could not qualify as a behavioural threshold even if there was a qualifying behavioural response, a session with Level 3 with 0.5 proportion spent swimming during also did not qualify as it was the first session presented and had borderline proportion.  ##### Variables * "Trial #" - Trial number (#) was the order of trials conducted. Trials 1-32 were with adult sea lamprey and trials 33-54 were with juvenile sea lamprey. * "Year" - Year of the experimental trial depicted as "20XX". The first group for adults in 2018 (shown as "2018"), only group for juveniles in 2018-2019, and second group of adults in 2019-2020 * "Date (Month_Day_Year)" - Date of the experimental trial, depicted as Month_Day_Year (M_DD_YYYY) * "Age" - Life stage of the lamprey tested. Either "Juvenile" meaning before spawning age or "Adult" meaning around spawning age. * "Frequency (Hz)" - using experimental tones of either 70 Hz or 90 Hz during trial * "Row Classification" - This column was to organize the rows for each trial conducted. Every two rows (for During or sound onset) or three rows (for After or sound offset) were dedicated for one trial. These rows were separated and labeled "Sound Level" for experimental tones played at varying levels on the MP3 player, (range from 0-15, labelled L0-L15 for "level 0 - level 15"). Each level played corresponded to a specific sound pressure level (129 – 169 dB re 1 µPa) and particle acceleration level (-29.95 to -34.75 dB re 1 m/s2), "% swim during" as the proportion of time spent swimming during the 1 min of sound (0.00-1.00), and "% swim after" as the proportion of time spent swimming after sound for one minute (0.00-1.00; only for "After" sheet). * "Session 1" to "Session 15" were the results of the sessions for each lamprey trial. Here would be the level played (range from 0-15, labelled L0-L15 for "level 0 - level 15") cell, followed underneath with a cell for proportion of time spent swimming either during or after. Behavioural thresholds qualifiers (positive responses) were identified in both spreadsheets with highlighted bright yellow cells, and faded yellow highlighted cells showing close to qualifying thresholds. Behavioural thresholds for sound offset (or "After") needed to have proportion of time spent swimming during sound equal to 0 and needed a proportion of time spent swimming after to be at or above 0.8. Behavioural thresholds for sound onset ("During") needed to have the proportion of time spent swimming during sound at 0.5 or above. Red lettering denotes the first session with a positive response and is therefore the behavioural threshold for that trial and sea lamprey.    #### File: Movement After\_Revised.xlsx **Description:** In total, 54 sea lamprey were used in this experiment, 22 juvenile sea lamprey and 32 adult sea lamprey. 32 trials were performed on adult sea lamprey with 17 trials with 70 Hz and 15 trials with 90 Hz. 22 trials were performed on juvenile sea lamprey, with 11 trials with 70 and 11 trials with 90 Hz tones. Behavioural threshold levels were determined by quantifying the proportion of time spent swimming after sound exposure with no movement during sound exposure. The behavioural threshold was quantified as the first decibel level of either frequency used (70 or 90 Hz) that elicited a positive response after sound. A “positive response,” defined as a sea lamprey that moved within the first 10 seconds after the offset and had to be 0.80 or higher proportion of swimming within 1 min after (48 of 60 seconds). Thresholds were then determined by examining all positive responses and finding the first and the lowest sound level leading to, maximum, one threshold per sea lamprey for after sound. Lamprey that did not have qualifying behaviours to sound offset were not included. ##### Variables * "Stage" - Life stage of the lamprey tested. Either "Juvenile" meaning before spawning age or "Adult" meaning around spawning age. * "Freq" - Frequency, two frequencies were used on different lamprey 70 Hz or 90 Hz (depicted as "70" or "90"). * "year" - Year of the experimental trial group. "1" meaning first group (2018 for adults and 2018-2019 for juveniles)  and "2" meaning second group (2019-2020 for adults) * "number in each group" - Groups, for organization, were organized by Stage, Frequency and year. This column counted how many lamprey responded in each group (example Juvenile, 90 Hz, year 1, responses 1, 2 and 3 were in this group).  * "threshold" - Threshold level using experimental tones played at varying levels on the MP3 player, (range from 0-15). * "Pressure" - Each level played corresponded to a specific sound pressure level ( range from 129 – 169 dB re 1 µPa) * "Acceleration - Each level played corresponded to a specific particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). #### File: Movement During\_Revised.xlsx **Description:** In total, 54 sea lamprey were used in this experiment, 22 juvenile sea lamprey and 32 adult sea lamprey. 32 trials were performed on adult sea lamprey with 17 trials with 70 Hz and 15 trials with 90 Hz. 22 trials were performed on juvenile sea lamprey, with 10 trials with 70 and 11 trials with 90 Hz tones. Behavioural threshold levels were determined by quantifying the proportion of time spent swimming during 1 minute of sound exposure. The behavioural threshold was quantified as the first decibel level of either frequency used (70 or 90 Hz) that elicited a “positive response” behaviour during sound. A “positive response” was quantified as the proportion of time spent moving/swiming during sound, if the sea lamprey was swimming 0.50 or more (30 sec of 60 sec) then it was characterized as a “positive response”. Thresholds were then determined by examining all positive responses and finding the first and the lowest sound level leading to, maximum, one threshold per sea lamprey for during sound. Lamprey that did not respond to sound, were not included. ##### Variables * "Stage" - Life stage of the lamprey tested. Either "Juvenile" meaning before spawning age or "Adult" meaning around spawning age. * "Freq" - Frequency, two frequencies were used on different lamprey 70 Hz or 90 Hz (depicted as "70" or "90"). * "year" - Year of the experimental trial. "1" meaning first group (2018 for adults and 2018-2019 for juveniles)  and "2" meaning second group (2019-2020 for adults) * "number in each group" - Groups, for organization, were organized by Stage, Frequency and year. This column counted how many lamprey responded in each group (example Juvenile, 90 Hz, year 1, responses 1, 2 and 3 were in this group).  * "threshold" - Threshold level using experimental tones played at varying levels on the MP3 player, (range from 0-15). * "Pressure" - Each level played corresponded to a specific sound pressure level ( range from 129 – 169 dB re 1 µPa) * "Acceleration - Each level played corresponded to a specific particle acceleration level (-29.95 to -34.75 dB re 1 m/s2). Version Changes June 9, 2025: Additional datasets included (Adult and Juvenile Proportion Movement Sound Threshold Determination.xlsx and Proportion of Twitches.xlsx) for additional background data information on sound threshold determinations for each trial conducted and summary data for proportion of twitches for the two significant data files Adult and Juvenile 70 Hz, in that order. Data for all other datasets (Movement During_Revised.xlsx, Movement After_Revised.xlsx, Juvenile_Twitch_Proportion.xlsx, and Adult_Twitch_Proportion.xlsx) were re-examined and small data corrections were made or addition of columns. Movement During had one trial data changed and two added for Adult 90 Hz. Movement After had one trial data removed from Juvenile 90 Hz, one trial data changed and one added to Adult 90 Hz and two trial data removed from Adult 70 Hz. Juvenile_Twitch_Proportion.xlsx and Adult_Twitch_Proportion.xlsx both had additional columns added for date of the trials (yyyy_mm_dd) and number of the trials and were organized by level and then date of trial. ## Code/software Excel was used to store all files. ANOVA in SPSS (IBM) was used to run the data.