Heat production in a feeding matrix formed on carrion by communally breeding beetles

Insects regulate their body temperature mostly behaviourally, by changing posture or microhabitat. These strategies may be ineffective in some habitats, for example on carrion. Carrion beetles create a feeding matrix by applying to cadaver surface anal or oral exudates. We tested the hypothesis that the matrix, which is formed on carrion by communally breeding beetle Necrodes littoralis L. (Silphidae), produces heat that enhances insect fitness. Using thermal imaging we demonstrate that heat produced in the matrix formed on meat by adult or larval beetles is larger than in meat decomposing without insects. Larval beetles regularly warmed up in the matrix. Moreover, by comparing matrix temperature and larval fitness in colonies with and without preparation of meat by adult beetles, we provide evidence that formation of a matrix by adult beetles has deferred thermal effects for larval microhabitat. We found an increase in heat production of the matrix and a decrease in development time and mortality of larvae after adult beetles applied their exudates on meat in the pre-larval phase. Our findings indicate that spreading of exudates over carrion by Necrodes larvae, apart from other likely functions (e.g. digesting carrion or promoting growth of beneficial microbes), facilitates thermoregulation. In case of adult beetles, this behaviour brings distinct thermal benefits for their offspring and therefore may be viewed as a new form of indirect parental care with an important thermal component.

production of the matrix during the larval feeding phase and eventually brings thermal 1 0 4 benefits for the larvae. We found an increase in heat of the matrix formed by larvae and a 1 0 5 decrease in larval development time and mortality, following the application of exudates by 1 0 6 adult beetles. Therefore, spreading the exudates by adult beetles had lagging thermal effects 1 0 7 for their offspring's microenvironment, which supports the hypothesis that N. littoralis 1 0 8 manifests an indirect thermal parental care. Heat emission in feeding matrix formed on carrion by Necrodes beetles.

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To test if feeding matrix produced by N. littoralis generates heat, we monitored with 1 1 3 thermal imaging conditions in colonies of adult (A) and larval (L) beetles subsequently 1 1 4 feeding on meat (M) (hereafter M+A+L setup) and in colonies of larval beetles only (hereafter 1 1 5 M+L setup), using as a reference the equivalent meat setup without the insects (hereafter M 1 1 6 setup, Fig. 1). We found that adult and larval beetles spread their exudates over meat to form 1 1 7 a greasy, feeding matrix that covers the meat and surrounding soil, the surface of which was 1 1 8 enlarging with colony age (Fig. 2). The matrix had a higher temperature than the background 1 1 9 (Fig. 3) and beetle larvae were regularly warming up on its surface (Fig. 4).
To investigate the effect of environmental temperature on heat emission in a matrix, 1 2 1 we compared larval colonies (M+L setup) reared under different temperatures, using as a 1 2 2 reference paired containers with meat only (M setup). The comparison revealed that the heat 1 2 3 production in the feeding matrix formed by larvae increased with a rearing temperature  By quantifying the average temperature of a matrix-covered surface (M+A+L and 1 2 8 M+L setups) and meat without the beetle-derived matrix (M setup), we found that heat 1 2 9 emission in the matrix was significantly larger than in meat alone (F 1,27 =122, N=30, P<0.001, 1 3 0 Fig. 6a, b). The temperature of the matrix revealed a steady increase until meat resources were 1 3 1 depleted and larvae stopped feeding (Fig. 6a). When adult beetles (M+A+L setup) applied 1 3 2 their exudates in the pre-larval phase, the temperature of the matrix was significantly higher in 1 3 3 the larval feeding phase, as compared to the colonies of larval beetles only (F 1,27 =154.8, 1 3 4 N=30, P<0.001, Fig. 6c). Moreover, heat production in the matrix during the larval feeding  Feeding matrix, larval fitness and parental care in Necrodes beetles. To test if spreading the exudates over meat by adult beetles affects the fitness of Nicrophorus beetles results in higher larval survival and mass, compared to broods without 3 2 1 parental care (27,67). Simple parental care, in the form of clearing carrion of fly larvae and 3 2 2 brood guarding was also described in Ptomascopus beetles (Nicrophorinae) (68, 69). Our 3 2 3 findings suggest that indirect forms of parental care, particularly carrion manipulations 3 2 4 bringing deferred benefits for the larvae, may be more common among carrion beetles. Feeding matrixes formed by other insect groups on various substrates may produce 3 2 6 heat, equally to the matrix formed on carrion by Necrodes beetles. Blow fly larvae (Diptera: Calliphoridae) while feeding on carrion elevate the temperature inside their aggregation (17, temperature when feeding in aggregation inside a beehive (5, 63). Thermogenesis may occur 3 3 0 in these cases in the feeding matrix that is formed on carrion or bee chambers by the larvae.

