Acoustic communication of American Crocodiles (Crocodylus acutus) is relatively understudied. Our overall aim was to determine the acoustic structure of wild American Crocodile distress calls, distinguish call differences among size classes (hatchling, juvenile, sub-adult, and adult), and investigate call production on a gradient of human disturbance. American Crocodile distress calls have strong frequency modulation and are comprised of multiple harmonics in a downsweeping pattern. Measured parameters (total duration, first quartile duration, maximal frequency, first quartile frequency, end frequency, slope of first quartile, slope of last quartiles) differed significantly among size classes (P < 0.05). Hatchling distress calls are higher in frequency and strongly modulated, whereas calls produced by sub-adults and adults showed little modulation, are lower in frequency, and have greater overall duration. Proportion of crocodiles that produced distress calls during capture differed by size class and sampling location, particularly adult distress calls which are reported here to be produced with undocumented frequency. We determined that American Crocodiles of all size classes produce distress calls at varying rates among study sites. Our results demonstrate that American crocodiles produce distress call more frequently at sites with higher anthropogenic activity. Measured call parameters of juveniles and hatchling American crocodiles also varied among sites in relation to human disturbance. Calls recorded at sites of high anthropogenic impact have increased duration and less modulation which may adversely affect response to emitted distress calls. Proportional and call parameter variances suggest anthropogenic activity as a driver for increased call production and alteration of call parameters at high human-impacted sites.

We recorded American Crocodiles on Ambergris Caye from May – August 2015, December 2015 – January 2016, and March of 2016.  Concurrent with ongoing population surveys, we collected recordings on Caye Caulker in January, March, and August 2016. We collected acoustic recordings from BAL June – August 2016. We recorded distress calls ad libitum in the field during capture events as recording conditions and difficulty of capture determined the actual number of recordings collected. Independent of acoustic recording, we noted incidence of call production for each individual capture as not all captures resulted in call production or successful recording. We collected distress call recordings from May 2015 – January 2016 using a Marantz PDM661 or Roland R-26 digital recorder coupled with a Senheiser ME67 shotgun directional microphone. During the March–August 2016 field seasons, we employed a Sony Zoom H5 digital recorder with an XY modular microphone capsule. We recorded all calls in .wav format at a sampling rate of 44.1 kHz and 24 bits per sample. Although equipment differed, sampling protocol remained consistent. We held hatchling and juvenile crocodiles in hand and collected recordings approximately 50 cm from the microphone.  We recorded sub-adult and adult crocodiles from 1–2 m from the microphone to ensure safety of personnel. We also recorded any calls or responses from nearby conspecifics, noting size class if possible, when we recorded distress calls of captured crocodiles.  

Sound Analysis    

We performed acoustic analysis to determine the structure of distress calls for each size class. We analyzed five call per individual, in one case only 3 calls were analyzed due to heavy background noise, and measured seven acoustic variables, two temporal and five spectral, using Raven Pro 1.5 acoustic analysis software (Bioacoustics Research Program, 2014).  We used spectrographic analysis (window size 1024, overlap 80%) of the fundamental frequency to determine maximal frequency (F_max, Hz), frequency at end of first quartile (F_1/4, Hz), and final frequency (F_end, Hz; Fig. 3A).  Using call oscillograms, we measured temporal properties for total duration (DT, s) and duration of the first quartile (D_1/4, s; Fig. 3B). Measurement windows were drawn around the fundamental frequency and the maximal frequency values at the beginning, end, and first quartile were recorded. We used frequency and temporal measurements to calculate call modulation of the first temporal quartile slope (Slope 1, Hz/s, calculated as (F_1/4−F_max)/D_1/4), and the slope of the remaining three temporal quartiles (Slope 2, Hz/s, calculated as (F_end−F_1/4)/( DT−D_1/4)) (Vergne et al. 2012). Concurrent to call measurements, we recorded number of calls produced by each individual for 10, 20, and 30 second intervals as total recording time varied between individual crocodiles.  We began call counts at the first recorded call for each individual We used size designation to organize and analyze distress call recordings by overall size class. 

We performed statistical analyses using RStudio version 0.99.902 (RStudio Team, 2015). Our call parameter data did not meet the assumptions of normality or homogeneity of variance (P < 0.05)(Boucher 2017).  Thus, we analyzed call parameter means (DT, D_1/4, F_max, F_1/4, Fend, Slope 1, Slope 2) among size classes (hatchling, juvenile, sub-adult, adult) by performing non-parametric Kruskal-Wallis (H) tests. We used Mann-Whitney post-hoc testing with Bonferroni correction to determine pairwise variance of call parameters between size classes following a significant Kruskal-Wallis test (P < 0.05).  We tested size class differentiation by call parameters using a principal component analysis cross-validated by discriminant function analysis which assigned each recorded individual to a size class based on call parameters. To determine variance in number of calls produced (10, 20, 30 second intervals) we performed one-way analysis of variance (ANOVA) tests as these data met assumptions of normality (P > 0.05) and equality of variance (P > 0.05).

We analyzed differences in call production by determining the total number of American Crocodiles captured throughout the study for each location and compared them by size class. To account for small sample size, we aggregated data for Caye Caulker and BAL sites.  We performed a 2-sample test for equality of proportions with continuity correction (Newcombe, 2008) on total call production proportions among size classes.  Designed for small sample sizes (< 5), we performed Fisher’s Exact Test (Agresti, 2002) to determine inequality in call production by size classes between Ambergris Caye, and the combined Caye Caulker and BAL sites.  As a compliment to proportional tests, we performed Mann-Whitney tests to determine variance of spectral parameters of hatchling and juvenile calls between Ambergris Caye and aggregated Caye Caulker and BAL sites.