Introduction
Ascaridoidea (sensu Hodda, Reference Hodda2022) is a widespread superfamily of nematodes comprising gastrointestinal parasites infecting mammals, including humans, birds, reptiles, amphibians, and fish (Hartwich, Reference Hartwich, Anderson, Chabaud and Willmott2009). This taxon probably emerged in the Carboniferous parasitizing terrestrial tetrapods and, subsequently, moving to aquatic environments and infecting other hosts like bony and cartilaginous fishes (Li et al., Reference Li, lü, Nadler, Gibson, Zhang, Chen, Zhao and Guo2018).
The Brazilian fossil record related to ascaridoids includes mostly eggs found in ancient remains collected in paleontological and archeological sites and, even though the egg morphology is rather uniform and unspecific, some species have been proposed based on these immature forms. For example Ascarites rufferi, was described from an egg found in a coprolite of Cynodontia, the taxonomic bridge between reptiles and mammals, which was recovered in the Santa Maria Formation, Paraná Basin, Brazil (Silva et al., Reference Silva, Borba, Dutra, Leles, Da-Rosa, Ferreira and Araujo2014). Bauruascaris cretacicus and B. adamantinensis were proposed based on eggs found in coprolites of Crocodilomorpha from the Adamantina Formation (Bauru Group, Brazil) (Cardia et al., Reference Cardia, Bertini, Camossi and Letizio2018; Reference Cardia, Bertini, Camossi and Letizio2019). In addition, Poinar & Boucot (Reference Poinar and and Boucot2006) described the species A. priscus and A. gerus from eggs in a single dinosaur coprolite. Ascaridoid parasitic forms have been more frequently reported in Cenozoic ancient remains dated from the Holocene (Gonçalves et al., Reference Gonçalves, Araújo and Ferreira2003; Leles et al., Reference Leles, Araújo, Ferreira, Vicente and Iñiguez2008; Camacho et al., Reference Camacho, Pessanha, Leles, Dutra, Silva, Souza and Araujo2013; Jaeger et al., Reference Jaeger, Taglioretti, Dias and Iñiguez2013a, Reference Jaeger, Taglioretti, Fugassa, Dias, Neto and Iñiguez2013b; Sianto et al., Reference Sianto, Souza, Chame, Luz, Guidon, Pessis and Araújo2014; Guedes et al., Reference Guedes, Borba, Camacho, Neto, Dias and Iñiguez2020; Iñiguez et al., Reference Iñiguez, Brito, Guedes and Chaves2022).
The Tremembé Formation (Oligocene of the Taubaté Basin), aged between 33.9 and 23.03 million years ago, is an intercalation between shale and clay layers with lacustrine origin, and it has become an important source for paleoparasitological studies in avian coprolites, providing different well-preserved morphotypes of helminth eggs, protozoan cysts, and non-sporulated oocysts (Carmo et al., Reference Carmo, Garcia, Vieira, Lima, Araújo-Júnior and Pinheiro2023; Carmo et al., Reference Carmo, Berto, Pereira, Lima, Araújo-Júnior and Pinheiro2024a). Currently, the helminth eggs from this geological unit have been reported only at a single cell stage of embryonic development (Carmo et al., Reference Carmo, Garcia, Vieira, Lima, Araújo-Júnior and Pinheiro2023). Here, we report the first egg of Ascaridoidea preserved containing morula, from a single bird coprolite recovered from the Paraíba Valley, as well as discuss some insights into the potential of its lower taxon of origin.
