Proteomic-components provide insights into the defensive secretion in termite workers of the soldierless genus Ruptitermes

Journal Pre-proof Proteomic-components provide insights into the defensive secretion in termite workers of the soldierless genus Ruptitermes

Ana Maria Costa-Leonardo, Iago Bueno da Silva, Silvana Beani Poiani, José Roberto Aparecido Dos Santos-Pinto, Franciele Grego Esteves, Luiza Helena Bueno da Silva, Mario Sergio Palma PII:

S1874-3919(19)30394-X

DOI:

https://doi.org/10.1016/j.jprot.2019.103622

Reference:

JPROT 103622

To appear in:

Journal of Proteomics

Received date:

29 July 2019

Revised date:

3 December 2019

Accepted date:

14 December 2019

Please cite this article as: A.M. Costa-Leonardo, I.B. da Silva, S.B. Poiani, et al., Proteomic-components provide insights into the defensive secretion in termite workers of the soldierless genus Ruptitermes, Journal of Proteomics (2019), https://doi.org/10.1016/ j.jprot.2019.103622

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© 2019 Published by Elsevier.

Journal Pre-proof Research article

Proteomic-components provide insights into the defensive secretion in termite workers

a, b,*

Ana Maria Costa-Leonardo

a

oo

f

of the soldierless genus Ruptitermes

b

, Iago Bueno da Silva , Silvana Beani Poiani , José Roberto

b

b

a

pr

Aparecido dos Santos-Pinto , Franciele Grego Esteves , Luiza Helena Bueno da Silva and b

e-

Mario Sergio Palma

a

Pr

Laboratório de Cupins, Departamento de Biologia, Instituto de Biociências, Univ Estadual

Paulista, UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista, Rio Claro, SP 13506-900, b

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Brazil; Center for the Study of Social Insects, Department of Biology, Inst itute of Biosciences of

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Rio Claro, Univ Estadual Paulista, UNESP, Rio Claro, São Paulo, Brazil.

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*Corresponding author: Dr. Ana Maria Costa-Leonardo, IBRC- UNESP, Av. 24A, nº 1515, Bela Vista - Rio Claro, SP, Brazil, CEP 13506-900, E-mail: [email protected], Phone: +55 19 3526-4138 (ORCID: 0000-0002-8874-5538).

Acknowledgements This

work

was

supported by

FAPESP

(Processes

2013/26451-9,

2016/16212-5,

and

2017/10373-0), CNPq (Processes 301656/2013-4, 150699/2017-4, and 305539/2014-0), and CAPES (Financial Code 001 and Ordinance No. 206/2018).

Conflicts of interest The authors declare no conflicts of interest in relation to this manuscript.

Journal Pre-proof Tables Table 1. Protein identification by gel-free shotgun proteomic approach of defensive secretion of Ruptitermes reconditus termite - (i) toxins, defensins and proteolytic enzymes, and (ii) sticky components and alarm communication. *PIP % - score, protein identification probability. (i) toxins, defensins and proteolytic enzym es Accession num ber

Protein nam e

Mass (kDa)

Taxonom y

Ankyrin repeat domain-containing Copidosoma protein floridanum

18

A0A067R095

Ankyrin repeat domain-containing Zootermopsis protein 54 nevadensis

60

UPI000767D 337

Ankyrin-2-like

Dufourea novaeangliae

A0A067R8Q 6

Phospholipase B1 membraneassociated

Zootermopsis nevadensis

XP_0219170 65.1

Probable chitinase 10

UPI00079ED 4DF

Chitotriosidase-1

A0A067R7D 1

UPI0006C9A E69

oo

f

UPI0006C95 6D6

Seque *PI nce P cover age LLINNGTNFHVLDK 24.68 100 (64), % % VDVNVSSDVVGTPL ITPVSQPTISTA PMK 9.56% 100 HR (78), (49), SIFAELRR (62), % FGMNLLHRR (59) AGVRAADLAPSCLL AR (68), CSLETGAGNNSCN K (81) LPALHIAAKK (71), 1.34% 99 LGYISVMEVLK (42), % SFLVSFLVDAR (85), APEKDTEIVSDTQIT ESDILEQK (70) WLDKPYQVAR (60), 8.55% 100 ATSQHFNSK (75), % GMSWSGGGESTW R (36), TDLELATSGR (47) VTGNQPYHVVCYV 1.84% 100 EGWAVYR (64), % SFTLTNSSITTPPAPI K (56), MGLGGGMVWALDL DDFR (41) EEASATGKPK (72), 7.60% 100 VNMGIPLYGR (37), % GNQWVAYDDEDSA R (62) IKAGFADR (51), 5.77% 100 SPTVVSFSDTFAR % (35), DDLDKALLRPGR (40), DWGMSEKVGLR (69) EVACVSKFLGVLK 15.89 100 (70), DADSENDVEK % % (41), ACRYSFLR (62) QNFTQYVELYNEAA 3.41% 100 K (32), % MFEMADEFFR (74), SSDPEELR (68), MFQTAEEFFK (43) NVTSQPSTK (51), 8.13% 100 VLGSGHCSGLIK % (72), NQGYWPSYNVAYF K (45), YGDWFTYER (38) HLTPRDIFAPVNPK 14.67 99 (61), % % GTEPQPEFAGK (35), IDGTETGSVIVTSR (74), FCTATTGGK (50) IEPLHQKQR (42), 3.78% 100 VNCITSSTMEGYSL % AR (56), LSNKHHGR Peptide sequences (íons-score)

Pr

e-

pr

457

al

Zootermopsis nevadensis

55

328

49

ATP-dependent metalloprotease YME1L1

Zootermopsis nevadensis

84

ADAMTS-like protein 4

Copidosoma floridanum

22

A0A0L7LS53 Angiotensin converting enzyme

Operophtera brumata

146

UPI00077FB FEA

putative phospholipase B-like 2

Parasteatoda tepidariorum

62

R4UVR3

Trypsin-like serine protease

Coptotermes formosanus

35

Q16WL3

Scavenger Receptor w ith Serine Protease domain

Aedes aegypti

128

Jo u

rn

Lygus hesperus

Accession

Journal Pre-proof

gi|13309282 01

Tissue inhibitor of metalloproteases

Cryptotermes secundus

UPI00077DC 438

Aminopeptidase N-like protein isoform X1

Tribolium castaneum

494

D6WBA9

Aminopeptidase (EC 3.4.11.-)

Tribolium castaneum

111

28

(37), QLGFNGTAEVR (62) DLMSRVYK (38), ASVLLKSGR (52), GFRLLYR (78) AGIHAYLEKHK (51), SVIYCNGLR (64), LPEGVIEVER (32), RLPEDSVK (47), AFPCFDEPSYK (35) VLQSTNEIK (48), TDEMYGFYK (36), VMEDWTGIK (55)

Jo u

rn

al

Pr

e-

pr

oo

f

(ii) Sticky com ponents and alarm com m unication VSDVVQNVQK (62), PSTSFTSGK (54), UPI0006C99 Copidosoma PTTYVYSDQPGFTR Mucin-17 247 6A1 floridanum (38), TSTAQASSPTYR (35) NSTTGSSQSK (51), TSDPASR (82), A0A067RE6 Zootermopsis Mucin-1 71 SSYDSSNAQR (75), 7 nevadensis HLEVLYMAK (43), LIESQQNLYR (37) NLTEAENAAER UPI0007E6A (35), SSSNETLK Mucin-5AC Drosophila kikkawai 110 743 (58), HQRTDSTR (46) SSESTK (62), UPI0006EAD Mucin-5AC-like Papilio machaon 233 GSESIDTLK (49), C2A AIEAIEAPTAK (55) VLGYIQIR (38), A0A0U2D76 NADH-ubiquinone Cethosia biblis 36 IGIMGLLQPFSDAIK 1 oxidoreductase chain 1 (67), LMYLCWK (44) YFMNISILIK (71), NADH-ubiquinone Amblyomma SNFWITYIFIYSLIIYK H9M761 37 oxidoreductase chain 2 fimbriatum (42), YSSVALIFLNK (38) SYNAGILTVLSNR gi|13309164 NADH-ubiquinone Cryptotermes (67), 46 47 oxidoreductase chain 5 secundus GSFEMTLISLLVVLA AITK (59) GDAVTIILDCNR (31), Trachymyrmex GDLYLGSLEMLK A0A195FK63 Collagen alpha-2(XI) chain 172 septentrionalis (82), GDTGAK (53), GFTGPSGPLGLDG K (66) LISGLQYAVFALSK (74), LYDIPYGFNHR A0A1I8QDC Odorant receptor Stomoxys calcitrans 42 (59), 5 HQIVLNIGQNVK (65), TILIIMIR (32) NMTLAVVLVLFFIK Helicoverpa R4JKG0 Olfactory receptor 18 47 (44), FAPLLDK (70), armigera SRQELR (39)

9.63%

100 %

1.12%

98 %

2.71%

100 %

2.00%

100 %

7.34%

99 %

2.57%

100 %

1.15%

100 %

9.46%

100 %

11.87 %

100 %

7.86%

100 %

2.60%

98 %

10.92 %

100 %

6.78%

99 %

Table 2. Protein identification by gel-free shotgun proteomic approach of defensive secretion of Ruptitermes pitan termite - (i) toxins, defensins and proteolytic enzymes, and (ii) sticky components and alarm communication. *PIP % - score, protein identification probability.

