Abstract

Variability in snake venom composition is well-documented and crucial for understanding snake ecology and predicting snakebites. In this study, we characterize the venom composition and biological activities of newborn female and male Bothrops moojeni and their mother. Our results reveal significant differences between the venom of newborn females and males, demonstrating a broad and diverse range of proteins. The venoms of newborn females showed higher serine protease effects, increased hemorrhagic activity, and greater lethality compared to the venom of newborn males. However, no differences were observed in phospholipase A₂ and coagulant activity. The differences in protein composition and toxic activities between maternal and neonatal venom, as well as between the venoms of newborn females and males, contribute to understanding the diverse outcomes of snakebites. These results underscore the importance of considering sex and ontogeny in understanding venom composition in snakes.

Keywords:
snakes; viperidae; Bothrops moojeni; newborn; venom

Resumo

A variabilidade na composição dos venenos de serpentes é bem documentada e crucial para a compreensão da ecologia das serpentes ou do prognóstico dos envenenamentos ofídicos. Nesse estudo, caracterizamos a composição e a atividade biológica do veneno de fêmeas e machos recém-nascidos e sua mãe. Nossos resultados revelaram diferenças significativas entre o veneno de fêmeas e machos recém-nascidos, demonstrando uma ampla e diversificada gama de proteínas. Os venenos de fêmeas recém-nascidas apresentaram mais efeitos de serina protease, aumento da atividade hemorrágica e maior letalidade em comparação ao veneno dos machos recém-nascidos. No entanto, nenhuma diferença foi observada na fosfolipase A₂ e na atividade coagulante. As diferenças na composição proteica e nas atividades tóxicas entre o veneno materno e neonatal, bem como entre os venenos de fêmeas e machos recém-nascidos contribuem para a compreensão dos diversos resultados dos acidentes ofídicos. Esses resultados ressaltam a importância de considerar o sexo e a ontogenia na compreensão da composição do veneno desses animais.

Palavras-chave:
serpentes; viperidae; Bothrops moojeni; récem-nascidos; veneno

1. Introduction

Over the past century, snakebites in Brazil have shown a consistent epidemiological pattern, with snakes of the genus Bothrops accounting for over 90% of reported cases and predominantly affecting the lower limbs of rural males (Bochner and Struchiner, 2003BOCHNER, R. and STRUCHINER, C.J., 2003. Epidemiologia dos acidentes ofídicos nos últimos 100 anos no Brasil: uma revisão. Cadernos de Saude Publica, vol. 19, no. 1, pp. 7-16. http://dx.doi.org/10.1590/S0102-311X2003000100002. PMid:12700779.
http://dx.doi.org/10.1590/S0102-311X2003... ; Brasil, 2023BRASIL. Ministério da Saúde, 2023. Sistema de Informação de Agravos de Notificação (SINAN). Brasília: Ministério da Saúde.). The highest incidence of snakebites is concentrated in central-western and northern Brazil, with the state of Tocantins ranking third in reported cases (Queirós et al., 2020QUEIRÓS, D.C., NUNES, A.I.S., SANTOS, G.C.A. and SEIBERT, C.S., 2020. Perfil epidemiológico dos acidentados e fatores ambientais que favorecem acidentes ofídicos botrópicos, no Estado do Tocantins, Brasil. Revista Desafios, vol. 7, no. 4, pp. 63-71. http://dx.doi.org/10.20873/uftv7-8851.
http://dx.doi.org/10.20873/uftv7-8851... ; Brasil, 2023BRASIL. Ministério da Saúde, 2023. Sistema de Informação de Agravos de Notificação (SINAN). Brasília: Ministério da Saúde.).

Bothrops species, commonly known as jararacas, are morphologically and ecologically diverse and are widely distributed in Brazil (Bernarde, 2014BERNARDE, P.S., 2014. Serpentes peçonhentas e acidentes ofídicos no Brasil. São Paulo: Anolis Books.; Guedes et al., 2023GUEDES, T.B., ENTIAUSPE-NETO, O.M. and COSTA, H.C., 2023. Lista de répteis do Brasil: atualização de 2022. Herpetologia Brasileira, vol. 1, pp. 56-161. http://dx.doi.org/10.5281/zenodo.7829013.
http://dx.doi.org/10.5281/zenodo.7829013... ; Duque et al., 2023DUQUE, B.R., BRUNO, S.F., FERREIRA, V., GUEDES, T.B., MACHADO, C. and HAMDAN, B., 2023. Venomous snakes of medical importance in the Brazilian state of Rio de Janeiro: habitat and taxonomy against ophidis. Revista Brasileira de Biologia, vol. 83, e272811. http://dx.doi.org/10.1590/1519-6984.272811. PMid:37909585.
http://dx.doi.org/10.1590/1519-6984.2728... ). Bothrops moojeni is a medically important species found in the Caatinga, Cerrado and Pantanal with records in the states of Tocantins, Mato Grosso, Mato Grosso do Sul, Goiás, Maranhão, Distrito Federal, Piauí, Bahia, Minas Gerais, São Paulo, and Paraná (Guedes et al., 2023GUEDES, T.B., ENTIAUSPE-NETO, O.M. and COSTA, H.C., 2023. Lista de répteis do Brasil: atualização de 2022. Herpetologia Brasileira, vol. 1, pp. 56-161. http://dx.doi.org/10.5281/zenodo.7829013.
http://dx.doi.org/10.5281/zenodo.7829013... ). Although four species of Bothrops occur in Tocantins (Silva et al., 2019SILVA, R.C.C., FREITAS, M.A., SANT’ANNA, S.S. and SEIBERT, C.S., 2019. Serpentes no Tocantins: guia ilustrado. São Paulo: Ekos Editora, 170 p.), B. moojeni is the main responsible for snakebites in the state.

Bothrops envenomation triggers local and systemic effects that can lead to serious sequelae, including death. Many studies have demonstrated the severity of the effects induced by B. moojeni venom, which include hemostatic disorders, local bleeding, and severe local manifestations, such as edema, ecchymosis, and blisters, sometimes evolving to local necrosis and sequelae in the affected area (Oliveira et al., 2013OLIVEIRA, L.M.F., ULLAH, A., MASOOD, R., ZELANIS, A., SPENCER, P.J., SERRANO, S.M.T. and ARNI, R.K., 2013. Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms. Toxicon, vol. 76, pp. 282-290. http://dx.doi.org/10.1016/j.toxicon.2013.10.016. PMid:24140922.
http://dx.doi.org/10.1016/j.toxicon.2013... ; Mamede et al., 2016MAMEDE, C.C.N., SOUSA, B.B., PEREIRA, D.F.C., MATIAS, M.S., QUEIROZ, M.R., MORAIS, N.C.G., VIEIRA, S.A.B., STANZIOLA, L. and OLIVEIRA, F., 2016. Comparative analysis of local effects caused by Bothrops alternatus and Bothrops moojeni snake venoms: enzymatic contributions and inflammatory modulations. Toxicon, vol. 117, pp. 37-45. http://dx.doi.org/10.1016/j.toxicon.2016.03.006. PMid:26975252.
http://dx.doi.org/10.1016/j.toxicon.2016... ).

