Sex ratio of black caiman (Melanosuchus niger) hatchlings in a sample of the wild managed population from the Mamirauá Reserve, Central AmazonABSTRACT
Sex determination in crocodilians is dependent on environmental temperature, in which extreme temperatures favor the development of females, while intermediate temperatures favor males. Understanding such temperature dependence in light of current climate change is urgent, as males are the main targets in species’ sustainable management. Here we analyze the sex ratio of newly-hatched Black caiman (Melanosuchus niger) hatchlings from a wild managed population in Central Amazonia. We captured 86 newly-hatched hatchlings and determined their sex through histological analysis of the gonads. We identified 58% males and 42% females among the 86 hatchlings analyzed (male-to-female ratio of 25:18) We identified 58% males and 42% females among the 86 hatchlings analyzed (male-to-female ratio of 25:18) suggesting that the sex ratio of Black caiman hatchlings in 2023 in the area was relatively balanced. This balance contrasts with expectations of a female bias under recent warming conditions and indicates the potential recruitment of males into the population, which is essential to ensure population equilibrium and the long-term viability of harvest quotas.
KEYWORDS:
crocodilians; sustainable management; nesting
RESUMO
A determinação do sexo em crocodilianos é dependente da temperatura do ambiente, com temperaturas extremas produzindo fêmeas e temperaturas intermediárias produzindo machos. Entender essa dependência da temperatura diante dos alertas atuais de mudanças climáticas é urgente, uma vez que os machos são os principais alvos do manejo sustentável da espécie. Nesse estudo, nós analisamos a proporção sexual de filhotes de jacaré-açu (Melanosuchus niger) recém-eclodidos em uma população silvestre e manejada na Amazônia Central. Capturamos 86 filhotes recém-eclodidos e determinamos o sexo por análise histológica das gônadas. Nós identificamos 58% de machos e 42% de fêmeas entre os 86 filhotes analisados (razão macho-fêmea de 25:18), o que sugere que a proporção sexual dos filhotes de jacaré-açu eclodidos em 2023 na área foi relativamente equilibrada. Esse equilíbrio contrasta com nossas expectativas de um viés feminino devido ao aquecimento ambiental recente e indica o recrutamento potencial de machos para a população, o que é essencial para garantir o equilíbrio populacional e a viabilidade de cotas de manejo a longo-prazo.
PALAVRAS-CHAVE:
crocodilianos; manejo sustentável; nidificação
Effectively-sustainable wildlife management requires knowledge of the population dynamics of managed species. This includes understanding and monitoring animal populations from birth, through the recruitment of young individuals that become reproductive, to adults that will determine quotas for future harvest. The sustainable management of Amazonian caimans for the commercialization of meat and by-products has been widely discussed in recent years (Marioni et al. 2021), with specific harvest planned in Brazilian laws (Amazonas 2011, Franco et al. 2019), especially for populations of Black caimans (Melanosuchus niger Spix 1825) from the middle Solimões river region, Central Amazonia.
Although the Black caiman is quite a conspicuous species, there is a lack of basic population dynamics data for its management. For example, it is known that only about 10% of the eggs and juveniles produced annually by females are recruited into the population and reach reproductive age (Grigg 2015), but, to the best of our knowledge, the sex ratio of hatchlings has not been documented.
Sex determination in crocodilians is temperature-dependent (Bull 1980, Lang and Andrews 1994). Environmental factors, including climatic conditions, can directly influence the exposure of eggs to temperature variation (Ferguson & Joanen, 1983; López-Luna et al., 2020). While high or low temperatures tend to favor the development of females, males are predominantly produced at intermediate temperatures (FMF pattern), although precise estimates of this pattern are unknown for Melanosuchus niger (González et al. 2019).
This temperature dependence raises concerns in light of climate change scenarios, as rising temperatures could lead to an increase in the proportion of females within the population. This shift could significantly impact the generation of new individuals in the population, reflecting on the established harvesting quotas, which primarily target males (Amazonas 2011).
We monitored nine nests built in three lakes within the Mamirauá Reserve, situated at the confluence of the Solimões and Japurá Rivers, Central Amazonia, Brazil (Figure 1). Mamirauá Reserve is covered by floodplain habitats subject to a large monomodal flooding pulse (Ramalho et al. 2009) and composed of a mosaic of forests and water bodies (Irion et al. 1997).