(i) toxins, defensins and proteolytic enzym es Protein nam e

Taxonom y

Mass

Peptide sequences (íons -score)

Sequence

*P

Journal Pre-proof num ber

(kDa)

coverage

0049D4983

POTE ankyrin domain family member D

Zootermopsis nevadensis

309

EGHHDIAAELK (38), HEGERENINVADK (63), SLLPLGSLK (44), GLTSTERSK (71)

1.52%

9

0049D6E1A

Phospholipase

Zootermopsis nevadensis

127

ASLSIPFPTR (68), TGSTQPGR (42), DYTNFIVK (55)

2.31%

10

0049E49A4

Phospholipase B1, membraneassociated

Zootermopsis nevadensis

54

GFTSGLLK (41), ATSQHFNSK (35), VNYKDDWK (67), WQHLKDNFTQIMR (52)

7.73%

10

40584.1

Serpin I2

Cryptotermes secundus

44

DGYSHLTR (71), VPTMHLKK (50), FYLLIILPNEK (64)

6.68%

10

006C956D6

Ankyrin repeat domain-containing protein

Copidosoma floridanum

18

LLINNGTNFHVLDK (49), FGMNLLHRR (68)

14.55%

10

VR3

Trypsin-like serine protease

Coptotermes formosanus

35

13.45%

10

Tissue inhibitor of metalloproteases

Cryptotermes secundus

28

13.65%

9

0K8R411

Putative metalloprotease

Ixodes ricinus

54

8.02%

10

067RJV3

A disintegrin and metalloproteinase w ith thrombospondin motifs 12

Zootermopsis nevadensis

96

5.36%

10

067R998

Aminopeptidase (EC 3.4.11.-)

Zootermopsis nevadensis

98

4.15%

9

067R9V2

Ankyrin-3

Zootermopsis nevadensis

118

3.09%

10

067RE67

Mucin-1

7.50%

10

0062394DB

Mucin-17-like

2.33%

9

021939617.1

Mucin-17-like isoform X3

00B8E9ED8

f

oo

pr

e-

Zootermopsis nevadensis

28

IADVGCGGGILAER (67), LDPTISER (45), ECVRILK (62)

25

SEVAPAKR (77), YGVVFRASPR (39), MAPALRK (65)

Pr

330916447

Lasius niger

Cryptotermes secundus

46

SYNAGILTVLSNR (45), GSFEMTLISLLVVLAAITK (62), ALLFMCAGLIIHTMR (34)

Zootermopsis nevadensis

24

DIGQRTFSADLMR (58), DELQLIAPSEVTK (41)

al

0049C3D01

Ubiquinone biosynthesis Omethyltransferase NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 NADH-ubiquinone oxidoreductase chain 5 Gustatory and odorant receptor 22like

rn

0J7KVZ7

(ii) Sticky com ponents and alarm com m unication NSTTGSSQSK (38), TSDPASR (69), HLEVLYMAK (49), Zootermopsis nevadensis 71 TFYSMKIELVR (57), LIESQQNLYR (48) LVPGCYQNEMR (63), VDIMYK (74), SALIAEDKK (36), Linepithema humile 158 QIPSKPAK (52) Zootermopsisnevadensis 193 TNDSNNTVPK (41), TSTSSDK (78), TTSATTPRPR (53)

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330928201

GTEPQPEFAGK (39), IDGTETGSVIVTSR (48), YVAGDAAGAR (69), FCTATTGGK (55) DLMSRVYK (42), ASVLLKSGR (31), PEETYLITGK (65), GFRLLYR (52) AVDSEFIRDR (37), GILNTSHSIQPENK (74), QAIVEER (51), ENEDDWAK (65) GPGASVDVNK (82), TNEGHYFIEPMK (41), VCNTSPCDPK (39), GYVKIATIPAGSMR (56) PLDINISVEDER (68), FAGVTQFEATDAR (32), DYFQIAYPLPK (74) QFLDSENSIYFR (51), QEVQLINEVK (79), ENKIQELIFK (38)

1.51%

10

12.00%

10

11.46%

10

11.54%

10

12.20%

9

Journal Pre-proof Table 3. Confrontation bioassays - Frequency of the response behaviors performed by R. reconditus workers during interactions with opponent species. Bioassay (n = 10) 1 2

Opponent species C. gestroi H. tenuis

R. reconditus response (mean ± SE) Biting Avoiding Bursting 22.2 ± 2.1 3.3 ± 0.8* ns 50.0 ± 6.2** 0.33 ± 0.16 ns

* P < 0.05; ** P < 0.01; ns - not significant

Table 4. Toxicity bioassays - Behavior performed by P. araujoi workers after contact with the defensive secretion samples of R. reconditus and R. pitan.

2

R. pitan (n = 7)

1h 6h 1h 6h

5 5 4 4

Observed behavior Difficulty Stucked to Moribund walking the glue

f

R. reconditus (n = 10)

Able to walk

oo

1

Observation time

Pr al rn Jo u

3 -

pr

Termite species

e-

Bioassays

5 -

4 1

Dead 1 2

Journal Pre-proof Proteomic-components provide insights into the defensive secretion in termite workers of the soldierless genus Ruptitermes

Abstract Termite soldiers constitute the defensive frontline of the colonies, despite workers also perform such tasks, especially within the Neotropical Apicotermitinae, in which all species are soldierless.

Workers of the genus

Ruptitermes display an extreme form of defense,

characterized by body rupture and release of a sticky secretion. Previous observations suggested that such behavior may be advantageous against enemies, but the chemical

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composition of this secretion has been neglected. Here we firstly provide the proteomic profile of the defensive secretion of Ruptitermes reconditus and Ruptitermes pitan workers.

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Additionally, the mechanisms of action of this behavior was evaluated through different

e-

bioassays. A total of 446 proteins were identified in R. reconditus and 391 proteins in R. pitan, which were classified into: toxins, defensins and proteolytic enzymes; sticky components/ alarm proteins

folding/conformation

and

related

to

detoxification

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communication;

post-translational

processes;

modifications;

proteins

housekeeping

involved proteins;

in and

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uncharacterized/hypothetical proteins. According to the bioassays, the self-sacrifice is triggered

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by a physical stimulus, and the defensive secretion may cause immobility and death of th e opponents. Assuming that termites are abundant in the tropics and therefore exposed to

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predators, suicidal behaviors seem to be advantageous, since the loss of an individual benefit the whole colony. Significance

Although recent studies have reported the biochemical composition of different weapons in soldiered species of termites, such efforts had not been applied to sordierless taxa up until now. Thus, this is the first report of the defensive mechanisms in soldierless termite species based on proteomic analysis. The diversity of compounds, which included toxin-like and mucin-like proteins, reflect the mechanisms of action of the defensive secretion released by termite workers, which may cause immobility and death of the opponents. Our findings may contribute to the knowledge regarding the development of defensive strategies in termites, especially in groups which lost the soldier caste during the evolution.

Journal Pre-proof Keywords: Apicotermitinae; sticky defensive secretion; toxin-like proteins; mucin-like proteins; LCMS-based proteomics; Isoptera

1. Introduction Insect defense mechanisms incorporate different strategies that include morphological, physiological, and behavioral adaptations to eliminate parasites, predators and competitors [1]. An extreme form of defense is the self-sacrifice mechanism adopted by several social insects, which has evolved by kin selection [2]. Termites, for example, developed plentiful defensive

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strategies against predators, which are usually associated to sterile individuals such as workers and soldiers [3, 4]. The soldier caste is undoubtedly the most specialized, equipped with both

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mechanical (phragmosis and mandibles) and chemical (salivary, labral, and frontal glands) apparatuses [1, 5, 6]. Meanwhile, workers are quite vulnerable due to their thin tegument and

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limited agility, however they contribute actively to the colony defense by biting enemies and

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grasping their legs [7]. Moreover, workers are fully responsible for the nest construction, which represent a passive defense [4]. In some taxa such as Neocapritermes, soldier rate is quite low

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inside the nest or in foraging sites, thus workers developed suicidal behavior associated with toxic compounds, which may trigger different responses in the enemies and contribute to colony

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defense [8, 9].

