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Article https://doi.org/10.1038/s41467-022-33127-w
Wax worm saliva and the enzymes therein
are the key to polyethylene degradation
by Galleria mellonella
A. Sanluis-Verdes
1,9
,P.Colomer-Vidal
1,9
,F.Rodriguez-Ventura
1
,
M. Bello-Villarino
1
, M. Spinola-Amilibia
2
,E.Ruiz-Lopez
3
,R.Illanes-Vicioso
3
,
P. Castroviejo
4
,R.AieseCigliano
5
, M. Montoya
6
,P.Falabella
7
,
C. Pesquera
8
,L.Gonzalez-Legarreta
8
,E.Arias-Palomo
2
,M.Solà
3
,T.Torroba
4
,
C. F. Arias
1
& F. Bertocchini
1
Plastic degradation by biological systems with re-utilization of the by-products
could be a future solution to the global threat of plastic waste accumulation.
Here, we report that the saliva of Galleria mellonella larvae (wax worms) is
capable of oxidizing and depolymerizing polyethylene (PE), one of the most
produced and sturdy polyolefin-derived plastics. This effect is achieved after a
few hours’exposure at room temperature under physiological conditions
(neutral pH). The wax worm saliva can overcome the bottleneck step in PE
biodegradation, namely the initial oxidation step. Within the saliva, we identify
two enzymes, belonging to the phenol oxidase family, that can reproduce the
same effect. To the best of our knowledge, these enzymes are the first animal
enzymes with this capability, opening the way to potential solutions for plastic
waste management through bio-recycling/up-cycling.
Polyethylene (PE) accounts for 30% of synthetic plastic production,
largely contributing to plastic waste pollution on the planet to-date1.
Together with polypropylene (PP), polystyrene (PS) and poly-
vinylchloride (PVC), PE is one of the most resistant polymers, with very
long C–C chains organized in a crystalline, dense structure. Given the
hundreds of million tons of plastic waste accumulating and the still
escalating pace of plastic production, re-utilization of plastic residues
is a necessary path to alleviate the gravity of the plastic pollution
problem, and at the same time to render available a huge potential
reservoir of carbon2. To-date, only mechanical recycling is being
applied at a large scale. Several factors, such as the low number of
plastic types prone to be mechanically recycled, and the low quality of
the secondary products severely restrict the potential of this solution
to the problem of plastic waste accumulation. Chemical recycling, as
an alternative procedure, is preferentially aiming at plastic upcycling,
e.g., decomposing polyolefin-derived plastics in order to take advan-
tage of smaller intermediates. Severaltechnologies have been applied
at a lab scale, although the high energetic cost might still impede the
scaling up of these technological tools3.
In addition to mechanical and chemical recycling, biodegrada-
tion is widely considered as a promising strategy to dispose of plastic
residues. Biodegradation refers to environmental degradation by
biological agents. The IUPAC defines biodegradation as the “break-
down of a substance catalyzed by enzymes in vitro or in vivo”4, later
modified “to exclude abiotic enzymatic processes”5. In the case of PE,
biodegradation requires the introduction of oxygen into the poly-
meric chain6,7; this causes the formation of carbonyl groups and the
subsequent scission of the long hydrocarbon chains with the
Received: 11 May 2022
Accepted: 2 September 2022
Check for updates
1
Centro de Investigaciones Biologicas-Margarita Salas (CIB)-Consejo Superior de Investigaciones Cientificas (CSIC), Department of Plant and Microbial
Biology, Madrid, Spain.
2
CIB-CSIC, Department of Structural and Chemical Biology, Madrid, Spain.
3
Department of Structural Biology, Molecular Biology
Institute of Barcelona (IBMB)-CSIC, Barcelona, Spain.
4
Department of Chemistry, Faculty of Science and PCT, University of Burgos, Burgos, Spain.
5
Sequentia
Biotech SL, Barcelona, Spain.
6
CIB-CSIC, Department of Molecular Biomedicine, Madrid, Spain.
7
Department of Sciences, University of Basilicata,
Potenza, Italy.
8
Department of Chemistry and Process & Resource Engineering, Inorganic Chemistry Group-University of Cantabria, Nanomedicine-IDIVAL,
Santander, Spain.
9
These authors contributed equally: A. Sanluis-Verdes, P. Colomer-Vidal. e-mail: tifar@ucm.es;federica.bertocchini@csic.es
Nature Communications | (2022) 13:5568 1
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production of smaller molecules, which can then be metabolized by
microorganisms8,9. The crucial first step of this chain of events, i.e.,
the oxidation of PE polymer, is usually carried out by abiotic factors
such as light or temperature6,8,10. Once the long polymeric molecules
are broken down, a process that takes years of exposure to envir-
onmental factors in the wild, bacteria or fungi intervene and continue
the job6,9,11,12. This is the current paradigm driving the research field in
biodegradation. Within this paradigm, several bacterial and fungal
strains have been identified as capable ofcarrying on a certain extent
of PE degradation. However, in most of the cases, such degradation
requires an aggressive pre-treatment of PE (heating, UV light, etc.)
that accelerates the incorporation of oxygen into the polymer,
making the abiotic oxidation the real bottleneck of the reaction12–16.
In the past decade, a few microorganisms have been described as
capable of acting on untreated PE17–23, although they require a sig-
nificantly longer incubation time compared to experimental condi-
tions with pre-oxidized PE.
The identification of enzymes from microorganisms capable of
degrading untreated PE has proven a difficult task. In fact, to our
knowledge, no such enzyme has been identified. Reported enzymes
capable of acting on polyolefin-derived plastics require a pretreat-
ment of the plastic material12,14. For example, two reported laccases,
able to chemically modify PE, necessitate an abiotic pretreatment24
or the addition of redox mediators such as 1-hydroxybenzotriazole25.
Abiotic pre-treatments such as radiation or heat cause oxidation
of the polymer, which is the crucial limiting step in the biodegrada-
tion chain12.
This scenario confirms that the synthetic nature of the compound,
together with the hydrophobicity and inaccessibility features, make
plastic a difficult target for animal, fungal or microbial-derived enzy-
matic activities. Nonetheless, some lepidopteran and coleopteran
insects revealed the capacity to degrade untreated PE and PS26–31.
The larvae of Galleria mellonella, also known as wax worms (ww), can
oxidize PE within one hour from exposure29,32–38,makingitthefastest
known biological agent capable of chemically modifying PE.
