Do host habitat use and cospeciation matter in the evolution of Oswaldocruzia (Nematoda, Molineidae) from neotropical amphibians?

The genus Oswaldocruzia Travassos, 1917 includes approximately 90 species that are parasitic on amphibians and reptiles around the world, of which 43 occur in the neotropical region. However, molecular data supporting the taxonomic status of most species of the genus are scarce, and their phylogenetic relationships remain unknown. Using specimens of four molineid taxa (Oswaldocruzia belenensis Santos, Giese, Maldonado Jr. and Lanfredi, 2008; Oswaldocruzia chabaudi Ben Slimane & Durette-Desset, 1996, Oswaldocruzia chambrieri Ben Slimane & Durette-Desset, 1996 and Kentropyxia hylae Feitosa, Furtado, Santos and Melo, 2015) from amphibian hosts collected in different regions of Pará, Brazil, we conducted morphological studies, molecular analyses and phylogenies (maximum likelihood and Bayesian inference) using the cytochrome c oxidase subunity I (Cox1) gene. The newly generated sequences were compared with those of ten publicly available Cox1 sequences of Oswaldocruzia from Mexico. Our findings demonstrated significant differences between the sequences of amazonian specimens and sequences from specimens collected in Mexico, and we suggest that host-parasite cospeciation or habitat use might be related to molineid evolution in amphibian hosts. Additionally, this work presents new hosts and new geographical records for species of Oswaldocruzia from the neotropics.

A new species of Metadelphis Travassos, 1944 (Digenea: Dicrocoeliidae) from the gall-bladder of Molossus molossus (Pallas) (Chiroptera: Molossidae) in the Brazilian eastern Amazon.

Metadelphis tkachi n. sp. is described based on material from the gall-bladder of Molossus molossus (Pallas) (Chiroptera: Molossidae) in the Brazilian eastern Amazon. The main differentiating characters of the new species include lobed, tandem testes and elongated clusters of vitelline follicles commencing at the level of the seminal receptacle. The new species can be distinguished from Metadelphis lenti (Santos & Gibson, 1998) and Metadelphis apharyngotrema (Marshall & Miller, 1979) by the shape and position of the testes; from Metadelphis evandroi Travassos, 1944 by having tandem, lobed testes and shorter clusters of vitelline follicles; from Metadelphis compactus Travassos, 1955 by having tandem testes and more elongated clusters of vitelline follicles, and from Metadelphis alverangai Travassos, 1955 by having smaller testes and body, and vitellarium with large follicles. New morphological data are reported for M. lenti based on light and scanning electron microscopy; intraspecific variation was observed.

Ellipsomyxa arariensis n. sp. (Myxozoa: Ceratomyxidae), a new myxozoan parasite of Pygocentrus nattereri Kner, 1858 (Teleostei: Characidae) and Pimelodus ornatus Kner, 1858 (Teleostei: Pimelodidae) from Marajó Island, in the Brazilian Amazon region.

Ellipsomyxa arariensis n. sp. was found in the gallbladder of Pygocentrus nattereri Kner, 1858 and Pimelodus ornatus Kner, 1858 from the Arari River on Marajó Island in Pará, Brazil. The new species has disporous plasmodium that varies in size and shape, with ellipsoidal mature spores in the sutural view that have a curved suture line. The spores are 12.6 (12.0-13.4) µm in length and 7.3 (6.7-8.0) µm in width. The two polar capsules present in the spore are pyriform and of equal size, with subterminal openings that project in opposite directions. The polar capsules are 3.5 (3.4-4.0) µm long and 2.6 (2.5-3.2) µm wide. Based on the partial sequences of the SSU rRNA gene of the Ellipsomyxa arariensis n. sp. spores found in Pygocentrus nattereri Kner, 1858 (1325 bps) and Pimelodus ornatus Kner, 1858 (1240 bps), the new species is clearly distinct from all the other myxozoan sequences deposited in GenBank. Based on Bayesian inference and p distances, the new species belongs to the "Ellipsomyxa clade", together with all the other Ellipsomxa species, reinforcing the monophyletic status of this genus. Overall, the morphological data and the partial sequences of the SSU rRNA gene provide a conclusive diagnosis of Ellipsomyxa arariensis n. sp. as a species distinct from all the other Ellipsomyxa species described previously.