The symptoms and severity of snakebites are modulated by the type and quantity of toxins in the venom. Due to the consequences of envenomations and the epidemiological importance of snakebites, numerous studies have sought to characterize the proteomic composition of the venom of various snake species (Sunagar et al., 2014SUNAGAR, K., UNDHEIM, E.A.B., SCHEIB, H., GREN, E.C.K., COCHRAN, C., PERSON, C.E. and FRY, B.G., 2014. Intraspecific venom variation in the medically significant Southern Pacific Rattlesnake (Crotalus oreganus helleri): biodiscovery, clinical and evolutionary implications. Journal of Proteomics, vol. 99, pp. 68-83. http://dx.doi.org/10.1016/j.jprot.2014.01.013. PMid:24463169.
http://dx.doi.org/10.1016/j.jprot.2014.0... ; Sunagar et al., 2016SUNAGAR, K., MORGENSTERN, D., REITZEL, A.M. and MORAN, Y., 2016. Ecological venomics: how genomics, transcriptomics and proteomics can shed new light on the ecology and evolution of venom. Journal of Proteomics, vol. 135, pp. 62-72. http://dx.doi.org/10.1016/j.jprot.2015.09.015. PMid:26385003.
http://dx.doi.org/10.1016/j.jprot.2015.0... ; Amazonas et al., 2018AMAZONAS, D.R., PORTES-JUNIOR, J.A., NISHIYAMA-JR, M.Y., NICOLAU, C.A., CHALKIDIS, H.M., MOURÃO, R.H.V., GRAZZIOTIN, F.G., ROKYTA, D.R., AMORIM, F.G., COSTA, T.R., BAIWIR, D., PAUW, E., QUINTON, L. and SAMPAIO, S.V., 2018. Proteoetpeptidomic, functional and immunoreactivity characterization of Bothrops moojeni snake venom: influence of snake gender on venom composition. Toxins, vol. 10, no. 5, pp. 177. http://dx.doi.org/10.3390/toxins10050177. PMid:29701671.
http://dx.doi.org/10.3390/toxins10050177... ). The proteome of B. moojeni venom is mainly composed of metalloproteinases, serine proteases, phospholipases A₂, L-amino acid oxidases, and cysteine-rich proteins (Amorim et al., 2017AMORIM, F.G., MORANDI-FILHO, R., FUJIMURA, T., UEIRA-VIEIRA, C. and SAMPAIO, S.V., 2017. New findings from the first transcriptome of the Bothrops moojeni snake venom gland. Toxicon, vol. 140, pp. 105-111. http://dx.doi.org/10.1016/j.toxicon.2017.10.025. PMid:29107670.
http://dx.doi.org/10.1016/j.toxicon.2017... ; Amorim et al., 2018AMORIM, F.G., COSTA, T.G., BAIWIR, D., DE PAUW, E., QUINTON, L. and SAMPAIO, S.V., 2018. Proteoetpeptidomic, functional and immunoreactivity characterization of Bothrops moojeni snake venom: influence of snake gender on venom composition. Toxins, vol. 10, no. 5, pp. 177. http://doi.org/10.3390/toxins10050177. PMid:29701671.
http://doi.org/10.3390/toxins10050177... ). Both ontogeny and sex influence the composition of Bothrops venom. Comparisons between young and adult B. jararaca, B. jararacussu, B. moojeni, and other congeners have revealed variations in biological activities and venom composition (Antunes et al., 2010ANTUNES, T.C., YAMASHITA, K.M., BARBARO, K.C., SAIKI, M. and SANTORO, M.L., 2010. Comparative analysis of newborn and adult Bothrops jararaca snake venoms. Toxicon, vol. 56, no. 8, pp. 1443-1458. http://dx.doi.org/10.1016/j.toxicon.2010.08.011. PMid:20816886.
http://dx.doi.org/10.1016/j.toxicon.2010... ; Freitas-de-Sousa et al., 2020FREITAS-DE-SOUSA, L.A., NACHTIGALL, P.G., PORTES-JUNIOR, J.A., HOLDING, M.L., NYSTROM, G.S., ELLSWORTH, S.A., GUIMARÃES, N.C., TIOYAMA, E., ORTIZ, F., SILVA, B.R., KUNZ, T.S., JUNQUEIRA-DE-AZEVEDO, I.L.M., GRAZZIOTIN, F.G., ROKYTA, D.R. and MOURA-DA-SILVA, A.M., 2020. Size matters: an evaluation of the molecular basis of ontogenetic modifications in the composition of Bothrops jararacussu snake ve. Toxins, vol. 12, no. 12, pp. 791. http://dx.doi.org/10.3390/toxins12120791. PMid:33322460.
http://dx.doi.org/10.3390/toxins12120791... ; Hatakeyama et al., 2021HATAKEYAMA, D.M., TASIMA, L.J., GALIZIO, N.C., SERINO-SILVA, C., RODRIGUES, C.F.B., STUGINSKI, D.R., SANT’ANNA, S.S., GREGO, K.F., TASHIMA, A.K., NISHIDUKA, E.S., MORAIS-ZANI, K. and AZEVEDO, A.M.T., 2021. From birth to adulthood: an analysis of the Brazilian lancehead (Bothrops moojeni) venom at different life stages. PLoS One, vol. 16, no. 6, e025305. http://dx.doi.org/10.1371/journal.pone.0253050. PMid:34111213.
http://dx.doi.org/10.1371/journal.pone.0... ). Moreover, juvenile snake venom has been shown to induce more severe coagulopathy in cases of human envenomation (Freitas-de-Sousa et al., 2020FREITAS-DE-SOUSA, L.A., NACHTIGALL, P.G., PORTES-JUNIOR, J.A., HOLDING, M.L., NYSTROM, G.S., ELLSWORTH, S.A., GUIMARÃES, N.C., TIOYAMA, E., ORTIZ, F., SILVA, B.R., KUNZ, T.S., JUNQUEIRA-DE-AZEVEDO, I.L.M., GRAZZIOTIN, F.G., ROKYTA, D.R. and MOURA-DA-SILVA, A.M., 2020. Size matters: an evaluation of the molecular basis of ontogenetic modifications in the composition of Bothrops jararacussu snake ve. Toxins, vol. 12, no. 12, pp. 791. http://dx.doi.org/10.3390/toxins12120791. PMid:33322460.
http://dx.doi.org/10.3390/toxins12120791... ).

Given the proteomic characteristics and differences between neonatal and maternal venoms (Amorim et al., 2018AMORIM, F.G., COSTA, T.G., BAIWIR, D., DE PAUW, E., QUINTON, L. and SAMPAIO, S.V., 2018. Proteoetpeptidomic, functional and immunoreactivity characterization of Bothrops moojeni snake venom: influence of snake gender on venom composition. Toxins, vol. 10, no. 5, pp. 177. http://doi.org/10.3390/toxins10050177. PMid:29701671.
http://doi.org/10.3390/toxins10050177... ), studies contributing to understanding the severity of snakebites are of great importance, especially for the state of Tocantins. In this context, we addressed the following questions: Is there sexual variation in the venom of siblings? Do the venom components of the mother and its offspring vary significantly? Thus, this study characterizes the composition and activity of the venom of a female B. moojeni and its litter.

2. Material and Methods

2.1. Venom source

A pregnant female B. moojeni (135 cm total length) was collected in the Araguaína municipality, Tocantins state (northern Brazil), and kept in captivity. After parturition, venom was obtained from both the mother and her litter. Capture authorization was granted by the Biodiversity Authorization and Information System (SISBIO, No. 52416-1). Procedures adhered to Normative Instruction No. 03/2014, and the biological material was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen No. A2C170C). Venom extraction, lyophilization, and storage (−20 ºC) were conducted at the Herpetology Laboratory of the Instituto Butantan (São Paulo state, southeastern Brazil) by one of us (Sávio Stefanini). The venom extracted from seven newborn female B. moojeni (28.8–31.2 cm total length) was pooled to create the newborn female venom pool, and the venom extracted from 10 newborn males (28.0–31.4 cm total length) was pooled to create the newborn male venom pool. The newborn venom was compared to the maternal venom.

2.2. Chemical characterization of venoms

2.2.1. Determination of protein content

Protein concentration was determined using the method described by Markwell et al. (1978)MARKWELL, M.A., HAAS, S.M., BIEBER, L.L. and TOLBERT, N.E., 1978. A modification of the Lowry procedures to simplify protein determination in membrane and lipoprotein saples. Analytical Biochemistry, vol. 87, no. 1, pp. 206-210. http://dx.doi.org/10.1016/0003-2697(78)90586-9. PMid:98070.
http://dx.doi.org/10.1016/0003-2697(78)9... . Bovine serum albumin was used as the standard, following the manufacturer’s protocol. Absorbance readings at 660 nm were used for quantification.

