J Insect Behav
https://doi.org/10.1007/s10905-024-09859-6

RESEARCH

Death Feigning in Larvae of Scorpionflies (Mecoptera:
Panorpidae): Frequency and Postural Changes Basedon Larval Instars
Ryo Ishihara · Kentarou Matsumura ·Takahisa Miyatake

Received: 2 March 2024 / Revised: 17 July 2024 / Accepted: 26 July 2024
© The Author(s) 2024

Abstract Death feigning is thought to have evolved
primarily as a predator avoidance behavior, and has
been reported in 10 of the 31 orders of insects. However, there have been no reports of death-feigning
behavior in Mecoptera species. We found that larvae of two scorpionfly species, Panorpa japonica
and P. pryeri, showed death feigning in response toexternal stimuli by brush poking stimulation. First,
we examined the frequencies of death-feigning postures. The two species showed two different postures of death feigning, “straight” and “ball.” Mostof the 1st instar larvae of P. japonica and P. pryeri
adopted the straight death-feigning posture. Next, we
examined duration of death feigning. As the larval
instar progressed, the death-feigning posture shiftedfrom straight to ball in both Panorpa species. In P.
japonica, the longest durations of death feigningwere found in the 2nd to 3rd instars, while the longest
duration of death feigning was found in the late 4th
instar in P. pryeri larvae.
Keywords Anti-predator behavior · freezing ·larvae · thanatosis · tonic immobility

R. Ishihara · K. Matsumura · T. Miyatake (*)
Graduate School of Environmental, Life, Natural Scienceand Technology, Okayama University, Okayama, Japane-mail: miyatake@okayama-u.ac.jp

Introduction
Death feigning is defined as immobilization in
response to external stimuli (e.g., Miyatake et al.
2004; Ruxton et al. 2018). The adaptive significanceof death feigning in insects has been much studied,and the behavior is thought to have evolved primarily as a predator avoidance strategy (e.g., Humphreys
and Ruxton 2018; Skelhorn 2018). For example, in
the red flour beetle, Tribolium castaneum, it has beenreported that the frequency of predation by Adanson’s jumper spider, Hasarius adansoni, was lower
for individuals of the strain with a longer death-feigning duration than for individuals of the strain with ashorter death-feigning duration (Miyatake et al. 2004,2009). This behavior of temporary immobility without postural change is called freezing, while temporary immobility with postural change is called death
feigning or thanatosis (Sakai 2021).
Postures of death feigning are sometimes uniqueand vary not only among species (Miyatake 2021),
but also between local populations (Ohba and Matsuda 2021). Furthermore, even within a species, theposture varies according to developmental stage; forexample, the immobile posture of the red flour beetle differs between adults and larvae, with larvae in astraight position, while adults turn over on their backs
and fold their legs (Miyatake et al. 2008; Matsumura
et al. 2017). However, to our knowledge, no otherstudy has reported on variations of death-feigning
postures during larval instars.
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Death feigning has been found in 10 of the 31
orders of insect taxa (Miyatake 2021): Odonata (Gyssels and Stoks 2005), Plecoptera (Moore and Williams 1990), Orthoptera (Nishino and Sakai 1996;
Honma et al. 2006), Phasmatodea (Godden 1972),
Neuroptera (Sendova-Franks et al. 2020), Mantodea(Edmunds 1972; Lawrence 1992), Hemiptera (Holmes 1906; Villet 1999; Kang et al. 2016; Ohba andMatsuda 2021), Coleoptera (Bleich 1928; Konishi
et al. 2020), Lepidoptera (Tojo et al. 1985; Dudley
1989; Larsen 1991) and Hymenoptera (Hölldobler
and Wilson 1990; van Veen et al. 1999; King and
Leaich 2006; Cassill et al. 2008; Cardoso et al. 2024).
We found that when larvae of two species ofMecoptera insects are stimulated, they adopted death
feigning in different postures depending on their larval instar. This paper is the first report of death feigning in Mecoptera. We also observed the frequency
and duration of death feigning in the two species ofMecoptera in the laboratory. We identified two typesof death-feigning posture in the scorpionfly larvae:
one where they stayed straight, and the other where
they curled up into a ball shape, and also found thatthe frequency of these postures varied with larvalstages.
Material and Methods
Insects
Two scorpionfly species (Mecoptera: Panorpidae),
Panorpa japonica and P. pryeri were used in theseexperiments (Fig. 1). These two species have a life
cycle during larval development in which they spend