Our work here begins with an initial observation that plastic
debris appears when a PE film is in contact with the recently formed
ww cocoon and mouth secretions. We then perform a detailed ana-
lysis of the saliva of the ww (GmSal), revealing the capacity of GmSal
to oxidize and break PE within a time frame of a few hours. This effect
is confirmed by Gel Permeation Chromatography (GPC) analysis of
GmSal-treated PE, showing the scission of long hydrocarbon chains
into small molecules. Using Gas Chromatography-Mass Spectro-
metry (GC-MS) we identify degradation products such as small oxi-
dized aliphatic chains, further confirming the breaking of the
polymer into shorter molecules. Proteomic analyses of GmSal reveals
the presence of a handful of enzymes belonging to the hexamerin/
prophenoloxidase family. Two of these enzymes are shown to oxi-
dize PE after a few hours’application at room temperature (RT). For
this reason, we named them PEases. These PEases are an arylphorin
here re-named Demetra, and an hexamerin here re-named Ceres. In
the case of Demetra, we show that PE oxidation is accompanied by
the deterioration of PE and the release of degradation by-products
similar to those obtained with the whole saliva.
To the best of our knowledge, these PEases are the first enzymes
capable of producing such modifications on a PE film working at
room temperature and in a very short time, embodying a promising
alternative to the abiotic oxidation of plastic, the first and most dif-
ficult step in the degradation process. The identification of inverte-
brate enzymes capable of oxidizing PE in a few hours represents an
alternative paradigm in the world of plastic degradation and more
widely in the plastic waste management fields, opening up various
possibilities which may helpto solve the plastic waste pollution issue,
along with expanding potential formulae/routes for synthetic poly-
mer production.
Results
Wax worm saliva oxidizes PE film
Saliva, broadly defined here as the juice present in the anterior portion
of the digestive apparatus, was collected from the ww mouth and
tested on a commercial PE film (Fig. 1A). After three consecutive
applications of 30 μl of GmSal for 90min each, Confocal Raman
microscopy/Raman spectroscopy (RAMAN) analysis indicated poly-
mer oxidation, accompanied by a general deterioration of the film
(Fig. 1B). This is evident in the overlapping with the PE control (Fig. 1C,
D), which reveals the expected PE signature profile. As a further con-
trol, the saliva of another lepidopteran larva, Samia cynthia,was
applied on the PE film, and no oxidation was generated (Fig. 1E). The
changes produced by the GmSal in a few hours-long applications are
similar to those generated by environmental factors after months or
years of exposure to weathering39,40. The Fourier Transformed Infrared
Spectroscopy (FTIR) analysis confirmed the oxidation profile (Fig. S1).
The changes in PE chemical composition revealed by the spectroscopy
techniques suggested that molecules other than the long PE polymeric
chain formed upon the contact with GmSal.
Wax worm saliva degrades PE
The molecular weight characteristics of PE before and after treatment
with GmSal were analyzed using High Temperature-Gel Permeation
Chromatography (HT-GPC). After a few hours’applications of GmSal
on PE film (15 applications, 90 min each), the molecular weight dis-
tribution became bi-modal, with a new peak in the low molecular
weight region, indicating the breaking of the C–C bonds with the
appearance of small compounds (Fig. 2A). Formation of C=O bonds as
a consequence of chain scission was shown by the increase of the
carbonyl index (CI, Fig. 2A). The weight average (Mw)wasonlyslightly
changed (from 207,100 to 199,500 g/mol) suggesting that the polymer
was not uniformly modified by the GmSal, with portions that have been
strongly depolymerized, while others remained still untouched41.The
same result was obtained using PE 4000 instead of PE film, with a
change in Mw from 4000 to 3900 g/mol and an increase in the car-
bonyl index in the GmSal-treated sample (Fig. 2B, black and blue
curves). These results show that PEwas oxidized and depolymerizedas
a consequence of GmSal exposure, with the formation of oxidized
molecules of lowmolecular weight. Inorder to analyze the dynamics of
changes in the PE after increased exposure to GmSal in time, we
doubled the exposure time of PE 4000 to GmSal (30 applications,
90 min each) (Fig. 2B, red curve). Breaking of la rge polymer chains with
the formation of smaller molecules notably increased at longer expo-
sure times, as showed by the comparison of the molecular weight
distributions and by the doubling of the CI (Fig. 2B). These experi-
ments confirmed the PE oxidation with depolymerization upon a few
hours’exposure to GmSal, an effect that increased with time, and
pointed to the presence therein of still unknown activities capable of
PE degradation.
Identification and analysis of the by-products of PE treated with
wax worm saliva
To have an insight into the degradation products resulting from PE-
saliva contact and released from the oxidized polymer, PE granules
(crystal polyethylene-PE 4000) were exposed to GmSal and subse-
quently analyzed by Gas Chromatography-Mass Spectrometry (GC-
MS) and identified by NIST11 library for untargeted compounds.
After 9 applications of 40 µL of GmSal for 90 min each at RT, new
compounds were detected in the experimental sample (Fig. 3).
The detected compounds comprised oxidized aliphatic chains, like
2-ketones from 10 to 22 carbons. Ketones from 10 to 18 carbons were
identified by comparing the fragmentgram of the ion m/z 58 from
methyl ketones that correspond with the transposition of the
McLafferty on the carbonyl located at the second carbon of
each ketone. Those not present in the library as 2-eicosanone and
Article https://doi.org/10.1038/s41467-022-33127-w
Nature Communications | (2022) 13:5568 2
Content courtesy of Springer Nature, terms of use apply. Rights reserved
2-docosanone showed the same fragmentgram m/z 58 and were
defined by the equidistance of the peaks along the retention time and
their molecular weight (Supplementary Table 1). Furthermore, the
presence of 2-ketones was confirmed with GC-MS/MS in MRM mode
with the ion with the highest m/z and the exclusive of each molecule
as 212 > 58 for 2-tetradecanone, 240 > 59 for 2-hexadecanone,
and 282 > 58 for 2-octadecanone. Also, butane, 2,3-Butanediol,
2-trimethylslyl (TMS) derivative, and sebacic acid, 2TMS derivative
were identified using sample silylation, indicating the deterioration
of the PE chain (Fig. 3C). At the same time, a small aromatic com-
pound recognizable as benzenepropanoic acid, TMS derivative, a
plastic antioxidant, was found. Derivative chemicals were confirmed
as well using GC-MS/MS with an m/z of 147 > 73, 331 > 73, and
104 > 75, respectively. The presence of this plastic antioxidant
suggests an “opening”in the polymeric structures, with the
release of small stabilizing compounds normally present in plastics
(plastic additives).