2.2.2. SDS-PAGE electrophoresis

Venom protein profiles were analyzed using polyacrylamide gel electrophoresis (SDS-PAGE) following Laemmli (1970)LAEMMLI, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, vol. 227, no. 5259, pp. 680-685. http://dx.doi.org/10.1038/227680a0. PMid:5432063.
http://dx.doi.org/10.1038/227680a0... . Venom samples (25 µg) were applied in the presence of β-mercaptoethanol and a molecular mass standard (14.4–94.0 kDa - GE Healthcare) was run in parallel. The electrophoresis gel was then stained with Coomassie Brilliant Blue R-250. Densitometric analysis was performed using the Gel Analyzer software.

2.2.3. High-performance liquid chromatography (HPLC)

Venoms were fractionated following Sousa et al. (2017)SOUSA, L.F., PORTES-JUNIOR, J.A., NICOLAU, C.A., BERNARDONI, J.L., NISHIYAMA-JR, M.Y., AMAZONAS, D.R., FREITAS-DE-SOUSA, L.A., MOURÃO, R.H.V., CHALKIDIS, H.M. and MOURA-DA-SILVA, A.M., 2017. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon. Journal of Proteomics, vol. 21, pp. 3-46. http://dx.doi.org/10.1016/j.jprot.2017.03.003. PMid:28274896.
http://dx.doi.org/10.1016/j.jprot.2017.0... using a semi-preparative reverse-phase column (C-18 column, Vydac, 10 µm, 4.6 mm i.d. × 250 mm). Five-milligram venom samples were dissolved in 500 μL of 0.1% trifluoroacetic acid in water. After centrifugation at 18,400×g for 10 minutes, the supernatant was applied to the column previously equilibrated with the same solution. Elution occurred at a flow rate of 2 mL/min using a gradient system of 0.1% trifluoroacetic acid in water (solution A) and acetonitrile (solution B) for 100 minutes. The gradient consisted of the following steps: 5% B for 5 min, followed by 5–15% B for 10 min, 15–45% B for 60 min, 45–70% B for 10 min, 70–100% B for 5 min, and 100% B for 10 min. Fractions were monitored with UV 214 nm detection.

2.2.4. Determination of coagulant activity on plasma

The minimum coagulant dose (MCD) was determined using a pool of citrated equine plasma. Venom samples were diluted in a 0.85% saline solution at a concentration of 1.0 mg/ml and then distributed in serial portions, ranging from 1000 to 0.2 µg of venom. Using a BBL Fibrosystem fibrometer®, 200 µL aliquots of the plasma solution (at 37 ºC) were combined with 100 µL of each venom solution. The test was conducted in triplicate, and the MCD was calculated using linear regression. MCD is defined as the lowest venom concentration capable of clotting plasma within 60 seconds at 37 ºC (Theakston and Reid, 1983THEAKSTON, R.D.G. and REID, H.A., 1983. Development of simple standard assay procedures for the characterization of snake venoms. Bulletin of the World Health Organization, vol. 61, no. 6, pp. 949-956. PMid:6609011.).

2.2.5. Serine protease activity

Serine protease activity was assessed following the method described by Knittel et al. (2016)KNITTEL, S., LONG, P.F., BRAMMALL, L., MARQUES, A.C., ALMEIDA, M.T., PADILLA, G. and MOURA-DA-SILVA, A.M., 2016. Characterising the enzymatic profile of crude tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Cnidaria: hydrozoa). Toxicon, vol. 119, pp. 1-7. http://dx.doi.org/10.1016/j.toxicon.2016.04.048. PMid:27169682.
http://dx.doi.org/10.1016/j.toxicon.2016... . This process involved adding 40 μL of 4 mM BAPNA dissolved in dimethyl sulfoxide (DMSO), 40 μL of 50 mM Tris-HCl (pH 8.0), and 20 μL of venom at a concentration of 5 μg to 96-well plates. The mixture was incubated at 37 °C for 40 minutes, and the release of the product (p-nitroaniline) was monitored using a microplate spectrophotometer (Kasuaki DR-200BS), at 405 nm. Results were expressed in Abs/min/mg of venom. The experiment was conducted in triplicate, with B. jararaca venom as the control.

2.2.6. Phospholipase A₂ activity

Phospholipase A₂ activity was assessed following Knittel et al. (2016)KNITTEL, S., LONG, P.F., BRAMMALL, L., MARQUES, A.C., ALMEIDA, M.T., PADILLA, G. and MOURA-DA-SILVA, A.M., 2016. Characterising the enzymatic profile of crude tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Cnidaria: hydrozoa). Toxicon, vol. 119, pp. 1-7. http://dx.doi.org/10.1016/j.toxicon.2016.04.048. PMid:27169682.
http://dx.doi.org/10.1016/j.toxicon.2016... . Twenty micrograms of each venom were incubated with 320 μM of chromogenic synthetic substrate (NOBA). After a 40-minute incubation at 37 °C, absorbance of the samples was measured using a microplate spectrophotometer (Kasuaki DR-200BS) at 425 nm. Phospholipase activity was expressed in nmoles/min of the released chromophore. The positive control for this assay was the venom of B. jararacussu, and the experiment was conducted in triplicate.

2.3. Biological activities

2.3.1. Animals

Experimental protocols for assessing biological activities in male Swiss mice (18–22 g) were approved by the Ethics Committee of the Butantan Institute (CEUAIB Protocol No. 3873030817). The procedures followed ethical standards and requirements for research involving laboratory animals.

2.3.2. Toxicity test

The toxicity of the venom samples was determined using the single-dose procedure as described by Aird and Kaiser (1985)AIRD, S.D. and KAISER, I.I., 1985. Comparative studies on three rattlesnake toxins. Toxicon, vol. 23, no. 3, pp. 361-374. http://dx.doi.org/10.1016/0041-0101(85)90020-0. PMid:4024144.
http://dx.doi.org/10.1016/0041-0101(85)9... . The individual dose was determined by averaging the last three LD₅₀ records from the Quality Control of the Herpetology Laboratory of the Butantan Institute, namely 135 μg/animal. Venom toxicity was assessed in mice groups (n = 4) through intraperitoneal injection of venom in 0.5 mL of saline solution. Animal mortality was monitored for 48 hours, and venom toxicity was assessed by recording the time and number of deaths at 24- and 48-hours post-venom injection (Finney, 1971FINNEY, D.J., 1971. Probit analysis. 3rd ed. Cambridge University Press: Cambridge, 488 p.).