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Termitidae is the largest family within Isoptera and comprises over 70% of the species [10], including Apicotermitinae, a subfamily in which most species lost the soldier caste. In Neotropical Apicotermitinae, all species are soldierless and workers may be considered the defense frontline of the colonies [4]. Thus, such workers developed particular morphological, physiological, and behavioral mechanisms to repel or combat enemies, even though such defensive strategies are still poorly comprehended within soldierless taxa [11, 12]. The genus Ruptitermes, which is included within Neotropical Apicotermitinae, is endemic to South America [13, 14]. Workers of this genus display open-foraging activity in leaf litter, often at night, and perform a suicidal behavior defense consisted of body rupture. When suicide workers burst, they break their body wall and release a translucent liquid, which becomes viscous in contact with the air [12, 15].

Journal Pre-proof Knowledge concerning the genus Ruptitermes is limited and few studies have described the basic biology and the defensive mechanisms of these termites [12, 15, 16]. Moreover, no study reported the biochemical composition of their defensive secretion so far. Thus, in the current investigation, we used a gel-free shotgun proteomic approach to provide a holistic overview of the proteomic profile of defensive secretion released by Ruptitermes reconditus and Ruptitermes pitan workers. In addition, the mechanism of action of this defensive secretion was evaluated through two different experimental setups: confrontation and toxicity on different opponents.

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2. Materials and methods

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2.1. Termites

y UNE

R

B z

47º 3 ’ 3 .6” W .

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2.2. Scanning electron microscopy (SEM)

º 3’ 43. ”

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U

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Termite workers of Ruptitermes reconditus (Silvestri, 1901) and Ruptitermes pitan

Defensive organs from five workers of each species were sectioned and fixed in

al

Karnovsky fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate

rn

buffer, pH 7.4) during 1 h. After being washed in distilled water, the samples were dehydrated in an ethanol series (70-100%) and through critical point drying (Balzers CPD 030). The samples

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were fixed on metallic stubs with double-sided copper sticky tape and sputtered with gold (Balzers SCD 050). The material was then examined and photographed under a Hitachi 3000 scanning electron microscope, operated at 15 kV.

2.3. Sample preparation for LCMS-based proteomics For this purpose, the sample preparation and the in-solution digestion methods used for the shotgun approach followed the methodology described by dos Santos -Pinto et al. [17]. The defensive secretion from 14 R. reconditus and 20 R. pitan workers were colletected and transferred to a solution containing a

. % p

hb

k

IGM F

T™

Inhibitor Cocktail Tablets, EDTA-Free, containing 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), Bestatin hydrochloride, Leupeptin, E -64, Aprotinin, Pepstatin A, and Phosphoramidon disodium salt) for protein extraction.

Journal Pre-proof

2.4. Shotgun proteomic approach The proteins (400 µg) from the defensive secretion samples were solubilized in 50 mM ammonium bicarbonate, pH 7.9, containing 7.5 M urea, for 2 h to denature the proteins. Proteins were then reduced with 10 mM DTT at 37ºC for 60 min, and thereafter alkylated with 40 mM iodoacetamide at 25ºC during 60 min in the dark. Samples were diluted five-fold with 100 mM ammonium bicarbonate, pH 7.8, and 1 M calcium chloride was added to the samples to a final concentration of 1 mM. Non-autolytic trypsin (Promega) was added to the denatured

f

protein solution (1:50 trypsin: protein, w/w) and incubated for 18 h at 37ºC. The digested

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samples were stored at -80 ºC until they were needed for analysis; the tryptic peptides were

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2.5. Mass spectrometry-based proteomic analysis

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solubilized in 50% ACN/0.5% TFA and submitted to mass spectrometry analysis .

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Peptide analyses were conducted using an HPLC system (Shimadzu) coupled to a µLC-ESI-micrOTOF-Q-III (Bruker Daltonics Bremen, Germany). Prior to the analyses, the mass spectrometer was calibrated with a Tune-Mix Electrospray Calibrant (Agilent) solution. We used

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a C18 column, 12 nm (3 mm x 100 mm, 2.2 µm) (Shim-pack XR-ODS, Shimadzu), in a gradient

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of 5-95% ACN for 120min. The HyStar v3.2 software (Bruker Daltonics) was used to control

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data acquisition and analysis over the mass range of m/z 100-3000. MS spectra were recorded, followed by the acquisition of five data-dependent CID MS/MS spectra generated from the three highest intensity precursor ions. MS/MS spectra were interpreted, and peak lists were generated using DataAnalysis 4.1 (Bruker Daltonics).

2.6. Database search proteomics We performed database searches against the latest available Termitoidae protein sequences deposited in the NCBInr database (blast.ncbi.nlm.nih.gov, on march 19, 2018) using the software MASCOT 2.2.06 (Matrix Science, London, UK). Thus, it was selected all 119,357 entries for Isoptera. For the proteins that could not be identified within this database, searches were performed against proteins from Arthropoda taxa, for which there were 6,448,695 entries. Search parameters were set as follows: taxonomy, enzyme selected as trypsin, 2 maximum

Journal Pre-proof missing cleavage sites allowed, peptide mass tolerance was 0.5 Da for the MS and 0.8 Da for the MS/MS spectra, carbamidomethyl (C) was specified as a fixed modification, while methionine oxidation was specified as variable modification. Afterwards, the identified proteins were subjected to additional filtering by Scaffold 4.3.2 (Proteome Software Inc., Portland, OR) to validate peptide identification. A false discovery rate (FDR) of less than 1% was calculated by requiring significant matches to at least two different sequences. Considering the Scaffold Local FDR algorithm, the peptide probability identification was set to a minimum of 99%, while the protein probability identification was set at 95%. The proteome data was deposited in the

f

ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier

pr

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PXD015893 and 10.6019/PXD015893.

2.7. Functional enrichment analysis and molecular biological interpretation

e-

In order to understand and interpret the proteomic data, the list of identified proteins

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was analyzed and classified according to the terms of Gene Ontology - GO [18]. Based on the functional classification using the GO (cellular component, biological proc ess, and molecular function) of these proteins, we proposed a general mechanism of functions for the proteins

rn

al

identified in the defensive secretion sampled from R. reconditus and R. pitan workers.

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2.8. Quantitative-based proteomics

Label-free quantitation was implemented using the Scaffold 4.3.2 (Proteome Software Inc., Portland, OR). The following parameters were used: maximum retention time alignment of 10 min. with a minimum S/N of 5 for feature linking mapping. Abundances were based upon precursor/peptide peak intensities. Normalization was performed to ensure that the total sum of the abundance was the same for all samples. Imputation was performed by replacing the missing values with random values from the lower 5% of the detected values.

2.9. Bioinformatic analysis tools The GlycoEP Server- Prediction of glycosites in eukaryotic glycoproteins [19] was used for N- and O-linked glycosylation prediction. The SWISS-MODEL server [20] was used for three-dimensional molecular model representative images. Venn analysis was performed by

Journal Pre-proof FunRich - Functional Enrichment analysis tool web server [21]. For signal peptide prediction was used the SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP/).

2.10. Confrontation bioassays Workers of R. reconditus (Silvestri, 1901) were confronted with forager workers and soldiers of two termite species, Coptotermes gestroi (Wasmann 1896) and Heterotermes tenuis

f

(Hagen 1858), and individuals of the ant Camponotus atriceps (Smith, 1858). All insects were

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collected at the Sao Paulo State University, Rio Claro, SP, Brazil. For observation of the

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defensive behavior, five R. reconditus workers (about 0.4 mm length) were selected for three different agonistic bioassays, according to the opponent insect: 1) two workers and one soldier

e-

of C. gestroi; 2) two workers and one soldier of H. tenuis; and 3) one individual of C. atriceps

Pr

(about 1 cm length). This ant specie was chosen due to its nocturnal foraging activities, which may facilitate encounters with R. reconditus under field conditions. For termites as opponents, a 6 cm Petri dish filled with moistened filter paper was used as arena, while for C. atriceps as

al

opponent, a 9 cm Petri dish was selected and prepared following the conditions described

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above. Video recording started right after the introduction of the opponents and lasted 8 min for

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termite species as opponents, and 5 min for C. atriceps (these periods of time were empirically determined). All bioassays were replicated 10 times, using individuals which had not participated in the previous treatments. The bioassays were analyzed with the software BORIS [22] and the frequency of the following behaviors exhibited by R. reconditus workers was recorded: (i) antennation, which is when they touch the opponent using the antennae; (ii) biting; (iii) avoidance, retreating after antennation, and (iv) bursting, which is the body rupture leading the release of a defensive secretion (suicidal behavior). The frequency of each behavior performed by R. reconditus workers against termite opponents was compared using a nonparametric Mann-Whitney test. A significance level (α) equal to 0.05 was adopted.