To verify if an increase in time exposure to GmSal caused an
increase in PE degradation, we repeated the experiment of PE 4000
exposure in sequential times, with four applications per day of 100 µL
of GmSal for 90 min each at RT, performed in 1, 2, 3 and 6 consecutive
days. The analysis of the supernatants revealed a progressive increase
in the formation of 2-decanone, 2-dodecanone, 2-tetradecanone, and
2-hexadecanone (Fig. S2). These data indicate an increase of at least
twice the relative abundance of degradation products with time (from
1 to 6 days) as a consequence of prolonged exposure to the GmSal.
Study of the wax worn saliva: enzymes identification and func-
tional studies
To understand the nature of the buccal juice, a GmSal sample was
analyzed by negative staining electron microscopy (EM), revealing a
high content of proteins or protein complexes (size: 10–15 nm)
(Fig. 4A). No other structures (such as vesicles or bacteria) were
detected. Electrophoretic analysis (SDS-PAGE) confirmed the presence
of proteins with a prominent band at around 75kDa (Fig. 4B).
Does GmSal contain enzymes responsible for the detected PE
modifications? To assess this, a proteomic analysis of GmSal contents
was carried out. More than 200 proteins were detected, including a
variety of enzymatic activities, transport and structural proteins, etc.
(Supplementary Data 1). To narrow down the number of potential
candidates, a saliva sample was analyzed by size exclusion chromato-
graphy (SEC). The elution profile showed a main single, wide peak
(Supplementary Fig. 3A). SDS-PAGE gel of the major fraction showed a
strong band at about 75 kDa (Supplementary Fig. 3C). The proteomic
profile of this band revealed the presence of proteins known in
arthropods as related to transport or storage.
Raman shift (cm-1)
Negative
control
500 1000 1500 2000 2500 3000 3500
0
20
40
60
80
100
120
500 1000 1500 2000 2500 3000 3500
0
200
400
600
800
Raman shift (cm-1)
Saliva
S. cynthia
on PE film
Collection of saliva
Capillary
Saliva PE
A
B
DE
Raman shift (cm-1)
500 1000 1500 2000 2500 3000 3500
0
0.2
0.4
0.6
0.8
1
C
Saliva
G. mellonella
on PE film
500 1000 1500 2000 2500 3000 3500
0
20
40
60
80
100
120
140
Raman shift (cm-1)
Raman intensityRaman intensity
Fig. 1 | Galleria mellonella saliva (GmSal) collection and functional study.
AScheme of saliva collection and application. B–ERAMAN analysis of PE film. BPE
film treated with GmSal: 3 applications of 90 min, 30 μl each. The peaks between
1500 and 2400 cm−1indicate different collective stretching vibrations due to the
presence of other organic compounds, sign of PE deterioration (red arrow). Oxi-
dation is indicated between 1600 and 1800 cm−1(carbonyl group) and
3000–3500 cm−1(hydroxyl group) (black arrows)78.CControl PE film. Brackets
indicate the peaks that characterizePE (PE signature), corresponding to the bands
at 1061, 1128, 1294, 1440, 2846, and 2880cm−1.DOverlapping profiles (Band C).
EPE film treated with Samia cynthia saliva. Source data are provided as a Source
Data file.
Article https://doi.org/10.1038/s41467-022-33127-w
Nature Communications | (2022) 13:5568 3
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To further identify if the wide peak contained subspecies, an ion
exchange chromatography (IEX) was run with a second saliva sample,
which showed four well-defined elution peaks (peaks 1 to 4 in Sup-
plementary Fig. 3B). Analysis by SDS-PAGE indicated that they all
contained proteins of similar molecular weight (Supplementary
Fig. 3D). To check which protein fractions of both the SEC and IEX
retained PE degradation activity, aliquots of the eluted fractions were
tested on a PE film. Using RAMAN spectroscopy, degradation activity
was analyzed from fractions of the IEX four peaks (Fig. 4C–G) and in
the SEC major peak (Supplementary Fig 4). Peaks indicated as 1,2 and 3
(as in Supplementary Fig. 3B) showed PE oxidation. High degradation
activity was also detected in the SEC main peak (peak 5 in Supple-
mentary Fig. 3A) (Fig. S4).
Proteomics of IEX peaks 1, 2, and 3 revealed the presence of a
handful of proteins, belonging to the arthropodan hexamerin/
prophenoloxidase superfamily (peaks 1, 2, and 3, respectively)
(Supplementary Data 2). This result on the one hand confirmed the
outcome of the SEC fraction proteomics (Supplementary Data 2, peak
5), and on the other refined it, reducing the number of potential can-
didates present in each peak. The fact that this family comprehends
oxidase activities, made them the obvious candidates for PE degra-
dation capacity within GmSal. These proteins, namely arylphorin
subunit alpha, arylphorin subunit alpha-like, and the hexamerin acidic
juvenile hormone-suppressible protein 1 were produced using a
recombinant expression system and tested for this ability.
Identification of wax worm enzymes as PE oxidizers
In order to assess the activity of these proteins, 5 μlofeachpurified
enzyme at a concentration of 1–5μg/μl, were applied separately
8 sequential times (90 min each) on PE films. While arylphorin subunit
alpha did not show any effect on PE film, arylphorin subunit alpha-like,
re-named Demetra (NCBI accession number: XP_026756396.1), caused
4 6 8 10 12 14 16 18
0
1
2
3
4
Time (min)
Abundance (x106)Abundance (x106)Abundance (x106)
Time (min)
Time (min)
IS
O
O
Si
Si
Ketones
O
( )n
O
Si
O
A
B
C
O
O
O
O
Si
Si
2 4 6 8 10 12 14 16 18
0.5
1.
1.5
2.
Time (min)
11 12 13
0.
0.5
1.
1.5
2.
7 8 9 10
0.
0.05
0.1
10C 12C
14C
16C
Ketones
O
( )n
18C 20C 22C
Sebacic acid,
2TMS derivative
Benzenepropanoic acid,
TMS derivative
2,3-Butanediol,
2TMS derivative
Fig. 3 | Identification of PE degradation by-products via GC-MS.
A–CChromatograms of PE treated with GmSal, indicating different compounds.