Fig. 1  Adults of two Mecoptera species used in this study: A:
Panorpa japonica and B: Panorpa pryeri
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the day underground and are active on the surface atnight. Predators of the larvae are unknown. The larvae pupate in the soil and then emerge above groundin early spring.
In the present study, 54 larvae were derived fromadults collected in April 2020 at a bank of the Asahikawa River, Okayama City (34° 6′ 8′′ N, 133° 9′ 3′′E) for P. japonica. For P. pryeri, larvae were derived
from adults collected in June 2022 at Koguri-yama
Mountain, Hirosaki City, Aomori Prefecture (40° 32′
44′′ N, 140° 28′ 39′′ E). Fifty-five larvae of P. pryeri
were used in the experiment, each larva was reared ina Petri dish (9 cm diameter × 2 cm high) lined witha moderately moistened nonfat cotton. The food wascommercially available dried sewage worms.
Observation of Death Feigning
Individual scorpionfly larvae were placed in a Petri
dish (9 cm diameter, 1.5 cm height) and lined withfilter paper and stimulated with a bristle handle end
brush (Pentel, ZBS 1–15, Tokyo). If the larvae did
not move after 5 s after stimulation while maintaining
the "straight" or "ball" posture described below, they
were considered to have entered death feigning; thosethat moved within 5 s were not considered to be indeath feigning. The duration of death feigning fromthe start to the end (when the larvae started moving
again) was measured with a stopwatch, and if thelarvae did not become inert after 10 or more stimulations, it was recorded as not feigning death. Bothspecies exhibited two types of postures while feigning
death, named “straight” and “ball” (Fig. 2).
Measurements of death feigning were taken at fivelarval instar stages: 1st, 2nd, 3rd, early 4th, and late
4th instars when the larva had matured to a white

Fig. 2  Death-feigning postures, straight (A) and ball (B).
Examples of P. japonica are shownJ Insect Behav

body color. Death-feigning posture and duration ofdeath feigning were compared for each larva. In thisexperiment, death feigning was observed in 39 P.
japonica and 44 P. pryeri, and five replicate measurements were taken for all individuals.
Statistics
A generalized linear mixed model was used to test therespective proportions of individuals that immobilized in a straight posture, those who immobilized in aballed posture, and those who did not immobilize. In
this analysis, we used an item response tree (IRTree)model (López-Sepulcre et al. 2015). This method
argues that instead of being analyzed using traditional
nonparametric statistics or a series of separate analyses split by response categories, this kind of data canbe more holistically analyzed using a generalized linear mixed model (GLMM) framework extended tobinomial response trees (Lopez-Sepulcre et al. 2015).
Whether the larvae of the scorpionfly feigned deathor not was considered as "node 1," and the postureof death feigning (straight or ball) was considered as"node 2." The presence or absence of each event was
analyzed as binary data with a binomial distribution(López-Sepulcre et al. 2015). In this analysis, species,
experiment, larval instar, and posture were used as afixed effect, and larval ID and observation were used
as a random effect.
In the analysis of the duration of death feigning,
the GLMM with a gamma distribution was used.
Other statistical models in this analysis were similar
to those for death-feigning frequency. If a significant
effect was found in each analysis, we conducted multiple comparison tests with Bonferroni correction.
All analyses were conducted in R ver.4.1.0 (R CoreTeam 2017) and using the statistical packages lme4
(Bates et al. 2015) and car (Fox and Weisberg 2019).
Results
Table 1 shows results of IRTree GLMM for test ofeffects of species, experiment, and larval instar on
frequency of death feigning. No significant effect oflarval instar was observed in the test results of node
1 (i.e., whether they feigned death: the percentage ofwhite bars vs. colored bars in Fig. 3). The estimate