2.3.3. Hemorrhagic activity

To assess hemorrhagic activity, a dose of 2 µg/animal was used (Lomonte and Gutiérrez, 1989LOMONTE, B. and GUTIÉRREZ, J., 1989. A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon, vol. 27, no. 7, pp. 725-733. http://dx.doi.org/10.1016/0041-0101(89)90039-1. PMid:2781572.
http://dx.doi.org/10.1016/0041-0101(89)9... ). The hemorrhagic effect of the venoms was assessed following Kondo et al. (1960)KONDO, H., KONDO, S., IKEZAWA, H., MURATA, R. and OHSAKA, A., 1960. Studies of the quantitative method for the determination of hemorrhagic activity of Habu snake venom. Japanese Journal of Medical Science & Biology, vol. 13, no. 1-2, pp. 43-51. http://dx.doi.org/10.7883/yoken1952.13.43. PMid:13853435.
http://dx.doi.org/10.7883/yoken1952.13.4... , with modifications by Gutiérrez et al. (1985)GUTIÉRREZ, J.M., GENÉ, A., RODAS, G. and CERDAS, L., 1985. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon, vol. 23, no. 6, pp. 887-893. http://dx.doi.org/10.1016/0041-0101(85)90380-0. PMid:3913055.
http://dx.doi.org/10.1016/0041-0101(85)9... . Groups of five mice were intradermally inoculated in the abdominal region with venom diluted in saline (100 µL). After 3 hours, the mice were euthanized, and their ventral skins were excised. Digital photographs were taken to examine the formation of the hemorrhagic halo and measure its size in pixels. The margin was traced, and the intensity of the hemorrhage was quantified. Hemorrhagic activity was assessed by calculating the mean and standard deviation (SD) of the diameters of the bleeding areas (mm²) and expressed in hemorrhagic units (HeU) (Jenkins et al., 2017JENKINS, T.P., SANCHEZ, A., SEGURA, A., VARGAS, M., HERRERA, M., STEWART, T.K., LEON, G. and GUTIERREZ, J.M., 2017. An improved technique for the assessment of venom-induced haemorrhage in a murine model. Toxicon, vol. 139, pp. 87-93. http://dx.doi.org/10.1016/j.toxicon.2017.10.005.
http://dx.doi.org/10.1016/j.toxicon.2017... ). The bleeding halos (mm²) and their color intensity (pixels - RGB) were measured using Inkscape software versions 0.92 and 0.93.

2.4. Statistical analysis

Data are expressed as mean ± SD. Statistical analyses were performed using one-way ANOVA, followed by Tukey’s test, with GraphPad PRISM software, version 8.0.

3. Results

The electrophoretic profile of newborn B. moojeni venoms revealed 12 protein bands ranging from 15 to 94 kDa (Figure 1). Notably, newborn venoms exhibited a higher concentration of proteins with electrophoretic mobility between 30 and 67 kDa, whereas the maternal venom contained three bands at approximately 50, 28, and 15 kDa. Interestingly, the 28 kDa band was nearly absent in newborn venoms.

Chromatographic analysis revealed 22 proteomic peaks in the venom of newborn females and 21 peaks in the venom of newborn males, with the most prominent peaks in both groups being 7, 14, 16, and 19. Fifteen peaks were identified in the maternal venom, with the most prominent ones being 6, 7, 14, 19, and 21 (Figure 2). The main proteomic families were identified based on their elution time. Proteins classified as disintegrins (Dis) were eluted between 25 and 35 minutes. Proteins from the alkaline phospholipase (K49-PLA₂) group were eluted between 55 and 60 minutes. Proteins from the alkaline and acidic phospholipase (D49-PLA₂) type, serine proteases (SVSPs), and metalloproteinases (SVMP-I) were eluted between 60 and 70 minutes. C-type lectins (CTLs) were eluted between 70 and 85 minutes, and mainly SVMP-III metalloproteases were observed after 80 minutes of elution.

When comparing the spectra of the neonatal venom, we observed distinct peaks in the regions corresponding to disintegrins, phospholipases A₂, serine proteases, and more prominently, C-type lectins and metalloproteases (Figure 2). It is important to note that peak 11 (eluted between 60 and 70 minutes) is likely a serine protease. This peak was noteworthy for its presence in both the mother and newborn venoms, although it is less abundant in female newborns. Conversely, peak 6 (eluted between 50 and 60 minutes) generally corresponded to K49-PLA₂ and was exclusive to maternal venom. In general, the maternal venom showed a simpler pattern in the elution region, eluting PIII-class SVMPs after 80 minutes. Figure 2 reveals an overlap between peaks 1, 2, 3, 11, 13, 14, 19, and 20 in the chromatogram of the tested samples.

The MCD of the venom from newborn females (0.20 ± 0.05 µg/mL) did not differ significantly from that of the venom from male newborns (0.40 ± 0.04 µg/mL). However, the MCD of the newborn venoms showed significantly higher coagulation activity than that of the maternal venom (16.70 ± 0.93 µg/mL; p < 0.001).

The serine protease activity of the venom from newborn females (0.34 ± 0.05 Abs/min/mg) was higher than that of the venom from newborn males (0.26 ± 0.03 Abs/min/mg; p < 0.001). However, the serine protease activity of both sexes was lower than that of the maternal venom (0.66 ± 0.03 Abs/min/mg; p < 0.0001; Figure 3). The phospholipase A₂ activity of the newborn venoms was similar to that of the maternal venom (Figure 4) but significantly lower than that of the control group (B. jararacussu).

The lethality of newborn venoms was confirmed in all groups of inoculated mice. However, the venom from newborn females was more lethal. After 48 hours, 100% mortality was observed in mice inoculated with the venom from newborn females, whereas the mortality rate was 50% for mice inoculated with the venom of newborn males and the mother.

The intradermal injection of 2 µg/animal of B. moojeni venom in mice (Figure 5) resulted in the formation of a hemorrhagic halo. The venom from newborn females caused a significantly larger halo (97.90 ± 7.49 HeU) than the venom of newborn males (47.40 ± 6.91 HeU; p < 0.05) and the mother (24.10 ± 19.88 HeU; p < 0.001).

4. Discussion

The composition of snake venom has been studied using omic tools to demonstrate variations within and among populations. These variations contribute to diverse damages and symptoms observed during the clinical evolution of snakebites (Gonçalves-Machado et al., 2016GONÇALVES-MACHADO, L., PLA, D., SANZ, L., JORGE, R.J.B., LEITÃO-DE-ARAÚJO, M., ALVES, M.L.M., ALVARES, D.J., MIRANDA, J., NOWATZKI, J., MORAIS-ZANI, K., FERNANDES, W., TANAKA-AZEVEDO, A.M., FERNÁNDEZ, J., ZINGALI, R.B., GUTIÉRREZ, J.M., CORRÊA-NETTO, C. and CALVETE, J.J., 2016. Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian atlantic rainfore. Journal of Proteomics, vol. 135, pp. 73-89. http://dx.doi.org/10.1016/j.jprot.2015.04.029. PMid:25968638.
http://dx.doi.org/10.1016/j.jprot.2015.0... ; Sunagar et al., 2016SUNAGAR, K., MORGENSTERN, D., REITZEL, A.M. and MORAN, Y., 2016. Ecological venomics: how genomics, transcriptomics and proteomics can shed new light on the ecology and evolution of venom. Journal of Proteomics, vol. 135, pp. 62-72. http://dx.doi.org/10.1016/j.jprot.2015.09.015. PMid:26385003.
http://dx.doi.org/10.1016/j.jprot.2015.0... ; Sousa et al., 2017SOUSA, L.F., PORTES-JUNIOR, J.A., NICOLAU, C.A., BERNARDONI, J.L., NISHIYAMA-JR, M.Y., AMAZONAS, D.R., FREITAS-DE-SOUSA, L.A., MOURÃO, R.H.V., CHALKIDIS, H.M. and MOURA-DA-SILVA, A.M., 2017. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon. Journal of Proteomics, vol. 21, pp. 3-46. http://dx.doi.org/10.1016/j.jprot.2017.03.003. PMid:28274896.
http://dx.doi.org/10.1016/j.jprot.2017.0... ; Amazonas et al., 2018AMAZONAS, D.R., PORTES-JUNIOR, J.A., NISHIYAMA-JR, M.Y., NICOLAU, C.A., CHALKIDIS, H.M., MOURÃO, R.H.V., GRAZZIOTIN, F.G., ROKYTA, D.R., AMORIM, F.G., COSTA, T.R., BAIWIR, D., PAUW, E., QUINTON, L. and SAMPAIO, S.V., 2018. Proteoetpeptidomic, functional and immunoreactivity characterization of Bothrops moojeni snake venom: influence of snake gender on venom composition. Toxins, vol. 10, no. 5, pp. 177. http://dx.doi.org/10.3390/toxins10050177. PMid:29701671.
http://dx.doi.org/10.3390/toxins10050177... ).