2.11. Toxicity bioassays

Journal Pre-proof For this purpose, workers of R. reconditus and R. pitan, and workers of Procornitermes araujoi (Emerson, 1952) were chosen to evaluate the topic effect of the defensive secretion of Ruptitermes spp. A single worker of R. reconditus and R. pitan were placed individually in Petri dishes (6 cm diameter) and disturbed with tweezers aiming to induce their body rupture. After the bursting, the defensive secretion was gently rubbed onto the tegument of P. araujoi workers (about 5 mm length). These individuals were kept in Petri dishes (6 cm diameter) with moistened filter paper, and observed after 1 h and 6 hs of the experiment beginning. The behavior of the workers was classified into: i) able to walk, ii) difficulty of walking, iii) stucked to

f

the released glue, iv) moribund (immobilized individual, but moving the antennae/legs), and v)

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dead. We performed 10 replications for R. reconditus and 7 for R. pitan. For each replication,

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we maintained a P. araujoi worker without secretion as control. 3. Results

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3.1. Scanning electron microscopy

Pr

The defensive secretion of R. reconditus and R. pitan is stored in paired structures, located between the thorax and the anterior abdomen. When disturbed, the workers burst the lateral region of the anterior abdomen and release a droplet, which quickly become viscous in

al

contact with the air (Figs. 1A-B). The ultramorphology of the defensive secretion showed that R.

rn

reconditus workers possess rounded secretory vesicles, which may be rough or smooth (Fig.

Jo u

1C). Otherwise, the defensive secretion stored by workers of R. pitan is non-rounded and homogeneous, arranged into polyhedral secretory units (Fig. 1D). In both species, a thin cytoplasm surrounds the secretory vesicles.

3.2. LCMS-based proteomics After mass spectrometry analyses and data processing, it was possible to identify a total of 446 proteins in the defensive secretion of R. reconditus and 391 proteins in the defensive secretion of R. pitan (considering redundant identifications for both samples). The protein searches were initially performed by comparing them to the Isoptera database, and the unidentified proteins were then compared to those found in other arthropods. All proteins were initially classified according to their functions, which were determined by analyses in the Gene Ontology (GO) database (cellular component, biological process and molecular function). It was

Journal Pre-proof also analyzed the taxonomic distribution of the best-matched organisms of the identified protein sequences (Fig. 2A). Despite the limited number of termite sequences in the public databases, 37% of the sequences best matched to Isoptera species, followed by 21% for Hymenoptera, 30% for other insects, 7% for Arachnidae, and 5% for Crustacea. Because the analyzed sample was a secretion released after the body wall rupture, the search for functional roles of proteins in GO databases predicted molecular function (when the information was available) based on reports of intracellular roles of these proteins. The roles of secreted proteins are poorly predicted by GO enrichment searches [17], thus the proteins were

f

classified using the functions attributed in the GO enrichment search, and then grouped

oo

according to their potential functional roles in the defensive secretion. Thus, the proteins were

pr

categorized into six functional groups: (i) toxins, defensins and proteolytic enzymes, (ii) sticky components and alarm communication, (iii) proteins related to detoxification process of secretion,

(iv) proteins

involved in folding/conformation and post -translational

e-

defensive

Pr

modifications (PTMs), (v) housekeeping proteins, and (vi) uncharac terized/hypothetical proteins. Due to the large amount of data generated, the complete list of functional groups is given in Supplementary Tables S1 to S12, while Tables 1 and 2 contain some proteomic data

al

which were specifically extracted from the tables mentioned above to highlight some proteins of

rn

interest in this study. In addition, we also provided the total protein distribution according to their

Jo u

general functions in the defensive secretion of both species (Fig. 2B). Overall, 3% of the total proteins were toxins, defensins and proteolytic enzymes; 2% were sticky components and alarm communication, 4% were proteins involved in the detoxification process of defensive secretion, 8% in the folding/conformation and PTMs, 55% were housekeeping proteins; and 28% of the proteins were identified as uncharacterized/hypothetical proteins. For this result, only nonredundant proteins identified in both sampled species were used to build the diagram. The functional groups are detailed below. The first group (i) is composed of a total of 26 toxins, defensins and proteolytic enzymes-like proteins such as different proteoforms of ankyrin-like proteins, phospholipase-like proteins,

aminopeptidase-like proteins,

serpin-like proteins,

trypsin-like serine protease,

metalloprotease-like proteins, and chitinase-like proteins. These proteins are listed in Tables 1 and 2, including compounds identified in three termite species belonging to three different

Journal Pre-proof families:

Zootermopsis

nevadensis

(Archotermopsidae),

Cryptotermes

secundus

(Kalotermitidae), and Coptotermes formosanus (Rhinotermitidae). Moreover, this functional group was analyzed by SignalP-5.0 Server for signal peptide prediction. This result is demonstrated in the Supplementary table S13 and corroborates the secretory protein condition of some proteins included in this group. The second functional group (ii) comprises a total of 17 sticky component-like proteins related to the adhesive function of defensive secretion, such as different mucin-like proteoforms, ubiquinone-like proteins, and collagen-like proteins. Moreover, this group also includes proteins involved in the alarm communication suh as odorant and

f

olfactory receptor-like proteins, and gustatory and odorant receptor-like proteins. These

oo

compounds are quoted in Tables 1 and 2, including some identified in the termites Z.

pr

nevadensis, and C. secundus. These two functional groups will be discussed in details later. The third group (iii) included 28 proteins related to the detoxification process of the

peroxidase-like

proteins,

catalase-like

proteins,

and

thioredoxin-like

proteins

Pr

proteins,

e-

defensive secretion such as different proteoforms of ubiquitin-like proteins, cytochrome-like

(Supplementary Tables S3 and S9). The fourth group (iv) is constituted of 59 proteins such as glycosidase-like, kinase-like, heat shock-like, phosphatase-like, and cellulase-like proteins,

al

which were related to the folding/conformation and PTMs of toxin-like and sticky components.

The fifth group (v) contains

441 housekeeping proteins,

disposed in

Jo u

S4 and S10.

rn

These proteins have been described in social insects and are listed in Supplementary Tables

Supplementary Tables S5 and S11. Although the defensive secretion of each species was mainly constituted of housekeeping proteins (55% of total proteins), this functional group did not play any apparent role in the defense mechanism developed by R. reconditus and R. pitan workers. The last group (vi) included 266 uncharacterized/hypothetical proteins (Supplementary Tables S6 and S12) and most of them have been previously described for Z. nevadensis, C. formosanus, and C. secundus. A Venn analysis was adopted to verify the shared proteins between the defensive secretion of R. reconditus and R. pitan. As shown in the Venn diagram (Fig. 2C), 83 proteins were shared by the studied species; most of them were housekeeping proteins (22 proteins) and uncharacterized/hypothetical proteins (55 proteins). From the total shared proteins, only six belonged to relevant functional groups: (a) ankyrin repeat domain-containing protein (accession

Journal Pre-proof number - UPI0006C956D6), (b) trypsin-like serine protease (accession number - R4UVR3), and (c) tissue inhibitor of metalloproteases (accession number - gi|1330928201) from the first group (i); (d) NADH-ubiquinone oxidoreductase chain 5 (accession number - gi|1330916447), and (e) mucin-1 (accession number - A0A067RE67) from the second group (ii); and (f) alphamannosidase 2 (accession number - A0A067R639) from the fourth group (iv). It is important to mention that, for this analysis, we considered the access codes of the different proteoforms that have been identified and deposited in the databases. Thus, the same protein may have different access numbers, and in our comparative analysis, only proteins with t he same access number

f

were designated as shared proteins between the species.

oo

Assuming the adhesive properties of defensive secretion and the relevance of the

pr

identification of mucin-like proteoforms in both species, the expression of these proteins was quantitatively verified by label-free strategy based on spectral counting. These results, showed

e-

in Fig. 3A, point that almost all mucin-like proteins were expressed in considerable levels in the

Pr

defensive secretion extracted from both species. Thus, the mucin-1 protein (designated A; accession number - A0A067RE67) from Z. nevadensis termite, which were common in both samples, showed to be expressed in large amounts in the R. reconditus samples. Given the

al

relevance of glycosylation in the mucin-like proteins, mucin-1 protein (accession number -

rn

A0A067RE67) as investigated by an algorithm that predicts the occurrence of glycosylation

Jo u

sites. Thus, a reconstruction of a mucin generic structure is presented in Fig. 3B, including the attachment of several oligosacharides (N- or O-linked carbohydrate) in the repetitive domain of the sequence. Additionally, Fig. 3C shows the mucin-1 sequence, highlighting the occurrence of 62 O-linked glycosylated sites and 4 N-linked glycosylated sites predicted by the GlycoEP Server [17]. Finally, a three-dimensional molecular model of the mucin-1 protein is given (Fig. 3D).

