Peptide Diversity in the Venom of the Social Wasp Polybia Paulista

Peptides 51 (2014) 122–130

CORE Metadata, citation and similar papers at core.ac.uk

Provided by Elsevier - Publisher Connector Contents lists available at ScienceDirect

Peptides

jo urnal homepage: www.elsevier.com/locate/peptides

Peptide diversity in the venom of the social wasp Polybia paulista

(Hymenoptera): A comparison of the intra- and inter-colony

compositions

Nathalia Baptista Dias, Bibiana Monson de Souza, Paulo Cesar Gomes,

∗

Mario Sergio Palma

UNESP – São Paulo State University, Center of Studies of Social Insects, Institute of Biosciences, Rio Claro, Brazil

r t i c l e i n f o a b s t r a c t

Article history: The venoms of the social wasps evolved to be used as defensive tools to protect the colonies of these

Received 22 August 2013

insects against the attacks of predators. Previous studies estimated the presence of a dozen peptide

Received in revised form 24 October 2013

components in the venoms of each species of these insects, which altogether comprise up to 70% of the

Accepted 25 October 2013

weight of freeze-dried venoms. In the present study, an optimized experimental protocol is reported that

Available online 13 November 2013

utilizes liquid chromatography coupled to electrospray ionization mass spectrometry for the detection

of peptides in the venom of the social wasp Polybia paulista; peptide proﬁles for both intra- and inter-

Keywords:

colonial comparisons were obtained using this protocol. The results of our study revealed a surprisingly

Polybia paulista

Venomics high level of intra- and inter-colonial variability for the same wasp species. We detected 78–108 different

peptides in the venom of different colonies of P. paulista in the molar mass range from 400 to 3000 Da;

Venom toxin

Peptide toxin among those, only 36 and 44 common peptides were observed in the inter- and intra-colony comparisons, LC/MS respectively.

1. Introduction chromatographic separation provides a global picture of the venom

showing both its complexity and an overview of the molecular

The evolution of venoms and their injection apparatus among nature of the venom components [11].

Insecta represents an evolutionary trend that contributed to their The conventional experimental approaches, such as large-scale

adaptation to many different terrestrial environments. As preda- fractionation techniques with off-line fraction collection, simply

tors, the stinging wasps have evolved to use their venoms as a concentrate the most abundant components and do not account for

chemical weapon for both the defense of their colony and for cap- the presence of minor constituents. Research involving proteomics

turing prey [3]. and peptidomics using advanced techniques such as LC–ESI-MS

The venoms of wasps are complex mixtures of biologically active and nano-ESI-MS/MS allows for a rapid and sensitive identiﬁ-

compounds, such as low molecular mass compounds, peptides, cation and characterization of proteins and peptides with high

and proteins [14,18]. Different types of inﬂammatory peptides are efﬁciency. Snake venoms are known to contain approximately

reported in these venoms, including mastoparans [7], protonectin- 100 different peptide components [2], while venoms of scorpi-

like peptides, chemotactic peptides, and wasp kinins [6]. ons, spiders, and marine snails of the genus Conus have been

Recent studies have demonstrated the analytical capabilities shown to contain between 300 and 1000 different components

of various mass spectrometry approaches in the detection and [1,5,12,19].

identiﬁcation of compounds present in very low concentrations Differences in the number and composition of the peptide com-

in animal venoms [6]. A combination of techniques such as ponents in animal venoms have been reported for snails [15,25],

high-performance liquid chromatography (HPLC) and mass spec- spiders [12,31] and scorpions [11,13,24]. The origin of these differ-

trometry contributed to the discovery of many components of ences may be multi-factorial, including differential processing of

animal venoms [10,11,17,21]. Venom proﬁling constitutes the the zymogens, differential gene expression, genetic polymorphism,

basic approach in global venom exploration by mass spectrom- and altered post-translational processing. The chemical diversity

etry. A venom proﬁling data set generated with or without of peptides from the Hymenoptera venoms is currently poorly

understood; in the venom of the social wasp Polybia paulista as

example, only six peptides are structurally and functionally known:

∗ the mastoparans Polybia-MP-I and -II, that are known to induce

Corresponding author. Tel.: +55 19 3526 4163; fax: +55 19 35348523.