Mamirauá Sustainable Development Reserve in the Middle Solimões, Central Amazonia, showing monitored nests of the black caiman Melanosuchus niger. (I) Lake Sarapião; (II) Lake Tracajá; and (III) Lake Tucunarezinho.
All lakes are located in the sector (a socio-political division used in the Reserve) named Jarauá, and are utilized in sustainable management actions for Melanosuchus niger (Franco et al. 2019). The nests monitored in this study are a subset (5% to 42% of the nests) of the those present in each lake: (I) Lake Sarapião, 42% (total nests in the lake = 7); (II) Lake Tracajá, 17% (total nests in the lake = 23); and (III) Lake Tucunarezinho, 5% (total nests in the lake = 38).
Monitoring was conducted during the nesting period (October 2022-January 2023) to prevent predation or flooding (Villamarin et al. 2011, Torralvo et al. 2017). During the estimated hatching period (January/2023), we revisited the lakes and, using forceps, we captured 86 hatchlings from nine nests distributed across three lakes (Figure 1). All hatchlings were already in the water, grouped together, where they were captured. Some were close to an adult caiman, which we presumed to be the mother. The relationship between the captured hatchlings and the nest identity, assigned during the monitoring stage, was determined used a GPS device according to their proximity. Monitored nests were spaced about 100 meters apart, and hatchlings stay near their nest after hatching, making nest origin identification reliable.
Vegetation cover above the nest influences nest temperature, as shaded nests tend to have lower temperatures (López-Luna et al., 2020). Therefore, we visually estimated the percentage of vegetation cover above the nest, 0% indicating no vegetation cover above the nest, up to 100% when the nest was completely covered by vegetation.
The captured hatchlings were translocated to the Reptiles Laboratory of the Mamirauá Institute, weighed using an analytical scale (500 g capacity, 5 g accuracy), had their total length (TL) measured with tape and head length with a digital caliper. They were subsequently euthanized for the removal of the Gonad-Adrenal-Mesonephros (GAM) complex, which was used for histological sex determination. Euthanasia was performed using the anesthetic Propofol and liquid Lidocaine, administered intravenously at a dose of 50 mg/kg. Death was confirmed by cardiac auscultation. The procedures were conducted under the authorizations registered with CEUA/IDSM (protocol no. 10/2022) and ICMBio/SISBIO (license no. 83966-1). The specimens are deposited in the Collections of the Mamirauá Institute.
Initially, the GAM was transversely cleaved and fixed for 24 hours in 10% formalin. After this fixation period, tissues were dehydrated in sequences of immersion in ethanol, clarified in xylene, and embedded in liquid paraffin. Finally, histological sections of 5-μm thickness were made, mounted on glass slides, and stained with Hematoxylin and Eosin (HE) (Pereira dos Santos et al. 2021). Sex determination was conducted through histological examination of the GAM complex, attached to the sexual duct. Differentiation was based on the presence or absence of ovarian and testicular structures, with females retaining the Müllerian duct and males exhibiting only its remnants (Stoker et al. 2003, Moore et al. 2010) (Figure 2). While adult individuals of the species M. niger exhibit sexual dimorphism in body size, little is known about this characteristic in their hatchlings (Verdade, 2003). Therefore, we also include morphometric data in the analyses of this study.
Photomicrographs (40x magnification) of the Gonad-Adrenal-Mesonephros (GAM) complex of juveniles Melanosuchus niger. A and B represent the GAM of a female, distinguishing the ovary (OV), mesonephros (ME), adrenal (A), mesosalpinx (MS), and Müllerian duct (DM). C and D represent the GAM of a male, distinguishing the testicle (TE), adrenal (A), mesonephros (ME), and the remnant Müllerian duct (DMR). Transverse sections stained with Hematoxylin and Eozin.