of developmental stage was negative (-0.29), indicating a trend that the number of individuals, which did
not feign death, increased (Fig. 3), but the effect was
not statistically significant (p = 0.09). Moreover, theeffect of species was not significant. In the results for
Node 2 (i.e., the death-feigning pose: the percentageof black bars vs gray bars in Fig. 3), the negative estimate of larval stage (-1.00) indicates that the proportion of "straight" death feigning individuals increased
with development, and the effect was statisticallysignificant (p = 0.02). The effect of the species-larval stage interaction was also statistically significant(p < 0.05), with an increasing proportion of P. pryeri individuals with "ball" death feigning with theirdevelopment (Fig. 3B).
Figure 4 shows the results of duration of death
feigning. Comparing the duration of death feigning between the two species regardless of their age
revealed that P. pryeri larvae showed significantly
longer duration than P. japonica larvae (Fig. 4A,
Table 2). Moreover, death feigning in the form of
a ball had a significantly longer duration than ina straight position, regardless of species (Fig. 4B,
Table 2). In these results, the effects of the interaction between species and posture were not significant (Table 2). Figure 4 C shows the death-feigning
duration of larvae in each instar of the two species.
The results of statistical analysis showed a significant
interaction between species and age with duration ofdeath feigning (Fig. 4C, Table 2). In both species,
the duration of death feigning increased with age up
to the third instar. In older P. pryeri larvae the duration of death feigning continued to increase with age,while it decreased in P. japonica larvae showed theduration of death feigning decreased at the fourth
instar (Fig. 4C, Table 2).
Discussion
We found that the posture and duration of death
feigning change as the larval instar progressed in the
two Mecoptera species examined. The relationshipbetween age and death feigning in the adult stage has
been studied in some insect species. For example, inthe fire ant Solenopsis invicta, where interspecific colony struggles are intense, workers, which have softer
exoskeletons, choose death feigning in the first few
days of life when they are more vulnerable to attack,Vol.: (0123456789)

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Table 1  IRTree GLMM
for test of effects of species,
experiment, and larval
instar on frequency ofdeath feigning. Results withp-value ≤ 0.05 are given in
bold

Intercept
Species
Larval stage
Species: larval stage

Node 1: feigned death or not

Node 2: pose of death feigning

Estimate

s.e

z

p

Estimate

s.e

z

p

2.61
1.18
-0.29
-0.15

1.03
0.86
0.17
0.23

2.54
1.37
-1.69
-0.65

0.01100.16940.09200.5186

2.91
1.26
-1.00
-0.56

1.05
0.88
0.43
0.29

2.76
1.43
-2.31
-1.97

0.00570.15220.02070.0492

while older workers are more likely to choose counterattack (Cassill et al. 2008). The braconid parasitoid
wasp Heterospilus prosopidis also has a long deathfeigning duration in young adults, but the duration
shortens with age (Amemiya and Sasakawa 2021).
These studies may be examples of changes in predator avoidance behavior as the body hardens. In adult
sweet potato weevils Cylas formicarius, adult virgin
females have longer death-feigning periods when they
are young, and after that the duration decreases withage (Kuriwada et al. 2011). In final instar (6th instar)larvae of the common cutworm Spodoptera litura,
the solitary phase showed longer duration of deathFig. 3  Proportions of eachpose of death-feigning in P.
japonica (A) and P. pryeri(B), respectively. Black andgrey bars show ball andstraight poses death feigning. White bar shows noresponse to stimulus