3.3. Confrontation and Toxicity bioassays The findings of each experimental setup is quoted in Table 3. During the confrontation bioassays, R. reconditus workers always started interactions with the opponents using antennal inspection, followed by retreating behavior or biting. During confrontations with H. tenuis workers and soldiers, R. reconditus were more aggressive when compared to confrontations

Journal Pre-proof with C. gestroi individuals as enemies. This aggressiveness was evaluated according to the biting frequency. Otherwise, retreating behavior was more frequent when the bursting workers confronted C. gestroi opponents (Table 3). The frequency of antennation performed by R. reconditus workers was not significantly different between the opponent species (P = 0.2530), as well as the frequency of bursting (P = 0.77), since workers of R. reconditus burst in all replicates. However, the frequency of this behavior was higher against soldiers of both species tested. Once released, the defensive secretion from R. reconditus workers was not able to completely immobilize soldiers of the opponent species, but these opponents showed some

f

difficulty in moving the mandibles and walking. It was observed that the defensive suicidal

oo

behavior of R. reconditus workers was triggered by a tactile-physical stimulus from the enemies

pr

on the anterior abdomen, leading to a lateral projection of the worker abdomen towards the opponent and subsequent body bursting. Such observations explain why R. reconditus workers

e-

burst more frequently when attacked by soldiers, since this caste is equipped with powerful

Pr

mandibles and could easily trigger this behavior.

The abdomen rupture released a liquid droplet, which became sticky in contact with the air, responsible for the immobilization of the opponents. We did not compare the frequencies of

al

behavior performed by R. reconditus workers against termites and C. atriceps as opponents.

rn

However, it was possible to observe that in all replicates with the ant, R. reconditus were

Jo u

vigorously attacked and retreated in most encounters. Body bursting also occurred after a tactile-physical stimulus on the abdomen, and despite the defensive secretion did not immobilize the opponent, an intense self-grooming was performed by C. atriceps right after the contact with the defensive secretion. The toxicity bioassays results are demonstrated in Table 4, which summarizes the behavior performed by Procornitermes araujoi workers after contact with the defensive secretion samples of R. reconditus and R. pitan workers. In general, opponents stucked to the glue or with difficult walking after 1h of experiment, were moribund or dead at the final of the bioassay. All control individuals were alive after 6h of the experiment.

4. Discussion

Journal Pre-proof Termites are often subjected to aggressive encounters, especially in tropical regions, where these insects have developed predation-prey interactions with other animals [23]. Additionally, agonistic behaviors are consequence of intra- and interspecific conflicts for nesting and f

[ 4].

Š b

ík

. [

]

m

p

defensive strategies, and although this function is mainly performed by soldiers, the role of workers in nest defense should not be underestimated. In the present study, the two Ruptitermes spp. investigated forage in the open and display the same ecological habits [13], thus are often exposed to predators, especially ants. The body rupture performed by these

f

species, followed by the release of a sticky secretion, seems to be a pertinent defensive

oo

strategy. Comparative analyses should be encouraged to fully understand the development of

pr

these defensive mechanisms and identify their occurrence in other soldierless taxa [1, 26, 27]. The defensive secretion reported for R. reconditus and R. pitan workers were

e-

functionally similar, characterized as a translucent fluid secretion that quickly becomes sticky

Pr

and immobilizes an enemy (Costa-Leonardo, personal communication). This entangling secretion activity was described as tanning [1], which uses protein binding to stiffen the secretion after its releasement. Beyond the sticky properties, other functional characteristics

al

have been recorded in termite defensive secretions, including Ruptitermes, such as irritant

rn

compounds, repellents, and true poisons [28–30].

Jo u

In the current investigation, the proteomic-components identified in the defensive secretion corroborate previous analyses on the genus Ruptitermes [12], in which the authors performed a preliminary protein analysis, but the biochemical profile of the defensive secretion was not characterized. According to our scientific outcomes, the defensive secretion of R. reconditus and R. pitan has not only adhesive compounds but also toxins, defensins and proteolytic enzymes. As mentioned before, all identified proteins were organized into six different functional groups, and assuming the importance of the toxic and sticky properties of the defensive secretion in Ruptitermes spp., these functional groups are highlighted below. The first group (i) included toxins, defensins and proteolytic enzymes, such as phospholipases B, which were described in animal venoms presenting toxin activity. These proteins have been identified in snake venoms as anticoagulants due their hydrolysis mechanism, although this is still unclear [31], and can display local miotoxicity activity [32, 33].

Journal Pre-proof Some proteins with protease, metalloendopeptidase, and metalloprotease activities were al so included. These proteins can be involved in the inflammatory process and systemic reactions, and have also been described for snake [34], and Hymenoptera venoms [35-37]. For example, tripsin-like serine protease can be involved in the toxin biosynthetic process [38, 39]. Another finding in the current investigation was chitinase, a protein classified as a "dissemination factor", since it facilitates molecular diffusion through the hydrolysis of chitin and other diffusive obstacles [40]. Thus, we suggest that this protein would facilitate the toxic action, hydrolyzing the chitin of an ant, since these insects are the main termite predators. The toxins reported in

f

the defensive secretion from R. reconditus and R. pitan may have a topical action and cause

oo

irritant effects, similar to those observed on ants [29]. Serpins are one of the most important

pr

protease inhibitors, which avoid degradation of proteinaceous toxins by proteases and may act as potential toxins [41, 42]. Additionally, we identified an ankyrin repeat domain, which acts as a

e-

defense response against pathogens [43]. The presence of these proteins in the defensive

Pr

secretions may be associated with colony asepsis against microorganism proliferation after termite death.

The adhesive properties of insect secretions are often composed of complex mixtures

al

[30], exhibiting glycoproteins and mucopolysacharides as constituents [44]. Therefore, it may

spp.,

included in the second group (ii),

sticky

components and alarm

Jo u

Ruptitermes

rn

explain the presence of different mucin-like proteoforms in the defensive secretion of

communication. This proteomic-component also corroborates previous histochemical analysis regarding the defensive mechanisms of Ruptitermes [12, 15]. Quantitative-based proteomics findings demonstrated that mucin-like proteins seem to be the most abundant sticky component in the defensive secretions of R. reconditus and R. pitan, and may be responsible for the viscous aspect of the released secretion. The compound mucin-

F . 3D

α-

helices and random structures enhancing its stability and function. These characteristics are probably due the presence of N- and O-linked carbohydrates in mucin-1, since this posttranslational modification may influence folding and protein conformation, stability, solubility, and biological activities, as well as protein protection against autolytic actions [45, 46]. Proteins involved in chemical communication, such as olfactory, gustatory, and odorant receptor-like proteins were also included into this group (ii). These components may be related

Journal Pre-proof to the alarm communication mechanisms and may be secreted during the bursting defensive behavior, since it is necessary an effective communication to recruit more defenders and allow the evasion of vulnerable individuals [25]. Therefore, an alerted termite may follow the chemical alarm released by nestmates and arrives at the combat site. The occurrence of proteins related to detoxification processes (group iii) in the defensive secretion of Ruptitermes spp. had already been suggested anteriorly [12]. According to Pasteels et al. [30], the biosynthesis of some chemical compounds of defensive secretions may irritate the tissue where it occurs. Thus, to avoid such an effect, the final steps of the

f

biosynthesis could occur in extracellular regions [47]. In other cases, where termite soldiers

preventing autotoxication [48]. Ubiquitin-like proteins,

pr

reductive detoxification processes,

oo

produce toxic compounds for colony defense, nestmates become immune to this effect due

potential detoxification compounds reported here, were identified as important components for

e-

protein binding and degradation in response to toxic exposure in invertebrates [49]. Thus, we

Pr

suggest that such proteins may prevent tissue damage caused by toxic constituents of the defensive secretion.

The presence of a series of proteins related to glycosylation PTMs (group iv), such as

al

alpha-mannosidase and beta-1,4-galactosyltransferase, may be involved in the glycosylation

rn

processing of mucins identified in the defensive secretions, contributing to their sticky property

Jo u

[50]. These proteins have also been identified in Hymenoptera venoms, and play an important role in protein transport among different cellular compartments, degradation of unstable proteins, protein folding, stability and maintenance of the integrity of toxins [35, 51]. The housekeeping proteins (group vi) are related to metabolic functions associated with energy production. In addition, they may be contaminants from the organ material derived from tissues and muscles delivered during the body rupture and release of the secretion [35, 36, 51] . The last functional group (iv) was composed of proteins which have been deposited in databases and designated as having unknown functions, due to the absence of homology with other known proteins and/or non-functional characterization through experimental assays [52]. Thus, this group still requires more studies to elucidate if they perform any important role during the defensive behavior developed by R. reconditus and R. pitan workers.

