Nesting activity of Olive ridley and effect of artificial shade at Cascajilloso Beach, a new inhabited nesting site in Pacific Panama

Sea turtle nesting activity on newly monitored beaches yields important data to support future regional and global conservation assessments. Here we report on nesting activity of Olive Ridley (Lepidochelys olivacea) at Cascajilloso Beach in Pacific Panama based on data from a new hatchery during seasons 2019, 2020 and 2021. Besides, we conducted a field experiment analyzing the effects of artificial shading on hatchlings’ biometric characteristics, hatching success and the incubation period. Nesting activity based on number of egg clutches transferred to the hatchery reached a peak between September (33%) and October (25%). Curved carapace length (CCL) of nesting females (64.3–66.2 cm) was similar to other populations of the Eastern Tropical Pacific (ETP). The number of egg clutches was 80 in 2019, 74 in 2020 and 108 in 2021, however, the clutch size was slightly smaller (91.5 to 94.5 eggs) compared with other populations of the ETP. This could be a phenotypic variation of nesting females at this beach. The ability to move egg clutches to the hatchery was affected during 2020 season due to COVID-19 restrictions. Heavy rainfall during the incubation period influenced the nest temperatures registered under artificial shade and unshaded treatments (maximum average 29.9 °C), potentially resulting in males. The warmer temperatures in our experimental nests produced heavier hatchlings contrary to general assumptions, but this was also related to straight carapace length only after a threshold value of 40 mm. Hatchlings under the artificial shade were able to grow to a certain size but potentially at the cost of storing less mass (trade-off). The ongoing beach patrolling and hatchery management techniques with long-term baseline data collection are needed to secure the nesting population of L. olivacea at this recently surveyed beach.

Methods
Study site
Cascajilloso Beach is located on the west coast of the Azuero Peninsula in Pacific Panama (7°22'N, 80°54'W, Figure 1). The beach length is 6.7 km and beach width is 30 m. L. olivacea, Chelonia mydas and Eretmochelys imbricata are reported to nest at the site (Flores et al. 2021). The sand is gray to black with the Pavo river mouth bordering the southern extreme of the beach. The vegetation of the beach includes: Cocos nucifera, Terminalia catappa, Canavalia rosea, Sphageticola trilobata and some grasses (Flores et al., 2021). 

Beach patrolling and nesting females
Beach patrols were conducted on foot every night when possible and depending on meteorological conditions during the nesting seasons. The patrol team was composed of two park rangers from MiAmbiente and occasionally a community member from Arenas town. Patrol duration (hours) was calculated from the time a beach survey started until the time it ended. We also calculated the per-kilometer patrol duration (hours per km), defined as the patrol duration for each night divided by the length (km) of the beach.

Each nesting female was measured for her curved carapace length (CCL) and curved carapace width (CCW) using a flexible measuring tape and Inconel flipper tags (Style 681, National Band and Tag Company, Newport, Kentucky, USA) were used to mark encountered females. Flipper tags were applied to the left front flipper (Balazs, 1999). Many nesting females left the beach before they could be measured yet their egg clutches were transferred to the hatchery.

Cascajilloso Beach hatchery
A hatchery with a maximum capacity of 176 nests was constructed in 2019. Inside the hatchery, an area of 0.25 m2 (50 cm x 50 cm) was used to transfer each relocated nest. The hatchery was protected with chain link mesh and a 30 cm high black plastic sheet all around to avoid the intrusion of crabs and other predators. Prior to the start of each season, the sand was cleaned, sieved and exposed to sunlight during 15 days prior to the transfer of the first nests. Only during the 2020 season, the sand was extra treated with a dilution of Clorox® as a disinfectant because of the presence of mites in some of the nests during 2019. The date that the egg clutch was laid was registered in a logbook and the predicted hatch date was calculated to be between 45 to 55 days. A circular anti-insect netting was placed on top of each nest to protect it from parasite wasps and other insects. Once hatched, hatchlings were immediately released into the sea to avoid being attacked by predators.

Hatching date was noted as the day disturbances were found on the surface of the nest caused by either hatchlings underneath or emerging hatchlings (see Castheloge et al., 2018). The number of dead and live hatchlings from each nest were counted and hatching success was determined as the number of hatchlings (dead and alive) divided by the total number of eggs in the nest. Incubation success was calculated as the number of hatchlings successfully released into the sea divided by the total number of eggs.  

Artificial shade experiment
During August 2020 an area 6 m2 and 2 m high inside the hatchery was used for the artificial shade treatment in which an 80% shade cloth was used above ground on top of a wooden post structure. The clutches of 11 nesting females were collected at the beach after females finished laying. In no less than two hours (Limpus et al., 1979) each clutch was transferred to the hatchery using a split-clutch design, in which half of the clutch was randomly transferred to the shaded area and the other half to the unshaded area of the hatchery. A humidity and temperature data logger sensor Tempo Disc® (Blue Maestro, London UK) was buried at the center of each experimental treatment clutch.. Each sensor was calibrated against an anemo-thermo-hygrometer Skywatch model ATMOS (JDC Electronic SA, Switzerland) and set to register temperature every 15 min. An air temperature and humidity CS125 sensor (Campbell Scientific, USA) connected to a CR200 datalogger (Campbell Scientific, USA) was also installed 1.5 m above ground near the hatchery to measure these variables every hour. Due to damage the datalogger stopped recording on day 61 of the overall incubation period. Rainfall data was downloaded for the location of Cascajilloso Beach from the PERSIANN (Precipitation Estimation from Remotely Sensed Information using Artificial Neural Networks) system developed by the Center for Hydrometeorology and Remote Sensing (CHRS) at the University of California, Irvine (https://chrsdata.eng.uci.edu/).

For each hatchling, the hatching date was recorded. Straight carapace length (SCL) was measured using a caliper Mitutoyo® (Mitutoyo American Corporation, USA). Body mass was measured using a digital scale AWS® model 100 CAL (American Weight Scales, Georgia USA). Each Tempo Disc datalogger was retrieved, the data were downloaded, and daily average temperature was calculated. However, due to the extremely wet conditions, 50 % of dataloggers failed to record data (five under unshaded treatment and six under shaded treatment). Hatching success and incubation success were also calculated for the experimental nests and were excluded from the overall 2020 season dataset. This is because the experimental clutches only had half of the eggs laid by females compared to those in the rest of the hatchery so as to allow comparison with seasons 2019 and 2021. After the biometric measurements were recorded the hatchlings were released into the sea. 

Statistical analysis
Prior to analyses, data was inspected for normality. When parametric assumptions were not met, non-parametric tests were performed. To compare the effect of artificial shade and direct sunlight on hatching success, incubation period and body mass, we used a Generalized Mixed Model (GLMM) with treatment as fixed factor and female tag number as a random factor and corresponding error structure. To test differences among nesting seasons of the measured variables we conducted non-parametric Kruskal-Wallis tests. Correlations among variables were tested using non-parametric Spearman rank correlation tests. P < 0.05 was considered statistically significant, and models were simplified by backward elimination starting with the interaction term(s) where appropriate. Statistical analyses were conducted using R v.4.1.2 (R Development Core Team, 2021). All values reported in the results are means ± SD, unless otherwise indicated.