E-mail address: [email protected] (M.S. Palma). mast cell degranulation [7–9]; the Polybines-I and -II that are

N.B. Dias et al. / Peptides 51 (2014) 122–130 123

Fig. 1. LC–ESI-MS total ion chromatograms and peptide proﬁles (peptide mass vs. retention time) of P. paulista venom from nests A (108 peptides), B (92 peptides), and C

(98 peptides). The reconstructed masses of each peptide are represented as different symbols according to the sample.

N-terminally acetylated peptides involved with a series of inﬂam- diversity of the venom toxins from the social wasp P. paulista was

matory actions [22]; the Polybia-CP, that is a chemotactic peptide investigated in our lab. Wasp workers were collected from same

for polymorphonucleated leukocytes [7,9,29], and the peptide pre- nest at different seasons (intra-nest study), as well from differ-

senting a disulﬁde bridge, that promotes insulin secretion from ent nests in the same season (inter-nest study), and samples were

␤-cells [20]. Hence, to shed some light on the subject, peptide characterized using an optimized LC–ESI-MS protocol.

124 N.B. Dias et al. / Peptides 51 (2014) 122–130

Fig. 2. Overlapping peptides detected in samples (nests) A–C, as observed by LC–ESI-MS. Different samples are represented by gray dots (nest A), diamonds (nest B), and

triangles (nest C). The peptides in the range from 400 to 3000 Da are shown.

2. Material and methods spectrometer was equipped with an ESI source in a positive ion

mode. The mass spectrometric analysis conditions were as follows:

2.1. Venom extraction and sample preparation detector voltage was adjusted to 1.70 kV; the curved desolvation

◦

line (CDL) temperature was set at 200 C, while the temperature

◦

For inter-nest comparisons, wasps were collected from three of the heat block temperature was 200 C; the nebulizing gas (N2)

different nests at the spring time. The nests were geo-referenced ﬂow was adjusted to 1.5 L/min, and the ﬂow of drying gas (N2) was

and found to be located in Rio Claro-SP/Brazil as follows: sample A, set at 100 L/h. Accurate mass determination was corrected by a

◦ ◦ ◦ ◦

22 23 43.0 S, 047 32 38.3 W; sample B, 22 24 48.0 S, 047 32 41.4 calibration using the sodium triﬂuoroacetate clusters as reference.

◦ ◦

W; and sample C, 22 23 50.0 S, 047 34 41.4 W. Samples were also Mass spectra were acquired over the range of m/z 50–3000 in the

collected during the summer season and used in a comparison of positive mode using LCMS solution software v.3.6 (Shimadzu Co.).

the inter-nest composition of the venom peptides. Samples for the Using the command “Mass Table”, the mass spectra were acquired

intra-nest comparisons were collected from a single nest, sample D and transferred to an Excel spreadsheet (Microsoft Ofﬁce 2010)

◦ ◦

(22 23 43.0 S, 047 32 32.8 W), at three different seasons: spring and then used to manually deconvolute multiply charged peaks

(sample D1), summer (sample D2), and autumn (sample D3). to obtain the masses for the peptides eluting between m/z 400

The venom reservoirs of 30 social wasp workers of P. paulista and 3000. All the peaks of m/z values presenting signal to noise

(Hymenoptera, Vespidae) were manually extracted from each nest relationship (S/N) ≥ 10 were considered in the analysis. A mass list

using microsurgical forceps. Once surgically removed, the venom was created with the software LC solution; all similar masses were

reservoirs were depressed using a glass rod with a rounded tip in manually checked to conﬁrm the identiﬁcation and/or to distin-

the presence of a solution of 50% (v/v) acetonitrile (Mallinckrodt) guish the isobaric peptide components. In each sample injection,

in bi-distilled water, containing 0.1% (v/v) triﬂuoroacetic acid and the occurrence of different peptide masses was considered within

a cocktail of proteinase inhibitors (SIGMAFAST Protease Inhibitor the time window of 1 min; in this study, the components with

Cocktail Tablets). Next, the collected material was centrifuged at reconstructed mass matches smaller than 0.5 Da and retention

◦

10,000 × g for 10 min at 4 C in a micro-centrifuge MSE (SANYO); times smaller than 1 min were considered as identical peptides.

this procedure was repeated two more times. The supernatant was The presence of carryover peptides was checked by the analysis

ﬁltered through ethyl cellulose membrane ﬁlter disks (0.45 ␮m of a blank run between each chromatographic run; each venom

pores) (Millipore), lyophilized (HETO, model MLW. LGA 05), and sample was run in triplicate. Graphs were constructed using

◦

kept at −20 C until use. Microsoft Excel 2010 and Corel software.