Journal Pre-proof Although unidentified proteins have been reported for termites as responsible for secretion stiffening after air exposure [53], the mucin-like proteins had not been reported up until now. The mechanism by which the secretion becomes sticky and solid on the opponent is not totally clear yet. However, the findings presented here suggest that mucin-like proteins are responsible for the stickiness of the defensive secretion, which presents stiffening property after air exposure. Due the presence of carbohydrate coating, the mucins have water-holding capacity and can exhibit a different behavior according to their surrounding environment [54]. Thus, when in contact with the air, occurs a process of drying that together with physical

f

mechanisms such as viscosity force [55], it is likely to induce the cross -links among mucin-like

oo

proteins to stiffen the secretion after its releasement. This mucin cross-linking process may be

pr

increased by oxidation as demonstrated by Yuan et al. [56]. Nevertheless, it seems that the nature of the sticky secretions in R. reconditus and R. pitan workers includes a great diversity of

e-

biochemical compounds developed by these species during the evolution of their defensive

Pr

mechanisms.

The results from the agonistic bioassays showed that R. reconditus performs aggressive responses towards interspecific termites and ants. The defensive suicidal behavior,

al

which is part of this agonistic repertoire, was characterized by a lateral projection of the

rn

abdomen towards the enemy, followed by the body rupture, and release of a secretion. Soldiers

Jo u

are specialized in defending the colony and have powerful mandibles, so they were capable to cause more damage to the suicide workers, which ultimately burst. Bordereau et al. [57] observed that under attack of ants, Globitermes sulphureus soldiers often tried to pierce the enemies with their mandibles and only very excited soldiers released a droplet, which became viscous, and could immobilize the enemies. Likewise, workers of Neocapritermes taracua perform

suicidal

behavior solely when they are seriously attacked [58]. Thus, these

observations suggest that some termites have a threshold of stimulus that triggers the selfdestructive behavior. Suicidal behavior performed by R. reconditus workers was effective to immobilize some opponent termites, but it was unable to stuck the large ants used in the experimental setups. Nevertheless, individuals of C. atriceps displayed intense self-grooming, corroborating previous data on Pseudomyrmex termitarius and Ectatomma sp. ants, whose repertoire behavior also

Journal Pre-proof included hyperactivity and paralysis when exposed to R. reconditus defensive secretion [29]. Similar irritant effects were observed in ants after contact with the secretion from the frontal gland of S. serrifer soldiers [59]. Our toxicity bioassays revealed that the defensive secretion of R. reconditus and R. pitan workers may cause difficulty walking, moribundity and even death of the treated opponents. All P. araujoi individuals which were stucked after 1h of experiment, died after 6h, suggesting that the mucin-like proteoforms were important to entangle the enemies and prevent their mobility. Under natural conditions, soil grains from the subterranean nests of R. reconditus and R. pitan would adhere to the defensive glue and this interaction may be

f

important to harm the mobility of an opponent, and even block nest galleries.

p

h

m

b

. Š b

ík

. [6 ]

h

h

b h

pr

y

oo

During the bioassays, it was observed that after bursting, workers of R. reconditus

characterizes an excitement signal in soldiers of Prorhinotermes canalifrons, which is elicited by

e-

alarm pheromone. The same behavior was also observed in reproductives of Hodotermes

Pr

mossambicus [61] and soldiers of Macrotermes michaelseni [62]. Since the defensive secretion of Ruptitermes spp. showed compounds related to alarm communication, it is possible that termite bursting and subsequent behaviors may alarm nestmates, and recruit additional

al

defenders to the combat area. Based on the proteomic findings, the defensive secretion of R.

rn

reconditus and R. pitan contains a complex mixture of adhesive compounds and also toxins,

Jo u

defensins, proteolytic enzymes, and alarm compounds, whose activity could explain previous reports [29] and new behavioral data of the current research. The suicidal behavior is an extreme altruistic mechanism [2, 63]. According to Sands [27] and Mill [64], self-rupture in worker castes is one of the most effective termite defenses, since the one-for-one encounter is an expensive cost for predator. Moreover, termite bursting may isolate individuals by blocking nest galleries. The altruistic behavior seems to be advantageous, since the personal fitness cost may benefit the colony as a whole [65]. Neotropical Apicotermitinae are soldierless [11], and the development of defensive mechanisms by workers, especially those of chemical nature, seems to be relevant considering the high predation rate suffered by termites in tropical regions, mainly during foraging expeditions.

Journal Pre-proof

5. Conclusions In summary, our scientific outcomes demonstrated that defensive secretion of Ruptitermes spp. workers is composed of a complex mixture of proteinaceous components involved in the defensive suicidal behavior. A general interpretation of the defensive mechanism of action in R. reconditus and R. pitan, involving the functional groups identified in the current study, is summarized in Fig. 4. Moreover, here we provide the first view of the toxic constituents in the defensive secretion of these termite workers, which probably allowed them to have some

f

success against predators in the Neotropical region. Another interesting contribution of this

oo

study is the identification of mucin-like proteoforms, which had not been reported so far. The

pr

presence of these proteins indicates that they may be responsible for secretion stiffening after air exposure, contributing to the secretion sticky-gel property. Further investigations on

e-

Apicotermitinae defensive mechanisms, including other Ruptitermes spp., will allow a proper

Pr

comparison among the different biochemical compositions associated with such fascinating

Acknowledgements work

was

supported by

FAPESP

rn

This

al

altruistic behavior.

(Processes

2013/26451-9,

2016/16212-5,

and

Jo u

2017/10373-0), CNPq (Processes 301656/2013-4, 150699/2017-4, and 305539/2014-0), and CAPES (Financial Code 001 and Ordinance No. 206/2018).

Appendix A. Supplementary data Supplementary data related to this article can be found at

6. References [1] J. Deligne, A. Quennedey, M.S. Blum, The enemies and defense mechanisms of termites, in: H.R. Hermann (Eds.), Social insects, Academic Press, New York, 1981, pp. 1-76. [2] J.R. Shorter, O. Rueppell, A review on self-destructive defense behaviors in social insects, Insectes Sociaux 59 (2012) 1-10. https://doi.org/ 10.1007/s 00040-011-0210-x. [3] G.D. Prestwich, Chemical defense and self-defense in termites, in: A. Rahman (Ed.), Natural

Journal Pre-proof Product Chemistry, Springer, Berlin, 1986, pp. 318-329. [4] C. Noirot, J.P. Darlington, Termite nests: architecture, regulation and defence, in: T. Abe, D.E. Bignell, M. Higashi, T. Higashi (Eds.), Termites: evolution, sociality, symbioses, ecology, Springer, Dordrecht, 2000, pp. 121-139. [5] G.D. Prestwich, Defense mechanisms of termites, Annu. Rev. Entomol. 29 (1984) 201-232. https://doi.org/10.1146/annurev.en.29.010184.001221. [6] A.M. Costa-Leonardo, I. Haifig, Labral gland in soldiers of the neotropical termite Cornitermes cumulans (Isoptera: Termitidae: Syntermitinae), Micron 64 (2014) 39-44.

f

https://doi.org/10.1016/j.micron.2014.03.014

oo

[7] B.L. Thorne, Termite-termite interactions: workers as an agonistic caste, Psyche: J.

[8] T. B

ík J. B b

k á Z. D m

á M. M

á D.

š Y. R

m-D

H. V

è

. B č k J. K

.M

m

h

á B. m

h

-

e-

Vy

J. Š b

pr

Entomol. 89 (1982) 133-150.

Pr

component suicidal weapon of Neocapritermes taracua old workers. Molecular Biology and Evolution 33 (2015) 809-819. https://doi.org/10.1093/molbev/ms v273.

al

[9] A.M. Costa-Leonardo, I.B. Silva, V. Janei, F.G. Esteves, J.R.A. Santos -Pinto, M.S. Palma. Worker defensive behavior associated to toxins in the Neotropical termite braziliensis

(Blattaria,

rn

Neocapritermes

Isoptera,

Termitidae,

Termitinae),

Journal

of

Jo u

Chemical Ecology 45 (2019) 755-767. https://doi.org/10.1007/s10886-019-01098-w. [10] M.S. Engel, D.A. Grimaldi, K. Krishna, Termites (Isoptera): their phylogeny, classification, and rise to ecological dominance, Am. Mus. Novit. 3650 (2009) 1-27. https://doi.org/10.1206/651.1. [

] T. B

J. Š b

ík

. .L. D h jö Y. R

, The soldierless Apicotermitinae:

insights into a poorly known and ecologically dominant tropical taxon, Insectes Sociaux 63 (2016) 39-50. https://doi.org/10. 1007/s00040-015-0446-y. [12] S.B. Poiani, A.M. Costa-Leonardo, Dehiscent organs used for defensive behavior of kamikaze termites of the genus Ruptitermes (Termitidae, Apicotermitinae) are not glands, Micron 82 (2016) 63-73. https://doi.org/10.1016/j.micron.2015.12.011. [13] A.A. Mathews, Studies on termites from the Mato Grosso state, Brazil, Academia Brasileira de Ciências, Rio de Janeiro, 1977.