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feigning compared to the gregarious phase (Tojoet al. 1985).
In insects, there are many examples in which predators control behavioral differences depending on
the larval instar. For example, larvae of the Colorado
potato beetle Leptinotarsa decemlineata show body
writhing and falling in response to predator contact
more frequently in the 3rd and 4th than in the 2nd
instar (Ramirez et al. 2010). On the other hand, in thegrasshopper Ageneotettix deorum, 3rd and 4th instar
nymphs showed antipredator behavior in response to
predator contact, while 5th instar larvae do not (Danner and Joern 2003). Ramirez et al. (2010) suggested

J Insect Behav

Fig. 4  Duration of death feigning in two scorpionfly species.
A and B show death-feigning duration between each species
and posture, respectively. C shows death-feigning duration ateach instar larva. In C, black and grey show P. japonica and P.

pryeri, respectively. There are significantly differences among
different alphabets (Bonferroni correction). In early and late
4th instars, duration of death feignings are longer in P. pryeri
than P. japonica. Error bars show standard error

Table 2  Results of GLMM for test of effects of species,
experiment, and larval instar on death-feigning duration (onlythe experiment 1)

that the frequency with which antipredator behavior isadopted, might be controlled by larval predation risk.
However, no study has been conducted of alternationsof death feigning as instars progress, and this is thefirst study to quantitatively show that death-feigning
posture changes during larval instars. Differences indeath-feigning posture among local populations havealso been reported in the ferocious water bug Appasus
japonicus (Ohba and Matsuda 2021).
Why do death-feigning postures change with age
and among species? One possibility is predation

Factord.f

FPSpeciesAge
Species*age
PostureSpecies*posture
Error

1
4
4
1
1
339

14.6533
2.36243.86634.13761.145

0.0002
0.05330.00440.04270.2854Vol.: (0123456789)

J Insect Behav

avoidance in the species’ habitat. Larvae of Panorpidae are found in the soil of mountainous areas(Byers and Thornhill 1983), and the larvae of P.
japonica and P. pryeri are also found in soil. When
predators such as mammals dig up the surface layer,
larvae of this species are at increased risk of predation. In such cases, larger-sized larvae may be able
to escape predators by curling up and rolling down
the slope. One of the authors has observed that thelarvae curl up and roll down on slopes when P.
japonica larvae are stimulated during rearing, (RI,
personal observation). On the other hand, smalllarvae are less conspicuous and may be less likely
to be detected by predators by becoming immobile rather than curling up to escape. That is, death
feigning in small body size larvae may function as
hiding, while the posture of death feigning in ball inlarge body size larvae may function as escaping byrolling down a slope. The different death-feigningposes depending on the larval instars among species
may be due to the different types of natural enemies
among the two species.
In addition, repeated exposure to external stimuli
may have influenced the death feigning. A previous
study showed that the death feigning in the Colorado
potato beetle L. decemlineata e subsequently changed
depending on their experience of repeated external stimulations at a younger age (Acheampong and
Mitchell 1997). In this study, stimulation was applied
repeatedly from the 1st instar larva to the late 4th instar
larva, so repeated experience of external stimulation ata younger age may have influenced the death feigning.
In Panorpidae, there are few examples of studies on larval strategies against predators, including physical andchemical defenses, and to our knowledge, only camouflage by larval body color has been suggested (Jiang
et al. 2019). This study indicates that Panorpidae larvae
may use death feigning as an anti-predator strategy.
Further work is, therefore, needed to confirm whether
the death feigning of Panorpidae is anti-predator behavior, and to investigate the significance of changes indeath-feigning posture during larval instars.
Author Contributions The study was conceived by Ryo
Ishihara, Kentarou Matsumura and Takahisa Miyatake. The
experiments were designed by Takahisa Miyatake and Ryo
Ishihara, and these were performed by Ryo Ishihara The data
was analyzed by Ryo Ishihara, and Kentarou Matsumura. The
manuscript was written by Ryo Ishihara and Takahisa Miyatake. All authors approved the final version prior to submission.
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Funding Open Access funding provided by Okayama University. This work was supported by JSPS KAKENHI Grant
Numbers 21H02568, 23K21343 to TM.
Data Availability Uploaded as related file.
Declarations
Ethical Approval This article does not contain any studies
with animals performed by any of the authors.
Informed Consent This article deals with insects and thus we
did not need Informed consent.
Conflict of Interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permitsuse, sharing, adaptation, distribution and reproduction in any
medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The
images or other third party material in this article are includedin the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is notincluded in the article’s Creative Commons licence and yourintended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directlyfrom the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.