2.2. LC/MS and MS analysis 3. Results

Liquid chromatography and electrospray ionization mass The colonies of social wasps are basically constituted of some

spectrometry experiments were performed on a LC-IT-ToF Mass queens, and thousands of workers (non-reproductive females

Spectrometer (Shimadzu). The chromatographic system consisted that gather food and protect the colony). Amongst the social

of a LC-20AD binary pump, DGU-14A degasser, SIL-20AHT autosam- Hymenoptera males do not produce venom, therefore only work-

pler, and a CTO-20A column oven. The system was equipped with a ers’ venoms was investigated.

XBrigde BEH300 C18 column 3.5 ␮m 2.1 mm × 100 mm (waters), Social wasps are extremely aggressive insects, that do not

◦

pore size 120 A,˚ which was run at 40 C. An optimized sample permit any manipulation, i.e., are absolutely wild; therefore, the

amount used in all analyses was adjusted to equal 40 ␮g of the colonies cannot be handled to synchronize the emergence, and do

venom extract. The mobile phase, delivered at 0.2 mL/min, con- not permit the selective production of queens (like is done for hon-

sisted of bi-distilled water containing 0.05% (v/v) triﬂuoroacetic eybees). It is known that the workers have a life span of about 35–40

acid (solvent A), and 95% (v/v) acetonitrile in water containing days, and due to the impossibility to handle the colonies, becomes

0.05% (v/v) trifluoroacetic acid (solvent B). The 90 min elution very difficult the identification of the just-born individual, followed

was performed using a gradient from 5% to 95% of solvent B; the by their labeling with an appropriated ink to permit follow then

elution was monitored by the light absorption at 214 nm. The mass until and speciﬁc age, when they could be collected. The way to

N.B. Dias et al. / Peptides 51 (2014) 122–130 125

Fig. 3. Histograms of the peptide abundance by mass (100 Da bins). The overlaid curves show cumulative total peptide numbers found in the venom of the social wasp, P.

paulista, in nests A–C.

126 N.B. Dias et al. / Peptides 51 (2014) 122–130

Fig. 4. The LC–ESI-MS total ion chromatograms and peptide proﬁles (peptide mass vs retention time) of P. paulista venom from a nest at three different collection times: (D1)

the spring (94 peptides), (D2) the summer (95 peptides), and (D3) the autumn (78 peptides). The reconstructed masses of each peptide are represented as different symbols

according to the sample.

know grossly workers’ age is the daily collection since their emer- the results. In the second part of the study, a comparison of the

gence and making the microscopic analysis of their wings wearing venom peptide proﬁles between the venom samples collected from

as function of time, which is very laborious, time consuming and the wasps of the same nest at different seasons of the year was

the results is not very reliable. Thus, it was not studied the effect of performed.

insects’ age in peptide diversity of their venom. The venom sample components were distributed over the molar

The initial analysis of peptide diversity between three differ- mass range of 400–3000 Da and together accounted for up to 70%

ent nests of P. paulista (inter-colony comparison) showed that of the dry weight of the venom; our analysis allowed us to obtain

even though all the nests were located in the same ecological venom proﬁles for different samples. The mass spectrometric anal-

region, there were sufﬁcient environmental differences to explain ysis were performed using ESI as ionization source both in the

N.B. Dias et al. / Peptides 51 (2014) 122–130 127

Fig. 5. Overlapping peptides detected in samples D1–D3, as observed by LC–ESI-MS. Different samples are represented by triangles (nest D1), squares (nest D2), and black

dots (nest D3). The peptides in the range between 400 and 3000 Da are shown.

positive and negative acquisition mode; however, considering that 45, 19, and 32 unique peptides were found in the venom samples

the peptide fraction of this venom is constituted exclusively of poly- from nests A, B and C, respectively. At the same time, nests A and B

cationic components, only the data obtained in the positive mode shared 17 common peptides; nests A and C shared 10; and nests B

were presented in this publication, which resulted in much more and C shared 20 common peptides.