A new species of Kentropyxia Baker, 1982 parasitic in the small intestine of Osteocephalus taurinus Steindachner (Anura: Hylidae) from the Brazilian Eastern Amazon.

Kentropyxia hylae n. sp. is described based on material from small intestine of Osteocephalus taurinus Steindachner from Caxiuanã National Forest, municipality of Melgaço, state of Pará, Brazil. The new species is easily distinguished from the type-species and only species of the genus, Kentropyxia sauria Baker, 1982, by its smaller dimensions, the presence of cervical alae, spicules divided into three parts: a robust, single pointed outer processes ending with small finger-like projections and latero-dorsal and latero-ventral processes branching at final portion of spicule into six main branches, each ending with 14 projections in the latero-dorsal process and 15 projections in the latero-ventral process and by the presence of a middle groove in bursa ray 4. This is the second species parasitic in hylids from the Brazilian Amazon and a new parasite for O. taurinus.

Description of Rhabdias breviensis n. sp. (Rhabditoidea: Rhabdiasidae) in two Neotropical frog species.

Nematodes of the genus Rhabdias Stiles & Hassall, 1905 (Rhabditoidea: Rhabdiasidae) have a dioecious free-living stage and a hermaphroditic stage that parasitises the lungs of amphibians and reptiles. Approximately 94 species of Rhabdias have been described. Because the similar morphological characteristics such as the labial structures, the location of the vulva and the shape of the tail of Rhabdias spp. hinder their identification, molecular biology techniques and scanning electron microscopy have been employed to diagnose species of this genus. This study describes Rhabdias breviensis n. sp., parasitic in the lungs of two Neotropical frog species Leptodactylus petersii Steindachner and Leptodactylus macrosternum Miranda-Ribeiro. The description of this species integrates classical taxonomy, scanning electron microscopy and a molecular analysis of the mitochondrial COI gene. The new species differs from all other Rhabdias species parasitic in Neotropical hosts in certain morphometric parameters, the position of the vulva, the host group and the cephalic characters.

Phylogeny and ultrastructure of Myxobolus rangeli n. sp. (Myxozoa, Bivalvulida), a histozoic parasite in Siluriformes fish from the Amazon region.

A new species of Myxobolus parasitizing the arterial bulb and cardiac musculature of the freshwater fish Pimelodus ornatus Kner, 1858, from the Arari river in the municipality of Cachoeira do Arari, island of Marajó, Pará, Brazil, was described. In the present study, the observed prevalence of myxozoan parasites in the heart tissue of the hosts was 20% (6/30). The myxozoans observed had mature biconvex spores, slightly rounded, an anterior end with two pyriform polar capsules and a posterior end with very evident sporoplasm, measuring 8 ± 0.2 µmin length. The spore width was 5.8 ± 0.4 µm, with a thickness of 3.4 ± 0.2µm. The length of the polar capsules was 3.6 ± 0.3 µm and the width was 1.2 ± 0.2µm, with 6 to 7 turns of the polar filament. The divergences observed, regarding the morphometric and genetic structure of SSU rDNA, in relation to other Myxobolidae already described in the literature, confirm the description of the new species Myxobolus rangeli n. sp.

Human intraocular filariasis caused by Dirofilaria sp. nematode, Brazil.

A case of human intraocular dirofilariasis is reported from northern Brazil. The nematode was morphologically and phylogenetically related to Dirofilaria immitis but distinct from reference sequences, including those of D. immitis infesting dogs in the same area. A zoonotic Dirofilaria species infesting wild mammals in Brazil and its implications are discussed.

Rhabdias paraensis sp. nov.: a parasite of the lungs of Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia.

The nematode parasites of Rhinella marina include species of the genus Rhabdias (Rhabdiasidae: Rhabditoidea). The present study describes Rhabdias paraensis sp. nov., which parasitizes the lungs of R. marina in Brazilian Amazonia. Of the more than 70 known species of this genus, 18 are parasites of bufonids, of which, eight are Neotropical. The new species described here is similar to Rhabdias alabialis in the absence of lips is different by the presence of conspicuous cephalic papillae. We describe details of the four rows of pores, which are distributed equally along the whole of the length of the body and connected with hypodermal cells, using histology and scanning electron microscopy. Other histological aspects of the internal structure of this nematode are also described.