References
Acheampong S, Mitchell BK (1997) Quiescence in the Colorado potato beetle, Leptinotarsa decemlineata. Entomol
Exp Appl 82:83–89
Amemiya M, Sasakawa K (2021) Factors affecting thanatosis
in the braconid parasitoid wasp Heterospilus prosopidis.
Insects 12:48. https://​doi.​org/​10.​3390/​insec​ts120​10048
Bates D, Maechler M, Bolker B, Walker S (2015) Fitting liner
mixed-effects models using lme4. J Stat Software 67:1–48
Bleich OE (1928) Thanatose und hypnose bei Coleopteren.
Berich Wiss Biol A 10:1–61
Byers GW, Thornhill R (1983) Biology of the Mecoptera.
Annu Rev Entomol 28:203–228
Cardoso DC, Alves ÍCC, Cristiano MP, Heinze J (2024) Deathfeigning in ants. Myrmecol News 34:45–56
Cassill DL, Vo K, Becker B (2008) Young fire ant workers
feign death and survive aggressive neighbors. Naturwissenschften 95:617–624
Danner BJ, Joern A (2003) Stage-specific behavioral responsesof Ageneotettix deorum (Orthoptera: Acrididae) in thepresence of lycosid spider predators. J Insect Behav
16:453–464

J Insect Behav
Dudley R (1989) Thanatosis in the neotropical butterfly Caligo
illioneus (Nymphalidae: Brassolinae). J Res Lepidoptera
28:125–126
Edmunds M (1972) Defensive behavior in Ghanaian praying
mantids. Zool J Linn Soc 51:1–32
Fox J, Weisberg S (2019) An [R] companion to applied regression. Third edition. Thousand Oaks CA: Sage. https://​socia​lscie​nces.​mcmas​ter.​ca/​jfox/​Books/​Compa​nion/
Godden DH (1972) The motor innervation of the leg musculature and motor output during thanatosis in the stick insect
Carausius morosus Br. J Comp Physiol 80:201–225
Gyssels FGM, Stoks R (2005) Threat-sensitive responses topredator attacks in a damselfly. Ethology 111:411–423
Hölldobler B, Wilson E (1990) The ants. Harvard University
Press, Cambridge, The Belknap Press
Holmes JS (1906) Death-feigning in Ranatra. J Comp Neurol
Psychol 16:200–216
Honma A, Oku S, Nishida T (2006) Adaptive significanceof death feigning posture as a specialized inducible
defence against gape-limited predators. Proc R Soc B
273:1631–1636
Humphreys RK, Ruxton GD (2018) A review of thanatosis(death feigning) as an anti-predator behaviour. Behav Ecol
Sociobiol 72:1–16
Jiang L, Hua Y, Hu GL, Hua BZ (2019) Habitat divergenceshapes the morphological diversity of larval insects:
insights from scorpionflies. Sci Re 9:12708
Kang C, Moon H, Sherratt TN, Lee SI, Jablonski PG (2016)
Multiple lines of anti-predator defence in the spotted lanternfly, Lycorma delicatula (Hemiptera: Fulgoridae). Biol
J Linn Soc 120:115–124
King BH, Leaich HR (2006) Variation in propensity to exhibit
thanatosis in Nasonia vitripennis (Hymenoptera: Pteromalidae). J Insect Behav 19:241–249
Konishi K, Matsumura K, Sakuno W, Miyatake T (2020)
Death feigning as an adaptive anti-predator behaviour:
further evidence for its evolution from artificial selection
and natural populations. J Evol Biol 33:1120–1128
Kuriwada T, Kumano N, Shiromoto K, Haraguchi D (2011)
Age-dependent investment in death-feigning behaviour inthe sweetpotato weevil Cylas formicarius. Physiol Entomol 36:149–154
Larsen TB (1991) The art of feigning death—thanatosis in
Euploea (Danainae) and other aposematic butterflies.
Entomol Rec 103:263–266
Lawrence SE (1992) Sexual cannibalism in the praying mantid,
Mantis religiosa: a field study. Anim Behav 43:569–583
López-Sepulcre A, De Bona S, Valkonen JK, Umbers KDL,
Mappes J (2015) Item response trees: a recommended
method for analyzing categorical data in behavioral studies. Behav Ecol 26:1268–1273
Matsumura K, Fuchikawa T, Miyatake T (2017) Decoupling of
behavioral trait correlation across life stages in two holometabolous insects. Behav Genet 47:459–467
Miyatake T (2021) Environmental, physiological, and genetic
effects on tonic immobility in beetles. In: Sakai M (ed)Death-feigning in insects: mechanism and function of
tonic immobility. Springer, Tokyo, pp 39–54