Journal Pre-proof [14] R. Constantino, Cupins do Cerrado, Technical Books, Rio de Janeiro, 2015. [15] A.M. Costa-Leonardo, A new interpretation of the defense glands of neotropical Ruptitermes (Isoptera, Termitidae, Apicotermitinae), Sociobiol. 44 (2004) 391-402. [16] A.N. Acioli, R. Constantino, A taxonomic revision of the neotropical termite genus Ruptitermes (Isoptera, Termitidae, Apicotermitinae), Zootaxa, 4032 (2015) 451-492. https://doi.org/10.11646/zootaxa.4032.5.1. [17] J.R.A. dos Santos-Pinto, A.M.C. Garcia, H.A. Arcuri, F.G. Esteves, H.C. Salles, G. Lubec, M.S. Palma, Silkomics: insight into the silk spinning process of spiders, J. Proteom.

f

Res. 15 (2016) 1179-1193. https://doi.org/10.1021/acs.jproteome.5b01056.

oo

[18] A. Schmidt, I. Forne, A. Imhof, Bioinformatic analysis of proteomics data. BMC Syst. Biol. 8

pr

(2014) S3. https://doi.org/10.1186/1752-0509-8-S2-S 3.

[19] J.S. Chauhan, a. Rao, g.p. Raghava, In silico platform for prediction of N-, O-and C-

e-

glycosites in eukaryotic protein sequences, PloS one 8 (2013) e67008.

Pr

https://doi.org/10.1371/journal.pone.0067008

[20] A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F.T. Heer, T.A.P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, SWISS-MODEL: homology modelling

al

of protein structures and complexes, Nucl. Acids Res. 46 (2018) 296-303.

rn

https://doi.org/10.1093/nar/gky427.

Jo u

[21] M. Pathan, S. Keerthikumar, C.S. Ang, L. Gangoda, C.Y. Quek, N.A. Williamson, D. Mouradov, O.M. Sieber, R.J. Simpson, A. Salim, A. Bacic, A.F. Hill, D.A. Stroud, M.T. Ryan, J.I. Agbinya, J.M. Mariadason, A.W. Burgess, S. Mathivanan, FunRich: An open access standalone functional enrichment and interaction network analysis tool, Proteom. 15 (2015) 2597-601. https://doi.org/10.1002/pmic.201400515. [22] O. Friard, M. Gamba, BORIS: a free, versatile open-source event-logging software for video/audio coding and live observations, Methods Ecol. Evol. 7 (2016) 1325-1330. https://doi.org/10.1111/2041-210X.12584 [23] K.H. Redford, Ants and termites as food, in: H.H. Genoways (Ed.), Current Mammalogy, Springer, Boston, 1987, pp. 349-399. [24] B.L. Thorne, N.L. Breisch, M.L. Muscedere, Evolution of eusociality and the soldier caste in termites: influence of intraspecific competition and accelerated inheritance, Proc. Natl.

Journal Pre-proof Acad. Sci. USA 100 (2003) 12808-12813. https://doi.org/10.1073/pnas.2133530100. [

] J. Š b

ík

.J

š á R. H

h m

m

J. Insect Physiol. 56

(2010) 1012-1021. https://doi.org/10.1016/j.jinsphys.2010.02.012. [26] W.A. Sands, The soldierless termites of Africa (Isoptera: Termitidae), Bull. Br. Mus. Nat. Hist. 18 (1972) 1-244. [27] W.A. Sands, Agonistic behavior of African soldierless Apicotermitinae (Isoptera: Termitidae), Sociobiol. 7 (1982) 61-72. [28] T. Eisner, Chemical defense against predation in arthropods, in: E. Sondheimer, J.B.

f

Simeone (Eds.), Chemical Ecology, Academic Press, New York, 1970, pp. 157-217.

oo

[29] A.E. Mill, Behavioural and toxic effects of termite defensive secretions on ants, Physiol.

pr

Entomol. 8 (1983) 413-418. https://doi.org/10.1111/j.1365-3032.1983.tb00375.x. [30] J.M. Pasteels, J.C. Grégoire, M. Rowell-Rahier, The chemical ecology of defense in

e-

arthropods, Annu. Rev. Entomol. 28 (1983) 263-289.

Pr

[31] H. Xiao, H. Pan, K. Liao, M. Yang, C. Huang, Snake Venom PLA 2, a Promising Target for Broad-Spectrum Antivenom Drug Development, Biomed. Res. International, 6592820 (2017) 1-10. https://doi.org/10.1155/2017/6592820.

al

[32] J.M. Gutiérrez, C.L. Ownby, Skeletal muscle degeneration induced by venom

rn

phospholipases A2: insights into the mechanisms of local and systemic

Jo u

myotoxicity, Toxicon 42 (2003) 915-931. https://doi.org/10.1016/j.toxicon.2003.11.005. [33] B. Lomonte, J. Rangel, J, Snake venom Lys49 myotoxins: from phospholipases A2 to nonenzymatic membrane disruptors, Toxicon, 60 (2012) 520-530. https://doi.org/10.1016/j.toxicon.2012.02.007. [34] M. Borja, E. Neri-Castro, G. Castañeda-Gaytán, J.L. Strickland, C.L. Parkinson, J. Castañeda-Gaytán, R. Ponce-López, B. Lomonte, A. Olvera-Rodríguez, A. Alagón, R. Pérez-Morales, Biological and Proteolytic Variation in the Venom of Crotalus scutulatus scutulatus from Mexico, Tox. 10 (2018) 1-19. http://dx.doi.org/10.3390/toxins10010035 [35] L.D. Santos, K.S. Santos, J.R.A. dos Santos-Pinto, N.B. Dias, B.M.D. Souza, M.F. Santos, J. Perales, G.B. Domont, F.M. Castro, J.E. Kalil, M.S. Palma, M.S., Profiling the prot eome of the venom from the social wasp Polybia paulista: a clue to understand the envenoming mechanism, J. Proteom. Res. 9 (2010) 3867-3877. https://doi.org/10.1021/pr1000829.

Journal Pre-proof [36] J.R.A. dos Santos Pinto, E.G. Fox, D.M. Saidemberg, L.D. Santos, A.R. Silva-Menegasso, E. Costa-Manso, E.A. Machado, O.C. Bueno, M.S. Palma, Proteomic view of the venom from the fire ant Solenopsis invicta Buren, J. Proteom. Res. 11 (2012) 4643-4653. https://doi.org/10.1021/pr300451g. [37] W. Bouzid, C. Klopp, M. Verdenaud, F. Ducancel, A. Vétillard, A., Profiling the venom gland transcriptome of Tetramorium bicarinatum (Hymenoptera: Formicidae): The first transcriptome analysis of an ant species, Toxicon 70 (2013) 70-81. https://doi.org/10.1016/j.toxicon.2013.03.010.

f

[38] S. Yuan, H. Duan, C. Liu, X. Liu, T. Liu, H. Tao, Z. Zhang, The role of thioredoxin and

oo

disulfide isomerase in the expression of the snake venom thrombin-like enzyme calobin in

https://doi.org/10.1016/j.pep.2004.08.004.

pr

Escherichia coli BL21 (DE3), Protein Expr. Purif. 38 (2004) 51-60.

e-

[39] S.M. Serrano, R.C. Maroun, Snake venom serine proteinases: sequence homology vs.

Pr

substrate specificity, a paradox to be solved, Toxicon 45 (2005) 1115-1132. https://doi.org/10.1016/j.toxicon.2005.02.020.