intense peaks than the data got in the negative mode. The total ion chromatograms for the intra-nest analysis (sam-

The total ion chromatogram of the crude venoms extracted from ples D1–D3) are shown in Fig. 4; the overlap of the masses and the

three different nests of P. paulista (Fig. 1) revealed a surprisingly retention times in these analyses is shown in Fig. 5, where the pep-

high level of inter- and intra-nest variation and allowed the detec- tides from sample D1 are represented by triangles, sample D2 by

tion of many peptides in the venom. In the mass spectrometric squares, and sample D3 by dots. This analysis revealed that 94 dif-

analysis for inter-nest comparison at the level of the monoiso- ferent peptides were detected in sample D1, 95 in sample D2, and

topic mass (±0.1 Da) and with a retention window of 1 min, were 78 in sample D3 (Fig. 4A–C).

detected a total of 179 different peptides, with 108 peptides in The distribution of the mass differences is presented in Fig. 6

nest A, 92 in nest B, and 98 in nest C. Fig. 1A–C show the recon- (A–C, corresponding to samples D1–D3). The peptides detection

structed masses of each peptide (represented by different symbols) are broken down to 100 Da intervals; the cumulative number of

for the samples collected in the spring. Despite the similarity in the peptide masses over time (binned in 1 min intervals) is displayed

UV chromatographic proﬁles (Fig. 1A–C), it was possible to detect as a black line. The analysis of the samples (D1–D3) also showed

numerous peptide mass differences. The overlap of peptide masses a multimodal distribution of the obtained masses, similarly to that

according to the elution time for the venom samples from the three already described above for the inter-nest analysis.

different nests is shown in Fig. 2. As found in the inter-nest analysis, there appear to be many

The molecular mass distribution of the peptides from different novel peptides identiﬁed in the peptide clusters derived from sam-

nests of P. paulista is shown in Fig. 3 (nests A–C, respectively). It ple D, given the relatively small number of peptides with known

was already known that the peptides comprised in mass range sequences in the P. paulista venom. Similarly to the strategy used

of 1200 and 1800 Da contain mixtures of known peptides as the described above for the inter-nest analysis, the detailed detection

mastoparans Polybia-MP- I [7,8,29] and Polybia MP-II [9,23], the of all peptides presenting S/N ≥ 10 in each time window of 1 min

Polybines-I and–II [22], the chemotactic peptide for polymor- in the total ion chromatograms (TICs) of the intra-nest analysis are

phonucleated leukocytes Polybia-CP [7,29], as well as unknown shown in Table S2 (Supplementary data). A Venn diagram of the

peptides, while the cluster of 2000–2900 Da corresponds to the peptides identiﬁed in the intra-nest comparisons (Fig. 7) shows the

disulﬁde bond-containing peptides, like the silverin-like compo- total number of 146 different peptides, with only 44 common com-

nents [20,29], amongst other unknown ones. ponents across the three samples (D1–D3), which represents 30%

A search across the chromatographic proﬁles in the last clus- of the total. Samples D1 and D2 share 18 common peptides; sam-

ter identiﬁed a number of peptides showing an increase of 2 Da ples D2 and D3 share 8 common peptides; and samples D1 and D3

in their molecular mass after the reaction with DTT, which corre- share 7 common peptides. At the same time, 69 unique peptides

sponds to the reduction of one disulﬁde bridge (results not shown). were detected in this analysis, with 25, 25, and 19 unique peptides

Considering the relatively small number of peptides with known found in samples D1, D2, and D3, respectively.

sequences in the P. paulista venom, the results presented above A few peaks in the total ion chromatograms both of inter- and

demonstrate that there are many novel peptides identiﬁed in these intra-nest analysis (Figs. 1A–C and 4A–C) do not presented any

clusters. The detailed detection of all peptides presenting S/N ≥ 10 molecular masses associated to them because they correspond to

in each time window of 1 min in the total ion chromatograms (TICs) non-peptide compounds presenting masses lower than 400 Da.

for the inter-nest analysis are shown in Table S1 (Supplementary

data), and these data were used to make the inter-nest comparison 4. Discussion

as a Venn diagram (Fig. 7) showing a total of 179 peptides identi-

ﬁed in the P. paulista venoms, with 36 peptides common among the Despite the fact that peptides constitute approximately 70% of

samples from the three nests (A, B and C), which corresponds to 20% the venom content of the social wasp P. paulista, only a few venom

of the total. It could also be observed from this Venn diagram that peptides have been sequenced and biochemically characterized

128 N.B. Dias et al. / Peptides 51 (2014) 122–130

Fig. 6. Histograms of the peptide abundance by mass (100 Da bins). The overlaid curves show cumulative total peptide numbers found in the venom of the social wasp P.

paulista in samples D1–D3.