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Although such thermogenesis is not known in other insect groups that form the matrix, e.g. burying beetles (23), our findings suggest that it may be more frequent, particularly among 3 3 3 species that feed on rich and bulky resources, e.g. carrion, fallen fruits or dung. Beetle colony maintenance and general rearing protocols 3 3 7 Laboratory colony was established using adult beetles sampled from pig carcasses in kept in rearing containers (20-30 insects per container, sex ratio about 1:1) on humid soil, at 3 4 0 room temperature (20-23°C) and humidity (50-60%). Three to five containers were usually 3 4 1 maintained at the same time in the laboratory. Colonies were provided with fresh pork meat 3 4 2 ad libitum (raw pork in pieces: shoulder, neck or ham).

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All experiments were performed in rearing containers with a volume of 1.5 l (initial was not autoclaved. On one side of container meat was placed, and on the opposite side, we 3 4 6 put wet cotton wool to maintain high humidity inside and water for the beetles (Fig. 3). To 3 4 7 prevent the meat from drying out and to mimic skin that covers carrion and below which N.  N. littoralis lays eggs (30-50 per batch) to the soil. There are three larval instars. After 3 5 0 the third instar larva ceases feeding, it buries itself in the soil, forms a pupal chamber where it 3 5 1 pupates. After emergence, adult beetle stays for some time in the chamber, then it digs itself 3 5 2 out fully coloured. Thermal imaging and temperature quantification 3 5 5 We monitored thermal conditions inside beetle colonies using thermal imaging camera 3 5 6 Testo 885-2 with 30° x 23° infrared lens (Testo, Germany), mounted to a tripod while making 3 5 7 images. Images were taken in room temperature and humidity (20-23°C, 50-60%), with 3 5 8 rearing containers taken outside of a temperature chamber and images made within no more 3 5 9 than 2 minutes. Based on initial tests with meat, all temperature measurements were made with the 3 6 1 emissivity set at 0.8 and reflected temperature set at 17°C. Heat production of the matrix-3 6 2 covered surface was defined as a difference between the average temperature of the surface 3 6 3 and the background. To obtain background surface temperatures, we measured average 3 6 4 temperature in an area of clean soil located in the meat-opposite side of the container. These 3 6 5 measurements were averaged for the first three days starting from the colony establishment. During these initial days, the matrix temperature was usually only a little higher than the 3 6 7 background temperature, so the heat production in the matrix had negligible effects on the 3 6 8 background temperature. Because the matrix enlarged with colony age, and at some time it 3 6 9 covered also the soil that surrounded meat, it was impossible to quantify heat production 3 7 0 always within the same area. Instead, we quantified the average temperature in the largest 3 7 1 possible circular or ellipsoidal area, depending on the shape of the surface covered with the 3 7 2 matrix. We used for this purpose in-built tools of the IRSoft 4.5 software (Testo, Germany). To determine the optimal number of larvae and the optimal quantity of meat, we 3 7 6 performed several initial trials. By comparing heat production in larval colonies of various abundance (10, 20, 30, 40, 50 and 100 larvae), we found that normal growth and heat 3 7 8 production were already present in colonies of 40 larvae ( Fig. 10 in ESM). Accordingly, we 3 7 9 used such colonies in the main experiments. During initial trials, we also tested setups with 3 8 0 different quantity of meat and decided to use 2 g per larva in Experiment 1 and 3.5 g per larva 3 8 1 in Experiment 2. In Experiment 2 we investigated the effect of heat in a feeding matrix on 3 8 2 larval fitness, so we decided to provide larvae with more meat to maintain their optimal 3 8 3 growth. Environmental temperature and heat production in feeding matrix (Experiment 1) To test the effect of rearing temperature on the heat production in the matrix, we larvae; 80-85 g of raw pork in pieces: shoulder, neck or ham) was compared against heat 3 9 0 production in paired containers with meat only (M setup, 80-85 g of pork). Ten pairs of 3 9 1 containers (replicates) were studied in each temperature (nine pairs in 20 °C). To establish 3 9 2 experimental colonies we sampled freshly hatched first instar larvae from our main colony. Containers were kept in temperature chambers (ST 1/1 BASIC or +, POL EKO, Poland). Two chamber. Once a day colonies were taken out of a chamber to make thermal images. Results Software Inc., US), with rearing temperature as an independent variable, container type (M+L  Heat production and beetle development in different colonies (Experiment 2) 4 0 5 Our main experiment compared thermal conditions and beetle development between 4 0 6 two types of colonies in a paired experimental design (Fig. 1). The first colony type had adult 4 0 7 beetles in the pre-larval phase and larvae in the larval feeding phase (M+A+L setup). The beetles (sampled at random from our main colony, sex ratio 1:1) were kept on meat for the  colonies were taken out of the chambers to make thermal images.