[40] L.H. Gremski, R.B. Silveira, O.M. Chaim, C.M. Probst, V.P. Ferrer, J. Nowatzki, H.C.

al

Weinschutz, H.M. Madeira, V. Gremski, H.B. Nader, A. Senff-Ribeiro, S.S. Veiga, A novel

rn

expression profile of the Loxosceles intermedia spider venomous gland revealed by

Jo u

transcriptome analysis, Mol. Biosyst. 6 (2010) 2403-2416. https://doi.org/10.1039/C004118A. [41] Z.Y. Chen, Y.T. Hu, W.S. Yang, Y.W. He, J. Feng, B. Wang, R.M. Zhao, J.P. Ding, Z.J. Cao, W.X. Li, Y.L. Wu., Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family, J. Biol. Chem. 287 (2012) 1381313821. [42] L.H. Gremski, D. Trevisan-Silva, V.P. Ferrer, F.H. Matsubara, G.O. Meissner, A.C. Wille, L. Vuitika, C. Dias-Lopes, A. Ullah, F.R. de Moraes, C. Chávez-Olórtegui, K.C. Barbaro, M.T. Murakami, R.K. Arni, A. Senff-Ribeiro, O.M. Chaim, S.S. Veiga, Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins, Toxicon 83 (2014) 91-120. https://doi.org/10.1016/j.toxicon.2014.02.023

Journal Pre-proof [43] M.S. Bulmer, R.H. Crozier, Variation in positive selection in termite GNBPs and Relish, Mol. Biol. Evol. 23 (2005) 317-326. https://doi.org/10.1093/molbev/msj037. [44] O. Betz, Adhesive exocrine glands in insects: morphology, ultrastructure, and adhesive secretion, in: J. von Byern, I. Grunwald. Biological adhesive systems: From Nature to Technical and Medical Application, Springer, Viena, 2010, pp. 111-152. [45] R.G. Spiro, R.G, Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds, Glycobiol. 12 (2002) 43-56. https://doi.org/10.1093/glycob/12.4.43R.

f

[46] D. Andrade-Silva, A. Zelanis, E.S. Kitano, I.L. Junqueira-de-Azevedo, M.S. Reis, A.S.

oo

Lopes, S.M. Serrano, Proteomic and glycoproteomic profilings reveal that post -

pr

translational modifications of toxins contribute to venom phenotype in snakes, J. Proteom. Res. 15 (2016) 2658-2675. https://doi.org/10.1021/acs.jproteome.6b00217.

e-

[47] G.M. Happ, Quinone and hydrocarbon production in the defensive glands of Eleodes

Pr

longicollis and Tribolium castaneum (Coleoptera, Tenebrionidae), J. Insect Physiol. 14 (1968) 1821-1837. https://doi.org/10.1016/0022-1910(68)90214-X. [48] S.G. Spanton, G.D. Prestwich, Chemical self-defense by termite workers: prevention of

al

autotoxication in two rhinotermitids, Sci., 214 (1981) 1363-1365.

rn

https://doi.org/10.1126/science.214.4527.1363.

Jo u

[49] M.L. Kelley, R.J. Van Beneden, Identification of an E3 ubiquitin-protein ligase in the softshell clam (Mya arenaria), Mar. Environ. Res. 50 (2000) 289-293. https://doi.org/10.1016/S0141-1136(00)00087-8. [50] F. Marin, G. Luquet, B. Marie, D. Medakovic, D., Molluscan shell proteins: primary structure, origin, and evolution, Curr. Topics Dev. Biol. 80 (2007) 209-276. https://doi.org/10.1016/S0070-2153(07)80006-8. [51] C.L. de Souza, J.R.A. dos Santos-Pinto, F.G. Esteves, A. Perez-Riverol, L.R.G. Fernandes, R.L. Zollner, M.S. Palma, Revisiting Polybia paulista wasp venom using shotgun proteomics - Insights into the N-linked glycosylated venom proteins, J. Proteom. 200 (2019) 60-73. doi: 10.1016/j.jprot.2019.03.012. [52] N. Nadzirin, M. Firdaus-Raih, Proteins of Unknown function in the Protein Data Bank (PDB): an inventory of true uncharacterized proteins and computational tools for their

Journal Pre-proof analysis, International J. Mol. Sci. 13 (2012) 12761-12772. https://doi.org/10.3390/ijms131012761. [53] B.P. Moore, B.P., Studies on the chemical composition and function of the cephalic gland secretion in Australian termites, J. Insect Physiol. 14 (1968) 33-39. https://doi.org/10.1016/0022-1910(68)90131-5. [54] M. Sumarokova, J. Iturri, A. Weber, M. Maares, C. Keil, H. Haase, J.L. Toca-Herrera, Influencing the adhesion properties and wettability of mucin protein films by variation of the environmental pH, Sci. Rep. 8 (2018) 9660. doi: 10.1038/s41598-018-28047-z.

oo

f

[55] O. Betz O, G. Kölsch, The role of adhesion in prey capture and predator defence in arthropods, Arthropod Struct. Dev. 33 (2004), 3-30. https://doi.org/10.1016/j.asd.2003.10.002.

pr

[56] S. Yuan, M. Hollinger, M. E. Lachowicz-Scroggins, S. C. Kerr, E. M. Dunican, B. M. Daniel,

e-

S. Ghosh, S. C. Erzurum, B. Willard, S. L. Hazen, X. Huang, S. D. Carrington, S. Oscarson, J. V. Fahy. Oxidation increases mucin polymer cross-links to stiffen airway mucus gels, Sci.

Pr

Transl. Med. 25 (2015), 276ra27. doi: 10.1126/scitranslmed.3010525. [57] C. Bordereau, A. Robert, V. Van Tuyen, A. Peppuy, Suicidal defensive behaviour by frontal

al

gland dehiscence in Globitermes sulphureus Haviland soldiers (Isoptera), Insectes

ík T. B

R. H

Jo u

[ 8] J. Š b

rn

Sociaux, 44 (1997) 289-297. https://doi.org/ 10. 1007/s 000400050049.

Foltynová, J. Preisler, J.

čk

Z. D m Y. R

Exp

á J. b

y

k á MM

kp

k

š

.

m

k

Sci. 337 (2012) 436. https://doi.org/10.1126/science.1219129. [59] A.M. Costa-Leonardo, K. Kitayama, Frontal gland dehiscence in the brazilian termite Serritermes serrifer (Isoptera, Serritermitidae), Sociobiol. 19 (1991) 333-338. [6 ] J. Š b

ík R H

Y. R

b h

h

m

Prorhinotermes

canalifrons (Isoptera: Rhinotermitidae), J. Insect Behav. 21 (2008) 521-534. https://doi.org/10. 1007/s10905 -008-9147-y. [61] J.C.C. Nel, Aggressive behaviour of the harvester termites Hodotermes mossambicus (Hagen) and Trinervitermes trinervoides (Sjöstedt), Insectes Sociaux, 15 (1968) 145-156. https://doi.org/10. 1007/BF02223463. [62] R. Sieber, R.H. Leuthold, Behavioural elements and their meaning in incipient laboratory

Journal Pre-proof colonies of the fungus-growing termite Macrotermes michaelseni (Isoptera: Macrotermitinae), Insectes Sociaux, 28 (1981) 371-382. https://doi.org/10. 1007/BF02224194. [63] J. Deligne, E. De Coninck, Suicidal defence through a dehiscent frontal weapon in Apilitermes longiceps soldiers (Isoptera: Termitidae), Belg. J. Entomol. 8 (2006) 3-10. [64] A.E. Mill, Exploding termites – an unusual defensive behaviour. Entomol. Mon. Mag. 120 (1984) 179-183.

f

[65] E.O. Wilson, Sociobiology, Harvard Press, Cambridge, 1975.

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Figure captions

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Fig. 1. A and B - Foraging workers of Ruptitermes reconditus and Ruptitermes pitan, respectively. Note the droplet expelled after body rupture (arrows). Scales = 1 mm. C -

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Scanning electron microscopy (SEM) of the rough (orange) and smooth (green) secretory

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vesicles observed in the defensive secretion of R. reconditus. Arrows indicate the thin strip cytoplasm (blue) surrounding the secretion. D - Scanning electron microscopy (SEM) of the non-rounded and homogeneous vesicles (orange) of the defensive secretion of R. pitan. Arrows

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indicate the thin strip cytoplasm (blue) involving the homogeneous secretion. Scales = 20 µm.

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Fig. 2. A - Taxonomic distribution of best-match identified protein sequences against Arthropoda databases. B - Total protein distribution identified in the defensive secretion samples of Ruptitermes spp. according to the functional role. C - Venn diagram showing shared proteins between the defensive secretion of R. reconditus and R. pitan.

Fig. 3. A - Quantitative label-free analysis of the mucin-like proteins identified in the defensive secretion of Ruptitermes reconditus and Ruptitermes pitan. Abundances were based upon precursor/peptide peak intensities. B - Representation of the generic structure of a mucin. C Mucin-1 sequence (Accession number - A0A067RE67), highlighting the presence of N- (yellow residues) and O-linked (blue residues) glycosylated sites that were predicted by GlycoEP Server. D - Three-dimensional molecular model of the mucin-1 from Zootermopsis nevadensis termite. The image was generated using SWISS-MODEL.

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Fig. 4. Schematized representation of the general defensive mechanism of action in workers of Ruptitermes reconditus and Ruptitermes pitan, according to the functional groups present in the secretion. A - R. pitan highlighting a secretion droplet released after body bursting (arrow). B Bursting behavior. C - Sticky components action on the opponent, represented by a soldier of

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Heterotermes tenuis.