DOI: 10.5061/dryad.3tx95x6ph
title: Synergistic effects of musking and autohemorrhaging on the duration of death feigning in dice snakes (Natrix tessellata)

description: Predation exerts a significant selection pressure on prey, shaping a
 multitude of traits that serve as antipredator defences. In turn, natural
 selection could favour combinations of antipredator defences with
 synergistic effects that enhance prey survival. An especially interesting
 antipredator defence is death feigning (DF), present in a wide variety of
 taxa and usually characterized by the prey lying motionless often along
 with defecation, musking, and auto-haemorrhaging. All these aspects of DF
 should work in conjunction with one another, intensifying the overall
 effect of the display and in turn facilitating quicker escape. To confirm
 this hypothesis, we tested 263 dice snakes (Natrix tessellata) directly in
 the field. We noted the occurrence of smearing faeces and musk and
 autohaemorrhaging and we measured the duration of DF, expecting to see a
 negative association between the occurrence of these behaviours and the
 duration of DF. Our results affirm our hypothesis: dice snakes that
 smeared themselves in musk and faeces before DF and had auto-haemorrhaging
 during DF spent significantly less time in DF. Our results highlight the
 functional integration of antipredator behaviors across different phases
 of predator-prey interactions, emphasizing the need for future research to
 prioritize studying the sequential display of behaviors.
description: Fieldwork was performed at Golem Grad Island (40°52′08′′ N,
 20°59′23′′ E, Prespa Lake, North Macedonia). We searched for dice snakes
 in the open or sheltered under natural cover and quickly captured them by
 hand. At capture, each snake (N=263) was always grabbed approximately at
 mid-body with one hand since it has been previously shown that holding
 snakes at different parts of the body elicits drastically different
 behavioural responses. During the first 30 seconds
 after capture, while holding the snake in the air, we noted the occurrence
 of smearing. Smearing was defined as the snake expelling the contents of
 the cloacal glands and defecating while at the same time using the tail to
 smear musk and faeces on its body. This was termed as the “capture test”.
 All authors captured the snakes and observed the occurrence of smearing as
 it was easily observable. Immediately after the capture
 test, we performed the “post-capture test”, which included a series of
 generalized predator-like actions to test for post-capture antipredator
 response. We assume that, although humans were proxies for natural
 predators, the same general principles apply to natural encounters between
 snakes and their predators. To standardize this procedure only one person
 handled the snakes (VB). This predator-like handling procedure involved a
 30-s handling sequence. Each snake was ﬁrst held for 10 seconds with both
 hands, and two ﬁngers were used to pinch the body at two ﬁxed points,
 behind the head and above the cloacal region. Then, the snake was passed
 from one hand to the other for 10 s and ﬁnally slightly stretched for 10 s
 to imitate a generalist predator handling its prey. The snake was then
 placed on its back, and the person handling the snake stepped out of view
 of the snake, mimicking predator hesitation or latency to eat the prey.
 Another observer (MM) remained motionless at 1 m (crouching) to record the
 snake’s behaviours. From the moment when the snake was put on its back
 (i.e., the start of the post-capture test), we recorded measurements of
 the following sequence of variables: (1) occurrence of death feigning (DF)
 – if the snake had its mouth open with a protruding tongue while remaining
 completely immobile we defined this as DF; (2) occurrence of
 auto-haemorrhaging (AH) – while in DF, if the snake had visible blood
 around the mouth area we considered this a display of AH; (3) duration of
 DF – if DF occurred, duration of DF was measured as the time elapsed from
 the onset of DF until the snake closed its mouth. After
 the snake completed both tests, we first measured the body temperature by
 inserting a probe thermometer into the cloaca while holding the snake by
 the tail. Then, we noted the dorsal colour morph (blotched, green or
 melanistic), sex (based on the shape of the tail or hemipenis eversion
 when necessary), measured the snout-vent length (SVL; performed by gently
 stretching the snake along a fixed tape measure) and determined the
 reproductive status in females (through palpation). In order to prevent
 pseudoreplication, animals were permanently marked with a unique scale
 clip ID (by notching the ventral scale with scissors) for later
 identification, and released at the site of capture. We determined size
 thresholds of sexually mature individuals (forming the variable age
 (adult/immature) based on a dataset containing over 6000 measured and
 marked Golem Grad dice snakes. The SVL of the smallest male found in
 copulation was 55 cm, whereas the SVL of the smallest gravid female in
 this population was 63 cm. To analyse our data we used
 R statistical software (ver. 4.2.2). We determined the distribution data
 using the Shapiro-Wilks test and visual inspection of the data. Then we
 modelled the variation in the occurrence of AH using binomial generalized
 linear models (GLMs). As AH only occurs during DF we subset the entire
 dataset to individuals who displayed DF during post-capture tests (N=92).
 We first built two sets of models; a null model, containing only the
 response variable (AH~1) and a full model containing all factors of
 interest (Morph*State+SVL+Smearing+Temperature). We then used stepwise
 regression to find the best-fit models (with
 the <em>step</em>() function from the MASS package in R). The
 same approach was performed with the duration of DF, except we used Gamma
 GLMs. After finding the best-fit models for all
 response variables we obtained model summaries (odds ratios, coefficients,
 confidence intervals and *<em>p</em> values) which we report
 in RSBL-2024-0058_ESM_model summaries. *We used the ggplot package to plot
 the figures. The reproducible code can be found in the accompanying R file
 as RSBL-2024-0058_script.
description: # Synergistic effects of musking and auto-hemorrhaging on the duration of
 death feigning in dice snakes (*Natrix tessellata*)
 [https://doi.org/10.5061/dryad.3tx95x6ph](https://doi.org/10.5061/dryad.3tx95x6ph) The database contains two sheets with one (titled “data”) being used to perform the statistical analyses and the other one (titled “graph”) was used to produce Figure 2. The columns in the data sheets are as follows: ### Sheet “data” * Smearing – occurrence of smearing as a capture behaviour (yes/no) * Recovery – duration of DF (in seconds). Individuals that did not exhibit DF have these cells left blank to not interfere with the accompanying code. * DF – occurrence of DF (yes/no) * AH – occurrence of AH (yes/no) * Temperature – cloacal temperatures of tested snakes (in °C) * SVL – snout-to-vent lengths of tested snakes (in cm) * Sex – sex of the tested snakes (males/females) * Age – age category of tested snakes (adult/juvenile) * State – variable that includes the reproductive state of female snakes (male/female/gravid) * Morph – dorsal colour morph of tested snakes (blotched/green/melanistic) ### Sheet “graph” * Duration of DF – duration of DF (in seconds) * Sequence – sequence of behaviours displayed during the tests (DF/DF+Hemo/Smearing+DF+Hemo), where Hemo represents AH