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After larvae stopped feeding and began to bury themselves, we counted and weighed Acknowledgments 4 4 0 The study was funded by the National Science Centre of Poland (grant no.  This manuscript describes laboratory experiments using carrion beetle species N. littoralis.

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The species is not under protection. No permission or approval from the Ethics Commission  The datasets supporting this article have been uploaded as part of the supplementary material. We have no competing interests to declare.   o  r  n  A  (  2  0  0  8  )  T  h  e  r  m  o  r  e  g  u  l  a  t  i  o  n  i  n  I  n  s  e  c  t  s  .  E  n  c  y  c  l  o  p  e  d  i  a  o  f  E  n  t  o  m  o  l  o  g  y  ,  (  S  p  r  i  n  g  e  r  4  6  2  N  e  t  h  e  r  l  a  n  d  s  ,  D  o  r  d  r  e  c  h  t  )  ,  p  p  3  7  5  7  -3  7  6  0  .  4  6  3  2  .  H  e  i  n  r  i  c  h  B  (  2  0  0  9  )  T  h  e  r  m  o  r  e  g  u  l  a  t  i  o  n  .  E  n  c  y  c  l  o  p  e  d  i  a  o  f  I  n  s  e  c  t  s  ,  (  E  l  s  e  v  i  e  r  ) , p p 9 9 3 -9 9 9 . 4 6 4 3 .   1  9  3  3  )  Ö  k  o  l  o  g  i  s  c  h  e  u  n  t  e  r  s  u  c  h  u  n  g  e  n  a  n  N  e  c  r  o  p  h  o  r  u  s  F  .  Z  e  i  t  s  c  h  r  i  f  t  f  ü  r  5  1  1  M  o  r  p  h  o  l  o  g  i  e  u  n  d  Ö  k  o  l  o  g  i  e  d  e  r  T  i  e  r  e  2  7  (  3  )  :  5  1  8  -5  8  6  .  5  1  2  2  5  .  V  o  g  e  l  H  ,  e  t  a  l  .  (  2  0  1  7  )  T  h  e  d  i  g  e  s  t  i  v  e  a  n  d  d  e  f  e  n  s  i  v  e  b  a  s  i  s  o  f  c  a  r  c  a  s  s  u  t  i  l  i  z  a  t  i  o  n  b  y  t  h  e  b  u  r  y  i  n  g  5  1  3  b  e  e  t  l  e  a  n  d  i  t  s  m  i  c  r  o  b  i  o  t  a  .  N  a  t  u  r  e  C  o  m  m  u  n  i  c  a  t  i  o  n  s  8  :  1  5  1  8  6  .  5  1  4  2  6  .  D  e  g  e  n  k  o  l  b  T  ,  D  ü  r  i  n  g  R  -A  ,  &  V  i  l  c  i  n  s  k  a  s  A  (  2  0  1  1  )  S  e  c  o  n  d  a  r  y  m  e  t  a  b  o  l  i  t  e  s  r  e  l  e  a  s  e  d  b  y  t  h  e  5  1 I  k  e  d  a  H  ,  K  a  g  a  y  a  T  ,  K  u  b  o  t  a  K  ,  &  A  b  e  T  (  2  0  0  8  )  E  v  o  l  u  t  i  o  n  a  r  y  r  e  l  a  t  i  o  n  s  h  i  p  s  a  m  o  n  g  f  o  o  d  h  a  b  i  t  ,  5  6  2  l  o  s  s  o  f  f  l  i  g  h  t  ,  a  n  d  r  e  p  r  o  d  u  c  t  i  v  e  t  r  a  i  t  s  :  l  i  f  e  -h  i  s  t  o  r  y  e  v  o  l  u  t  i  o  n  i  n  t  h  e  S  i  l  p  h  i  n  a  e  (  C  o  l  e  o  p  t  e  r  a  :  5  6  3  S  i  l  p  h  i  d  a  e  )  .  E  v  o  l  u  t  i  o  n  ;  i  n  t  e  r  n  a  t  i  o  n  a  l  j  o  u  r  n  a  l  o  f  o  r  g  a  n  i  c  e  v  o  l  u  t  i  o  n  6  2  (  8  )  :  2  0  6  5  -2  0  7  9  .  5  6  4  4  6  .  K  i  n  g  J  E  ,  R  i  e  g  l  e  r  M  ,  T  h  o  m  a  s  R  G  ,  &  S  p  o  o  n  e  r  -H  a  r  t  R  N  (  2  0  1  5  )  P  h  y  l  o  g  e  n  e  t  i  c  p  l  a  c  e  m  e  n  t  o  f  A  5  6  5  u  s  t  r  a  l  i  a  n  c  a  r  r  i  o  n  b  e  e  t  l  e  s  (  C  o  l  e  o  p  t  e  r  a  :  S  i  l  p  h  i  d  a  e  ) .  