The sex ratio of the hatchlings was presented as the percentages of males and females found in nests, in lakes, as well as for the area considering all lakes together. For the general analysis of how the male-female sex ratio differ from 1:1 we used a chi-square test. We also compared total length (cm) and body weight (g) between males and females using the Kruskal-Wallis test, and head length (cm) using the Student’s t-test. Additionally, we tested the relationship between the sex ratio of the hatchlings and the percentage of vegetation cover of each nest. For this, we used a binomial GLM with a quadratic term to capture non-linear effects, expecting extreme values of vegetation cover (colder or warmer due to solar exposure) to favor female hatching, and intermediate values to favor males, consistent with the FMF pattern.
We identified a total of 58% males and 42% females in the 86 analyzed hatchlings from nine nests, with a male-to-female ratio of 25:18, as derived from the 1:1 sex ratio analysis. However, this deviation from a 1:1 sex ratio was not statistically significant (χ² = 2.28, df = 1, p = 0.13). The sex ratio of hatchlings varied between 40-75% males and 25-60% females among lakes, while individual nests showed wider variation, ranging from 0 to 100% for both sexes (Table 1).
Sex ratio of Black Caiman (Melanosuchus niger) hatchlings and vegetation cover above the nest from nine nests located across three lakes in the Mamirauá Sustainable Development Reserve, Central Amazon.
There was a difference in body weight between the sexes of the hatchlings (H = 4.67, p = 0.03), with males (84.1 g ± 6.5 SD) being heavier than females (81.9 g ± 5.6 SD). They also differ by mean total length (H = 5.78, p = 0.01); females had and average and SD of 32.9 ± 1.3 cm and males 33.6 ± 0.9 cm, but not by head length (t = -1.29, p > 0.05; females with 4.5 ± 0.19 cm and males 4.6 ± 0.1 cm) (Figure 3). However, there was no significant relationship between the sex ratio of each nest and vegetation cover above nests (linear term: β = 0.046, p = 0.77; quadratic term: β = -0.00017, p = 0.94).
Morphological traits of Black caiman hatchlings in relation to sex: (A) Mass and (B) Size compared using Kruskal-Wallis test, and (C) Head length compared with Student’s t-test.
Temperature-dependent sex determination (TSD) in crocodilians has been the focus of research for over three decades and is supported by strong evidence (González et al., 2019). Although this study did not include temperature data, the region has faced extreme droughts and rising temperatures in recent years (Fleischmann et al. 2025). We therefore expected a female-biased sex ratio, but our results indicate a relatively balanced proportion of males and females hatched in 2023 at the study site.
The presence of males observed in our sample represents recruitment of potential breeders for the population, maintaining the replacement of individuals harvested. Survival and reproductive success of hatchlings depend on multiple factors, making the annual sex ratio a key metric for management. To support this, ongoing studies are collecting nest temperature data absent from this study. Together, this information contributes to the understanding of the species’ population dynamics and should underpin actions for effective sustainable management and conservation efforts.
ACKNOWLEDGMENTS
The authors thank João da Silva Carvalho (João Jacaré) for his assistance with the captures and the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) for the postdoctoral fellowship awarded to KT through call No. 003/2022 - PRODOC/FAPEAM.
REFERENCES
-
Amazonas. Secretaria de Estado do Meio Ambiente - SEMA. 2011. Resolução CEMAAM nº 008, 27 de junho de 2011. Estabelece critérios para o manejo de jacarés no Estado do Amazonas. Diário Oficial do Estado do Amazonas, Brasil. Available at: https://www.sema.am.gov.br/wp-content/uploads/2024/03/08.-RESOLUCAO-008-11-DOU-MANEJO-DE-JACARE-mesclado.pdf. Acessado em 21 de setembro de 2025
» https://www.sema.am.gov.br/wp-content/uploads/2024/03/08.-RESOLUCAO-008-11-DOU-MANEJO-DE-JACARE-mesclado.pdf. Acessado em 21 de setembro de 2025 - Bull, J.J. 1980. Sex determination in reptiles. The Quarterly Review of Biology 55: 3-21.
- Ferguson, M.W.J.; Joanen, T. 1983. Temperature-dependent sex determination in Alligator mississippiensis Journal of Zoology 200: 143-177.