Materials and methods
Three coprolites were recovered from sedimentary rocks of the Tremembé Formation, Taubaté Basin, in 2023 at the outcrop of Aligra Comércio de Argila S/A, Taubaté municipality (State of São Paulo). Samples were rehydrated in 10 mL of 0.5% trisodium phosphate (Na3PO4) solution for 72 h (Callen, Reference Callen, Macneish and Byers1967; Fugassa et al., Reference Fugassa, Araújo and Guichón2006) and subjected to spontaneous sedimentation for 24 h (Hoffman et al., Reference Hoffman, Pons and Janer1934). A drop of sediment was placed on a glass slide, together with a drop of glycerine, covered with a coverslip (Ferreira et al., Reference Ferreira, Reinhard and Araújo2014), and observed using a light microscope Nikon Eclipse Ei with image capture system PrimeCam Intervision 12. A total of 20 slides were mounted for each sample of coprolite. Parasitological identification was performed based on morphology and morphometry (Ferreira et al., Reference Ferreira, Reinhard and Araújo2014); because there is no general literature for taxonomic identification of nematode eggs, the present identification was based on the expertise of the authors and comparisons with specific literature (see results and discussion for details). The avian origin of the coprolite was interpreted based on criteria adopted by Castro et al. (Reference Castro, Fernandes and Carvalho1988), Souto (Reference Souto2017), and Carmo et al. (Reference Carmo, Garcia, Vieira, Lima, Araújo-Júnior and Pinheiro2023). Two females of Contracaecum Railliet & Henry, 1912 (Ascaridoidea, Anisakidae) were collected from a single bird of the species Nannopterum brasilianum, and their uteri were dissected to obtain eggs that were used for comparison with the fossilized material. The genus Contracaecum was identified based on the following criteria: three well-developed labia, excretory pore at the base of subventral labium, presence of ventriculus with posterior appendix and intestinal caecum (Hartwich, Reference Hartwich, Anderson, Chabaud and Willmott2009; Gibbons, Reference Gibbons2010; Supplementary Figure S1). We also provided an micrography of an eimeriid oocyst (Fig. 1B), found by our research group in a previously analyzed sample, from the same geological formation, for comparisons.
Results and discussion
Coprolites were ovoid, grayish-yellow in colour, 10.80- to 28-mm long and 6.93- to 16.30-mm wide, containing fragmented remains of fishes (Fig. 1A). Based on morphological and morphometrical features, diet remains, and zoopaleontological context, the samples were assigned to piscivorous birds (Avialae, Neornithes). Some bird species may have benefited from periodic fish mortality because of seasonality (dry and wet seasons), which possibly supported the great success of this feeding habit in the Paraíba Valley during the Oligocene (Olson & Alvarenga, Reference Olson and Alvarenga2002).
Avian coprolite with fish bone fragments (A), eimeriid oocyst (B), Contracaecum egg (C), and fossilized egg of Ascaridoidea (C) from the Tremembé Formation, Taubaté Basin, State of São Paulo, Brazil.
A single slide from a single coprolite was positive for the parasite form, which was represented as a spherical egg, measuring 34.21 × 32.44 μm, containing morula, translucent, with somewhat thick and ornamented shell (Fig. 1D). The identification of immature parasitic forms (i.e., eggs and larvae), preserved without adult worms or genetic material, is rather difficult and generalist because these forms hold ancestral ontogenetic traces that are common within a higher taxa (i.e., family, order), show homogeneous morphology and lack specific traits (Carmo et al., Reference Carmo, Lima, Araújo-Júnior and Pereira2024b). Nevertheless, the egg exhibited morphological features typical of the Ascaridoidea nematodes (i.e., the shell with ornamentations [resembling a mammillated layer] and the pronounced spherical form). These characteristics are especially common in eggs from representatives of Ascarididae, Anisakidae, and Raphidascarididae, all belonging to Ascaridoidea (Koie & Fagerholm, Reference Koie and Fagerholm1995; Anderson, Reference Anderson2000; Carrera-Játiva et al., Reference Carrera-Játiva, Rodríguez-Hidalgo, Sevilla and Jiménez-Uzcátegui2014; Carvalho, Reference Carvalho, Santana, Gonçalves, Pinheiro and Giese2020).
The family Anisakidae was of particular interest in the present context because it includes some parasites that use piscivorous birds as definitive hosts and fish as intermediate or paratenic hosts, as well as produce eggs with similar morphometry and morphology as that found in the present study (Koie & Fagerholm, Reference Koie and Fagerholm1995; Anderson, Reference Anderson2000; Carrera-Játiva et al., Reference Carrera-Játiva, Rodríguez-Hidalgo, Sevilla and Jiménez-Uzcátegui2014; Carvalho, Reference Carvalho, Santana, Gonçalves, Pinheiro and Giese2020). The present egg was quite similar to those reported by Carrera-Játiva et al. (Reference Carrera-Játiva, Rodríguez-Hidalgo, Sevilla and Jiménez-Uzcátegui2014), and mainly to the morulated stages reported by Huizinga (Reference Huizinga1967), all assigned to the genus Contracaecum.