A,BKetones of different length, indicated by the number of carbon atoms. C2,3-
Butanediol 2TMS derivative, benzenepropanoic acid TMS derivative, sebacic acid
sTMS derivative. Compounds in C were identified with silylation (see “Methods”).
IS: internal standard. Source data are provided as a Source Data file.
Log M
dWf/dLogM
CI = 0.0106
CI = 0.0500
CI = 0.1039
CI = 0
CI = 0.0882
PE 4000PE film
2.0 2.5 3.0 3.5 4.0 4.5
0.2
0.4
0.6
0.8
1.0
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7
0.1
0.2
0.3
0.4
0.5
Log M
AB
Fig. 2 | HT-GPC analysesof PE treated with GmSal. Molecular weight distribution
of PE film (A) and PE 4000 (B) are indicated. AControl PE (in black) and PE-treated
(in red) are compared. The carbonyl index (CI) of the control and experimentalare
indicated. M
w
control (red): 0; M
w
experimental (black): 0.0882. BControl PE
(black), treated-PE (15 applications) (blue), treated-PE (30 applications) are com-
pared. The CI of each sample is indicated. Arrows indicate compounds with low
molecular weight in the treated PE. Sourcedata are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-022-33127-w
Nature Communications | (2022) 13:5568 4
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PE deterioration with occasional conspicuous visual effect on the film
itself (Fig. 5A–E and Supplementary Fig. 5). RAMAN confirmed oxida-
tion and a damagedPE signature (Fig. 5F–H). The hexamerin, re-named
Ceres (NCBI accession number: XP_026756459.1), caused PE oxidation/
deterioration (Supplementary Fig. 6), but without the visual effect
caused by Demetra. The same experiment performed with inactivated
enzymes did not show any modification on the PE film, as indicated by
RAMAN analysis (Supplementary Fig. 7). The FTIR of PE treated with
Demetraand Ceres also showed differences in the extent of oxidation
between the two PEases (Supplementary Fig. 8). These results support
the original hypothesis based on the idea that the buccal secretion is
the main source of plastic degradation activities in the ww.
To analyze the potential ofthe saliva proteins in oxidizing PE, GC-
MS was performed on PE granules (PE 4000) exposed to Demetra or
Raman intensity
Raman shift (cm-1)
0 500 1000 1500 2000 2500 3000 3500
200
400
600
800
1000
0 500 1000 1500 2000 2500 3000 3500
200
400
600
800
1000
1200
Raman intensity
Raman intensityRaman intensity
Raman shift (cm-1)
Raman shift (cm-1)
Raman shift (cm-1)
0 500 1000 1500 2000 2500 3000 3500
0
100
200
300
400
500
0 500 1000 1500 2000 2500 3000 3500
0
200
400
600
800
Negative control Demetra
Demetra
CDE
Demetra Demetra
FG
HI
AB Demetra
1mm
ControlControl
1mm 1mm 1mm 1mm
Fig. 5 | Demetra effect on PE film. A,BControl PE film. C-E. Demetra on PE film.
F–IRAMAN spectroscopy of control (F) and Demetra treated PE film (G,H).
FControl PE film (see Fig.1for details). G,HTwo different punctual analyses within
the crater as showed in E, indicating PE deterioration (same experimental
conditions). See Fig. 1for details. Moreover, the bands at 845 and 916 cm−1corre-
spond to C–O–C and C–COO groups, respectively. The presence of PE is insignif-
icant in G. Experiments in figures A–Ewere performed multiple times (>5). Source
data are provided as a Source Data file.
0500 1000 1500 2000 2500 3000 3500
0
50
100
150
200
250
300
350
400
Ion exchange
Fraction 34
Activity
0 500 1000 1500 2000 2500 3000 3500
0
200
400
600
800
0 500 1000 1500 2000 2500 3000 3500
0
50
100
150
200
250 Ion exchange
Fraction 29
Activity
Ion exchange
Fraction 9
No activity
Raman intensity
0500 1000 1500 2000 2500 3000 3500
0
200
400
600
800
ABC
200 nm
D
Raman intensity
GFE
0500 1000 1500 2000 2500 3000 3500
0
20
40
60
80
100 Ion exchange
Fraction 44
Activity
Raman shift (cm-1) Raman shift (cm-1)
Raman shift (cm-1) Raman shift (cm-1)Raman shift (cm-1)
Fig. 4 | GmSal content functional characterization. A Electron microscopy
negative staining of a saliva sample, dilution 1:500. Protein complexes (10–15 nm
size) are indicatedin the top right square. BSDS-PAGE of a GmSal sample, dilution
1:50. Molecular weight standards are listed on the left. The arrow indicatesthe
major band at 75 kDa. C–F. RAMAN of PE treated with IEX fractions (see Fig. S4).
CFraction 9, no activity (control PE film, see Fig. 1for details). D–FProfile
corresponding to IEX peak 1 (fraction 29), 2 (fraction 34) and 3 (fraction 44) (see
Fig. 1for details). The intense peak at 1085cm−1in E indicates amorphous PE. The
decrease of the peaks at 2845 and 2878 cm−1indicates a less significant presence of
PE. GOverlapping of fractions Cto F. Experiments in figures A,Bwere performed
multiple times (>5). Source data are provided as a Source Data file.
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Ceres. After 24 applications of Demetra (10 µl at 1.2 mg/mL, 90 min
each), 2-ketones from 10 to 22 carbons were detected in the super-
natant using GC-MS, with the fragment m/z of 58, and retention time
used for identification (Fig. 6A). Increasing the treatment (ten versus
five applications, 90 min each) showed an increase of 2-ketones of 10
to 20 carbons in relative abundance, and the appearance of
2-docosanone which was not detected after five applications (Fig. 6B).
On the other hand, after PE 4000 treatment with Ceres, no by products
were detected in the supernatant, suggesting substantial difference
between the two proteins, despite the fact that they sharethe capacity
to oxidize PE. Both enzymes present the same functional domains as
haemocyanins’(Fig. S9A, B). However, the sequence comparison
between the two enzymes shows only 30% of identity (Fig. S9C).
Moreover, in silico analysis (Fig. S9D) indicates that Demetra is more
stable than Ceres, which could be one of the reasons contributing to
the observed differences.