Miyatake T, Katayama K, Takeda Y, Nakashima A, Sugita A,
Mizumoto M (2004) Is death-feigning adaptive? Heritablevariation in fitness difference of death-feigning behaviour.
Proc R Soc B 271:2293–2296
Miyatake T, Tabuchi K, Sasaki K, Okada K, Katayama K,
Moriya S (2008) Pleiotropic antipredator strategies, fleeing and feigning death, correlated with dopamine levels in
Tribolium castaneum. Anim Behav 75:113–121
Miyatake T, Nakayama S, Nishi Y, Nakajima S (2009) Tonically immobilized selfish prey can survive by sacrificing
others. Proc R Soc B 276:2762–2767
Moore KA, Williams DD (1990) Novel strategies in the complex defense repertoire of a stonefly (Pteronarcys dorsata)nymphs. Oikos 57:49–55
Nishino H, Sakai M (1996) Behaviorally significant immobile
state of so-called thanatosis in the cricket Gryllus bimaculatus DeGeer: its characterization, sensory mechanismand function. J Comp Physiol A 179:613–624
Ohba S, Matsuda I (2021) Geographical variation of deathfeigning in Appasus japonicus (Heteroptera: Belostomatidae). Rostria: Trans Hemipterol Soc Japan, 66: 22–25
R Core Team (2017) R: A language and environment for statistical computing. – R Foundation for Statistical Computing, Vienna, Austria. R version 3.4.3
Ramirez RA, Crowder DW, Snyder GB, Strand MR, Snyder
WE (2010) Antipredator behavior of Colorado potato
beetle larvae differs by instar and attacking predator. Biol
Control 53:230–237
Ruxton GD, Allen WL, Sherratt TN, Speed MP (2018) Avoiding attack: the evolutionary ecology of crypsis, aposematism, and mimicry. Oxford University Press
Sakai M (2021) Death-feigning in insects: mechanism andfunction of tonic immobility. Springer Nature, Tokyo
Sendova-Franks AB, Worley A, Franks NR (2020) Post-contact
immobility and half-lives that save lives. Proc R Soc B
287:20200881
Skelhorn J (2018) Avoiding death by feigning death. Curr Biol
28:R1121–R1142
Tojo S, Morita M, Himura K (1985) Effects of juvenile hormone on some phase characteristics in the cotton cutworm, Spodoptera litura. J Insect Physiol 31:243–249
van Veen JW, Sommeijer MJ, Aguilar Monge I (1999) Behavioural development and abdomen inflation of gynes andnewly mated queens of Melipona beecheii. Insectes Soc
46:361–365
Villet MH (1999) The cicada genus Nyara n. gen. (Homoptera:
Cicadidae): Systematics, behavior and conservation status. Trop Zool 12:157–163
Publisher’s Note Springer Nature remains neutral with regard
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