Lanfrediella amphicirrus gen. nov. sp. nov. Nematotaeniidae (Cestoda: Cyclophyllidea), a tapeworm parasite of Rhinella marina (Linnaeus, 1758) (Amphibia: Bufonidae).

The family Nematotaeniidae, tapeworms commonly found in the small intestines of amphibians and reptiles, includes 27 recognised species distributed among four genera: Bitegmen Jones, Cylindrotaenia Jewell, Distoichometra Dickey and Nematotaenia Lühe. The taxonomy of these cestodes is poorly defined, due in part to the difficulties of observing many anatomical traits. This study presents and describes a new genus and species of nematotaeniid parasite found in cane toads (Rhinella marina) from eastern Brazilian Amazonia. The cestodes were collected during the necropsy of 20 hosts captured in the urban area of Belém, Pará. The specimens were fixed and processed for light microscopy, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. Samples were also collected for molecular analyses. The specimens presented a cylindrical body, two testes and paruterine organs. However, they could not be allocated to any of the four existing nematotaeniid genera due to the presence of two each of dorsal compact medullary testes, cirri, cirrus pouches, genital pores, ovaries and vitelline glands per mature segment. Lanfrediella amphicirrus gen. nov. sp. nov. is the first nematotaeniid studied using Historesin analysis, SEM and 3D reconstruction, and it is the second taxon for which molecular data have been deposited in GenBank.

Contracaecum larvae: morphological and morphometric retrospective analysis, biogeography and zoonotic risk in the amazon.

In Brazil there are several records of parasitic nematodes of fish with zoonotic potential, especially those belonging to the family Anisakidae. This study considers the morphology, morphometry and prevalence of Contracaecum in Astronotus ocellatus , fish consumed in the Amazon and sold as ornamental and it also performs a retrospective analysis of the diversity of fish with larvae of Contracaecum, in studies carried out in Brazil over a period of 90 years. 40 specimens of A. ocellatus were necropsied, and the nematodes were collected and fixed in 93 parts 70% ethyl alcohol, 5 parts formaldehyde, and 2 parts glacial acetic acid (AFA) for morphological analysis under light microscopy and scanning electron microscopy. Of the 40 fish collected during this work, 27 were parasitized by Contracaecum larvae with a total intensity of 150 larvae. Retrospective analysis of intermediate host diversity for Contracaecum larvae resulted in 16 orders, 49 families, 96 genera, 140 species and a hybrid morphotype. In the retrospective study, half of the fish were from freshwater, with the order Perciformes being the most representative, with 16 families, 30 genera and 37 species. In Brazil, the occurrence of larvae of Contracaecum in fish was reported in 15 of the 26 states, with Rio de Janeiro presenting the most information regarding fish harboring Contracaecum larvae.

Occurrence of Dipetalonema gracile in a wild population of woolly monkey Lagothrix poeppiigii in the northeastern Peruvian Amazon.

Dipetalonema gracile (Rudolphi, 1809) (Filarioidea: Onchocercidae) is one of six species of cavities filarial parasites of Neotropical non-human primates. The present study recorded the occurrence of D. gracile, provides morphological and morphometric data and extends the geographical distribution. Adult filariae were obtained from the thoracic and abdominal cavities of 38 specimens of woolly monkey, which were used for local human consumption, in the northeastern Peruvian Amazon. Male and female filarids were processed and analysed using light and scanning electron microscopy. Details of the cephalic papillae, post-cloacal bands and papillae, vulva, phasmid position and lateral appendages are showed by scanning electron microscopy and is recorded the occurrencce of Lagothrix poeppigii monkey as a new host of this filaria in the Yavari-Mirin river basin, Peruvian Amazon.

Canine filariasis in the Amazon: Species diversity and epidemiology of these emergent and neglected zoonoses.