Discussion
This study reports that the saliva of the ww oxidizes and depolymerizes
PE, with ww enzymes therein capable of reproducing the effect
observed with the whole saliva. To the best of our knowledge, this is
the first report of an enzymatic activity capable of attacking the PE
polymer without any previous abiotic treatment. This capacity is
achieved by animal enzymes working at room temperature and in
aqueous solution with a neutral pH. Under these conditions, the
enzymatic action of the ww saliva overcomes in a few hours a recog-
nized bottleneck step (i.e., oxidation) in PE degradation6,8–10.
The capacity of Galleria mellonella as well as other Coleoptera and
Lepidoptera to degrade sturdy polyolefin-derivedpolymersasPEorPS
has been extensively documented in the past few years, with no pre-
treatment required for the plastic polymer to be degraded26–29,31–38,42–47.
If this capacity resides in the microorganisms of the worm gut, in the
invertebrate itself, or in a complementation of the two, is still an object
of debate. The gut microbiome has traditionally been considered
the culprit of plastic degradation by insects. However, despite the
numerous reports appeared lately about microbe species being
potentially responsible for insect-driven plastic biodegradation, no
consensus on specific species or genera of bacteria/fungi colonizing
theLepidopteraandColeopteragutandinvolvedinplasticdegrada-
tion has been reached26–28,30,31,34–38,43. Indeed, the exclusive involvement
of microorganisms in this process has been recently questioned32,48.As
for G. mellonella, the fast degradation makes it improbable for the gut
microbiota to be the sole player in the observed modification of PE
chemical structure, as already suggested32. This study provides solid
evidence in favour of this argument and confirms the key role played
by the larvae of G. mellonella in PE degradation: the saliva of the ww,
and the enzymes it contains, oxidizes and depolymerizes PE. The
action of the enzymes present in the saliva of G. mellonella on PE is
therefore equivalent to that of abiotic pretreatments. The saliva-
dependent oxidation of PE could thus provide a suitable substrate for
further biological attack by causing the scission of the long polymer
chains into smaller molecules that could then be metabolized and
assimilated along the insect’s digestive system (by the microbiome
and/or by the insect’s cells).
The current paradigm/hypothesis of PE biodegradation stands on
the breaking of the C–C bond via the same mechanism that bacteria
deploy to break alkanes49,50.Thewaxwormsliveandgrowinthe
honeycombs of the beehives. They feed among other things (pollen,
larvae, etc.) on beeswax. Given the similarity between plastics and
beeswax, it is conceivable that the observed effect on PE is a con-
sequence of the worm’s capability to degrade wax. However, the two
PEases, Demetra and Ceres, are an arylphorin and an hexamerin,
respectively51, and they are phylogenetically related to phenol-
oxidases (enzymes targeting aromatic rings) and hemocyanins, oxy-
gen transport proteins that also present phenoloxidase activity52,53.In
the literaturediverse functions are attributed to this family of proteins,
such as storage54, immunity55, and defence against plant phenolics56.
Thepresenceofthistypeofenzymesinthesalivaofinsects,already
described in the literature57–59, has a clear functional interpretation as
the first line of defence against pathogens and plant anti-herbivore
mechanisms57–59. The ability of insect enzymes to detoxify plant
phenolics56 suggests the potential existence of an alternative way to
degrade PE. Polymeric chains are not the onlycomponents of plastics,
as a number of small molecules, generically known as additives, are
normally used to confer the desired properties to plastic objects. The
appearance of a small aromatic compounds recognizable as a plastic
additive such as benzenepropanoic acid, TMS derivative among the
identified degradation products raises the possibility that this com-
pound could become the target of the phenoloxidase-like activity of
the ww enzymes. A consequence of this potential action of the ww
enzymes on aromatic additives could be the formation of free radicals
leading to the initiation of the autoxidative chain reaction6. The idea of
the formation of free radicals as a first step to trigger autooxidation is
not new, having been proposed as a way in which some bacteria might
initiate plastic biodeterioration, via still unknown enzymes14,20.
The ecological niche of G. mellonella larvae implies that they must
deal with the presence in the beehive (wax, propolis, pollen…)ofan
extended variety of phenols60–62. This might provide an explanation to
their capacity of PE degradation.
However, this will remain a speculation until the presence/for-
mation of radicals is verified. Further studies will be required to get
deeper insights into the functional modality of these ww enzymes,
their diversity (as already indicated by the differences in the experi-
mental outcomes between Demetra and Ceres), and the molecular
mechanisms acting on PE.
Limited information is available about closely related proteins.
A sequence comparison shows that both enzymes exhibit some degree
of sequence identity with only a few proteins in the NCBI database
(Supplementary Data 3 and 4). Of these proteins, only a handful
(two arylphorins and one storage protein) have been structurally
characterized to-date, and none of them shows a high degree of
sequence identity with Demetra and Ceres (between 30 and 57%)
(Supplementary Table 2). Further structural, biochemical, and func-
tional studies will be necessaryto have anexhaustive understanding of
10C
12C
4 6 8 10 12 14 16 18
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Time (min)
Abundance (x106)
16C
18C
20C
22C
14C Ketones
O
( )n
AB
Ketone (number of C)
0
0.1
0.3
0.4
10 12 14 16 18 20 22
Relative
abundance
5 applications
10 applications
0.2
Fig. 6 | Generation of PE degradation by-products by Demetra. A GC-MS chro-
matogram of PE treated with Demetra. The arrows indicated the peaks corre-
sponding to ketones with different number of carbons. BIncrease of ketones
formation as degradation products from five to ten applications of Demetra to PE.
Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-022-33127-w
Nature Communications | (2022) 13:5568 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved
the mechanisms of action of these PEases, the first of their kind to be
described, to the best of our knowledge.
The existence of such enzymes, secreted from the mouth and
evolved to work at room temperature and neutral pH on plastic, pro-
vides a promising alternative paradigm for the biological degradation
of PE. This framework goes beyond the current definition of biode-
gradation, which is exclusively based on the conversion of plastic to
CO
2
through the metabolic activity of microorganisms. On one hand,
the observed oxidation and deterioration of PE does not depend on
any microbial activity; on the other hand, the easy working conditions
and the appearance of degradation products such as ketones and
additives suggest the potential use of these enzymes for plastic waste
degradation and recycling or upcycling of plastic components. This
could be used either as an alternative to the metabolic conversion of
plastic to CO
2
, or as the initial oxidative step in combination with
standard microbial degradation pathways.
Further, this study suggests that insect saliva might result as a
depository of degrading enzymes (plastic, cellulose or lignin to men-
tion some) which could revolutionize the bioremediation field.