N.B. Dias et al. / Peptides 51 (2014) 122–130 129

each colony makes the defensive arsenal represented by these tox-

ins extremely important. The polymorphism helps to create novel

toxins with different potencies and selectivity, thus widening the

spectrum of predators against which these toxins may be effective.

The proteomic composition of the venom of P. paulista was char-

acterized using a combination of classical biochemical approaches,

2D-SDS-PAGE, and mass spectrometry [27,28]. Despite the power

of this method, only individual characterizations of a few pep-

tides from P. paulista venom have been achieved previously. To our

knowledge, the present study is the ﬁrst to reveal the complexity

of the peptide composition of the venom of a social wasp.

Fig. 7. Venn diagrams show the number of shared and distinct peptides (0.5 Da,

1 min) for the samples from different nests, labeled as A, B and C; the samples from

5. Conclusion

the same nest are labeled as D1, D2, and D3.

The present study showed that the chemical diversity of the

to date; they include the mastoparans Polybia-MP-I and MP-II, venom of the social wasp, P. paulista, is very rich, and its potential

Polybine-I and -II, Polybia-CP and the silverin-like peptide contain- remains to be explored. With respect to P. paulista, the six peptides

ing a disulﬁde bridge [7–9,22,23,29]. Based on the analysis of the known prior to this study represent less than 3% of the total num-

molecular masses and retention times of the compounds present in ber of peptides detected in our study. Considering the occurrence

different samples in this study, we revealed the richness of the pep- of the ﬁve known peptides in P. paulista by another perspective the

tide composition of the venom of P. paulista. Taking into account mastoparans individually represent from 0.7% to 1.2% of the dry

that the venom was extracted in the presence of a cocktail of pro- mass of crude venom, while the polybines and chemotactic pep-

teinase inhibitors, the richness of peptides observed in the present tide individually represent about 0.5% and the peptide presenting

study probably is not related to artifacts of sample manipulation disulﬁde bond represent about 0.3% of the dry crude venom. This

in the presence of endogenous proteases of wasp venom. means that the use of highly sensitive analytical techniques, such as

The results reported above clearly showed higher (albeit small) the combination of high-performance LC protocols with mass spec-

peptide variability between the venoms from three different nests trometry, may pave the way for detecting and sequencing many

compared with the intra-nest variability. In total, 179 different novel peptides from Hymenoptera venoms.

peptides were detected in the inter-nest comparison vs. 146 pep-

tides in the intra-nest analysis. Only 20% and 30% of all peptides Acknowledgments

were commonly observed in the inter-nest and intra-nest analyses,

respectively. In either case, the variability of peptide composition This research is supported by grants from BIOprospecTA-FAPESP

was high in both comparisons, even when the same colony of wasps (Proc. 2011/51684-1 and 09/11155-0), CNPq and Instituto Nacional

was monitored seasonally (the intra-nest comparison). A careful de Ciência e Tecnologia em Imunologia (INCT-iii/CNPq-MCT). MSP

observation of the data presented in Tables S1 and S2 (supple- is a researcher for the Brazilian Council for Scientiﬁc and Techno-

mentary data) reveals that in general the magnitude of the relative logical Development (CNPq). We thank Antonio Sergio Pascon and

intensity of the same peptide component is maintained for the most Andrigo Monroe Pereira for wasp collection.

components in all the samples; however, sometimes the same com-

ponent was detected with relative intensity differences higher than Appendix A. Supplementary data

10 times-fold from one sample to another, reﬂecting very different

levels of expression from one colony to another, or even between Supplementary data associated with this article can be found,

different samples within the same colony, at different time seasons in the online version, at http://dx.doi.org/10.1016/j.peptides.

of year. 2013.10.029.