Sunagar et al. (2014)SUNAGAR, K., UNDHEIM, E.A.B., SCHEIB, H., GREN, E.C.K., COCHRAN, C., PERSON, C.E. and FRY, B.G., 2014. Intraspecific venom variation in the medically significant Southern Pacific Rattlesnake (Crotalus oreganus helleri): biodiscovery, clinical and evolutionary implications. Journal of Proteomics, vol. 99, pp. 68-83. http://dx.doi.org/10.1016/j.jprot.2014.01.013. PMid:24463169.
http://dx.doi.org/10.1016/j.jprot.2014.0... suggested that the molecular evolution of venom may have been influenced by several factors, including ontogeny and sex. This is because the protein patterns of venoms from newborns differ from those of adults. It is important to highlight that the Brazilian system for reporting snakebites does not distinguish the snakes by life stage. Consequently, analyzing epidemiological data on these aspects becomes challenging. However, knowledge of the ontogenetic variation of the venom of these animals is necessary to understand the clinical outcomes of envenomations. This knowledge is vital for assessing the effectiveness of antivenoms and prospecting molecules with potential pharmacological applications, particularly considering that bites by adult snakes lead to more necrosis than those involving juvenile snakes (Nicoleti et al., 2010NICOLETI, A.F., MEDEIROS, C.R.D., DUARTE, M.R. and FRANÇA, F.O.D.S., 2010. Comparison of Bothropoides jararaca bites with and without envenoming treated at the Vital Brazil Hospital of the Butantan Institute, State of São Paulo, Brazil. Revista da Sociedade Brasileira de Medicina Tropical, vol. 43, no. 6, pp. 657-661. http://dx.doi.org/10.1590/S0037-86822010000600011. PMid:21181018.
http://dx.doi.org/10.1590/S0037-86822010... ; Wray et al., 2015WRAY, K.P., MARGRES, M.J., SEAVY, M. and ROKYTA, D.R., 2015. Early significant ontogenetic changes in snake venoms. Toxicon, vol. 96, pp. 74-81. http://dx.doi.org/10.1016/j.toxicon.2015.01.010. PMid:25600640.
http://dx.doi.org/10.1016/j.toxicon.2015... ).

This is the first study to examine sexual and ontogenetic differences in the venom of a snake species from the state of Tocantins. Our proteomic analysis revealed that the venoms of newborn male and female B. moojeni exhibit over 20 protein peaks in the chromatographic profile. In contrast, maternal venom exhibited 15 major protein peaks, i.e., five peaks fewer than those of the newborns. Chromatographic analysis further revealed that disintegrating enzymes, such as phospholipase A₂, serine proteases, and metalloproteases, are common to the venom of both the mother and the young.

Bothropic venom is mainly composed of classes of metalloprotease-like proteases, serine proteases, C-type lectins, and phospholipases (Sousa et al., 2013SOUSA, L.F., NICOLAU, C.A., PEIXOTO, P.S., BERNARDONI, J.L., OLIVEIRA, S.S., PORTES-JUNIOR, J.A., MOURÃO, R.H., LIMA-DOS-SANTOS, I., SANO-MARTINS, I.S., CHALKIDIS, H.M., VALENTE, R.H. and MOURA-DA-SILVA, A.M., 2013. Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of Bothrops complex. PLoS Neglected Tropical Diseases, vol. 12, no. 7, e244. http://dx.doi.org/10.1371/journal.pntd.0002442. PMid:24069493.
http://dx.doi.org/10.1371/journal.pntd.0... ). Metalloproteases are present in the range of 20 to 110 kDa (Fox and Serrano, 2008FOX, J.W. and SERRANO, S.M.T., 2008. Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity. The FEBS Journal, vol. 275, no. 12, pp. 3016-3030. http://dx.doi.org/10.1111/j.1742-4658.2008.06466.x. PMid:18479462.
http://dx.doi.org/10.1111/j.1742-4658.20... ). The 50 kDa range, found in venoms, is associated with the presence of PIII class metalloproteases (Morais et al., 2012MORAIS, N., MAMEDE, C.C.N., FONSECA, K.C., QUEIROZ, M.R., GOMES-FILHO, S.A., SANTOS-FILHO, N.A., BORDON, K.C.F., BELETTI, M.E., SAMPAIO, S.V., ARANTES, E.C. and OLIVEIRA, F., 2012. Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom. Toxicon, vol. 60, no. 7, pp. 1251-1258. http://dx.doi.org/10.1016/j.toxicon.2012.08.017. PMid:22975266.
http://dx.doi.org/10.1016/j.toxicon.2012... ). The 28 kDa range is associated with PI class metalloproteinases, while the 15 kDa range is associated with phospholipase A₂ (Valente et al., 2009VALENTE, R.H., GUIMARÃES, P.R., JUNQUEIRA, M., NEVES-FERREIRA, A.G.C., SOARES, M.R., CHAPEAUROUGE, A., TRUGILHO, M.R., LEÓN, I.R., ROCHA, S.L., OLIVEIRA-CARVALHO, A.L., WERMELINGER, L.S., DUTRA, D.L., LEÃO, L.I., JUNQUEIRA-DE-AZEVEDO, I.L., HO, P.L., ZINGALI, R.B., PERALES, J. and DOMONT, G.B., 2009. Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data. Journal of Proteomics, vol. 72, no. 2, pp. 241-255. http://dx.doi.org/10.1016/j.jprot.2009.01.001. PMid:19211044.
http://dx.doi.org/10.1016/j.jprot.2009.0... ). These findings closely align with compounds identified in regions similar to those in our study. Therefore, we conclude that we detected the same classes previously described. Furthermore, SDS-PAGE analysis showed prominent bands at 30, 90, and 12 kDa. In the venom of B. moojeni, researchers isolated a phospholipase in the 13.8 kDa band (Silveira et al., 2013SILVEIRA, L.B., MARCHI-SALVADOR, D.P., SANTOS-FILHO, N.A., SILVA JUNIOR, F.P., MARCUSSI, S., FULYF, A.L., NOMIZO, A., SILVA, S.L., STÁBELI, R.G., ARANTES, E.C. and SOARES, A.M., 2013. Isolation and expression of a hypotensive and anti-platelet acidic phospholipase A2 from Bothrops moojeni snake venom. Journal of Pharmaceutical and Biomedical Analysis, vol. 73, pp. 35-43. http://dx.doi.org/10.1016/j.jpba.2012.04.008. PMid:22571953.
http://dx.doi.org/10.1016/j.jpba.2012.04... ), a serine protease between 32 and 35 kDa (Oliveira et al., 2013OLIVEIRA, L.M.F., ULLAH, A., MASOOD, R., ZELANIS, A., SPENCER, P.J., SERRANO, S.M.T. and ARNI, R.K., 2013. Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms. Toxicon, vol. 76, pp. 282-290. http://dx.doi.org/10.1016/j.toxicon.2013.10.016. PMid:24140922.
http://dx.doi.org/10.1016/j.toxicon.2013... ), and a metalloprotease at 45 kDa (Morais et al., 2012MORAIS, N., MAMEDE, C.C.N., FONSECA, K.C., QUEIROZ, M.R., GOMES-FILHO, S.A., SANTOS-FILHO, N.A., BORDON, K.C.F., BELETTI, M.E., SAMPAIO, S.V., ARANTES, E.C. and OLIVEIRA, F., 2012. Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom. Toxicon, vol. 60, no. 7, pp. 1251-1258. http://dx.doi.org/10.1016/j.toxicon.2012.08.017. PMid:22975266.
http://dx.doi.org/10.1016/j.toxicon.2012... ).