Although further studies will be necessary to obtain a deeper under-
standing of the step-by-step evolution of plastic in contact with ww
saliva enzymes, this discovery introduces another potential approach
for dealing with plastic degradation. In a circular economy frame, this
study opens up a potential field both in plastic upcycling, and in
manufacturing the plastic of the future, with ad-hoc formulations
prone to facilitate degradation by selected enzymes.
Methods
Resource availability
Further information and requests for resources and reagents should be
directed to and will be fulfilled by the lead contact, Federica Bertoc-
chini (federica.bertocchini@csic.es).
Experimental model and subject details
Galleria mellonella larvae colony was maintained in an incubator at
28 °C in the dark, and fed with beeswax from beehives.
Wax worm saliva collection
Larvae of 150–300 mg were used for saliva collection. Briefly, a glass
capillary connected to a mouth pipet was placed at the buccal opening
and the liquid was collected. Five to ten microliters of saliva were
collected from each worm.The concentration of proteins in the saliva
measured via Bradford methodology varies between 20 and 30 mg/ml.
Saliva for PE application was immediately used. Occasionally, frozen
saliva-only can be utilized. For electron microscopy, saliva was diluted
1:1 in the following buffer: 10mM Tris-Cl pH 8, 50mM NaCl. For pro-
teomic analyses, saliva was diluted 1:1 in 10 mM Tris-Cl pH 8, 50mM
NaCl, 2mM DTT, 20% glycerol. For control, Samia cynthia larvae
(kindly provided by InsectPark-Microfauna S.L. (El Escorial,Madrid)) at
the last stage were used to collect saliva as described above.
RAMAN and FTIR analyses
PE film was treated with 30 μl of GmSal for 90 min, three applications
for RAMAN. PE film was treated with 5 μlofGmSalfor90min,nine
applications for FTIR. Recombinant proteins were applied as follows:
5μl of protein (concentration between 1 and 5 μg/ml) were applied
eight times on PE film 90min each time, 16 applications for FTIR. For
the control with inactivated proteins, recombinant proteins were
denatured at 100 degrees centigrade for 10min. SEC and IEX peak
aliquots were applied six times, 30 min each, and left overnight.
Treated and control films were washed with water and ethanol.
RAMAN analyses were performed on (treated and control) PE films
using Alpha300R—Alpha300A AFM Witec equipment with 5 mW
power, 50× (NA0.8) objective, integration time 1, accumulation 30,
wavelength 532nm. FTIR analyses were performed with a Jasco LE-
4200 equipment, with the following features: interval 4000–400 cm−1,
Resolution 4 cm−1, scan 264.
High temperature-gel permeation chromatography (HT-GPC)
analysis
HT-GPS was performed by Polymer Chart, Valencia, Spain.
Briefly, the experimental conditions are the following: Equipment:
GPC-IR5_I Polymer Char; solvent: TCB stabilized with 300 ppm of BHT;
dissolution temperature, detectors temperature, columns tempera-
ture: 160°; volume: 8 ml; weight: 8mg; dissolution time: 60min;
injected volume: 200 μl; injection tim e: 55 mi n; flow: 1 ml/min; col-
umns: 3 × PL gel Olexis Mix-Bed columns (13 microns), 300× 7.5 mm +
guard column
For the carbonylindex analysis, GPC_IR6 was used, with dissolvent
o-DCB and temperature at 150°.
PE film and PE 4000 (20 mg)were treated with 100 μlofGmSalfor
90 min. The treatment was repeated 15 times (film and PE 4000) and
30 times (PE 4000).
Gas Chromatography Mass Spectroscopy (GC-MS) and Tandem
analysis
An amount of 20 mg of PE 4000 was placed in a 1.5 ml Eppendorf tube.
PE was exposed to 40 µlofG. mellonella saliva 9 times for 90 min each
at room temperature and avoiding light. For prolonged treatments
(days 1, 2, 3, and 6), three applications of 100µl of saliva for 90 min
each at room temperature were carried out each day. PE controls were
performed using Milli-Q water in substitution of the saliva of G. mel-
lonella larvae, as wellas saliva of G. mellonella larvae only. Also, PE was
exposed to 10 µl (1.2 mg/mL) of Demetra 24 times for 90 min. Pro-
longed treatment was performed as well for Demetra (days 1 and 2),
five applications per day of 10 µl(1.2mg/mL)for90mineach.As
control, the same experiment was repeated using the protein buffer.
Afterward, samples were centrifuged with an Eppendorf centrifuge
5810R at 19083 × gfor 30 s and the subnatant was transferred to a new
1.5ml Eppendorf tube. Samples and controls were extracted using a
QuEChERS (quick, easy, cheap, effective, and safe) method63 based
on64 with some modifications. Briefly, 50 µlofdiphenylphthalate
(Internal Standard; IS) at a concentration of 1 mg/ml was added at each
sample and extracted with 300µl of dichloromethane (DCM) and 5%
(v/m) of NaCl. The tube was vortexed for 30 s and sonicated in a bath
(50/60 Hz) for 15 min at room temperature, followed by centrifugation
with an Eppendorf centrifuge 5810R at 20 °C and 19083 × gfor 10 min.
Finally, DCM located as the subnatant was collected and placed in an
insert before analysis. Silylation reaction with N, O-Bis(trimethylsilyl)
trifluoroacetamide (BSTFA) was performed to determine the low-
volatility polar compounds which show low detection sensibility. A
fraction of 50 µlofeachsamplewith50µl of BSTFA was incubated for
20 min at 60 °C before the analysis.
Dichloromethane (DCM; CAS-No: 75-09-2) for gas
chromatography-mass spectrometry (GC-MS) was SupraSolv grade
purity and obtained from Sigma-Aldrich (Darmstadt, Germany).
Sodium chloride (NaCl; ≥99.5%; CAS-No: 7647-14-5) and ultrapure
water from a Milli-Q system were supplied from Merck (Darmstadt,
Germany).
Crystalline granular powder polyethylene (PE 4000; CAS-No:
9002-88-4, specification sheet in https://www.sigmaaldrich.com/
ES/es/product/aldrich/427772) was supplied by Sigma-Aldrich (Saint
Louis, USA).