To understand the reasons behind these observations, it is

important to consider the reproductive behavior of the eusocial References

vespids such as P. paulista. The colonies of these species possess

several queens, who mate individually with different numbers of [1] Biass D, Dutertre S, Gerbault A, Menou JL, Offord R, Favreau P, et al. Comparative

proteomic study of the venom of the piscivorous cone snail Conus consors. J

males [16,30]. As a result, the sperm from different males ends

Proteom 2009;72:210–8.

up mixed within the spermatheca in unknown proportions and is

[2] Calvete JJ, Juarez P, Sanz L. Snake venomics: strategy and applications. J Mass

used continuously over the life span of each queen. In this case, Spectrom 2007;42:1405–14.

[3] Chen W, Yang X, Yang X, Zhai L, Lu Z, Liu J, et al. Antimicrobial peptides from

some paternal genotypes may be responsible for the most fertiliza-

the venoms of Vespa bicolor Fabricius. Peptides 2008;29:1887–92.

tion during some periods, while other genotypes are used mostly

[4] Cowan DP. The solitary and pre-social Vespidae. In: Ross KG, Matthews RW,

at another time [4,26]. Let us consider the following factors: (i) editors. The social biology of wasps Ithaca: Comstock Publishing Associates;

1991. p. 33–73.

the social wasp P. paulista lives in polygynic colonies, i.e., it fre-

[5] Davis J, Jones A, Lewis RJ. Remarkable inter- and intra-species complexity of

quently recruits young queens; (ii) the life span of the queens from

conotoxins revealed by LC/MS. Peptides 2009;30:1222–7.

the social neotropical Polybiinae may last up to one year; and (iii) [6] De Graaf DC, Alerts M, Danneels E, Devreese B. Bee, wasp and ant venomics

pave the way for a component resolved diagnosis of sting allergy. J Proteom

the queens are continuously egg laying [26]. Thus, it is possible to

2009;72:145–54.

assume that there always will be several fertilized queens laying

[7] De Souza BM, Mendes MA, Santos LD, Marques MR, César LMM, Almeida

eggs, introducing new genotypes into the colony. It appears that a RNA, et al. Structural and functional characterization of two novel peptide

relatively large genetic variability introduced by this reproductive toxins isolated from the venom of the social wasp Polybia paulista. Peptides

2005;26:2157–64.

behavior is beneﬁcial to the wasp society and seems to be responsi-

[8] De Souza BM, Palma MS. Peptides from hymenoptera venoms: biochemistry,

ble for the signiﬁcant peptide polymorphism observed in both the

pharmacology and potential applications in health and biotechnology. In: De

inter- and intra-nest comparisons. Considering the large number of Lima ME, Pimenta AMC, Martin-Euclaire MF, Zingali RB, editors. Animal toxins:

the state of art – perspectives on health and biotechnology Belo Horizonte:

vertebrate predators of social wasps and that the venom of social

Editora UFMG; 2009. p. 273–97.

insects is used only for defensive purposes [20], the existence of a

[9] De Souza BM, Silva AVR, Resende VMF, Arcuri HA, Dos Santos Cabrera

large library of natural peptides in the venom from the workers of MP, Ruggiero Neto J. Characterization of two novel polyfunctional

130 N.B. Dias et al. / Peptides 51 (2014) 122–130

mastoparan peptides from the venom of the social wasp Polybia paulista. Pep- [21] Pimenta AM, Rates B, Bloch Jr C, Gomes PC, Santoro MM, De Lima ME,

tides 2009;30:1387–95. et al. Electrospray ionization quadrupole time-of-ﬂight and matrix-assisted

[10] Escoubas P, Celerier ML, Nakajima T. High-performance liquid chromatography laser desorption/ionization tandem time-of-ﬂight mass spectrometric anal-

matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrome- yses to solve micro-heterogeneity in post-translationally modiﬁed peptides

try peptide ﬁngerprinting of tarantula venoms in the genus Brachypelma: from Phoneutria nigriventer (Aranea, Ctenidae) venom. Rapid Commun Mass

chemotaxonomic and biochemical applications. Rapid Commun Mass Spec- Spectrom 2005;19:31–7.

trom 1997;11:1891–9. [22] Ribeiro SP, Mendes MA, Santos LD, Souza BM, Marques MR, Azevedo Jr.