The Contracaecum eggs (54.36 – 61.27 × 43.82 – 50.04 μm; Fig. 1C) recovered from the dissected females were found in an initial stage of development, before morulation, similar to those reported by Huizinga (Reference Huizinga1967). Although the stages of development from these eggs were different, they showed morphological similarities such as an oval to spherical shape and an ornamented and relatively thinned shell that is typical from nematodes that have aquatic life cycles. Moreover, differences observed in the egg size between the fresh and the fossilized materials may be accounted for by intrinsic features of females from each species. According to Herreras et al. (Reference Herreras, Montero, Marcogliese, Raga and Balbuena2007), spatial constrictions imposed by uterine size may influence the number and size of eggs produced by females of anisakid nematodes.
Although the parasitic form reported here may have a size similar to that of some oocysts from Eimeriidae, previously found in the Tremembé Formation (Carmo et al., Reference Carmo, Berto, Pereira, Lima, Araújo-Júnior and Pinheiro2024a; Fig. 1B), it lacks common traits such as micropyle and micropyle cap. Moreover, we believe that the internal embryonic structure represents morula rather than developing sporocysts. In fact, more than 10 oocyst morphotypes were described in the Tremembé Formation, and none had preserved sporocysts or sporozoites (Carmo et al., Reference Carmo, Berto, Pereira, Lima, Araújo-Júnior and Pinheiro2024a). In this sense, sporocysts or sporozoites most likely degrade before feces fossilization in this paleoenvironment, or the abiotic conditions essential of sporulation (e.g., heat, humidity, oxygenation) were absent (Fayer, Reference Fayer1980; Berto et al., Reference Berto, Mcintosh and Lopes2014).
Fossilization is a singular phenomenon that takes place under specific conditions, which partially explains the lack of parasitological studies on material found in paleontological sites (Dentzien-Dias et al., Reference Dentzien-Dias, Poinar, Figueiredo, Pacheco, Horn and and Schultz2013; Carmo et al., Reference Carmo, Lima, Araújo-Júnior and Pereira2024b). Although we found only one egg in the present coprolites, the record of a morula reveals good potential for preservation of parasitic forms in the Tremembé Formation. In addition, the identification of the Ascaridoidea egg provides insights on the biogeographic distribution of this taxon, in the Brazilian territory, during the Oligocene.
The paleoparasitological information associated with the paleofaunistic diversity of birds and fishes from the Tremembé Formation (Carmo et al., Reference Carmo, Lima, Araújo-Júnior, Pinheiro, Melo and Couto-Ribeiro2024c) indicate that the ancient Brazilian paleoenvironments provided conditions for the rising and success of nematodes infecting such hosts during the Paleogene.
Previous paleoparasitological studies from the Tremembé Formation were performed in coprolites exclusively from the site Fazenda Santa Fé (Carmo et al., Reference Carmo, Garcia, Vieira, Lima, Araújo-Júnior and Pinheiro2023). Therefore, this is the first parasitological analysis in trace fossils from the outcrop of Aligra Comércio de Argila S/A, providing new perspectives related to the paleoparasitological studies in this lithostratigraphic unit, and expanding the knowledge on helminth infections in birds that inhabited the Paraiba Valley millions of years ago.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000610.
Conflict of interest
The authors declare no conflicts of interest.
Author contribution
G.M.d.C.: conceptualization, methodology, investigation, writing-original draft, writing-review & editing, funding acquisition. A.H.d.S.G.L.: methodology, investigation, writing-original draft. J.F.P.: writing-original draft. S.d.S.L.: validation, resources, supervision, funding acquisition. H.I.d.A.-J.: validation, resources, supervision. F.B.P.: validation, resources, supervision, funding acquisition.
Funding
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.