The similarity in venom components of B. moojeni from Tocantins with the venom from conspecifics from other regions is relevant information. This similarity may be linked to the composition of venom in both adult and newborn snakes. Despite geographic distances and varying environmental factors, fundamental proteins such as metalloproteinases, serine proteases, and phospholipases exhibit consistent expression (Assakura et al., 1985ASSAKURA, M.T.A.P., ASPERTI, M.C.A. and MANDELBAUM, F.R., 1985. Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca). Toxicon, vol. 23, no. 4, pp. 691-706. http://dx.doi.org/10.1016/0041-0101(85)90374-5. PMid:3933145.
http://dx.doi.org/10.1016/0041-0101(85)9... ; Amorim et al., 2017AMORIM, F.G., MORANDI-FILHO, R., FUJIMURA, T., UEIRA-VIEIRA, C. and SAMPAIO, S.V., 2017. New findings from the first transcriptome of the Bothrops moojeni snake venom gland. Toxicon, vol. 140, pp. 105-111. http://dx.doi.org/10.1016/j.toxicon.2017.10.025. PMid:29107670.
http://dx.doi.org/10.1016/j.toxicon.2017... ).

Similarly, variations in the protein profile between the venoms of newborns and adults also occur in Tocantins snakes. Adult snakes predominantly express the phospholipase A₂ protease, whereas newborns express different metalloproteinase isoforms, consistent with previous studies (Amorim et al., 2018AMORIM, F.G., COSTA, T.G., BAIWIR, D., DE PAUW, E., QUINTON, L. and SAMPAIO, S.V., 2018. Proteoetpeptidomic, functional and immunoreactivity characterization of Bothrops moojeni snake venom: influence of snake gender on venom composition. Toxins, vol. 10, no. 5, pp. 177. http://doi.org/10.3390/toxins10050177. PMid:29701671.
http://doi.org/10.3390/toxins10050177... ; Freitas-de-Sousa et al., 2020FREITAS-DE-SOUSA, L.A., NACHTIGALL, P.G., PORTES-JUNIOR, J.A., HOLDING, M.L., NYSTROM, G.S., ELLSWORTH, S.A., GUIMARÃES, N.C., TIOYAMA, E., ORTIZ, F., SILVA, B.R., KUNZ, T.S., JUNQUEIRA-DE-AZEVEDO, I.L.M., GRAZZIOTIN, F.G., ROKYTA, D.R. and MOURA-DA-SILVA, A.M., 2020. Size matters: an evaluation of the molecular basis of ontogenetic modifications in the composition of Bothrops jararacussu snake ve. Toxins, vol. 12, no. 12, pp. 791. http://dx.doi.org/10.3390/toxins12120791. PMid:33322460.
http://dx.doi.org/10.3390/toxins12120791... ; Hatakeyama et al., 2021HATAKEYAMA, D.M., TASIMA, L.J., GALIZIO, N.C., SERINO-SILVA, C., RODRIGUES, C.F.B., STUGINSKI, D.R., SANT’ANNA, S.S., GREGO, K.F., TASHIMA, A.K., NISHIDUKA, E.S., MORAIS-ZANI, K. and AZEVEDO, A.M.T., 2021. From birth to adulthood: an analysis of the Brazilian lancehead (Bothrops moojeni) venom at different life stages. PLoS One, vol. 16, no. 6, e025305. http://dx.doi.org/10.1371/journal.pone.0253050. PMid:34111213.
http://dx.doi.org/10.1371/journal.pone.0... ).

As with the venom from young B. jararaca in southeastern Brazil (Zelanis et al., 2011ZELANIS, A., TASHIMA, A.K., PINTO, A.F.M., LEME, A.F.P., STUGINSKI, D.R., FURTADO, M.F. and SHERMAN, N.E., 2011. Bothrops jararaca venom proteome rearrangement upon neonate to adult transition. Proteomics, vol. 11, no. 21, pp. 4218-4228. http://dx.doi.org/10.1002/pmic.201100287. PMid:21928397.
http://dx.doi.org/10.1002/pmic.201100287... ), the venom of newborn B. moojeni from Tocantins also exhibits higher expression of metalloproteinases and does not differ from adults in terms of phospholipase A₂ expression. This proteomic class undergoes ontogenetic changes, transitioning from a PIII-rich profile in newborns to a PI-rich profile in adults. Simultaneously, serine proteases are produced in higher quantities in adult venom. This explanation allows us to satisfactorily understand the differences in the effects of envenomation by newborn or adult snakes.

The coagulant activity of the newborns’ venom was also higher than that of the mother’s venom. These results corroborate other studies conducted with Bothrops. Saldarriaga et al. (2003)SALDARRIAGA, M.M., OTEROA, R., NUÑEZA, V., TOROA, M.F., DIAZA, A. and GUTIÉRREZ, J.M., 2003. Ontogenetic variability of Bothrops atrox and Bothrops asper snake venoms from Colombia. Toxicon, vol. 42, no. 4, pp. 405-411. http://dx.doi.org/10.1016/S0041-0101(03)00171-5. PMid:14505941.
http://dx.doi.org/10.1016/S0041-0101(03)... studied the venom of B. atrox from the Colombian Amazon and found that the venom of newborns and juveniles is more coagulant than that of adults. Zelanis et al. (2010)ZELANIS, A., TASHIMA, A.K., ROCHA, M.M.T., FURTADO, M.F., CAMARGO, A.C.M., HO, P.L. and SERRANO, S.M.T., 2010. Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey. Journal of Proteome Research, vol. 9, no. 5, pp. 2278-2291. http://dx.doi.org/10.1021/pr901027r.
http://dx.doi.org/10.1021/pr901027r... studied the venom of newborn B. jararaca and found that the coagulant activity is ten times more potent than that of adult conspecifics. Similarly, Hatakeyama et al. (2021)HATAKEYAMA, D.M., TASIMA, L.J., GALIZIO, N.C., SERINO-SILVA, C., RODRIGUES, C.F.B., STUGINSKI, D.R., SANT’ANNA, S.S., GREGO, K.F., TASHIMA, A.K., NISHIDUKA, E.S., MORAIS-ZANI, K. and AZEVEDO, A.M.T., 2021. From birth to adulthood: an analysis of the Brazilian lancehead (Bothrops moojeni) venom at different life stages. PLoS One, vol. 16, no. 6, e025305. http://dx.doi.org/10.1371/journal.pone.0253050. PMid:34111213.
http://dx.doi.org/10.1371/journal.pone.0... reported higher procoagulant and lower proteolytic activities in the venom of neonatal B. moojeni (up to one year old). From the age of two, the venom starts to show greater proteolytic activity, as in adult snakes.

Disturbances in the hemostatic system have been associated with specific serine proteases found in the venom of B. moojeni, exhibiting a molecular weight between 28–40 kDa and increased coagulant activity (Zingali et al., 1993ZINGALI, R.B., JANDROT-PERRUS, M., GUILLIN, M.C. and BON, C., 1993. Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition. Biochemistry, vol. 32, no. 40, pp. 10794-10802. http://dx.doi.org/10.1021/bi00091a034. PMid:8399228.
http://dx.doi.org/10.1021/bi00091a034... ; Nishida et al., 1994NISHIDA, S., FUJIMURA, Y., MIURA, S., YOSHIDA, E., SUGIMOTO, M., YOSHIOKA, A., FUKUI, H., OZAKI, Y. and USAMI, Y., 1994. Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from venom of Bothrops jararaca. Biochemistry, vol. 33, no. 7, pp. 1843-1849. http://dx.doi.org/10.1021/bi00173a030. PMid:8110787.
http://dx.doi.org/10.1021/bi00173a030... ). Serrano et al. (1993)SERRANO, S.M.T., MATOS, M.F.C., MANDELBAUM, F.R. and SAMPAIO, C.A.M., 1993. Basic proteinases from Bothrops mooojeni (Caissaca) venom-I. Isolation and Activity of two Serine proteinases, MSP1 and MSP2, on syntetic substrates and platelet aggregation. Toxicon, vol. 31, no. 4, pp. 471-481. http://dx.doi.org/10.1016/0041-0101(93)90182-I. PMid:8503135.
http://dx.doi.org/10.1016/0041-0101(93)9... described a 38 kDa serine protease (MSP2) in B. moojeni venom with coagulant activity. Oliveira et al. (2013)OLIVEIRA, L.M.F., ULLAH, A., MASOOD, R., ZELANIS, A., SPENCER, P.J., SERRANO, S.M.T. and ARNI, R.K., 2013. Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms. Toxicon, vol. 76, pp. 282-290. http://dx.doi.org/10.1016/j.toxicon.2013.10.016. PMid:24140922.
http://dx.doi.org/10.1016/j.toxicon.2013... identified serine proteases in the venom of adults from other Bothrops species, with coagulant activity and molecular masses between 32 and 35 kDa.