-
Fleischmann, A.S.; Rabelo, R.; Tregidgo, D.J.; Benitz, T.; Viana, F.M.F.; Franco, D.L.; et al 2025. Unprecedented social-ecological impacts of the 2023 extreme drought in the Central Amazon. EarthArXiv [preprint]. Available at: https://doi.org/10.31223/X5HH88
» https://doi.org/10.31223/X5HH88 - Franco, D.L.; Botero-Arias, R.; Vital, T.W. 2019. Evolução das políticas para o uso sustentável da fauna no Brasil: o caso do manejo comunitário de jacarés no Amazonas. Brazilian Journal of Development 5: 16319-16339.
- González, E.J.; Martínez-López, M.; Morales-Garduza, M.A.; García-Morales, R.; Charruau, P.; Gallardo-Cruz, J.A.; et al 2019. The sex-determination pattern in crocodilians: A systematic review of three decades of research. Journal of Animal Ecology 88: 1417-1427.
- Grigg, G. 2015. Biology and evolution of crocodylians. Clayton: CSIRO Publishing.
- Irion, G.; Junk, W.J.; De Mello, J.A.S.N. 1997. The large Central Amazonian river floodplains near Manaus: geological, climatological, hydrological and geomorphological aspects. In: Junk, W.J. (Ed.). The Central Amazon Floodplain: Ecology of a Pulsing System. Berlin, Heidelberg: Springer-Verlag. p. 23-46. (Ecological Studies; vol. 126).
- Lang, J.W.; Andrews, H.V. 1994. Temperature-dependent sex determination in crocodilians. Journal of Experimental Zoology 270: 28-44.
- López-Luna, M.A.; González-Soberano, J.; González-Jáuregui, M.; Escobedo-Galván, A.H.; Suárez-Domínguez, E.A.; et al 2020. Nest-site selection and nest size influence the incubation temperature of Morelet’s crocodiles Journal of Thermal Biology 91: 102624.
- Marioni, B.; Barão-Nóbrega, J.A.L.; Botero-Arias, R.; Muniz, F.; Campos, Z.; Da Silveira, R.; et al 2021. Science and conservation of Amazonian crocodilians: a historical review. Aquatic Conservation: Marine and Freshwater Ecosystems 31: 1056-1067.
- Moore, B.C.; Hamlin, H.J.; Botteri, N.L.; Lawler, A.N.; Mathavan, K.K.; Guillette, L.J. 2010. Posthatching development of Alligator mississippiensis ovary and testis. Journal of Morphology 271: 580-595.
- Pereira dos Santos, K.R.; Amanajás de Aguiar Júnior, F.C.; Antonio, E.A.; Silva, F.R.; Silva, K.T.; Nascimento Marinho, K.S.; et al 2021. Manual de técnica histológica de rotina e de colorações. Vitória de Santo Antão: BIBCAV/UFPE. 32 p.
- Ramalho, E.E.; Macedo, J.; Vieira, T.M.; Valsecchi, J.; Calvimontes, J.; Marmontel, M.; et al 2009. Ciclo hidrológico nos ambientes de várzea. Uakari 5: 61-87.
- Stoker, C.; Rey, F.; Rodriguez, H.; Ramos, J.G.; Sirosky, P.; Larriera, A.; et al 2003. Sex reversal effects on Caiman latirostris exposed to environmentally relevant doses of the xenoestrogen bisphenol A. General and Comparative Endocrinology 133: 287-296.
- Torralvo, K.; Botero-Arias, R.; Magnusson, W.E. 2017. Temporal variation in black caiman nest predation in várzea of central Brazilian Amazonia. PLoS ONE 12: e0185568.
- Verdade, L. 2003. Cranial sexual dimorphism in captive adult broad-snouted caiman (Caiman latirostris). Amphibia-Reptilia 24: 92-99.
- Villamarín, F.; Marioni, B.; Thorbjarnarson, J.B.; Nelson, B.W.; Botero-Arias, R.; Magnusson, W.E.; et al 2011. Conservation and management implications of nest-site selection of the sympatric crocodilians Melanosuchus niger and Caiman crocodilus in Central Amazonia, Brazil. Biological Conservation 144: 913-919.
Data availability
The data that support the findings of this study were published in this article.
Edited by
-
ASSOCIATE EDITOR:
Rafael de Fraga https://orcid.org/0000-0002-9900-4276