[11] Escoubas P, Quinton L, Nicholson GM. Venomics: unravelling the complex- WF. Structural and functional characterization of N-terminally blocked pep-

ity of animal venoms with mass spectrometry. J Mass Spectrom 2008;43: tides isolated from the venom of the social wasp Polybia paulista. Peptides

279–95. 2004;25:2069–78.

[12] Escoubas P, Sollod BL, King GF. Venom landscapes: mining the complexity of [23] Rocha T, De Souza BM, Palma MS, Cruz-Höﬂing MA. Myotoxic effects of

spider venoms via a combined cDNA and mass spectrometric approach. Toxicon mastoparan from Polybia paulista (Hymenoptera, Epiponini) wasp venom in

2006;47:650–63. mice skeletal muscle. Toxicon 2007;50:589–99.

[13] Favreau P, Menin L, Michalet S, Perret F, Cheneval O, Stocklin M, et al. Mass [24] Rodriguéz De La Vega RC, Schwartz EF, Possani LD. Mining on scorpion venom

spectrometry strategies for venom mapping and peptide sequencing from biodiversity. Toxicon 2010;56:1155–61.

crude venoms: case applications with single arthropod specimen. Toxicon [25] Romeo C, Di Francesco L, Oliverio M, Palazzo P, Massilia GR, Ascenzi P, et al.

2006;47:676–87. Conus ventricosus venom peptides proﬁling by HPLC-MS: a new insight in the

[14] Hoffman DR. Hymenoptera venom allergens. Clin Rev Allergy Immunol intraspeciﬁc variation. J Sep Sci 2008;31:488–98.

2006;30:109–28. [26] Ross KG. Kin selection and the problem of sperm utilization in social insects.

[15] Jakubowski JA, Kelley WP, Sweedler JV, Gilly WF, Schulz JR. Intraspe- Nature 1986;323:798–800.

ciﬁc variation of venom injected by ﬁsh-hunting Conus snails. J Exp Biol [27] Santos LD, Menegasso ARS, Pinto JRSP, Santos KS, Castro FM, Kalil JE, et al.

2005;208:2873–83. Proteomic characterization of the multiple forms of the PLAs from the venom

[16] Kenneth G, Ross KG, Carpenter JM. Population genetic structure, relatedness, of the social wasp Polybia paulista. Proteomics 2010;11:1403–12.

and breeding systems. In: Ross KG, Matthews RW, editors. The social biology [28] Santos LD, Santos KS, Pinto JR, Dias NB, De Souza BM, Dos Santos M, et al.

of wasps Ithaca: Comstock Publishing Associates; 1991. p. 451–79. Palma MS proﬁling the proteome of the venom from the social wasp Polybia

[17] Krishnamurthy T, Prabhakaran M, Long SR. Mass spectrometric investigations paulista: a clue to understand the envenoming mechanism. J Proteome Res

on Conus peptides. Toxicon 1996;34:1345–59. 2011;9:3867–77.

[18] Nakajima T. Pharmacological biochemistry of vespid venoms. In: Piek T, editor. [29] Souza BM, Marques MR, Tomazela DM, Eberlin MN, Mendes MA, Palma MS.

Venoms of the Hymenoptera: biochemical, pharmacological and behavioural Mass spectrometric characterization of two novel inﬂammatory peptides from

aspects London: Academic Press Inc. Ltd.; 1986. p. 309–27. the venom of the social wasp Polybia paulista. Rapid Commun Mass Spectrom

[19] Newton KA, Clench MR, Deshmukh R, Jeyaseelan K, Strong PN. Mass ﬁngerprint- 2004;18:1095–102.

ing of toxic fractions from the venom of the Indian red scorpion, Mesobuthus [30] Spradbery JP. Evolution of queen number and queen control. In: Ross KG,

tamulus: biotope-speciﬁc variation in the expression of venom peptides. Rapid Matthews RW, editors. The social biology of wasps Ithaca: Comstock Pub-

Commun Mass Spectrom 2007;21:3467–76. lishing Associates; 1991. p. 337–88.

[20] Palma MS. Hymenoptera venom peptides. In: Kastin A, editor. Handbook of [31] Wilson D, Alewood PF. Taxonomy of Australian Funnel-web spiders using rp-

biologically active peptides San Diego: Academic Press; 2013. p. 416–22. HPLC/ESI–MS proﬁling techniques. Toxicon 2006;47:614–27.