Chromatographic analyses were performed with a gas
chromatography-mass spectrometry system (GC-MS) 7980A-5975C
from Agilent Technologies. Separation of the metabolites was per-
formed on a DB-5th Column coated with polyimide (30 m length,
0.25 mm inner diameter, and 0.1 µmfilm thickness; Agilent Technolo-
gies, USA) for proper separation of substances, and Helium (He) was
utilized as a carrier gas. The analysis was performed using a split
Article https://doi.org/10.1038/s41467-022-33127-w
Nature Communications | (2022) 13:5568 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved
injector at 350 °C and an injection volume of 1 µl. The ion source
temperature was 230°C, the °C mass spectral analysis was performed
in scan mode, the quadrupole temperature of 150°C, and a fragmen-
tation voltage of 70 eV. The oven programme started at 60 °C for
3 min, then 20°C/min to 350 °C for 1 min. The total run time was
18.5 min and 19.5 min for derivatized samples. The resulting chroma-
tograms were processed using the software MSD ChemStation
E.01.00.237 from Agilent Technologies, Inc while for the identification
NIST11 library was used.
The evaluation of the prolonged treatment was based on the
relative abundance of each untargeted compound, which consists of
the quotient of the area under the peak of each compound divided by
the area under the peak of the IS.
Gas Chromatography/Tandem Mass Spectrometry was used for
confirmation of the non-target compounds by a BRUKER 456-GC
SCION TQ. This experiment was performed by the Elemental and
Molecular Analysis facility, University of Extremadura, Spain. Briefly,
the injector port was set at 230°C in Split mode. Separation was
achieved using a column HP 5MS, 30 m, 0.25 mm, and 0.25µm. Helium
(He) was utilized as a carrier gas. The column oven was programmed in
the following conditions: 60 ° C for 3 min, increase of 20 °C/min to
325 °C for 1 min. The collision energy was 15 eV.
Electron Microscopy analysis
Larvae saliva samples were diluted 1:50 in the proper buffer (see “Wax
worm saliva collection”).
Samples were analyzed by electron microscopy (EM) after being
adsorbed to glow-discharged carbon coated grids and stained with 2%
uranyl acetate. Grids were observed using a JEOL JEM-1230 EM oper-
ated at 100 kV and a nominal magnification of 40000. EM images were
taken under low dose conditions with a CMOS Tvips TemCam-F416
camera, at 2.84 Å per pixel.
Protein Chromatography analyses
For the size exclusion chromatography, 40 μlofwwsalivainthe
proper buffer (see “Wax worm saliva collection”) was thawed, pooled,
and centrifuged. The supernatant was filtered (0.45 µm cutoff, Ultra-
free Millipore) and loaded to a size exclusion chromatography column
Superdex 200 5-150 (Cytiva) equilibrated with 10 mM Tris-Cl, 50 mM
NaCl, 2 mM DTT.
For the ion exchange chromatography, upon thawing, the sample
was diluted to 100 µl with 10 mM Tris-Cl at pH 8, centrifuged, filtered
and the supernatant loaded to a monoQ 5/50 GLion exchange column
(Cytiva). After a wash step, a 40 ml gradient with buffer A (10 mM Tris-
Cl pH8), and buffer B (same as A supplemented with 500mM NaCl)
was applied.
Proteomic analysis
Liquid Chromatography Mass Spectrometry (LC-MS) analysis. All
peptide separations were carried out on an Easy-nLC 1000 nano sys-
tem (Thermo Fisher Scientific). For each analysis, the sample was loa-
ded into a precolumn Acclaim PepMap 100 (Thermo Fisher Scientific)
andelutedinaRSLCPepMapC18,15cmlong,50µm inner diameter
and 2 µm particle size (Thermo Fisher Scientific). The mobile phase
flow rate was 300nl/min using 0.1% formic acid in water (solvent A)
and 0.1% formic acid and 100% acetonitrile (solvent B). The gradient
profile was set as follows: 5–35% solvent B for 45 min, 35–100% solvent
B for 5min, 100% solvent B for 10 min. Four μl of each sample were
injected.
MS analysis was performed using a Q Exactive mass spectrometer
(Thermo Fisher Scientific). For ionization, 1900 V of liquid junction
voltage and 270°C capillary temperature was used. The full scan
method employed a m/z 400–1500 mass selection, an Orbitrap reso-
lution of 70,000 (at m/z 200), a target automatic gain control (AGC)
value of 3e6, and maximum injection times of 100 ms. After the survey
scan, the 15 most intense precursor ions were selected for MS/MS
fragmentation. Fragmentation was performed with a normalized col-
lision energy of 27eV and MS/MS scans were acquired with a starting
mass of m/z 100, AGC target was 2e5, resolution of 17,500 (at m/z 200),
intensity threshold of 8e4, isolation window of 2 m/z units and max-
imum IT was 100 ms. Charge state screening was enabled to reject
unassigned, singly charged, and equal or more than seven protonated
ions. A dynamic exclusion time of 20 s was used to discriminate against
previously selected ions.
MS data analysis. Mass spectra *.raw files were searched against an
in-house specific database against Galleria_Proteins (12715 proteins
entries) extracted from the genome annotation available in Mendeley
(https://doi.org/10.17632/t7b5s58vxt.3), using the Sequest search
engine through Proteome Discoverer (version 1.4.1.14) (Thermo Sci-
entific). Search parameters included a maximum of two missed clea-
vages allowed, carbamidomethyl of cysteines as a fixed modification
and oxidation of methionine as variable modifications. Precursor and
fragment mass tolerancewere set to 10 ppm and 0.02 Da, respectively.
Identified peptides werevalidated using the Percolator algorithm with
aq-value threshold of 0.01. The protein identification by nLC-MS/MS
was carried out in the Proteomics and Genomics Facility (CIB-CSIC),
a member of ProteoRed-ISCIII network65. The mass spectrometry
proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE66 partner repository with the identifiers
PXD035476 and PXD035479.
Recombinant protein production and utilization
Arylphorin, arylphorin subunit alpha-like (Demetra) and hexamerin
(Ceres) were produced by Genscript, utilizing the baculovirus
expression system in insect cells, according to the manufacturer.
Briefly, Sf9 cells were infected with P2 baculovirus, flasks were incu-
bated at 27 °C for 48–72 h and media harvested. Then cells were
removed, and transfection medium was applied for purification. The
produced proteins were resuspended in 150 mM NaCl, 20mM HEPES,
5% glycerol and used for the degradation assay. The same buffer alone
was used as negative control.