Our results support previous studies, as protein bands ranging from 30 to 40 kDa were detected in the SDS-PAGE electrophoresis of newborn venom (Figure 1). Notably, the venom of newborn females exhibited higher activity of serine protease, greater hemorrhagic effects, and increased lethality compared to the venom of newborn males.

Metalloproteases induce changes in microvessels, increasing permeability and promoting extravasation, leading to symptoms such as edema and hemorrhage (Fox and Serrano, 2008FOX, J.W. and SERRANO, S.M.T., 2008. Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity. The FEBS Journal, vol. 275, no. 12, pp. 3016-3030. http://dx.doi.org/10.1111/j.1742-4658.2008.06466.x. PMid:18479462.
http://dx.doi.org/10.1111/j.1742-4658.20... ; Markland Junior and Swenson, 2013MARKLAND JUNIOR, F.S. and SWENSON, S., 2013. Snake venom metalloproteinases. Toxicon, vol. 62, pp. 3-18. http://dx.doi.org/10.1016/j.toxicon.2012.09.004. PMid:23000249.
http://dx.doi.org/10.1016/j.toxicon.2012... ). In B. moojeni, the local effects of the venom can be triggered and intensified by type A₂ phospholipases (Dennis et al., 2011DENNIS, E.A., CAO, J., HSU, Y.-H., MAGRIOTI, V. and KOKOTOS, G., 2011. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chemical Reviews, vol. 111, no. 10, pp. 6130-6185. http://dx.doi.org/10.1021/cr200085w.
http://dx.doi.org/10.1021/cr200085w... ; Souza et al., 2015SOUZA, L.L., STRANSKY, S., GUERRA-DUARTE, C., FLOR-SÁ, A., SCHNEIDER, F.S., KALAPOTHAKIS, E. and CHÁVEZ-OLÓRTEGUI, C., 2015. Determination of toxic activities in Bothrops spp. Snake venoms using animal-free approaches: correlation between in vitro versus in vivo assays. Toxicological Sciences, vol. 147, no. 2, pp. 458-465. http://dx.doi.org/10.1093/toxsci/kfv140. PMid:26160116.
http://dx.doi.org/10.1093/toxsci/kfv140... ; Mamede et al., 2016MAMEDE, C.C.N., SOUSA, B.B., PEREIRA, D.F.C., MATIAS, M.S., QUEIROZ, M.R., MORAIS, N.C.G., VIEIRA, S.A.B., STANZIOLA, L. and OLIVEIRA, F., 2016. Comparative analysis of local effects caused by Bothrops alternatus and Bothrops moojeni snake venoms: enzymatic contributions and inflammatory modulations. Toxicon, vol. 117, pp. 37-45. http://dx.doi.org/10.1016/j.toxicon.2016.03.006. PMid:26975252.
http://dx.doi.org/10.1016/j.toxicon.2016... ).

Collectively, our results on the activity of serine proteases, phospholipases, and metalloproteinases agree with López-Lozano et al. (2002)LÓPEZ-LOZANO, J.L., SOUSA, M.V., RICART, C.A.O., CHÁVEZ-OLORTEGUE, C., SANCHEZ, E.F., MUNIZ, E.G., BÜHRNHEIM, P.F. and MORHY, L., 2002. Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest. Toxicon, vol. 40, no. 7, pp. 997-1006. http://dx.doi.org/10.1016/S0041-0101(02)00096-X. PMid:12076654.
http://dx.doi.org/10.1016/S0041-0101(02)... . Their proteome analysis of juvenile, sub-adult, and adult B. atrox from the Manaus region revealed variations in venom composition during ontogeny, potentially influencing coagulation.

Saldarriaga et al. (2003)SALDARRIAGA, M.M., OTEROA, R., NUÑEZA, V., TOROA, M.F., DIAZA, A. and GUTIÉRREZ, J.M., 2003. Ontogenetic variability of Bothrops atrox and Bothrops asper snake venoms from Colombia. Toxicon, vol. 42, no. 4, pp. 405-411. http://dx.doi.org/10.1016/S0041-0101(03)00171-5. PMid:14505941.
http://dx.doi.org/10.1016/S0041-0101(03)... demonstrated that venoms from newborn B. asper and B. atrox in Colombia have greater lethality and higher hemorrhagic and coagulant activity than adult venom. These variations in activity were attributed to an increased quantity of proteins, likely metalloproteinases. The myotoxic activity of this protein class has been previously reported in the venom of B. alternatus and B. leucurus (Costa et al., 2010COSTA, J.O., FONSECA, K.C., MAMEDE, C.C.N., CBELETTI, C.M.A., SANTOS-FILHO, N.A., SOARES, A.M., ARANTES, E.C., HIRAYAMA, S.N.S., SELISTRE-DE-ARAÚJO, H.S., FONSECA, F., HENRIQUE-SILVA, F., PENHA-SILVA, N. and OLIVEIRA, F., 2010. Bhalternin: functional and structural characterization of a new thrombinlike enzyme from Bothrops alternatus snake venom. Toxicon, vol. 55, no. 7, pp. 1365-1377. http://dx.doi.org/10.1016/j.toxicon.2010.02.014. PMid:20184912.
http://dx.doi.org/10.1016/j.toxicon.2010... ; Gomes et al., 2011GOMES, M.S.R., QUEIROZ, M.R., MAMEDE, C.C.N., MENDES, M.M., HAMAGUCHI, A., HOMSI-BRANDEBURGO, M.I., SOUSA, M.V., AQUINO, E.N., CASTRO, M.S., OLIVEIRA, F. and RODRIGUES, V.M., 2011. Purification and functional characterization of a new metalloproteinase (BleucMP) from Bothrops leucurus snake venom. Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP, vol. 153, no. 3, pp. 290-300. http://dx.doi.org/10.1016/j.cbpc.2010.11.008. PMid:21130897.
http://dx.doi.org/10.1016/j.cbpc.2010.11... ). This protein class is also present in the venom of B. moojeni from Tocantins.

Our results show higher serine protease and hemorrhagic activity in the venom from newborn females. Furthermore, the proteolytic activity of metalloproteinases seems to be more important. Bothrops moojeni venom is considerably more potent in inducing local effects than, for example, B. alternatus venom (Mamede et al., 2016MAMEDE, C.C.N., SOUSA, B.B., PEREIRA, D.F.C., MATIAS, M.S., QUEIROZ, M.R., MORAIS, N.C.G., VIEIRA, S.A.B., STANZIOLA, L. and OLIVEIRA, F., 2016. Comparative analysis of local effects caused by Bothrops alternatus and Bothrops moojeni snake venoms: enzymatic contributions and inflammatory modulations. Toxicon, vol. 117, pp. 37-45. http://dx.doi.org/10.1016/j.toxicon.2016.03.006. PMid:26975252.
http://dx.doi.org/10.1016/j.toxicon.2016... ). This feature has been attributed to the central role of metalloproteases and phospholipases A₂, which cause local damage, and the contribution of serine proteases that induce and exacerbate inflammation. Venoms containing a higher amount of these metalloproteinases are more lethal and exhibit more pronounced hemorrhagic activity (Massey et al., 2012MASSEY, D.J., CALVETE, J.J., SÁNCHEZD, E.E., SANZC, L., RICHARDSA, K., CURTISA, R. and BOESENA, K., 2012. Venom variability and envenoming severity outcomes of the Crotalus scutulatus scutulatus (Mojave rattlesnake) from Southern Arizona. Journal of Proteomics, vol. 75, no. 9, pp. 2576-2587. http://dx.doi.org/10.1016/j.jprot.2012.02.035. PMid:22446891.
http://dx.doi.org/10.1016/j.jprot.2012.0... ).