T  r  u  m  b  o  S  T  ,  K  o  n  M  ,  &  S  i  k  e  s  D  (  2  0  0  1  )  T  h  e  r  e  p  r  o  d  u  c  t  i  v  e  b  i  o  l  o  g  y  o  f  P  t  o  m  a  s  c  o  p  u  s  m  o  r  i  o  ,  a  6  1  3  b  r  o  o  d  p  a  r  a  s  i  t  e  o  f  N  i  c  r  o  p  h  o  r  u  s  .  J  o  u  r  n  a  l  o  f  Z  o  o  l  o  g  y  2  5  5  (  4  )  :  5  4  3  -5  6  0  .  6  1  4  6  9  .  S  u  z  u  k  i  S  &  N  a  g  a  n  o  M  (  2  0  0  6  )  R  e  s  o  u  r  c  e  g  u  a  r  d  i  n  g  b  y  P  t  o  m  a  s  c  o  p  u  s  m  o  r  i  o  :  S  i  m  p  l  e  p  a  r  e  n  t  a  l  c  a  r  e  6  1  5  i  n  t  h  e  N  i  c  r  o  p  h  o  r  i  n  a  e  (  C  o  l  e  o  p  t  e  r  a  :  S  i  l  p  h  i  d  a  e  ) .  Fig. 1. Experimental design to study the heat production of a feeding matrix formed on meat by carrion beetle Necrodes littoralis and the development of its larvae in colonies with and without adult beetles presence in the pre-larval phase (respectively M+A+L and M+L setups).
Trials with meat only (M setup) were used as the reference.   larvae is much lower than temperature of the matrix in their vicinity and temperature of the 'warmed' larvae is the same or only slightly lower. There is a clear heat gradient from the outside to the inside in the 'warming' larvae, which indicates they are warming up in the matrix. There are also 'cooling' (↓) larvae, with higher body temperature than in their vicinity and a heat gradient from the inside to the outside. However, they are present only outside of the matrix. Assuming that larvae contribute endothermically to the heat of the matrix, the 'cooling' larvae should be present on the surface covered with the matrix. As this is not the case, the images demonstrate that heat is not endothermically generated by the larvae, but is produced in the matrix. Moreover, larvae are warming up while staying on the matrix. Fig. 6. Longitudinal thermal profiles (a), differences in the average heat production of the feeding matrix during the pre-larval phase (b) and the larval feeding phase (c) between Necrodes littoralis colonies differing in the presence of adult beetles and in the length of the pre-larval phase. All colonies were reared at constant 23°C. As a no-beetle reference in (a), we used results for 23°C from the environmental temperature experiment. Thermal profiles were fitted to the data using the distance-weighted least-squares smoothing procedure.
Symbolsmeans, whiskers -95% confidence intervals, different letters denote significant differences in pairwise comparisons. phases. Symbolsmeans, whiskers -95% confidence intervals, different letters denote significant differences in pairwise comparisons. Necrodes littoralis colonies differing in the presence of adult beetles during the pre-larval phase and in the duration of this phase. Symbolsmeans, whiskers -95% confidence intervals, different letters denote significant differences in pairwise comparisons.