Galleria mellonella genome annotation
In order to obtain the most useful information from mass spectro-
metry analysis of proteins extracted from ww saliva, it is pivotal to use
a representative database of protein sequences. In addition to the
NCBI official annotation of Galleria mellonella protein sequences, a
new annotation was produced exploiting also the information of Gal-
leria mellonella salivary glands RNA-seq data. Galleria mellonella gen-
ome annotation was performed using the genome sequence available
at NCBI. A specific pipeline was developed to combine the information
from RNA-seq with ab initio predictors in order to obtain the most
accurate annotation. Briefly, the RNA-seq data was mapped on the
reference genome using STAR (version 2.5 0c)67 in local mode and used
to perform a reference-guided transcriptome assembly with Trinity
(v2.11.0)68. The obtained transcripts and the mapping files wereused as
input for the Braker2 pipeline69 to combine AUGUSTUS ab initio
annotation70 with the transcriptomeassembly to obtain the annotation
in GFF format, together with transcript and protein sequences. Pro-
teins were used as input for the PANNZER2 pipeline to obtain
descriptions71, Gene Ontology and KEGG annotations. About 32000
genes could be annotated in the Galleria genome. The corresponding
proteins were analyzed to assess their completeness performing a
BLASTP alignment against the UniRef90 database and calculating the
percentage of alignment. A similarity search against the UniRef90
(Nov2018) database showed that about 50% of the predicted proteins
covered 100% of the corresponding hits (i.e., full length) and that
about 80% of the predicted proteins covered at least 50% of the cor-
responding hits. As a further control, the proteins and the genome
were evaluated with the BUSCOv2 pipeline72. The BUSCO database
Article https://doi.org/10.1038/s41467-022-33127-w
Nature Communications | (2022) 13:5568 8
Content courtesy of Springer Nature, terms of use apply. Rights reserved
contains sets of single-copy highly conserved genes across different
taxa (i.e., Eukaryota or Insects). By performing an analysis with the
BUSCO database it is possible to assess the completenessof a genome/
proteome, the presence of duplications and/or fragmentations. This
analysis was performed using the predicted proteome and also the
unannotated genome for a comparison. By comparing the results of
the unannotated with the annotated genome, we can see a small
fraction of missing genes which are probably absent from the genome
assembly and that cannot be recovered from the current genome
sequence. This explains some missing genes present in the later NCBI
annotation.
The RNA-seq data that was used for the G. mellonella genome
annotation has been deposited in NCBI under the BioProjectaccession
number PRJNA822887. In addition, the newly generated annotation in
GFF3/GTF format was deposited to Data Mendeley (https://doi.org/10.
17632/t7b5s58vxt.3)
Analyses of the wax worm PEases sequences
The domain composition of the proteins was predicted with the
SMART algorithm73 selecting the options: Outlier Homologues, PFAM,
signal peptides, and internal repeats. In order to retrieve similar pro-
teins with a resolved 3D structure, a BLAST search was performed on
the PDB database74 using an e-value cutoff of 0.001 for both the pro-
teins, whereas a minimum sequence identity of 50% was selected for
Demetra (XP_026756396.1)and30%forCeres(XP_026756459.1). The
two protein sequences were aligned using Clustal Omega75 and then
visualized with Jalview76. Finally, the prediction of the biochemical
characteristics of the two proteins was performed with Expasy’sProt-
Param tool77.
Statistics and reproducibility
No statistical methods were used in this work. No statistical method
was used to predetermine sample size. No data were excluded from
the analyses.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The dataused in the figuresare available in theSource Data and Source
Data Supplementary Material files. The RNA-seq data that was used for
the G. mellonella genome annotation have been deposited in NCBI
under the BioProject accession number PRJNA822887. In addition, the
newly generated annotation in GFF3/ GTF format was deposited in Da ta
Mendeley (https://doi.org/10.17632/t7b5s58vxt.3). The mass spectro-
metry proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE partner repository with the dataset identi-
fiers PXD035476 and PXD035479. Source data are provided with
this paper.
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Acknowledgements
We thank the Proteomics and Genomics, Electron Microscopy and Gas
Chromatography facilities of the CIB for the excellent technical support.
We thank Paloma Delgado and Pedro Velasco from InsectPark-
Microfauna S.L., El Escorial, Madrid, Spain (https://insectpark.es/)for
providing Samia cynthia larvae. We thank the Centres Cienti
fics i Tec-
nologics Unitat Cromatografia de Gases-Espectrometria de Masses
Aplicada (CCIT), University of Barcelona, Spain for the support provided
in the GC-MS experiments. We thank the Roechling foundation for
supporting and sponsoring this work. This work has been funded as
follows: Roechling Stiftung to F.B., Consejo Superior de Investigacion
Cientifica (CSIC) to F.B., NATO Science for Peace and Security Pro-
gramme (Grant SPS G5536) to T.T., Junta de Castilla y León, Consejería
de Educación y Cultura y Fondo Social Europeo (Grant BU263P18) to T.T.,
Ministerio de Ciencia e Innovación (Grant PID2019-111215RB-100) to T.T.,
The Generalitat de Catalunya (2017 SGR 1192) to M.S., and Ministerio de
Ciencia e Innovación (Grant PID2020-120275GB-I00) to E.A.-P.
Author contributions
Conceptualization: F.B. and C.F.A. Methodology: A.S.-V., P.C.-V., F.R.-V.,
M.B.-V., M.S.-A., E.R.-L., R.I.-V., P.C., R.A.C., M.M., P.F., C.P., L.G.-L.,
E.A.-P., M.S., T.T., C.F.A., and F.B. Investigations: A.S.-V., P.C.-V., F.R.-V.,
M.B.-V., M.S.-A., E.R.-L., R.I.-V., P.C., R.A.C., M.M., P.F., C.P., L.G.-.L,
E.A.-P., M.S., T.T., C.F.A., and F.B. Visualization: F.B., C.F.A., P.C.V., L.G.,
M.S., and E.A.-P. Funding acquisition: F.B., T.T., M.S., and E.A.-P. Project
administration: F.B. Writing—original draft: F.B. Writing—review & edit-
ing: A.S.-V., P.C.-V., F.R.-V., M.B.-V., M.S.-A., E.R.-L., R.I.-V., P.C., R.A.C.,
M.M., P.F., C.P., L.G.-L., E.A.-P., M.S., T.T., C.F.A., and F.B.
Competing interests
The authors declare no competing interests.
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