The correlation study conducted by Souza et al. (2015)SOUZA, L.L., STRANSKY, S., GUERRA-DUARTE, C., FLOR-SÁ, A., SCHNEIDER, F.S., KALAPOTHAKIS, E. and CHÁVEZ-OLÓRTEGUI, C., 2015. Determination of toxic activities in Bothrops spp. Snake venoms using animal-free approaches: correlation between in vitro versus in vivo assays. Toxicological Sciences, vol. 147, no. 2, pp. 458-465. http://dx.doi.org/10.1093/toxsci/kfv140. PMid:26160116.
http://dx.doi.org/10.1093/toxsci/kfv140... showed that the venom of Bothrops species from Brazil varies inter- and intraspecifically (Amorim et al., 2018AMORIM, F.G., COSTA, T.G., BAIWIR, D., DE PAUW, E., QUINTON, L. and SAMPAIO, S.V., 2018. Proteoetpeptidomic, functional and immunoreactivity characterization of Bothrops moojeni snake venom: influence of snake gender on venom composition. Toxins, vol. 10, no. 5, pp. 177. http://doi.org/10.3390/toxins10050177. PMid:29701671.
http://doi.org/10.3390/toxins10050177... ), as well as ontogenetically (López-Lozano et al., 2002LÓPEZ-LOZANO, J.L., SOUSA, M.V., RICART, C.A.O., CHÁVEZ-OLORTEGUE, C., SANCHEZ, E.F., MUNIZ, E.G., BÜHRNHEIM, P.F. and MORHY, L., 2002. Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest. Toxicon, vol. 40, no. 7, pp. 997-1006. http://dx.doi.org/10.1016/S0041-0101(02)00096-X. PMid:12076654.
http://dx.doi.org/10.1016/S0041-0101(02)... ; Saldarriaga et al., 2003SALDARRIAGA, M.M., OTEROA, R., NUÑEZA, V., TOROA, M.F., DIAZA, A. and GUTIÉRREZ, J.M., 2003. Ontogenetic variability of Bothrops atrox and Bothrops asper snake venoms from Colombia. Toxicon, vol. 42, no. 4, pp. 405-411. http://dx.doi.org/10.1016/S0041-0101(03)00171-5. PMid:14505941.
http://dx.doi.org/10.1016/S0041-0101(03)... ; Zelanis et al., 2010ZELANIS, A., TASHIMA, A.K., ROCHA, M.M.T., FURTADO, M.F., CAMARGO, A.C.M., HO, P.L. and SERRANO, S.M.T., 2010. Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey. Journal of Proteome Research, vol. 9, no. 5, pp. 2278-2291. http://dx.doi.org/10.1021/pr901027r.
http://dx.doi.org/10.1021/pr901027r... ; Antunes et al., 2010ANTUNES, T.C., YAMASHITA, K.M., BARBARO, K.C., SAIKI, M. and SANTORO, M.L., 2010. Comparative analysis of newborn and adult Bothrops jararaca snake venoms. Toxicon, vol. 56, no. 8, pp. 1443-1458. http://dx.doi.org/10.1016/j.toxicon.2010.08.011. PMid:20816886.
http://dx.doi.org/10.1016/j.toxicon.2010... ). Interpopulation and interindividual variability in the composition of bothropic venom can influence the evolution of clinical conditions resulting from envenomation (Gonçalves-Machado et al., 2016GONÇALVES-MACHADO, L., PLA, D., SANZ, L., JORGE, R.J.B., LEITÃO-DE-ARAÚJO, M., ALVES, M.L.M., ALVARES, D.J., MIRANDA, J., NOWATZKI, J., MORAIS-ZANI, K., FERNANDES, W., TANAKA-AZEVEDO, A.M., FERNÁNDEZ, J., ZINGALI, R.B., GUTIÉRREZ, J.M., CORRÊA-NETTO, C. and CALVETE, J.J., 2016. Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian atlantic rainfore. Journal of Proteomics, vol. 135, pp. 73-89. http://dx.doi.org/10.1016/j.jprot.2015.04.029. PMid:25968638.
http://dx.doi.org/10.1016/j.jprot.2015.0... ; Sousa et al., 2017SOUSA, L.F., PORTES-JUNIOR, J.A., NICOLAU, C.A., BERNARDONI, J.L., NISHIYAMA-JR, M.Y., AMAZONAS, D.R., FREITAS-DE-SOUSA, L.A., MOURÃO, R.H.V., CHALKIDIS, H.M. and MOURA-DA-SILVA, A.M., 2017. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon. Journal of Proteomics, vol. 21, pp. 3-46. http://dx.doi.org/10.1016/j.jprot.2017.03.003. PMid:28274896.
http://dx.doi.org/10.1016/j.jprot.2017.0... , 2018SOUSA, L.F., ZDENEK, C.N., DOBSON, J.S., OP-DEN-BROUW, B., COIMBRA, F., GILLETT, A., DEL-REI, T.H.M., CHALKIDIS, H.M., SANT’ANNA, S., TEIXEIRA-DA-ROCHA, M.M., GREGO, K., TRAVAGLIA-CARDOSO, S.R., MOURA-DA-SILVA, A.M. and FRY, B.G., 2018. Coagulotoxicity of Bothrops (Lancehead Pit-Vipers) Venoms from Brazil: differential biochemistry and antivenom efficacy resulting from prey-driven venom variation. Toxins, vol. 10, no. 10, pp. 411. http://dx.doi.org/10.3390/toxins10100411. PMid:30314373.
http://dx.doi.org/10.3390/toxins10100411... ).

The results of Antunes et al. (2010)ANTUNES, T.C., YAMASHITA, K.M., BARBARO, K.C., SAIKI, M. and SANTORO, M.L., 2010. Comparative analysis of newborn and adult Bothrops jararaca snake venoms. Toxicon, vol. 56, no. 8, pp. 1443-1458. http://dx.doi.org/10.1016/j.toxicon.2010.08.011. PMid:20816886.
http://dx.doi.org/10.1016/j.toxicon.2010... showed important ontogenetic changes in the venom of B. jararaca, particularly concerning hemostatic, hemorrhagic, proteolytic, lethal, and inflammatory activities. In this sense, the sources of venom variation demonstrated in our study are relevant to understanding snake venom and the potential clinical outcomes of human envenomation, given the diverse physiological and toxic effects these venoms trigger.

5. Conclusion

We show significant biochemical variabilities in the composition of B. moojeni venom. This variability interferes with the intensity and nature of biological activities and may be closely associated with sexual dimorphism, body size, and body mass. The venom of newborn females exhibited higher serine protease activity, as well as greater hemorrhagic and lethal effects than the venom of newborn males. This suggests that envenomations caused by female newborns of B. moojeni may be more severe. Additionally, the maternal venom exhibited higher serine protease activity. The intraspecific variation in venom composition and activity reinforces the importance of considering ontogeny and sex as variables in antivenom production. These factors are pivotal in guiding studies aimed at identifying new medicines and biotechnological tools for snakebite treatment.

Acknowledgements

This study was funded by the Coordination for the Improvement of Higher Level Education Personnel-CAPES (PROCAD 2013), the Tocantins State Research Support Foundation/FAPT (calls 01/2018 and 01/2019), PPGCiamb/UFT (calls 33/2020 and 21/2023), Social Demand Scholarship provided by CAPES (2018/2020), and PROPESQ/UFT (call 19/2023).

References

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