Dirofilaria immitis and Acanthocheilonema reconditum are common parasites in dogs but have also been reported parasitizing humans. The differential diagnosis and epidemiological evaluation of these zoonoses are important to the development of efficient public health policies and control strategies. The purpose of this study was to carry out an epidemiological survey by using molecular methods for the specific identification of filarid parasites of domestic dogs in the Marajó mesoregion, State of Pará (PA), Brazil. A total of 418 canine blood samples from Marajó mesoregion (Northern Brazil) were collected, submitted to DNA extraction, polymerase chain reaction (PCR) with "pan filarial" primer, subsequent sequencing and sequence analysis using BLASTn software comparison with previously deposited sequences in GenBank. After that, a phylogenetic analysis by Maximum Parsimony was performed to aid the specific diagnosis. The obtained sequences showed the occurrence of 9 (2.15%) dogs infected with D. immitis and 30 (7.18%) by A. reconditum, with a confidence interval of 95%, there were no cases of co-infection. We observed that male dogs were more likely to D. immits and A. reconditum infection. However, age was not significant to both infections. This study reports for the first time the occurrence of A. reconditum in the northern region of Brazil and confirmed the presence of D. immitis in the Marajó mesoregion.

Biochemical properties of the major proteins from Rhodnius prolixus eggshell.

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.

Choledocystus elegans (Digenea: Plagiorchiidae) of Leptodactylus paraensis (Amphibia: Leptodactylidae) from the Brazilian Amazon.

The trematodes are parasites of the several vertebrates including amphibians, however the knowledge about of the taxonomy these parasites is still confuse. The trematode Choledocystus elegans was found in the small intestine of the Leptodactylus paraensis in eastern Amazon and presents the following characteristics: several pointed tegumentary spines, papillae on the outer and inner edges of the oral and ventral suckers, a round, well-developed cirrus sac, a well-developed cirrus, oblique testicles, a ovary right side, uterine loops extending between the testicles, follicular vitellaria distributed throughout the body, starting at the genital pore region and caeca close the end of the body. For the first time, this study identified C. elegans parasitizing L. paraensis and describes morphological aspects never characterized using light and scanning electron microscopy.

Rhinella marina (Amphibia: Bufonidae) Versus Rhabdias paraensis (Nematoda: Rhabdiasidae): Expanding the View on a Natural Infection.

Amphibian and reptile lungs are frequently infected with Rhabdias parasites, and this condition ultimately leads to reduced survival, performance, and growth because of granulomatous inflammation, nodule formation, and nematodal pneumonia onset. Here we investigate the histopathological features of naturally infected Rhinella marina by the lung nematode Rhabdias paraensis. A total of 10 host animals were captured in peridomiciliar areas in the eastern Brazilian Amazon, and anatomic-histological analyses were performed on both the infected and non-infected lungs of these amphibians. Helminths were usually found within the secondary and primary septa of infected lungs whereas parasites were not detected within vessels or adhering to tissues. In addition, we observed discrete erythrocytes, diapedesis foci, few granulocytes and erythrocytes in the interseptal spaces, discrete cell infiltration, and a small number of melanomacrophages, and no granulomas or cysts were observed. New aspects related to changes in tissue and helminth-host interactions are discussed for the relationship of R. paraensis × Rhi. marina from the Amazon region.

Morphological characterization of Eustrongylides sp. larvae (Nematoda, Dioctophymatoidea) parasite of Rhinella marina (Amphibia: Bufonidae) from Eastern Amazonia.

Absctract Eustrongylides spp. nematodes have birds as final hosts and uses other vertebrates as intermediate/paratenic host (fish, amphibians and reptiles) and have zoonotic potential. In amphibians, the larvae may be located in the subcutaneous tissues, liver and mesentery, between the muscle fibres, especially in the lower limbs. Rhinella marina, which is widely observed in Brazil, has exhibited complex diversity in its helminth fauna, reflecting the unique habitat of the Amazon biome. For the first time, this study describes the morphological aspects of third-stage larvae of Eustrongylides sp. in Rhinella marina from Santa Cruz do Ararí, Marajó Archipelago, Eastern Amazonia, using light and scanning electron microscopy.

Squamasnema amazonica n. gen. n. sp. (Heligmonellinae): A new parasite of Proechimys roberti (Rodentia: Echimyidae) in the Brazilian Amazon.

A new species of nematode, Squamasnema amazonica n. gen. n. sp., is described based on specimens found parasitizing the small intestine of Proechimys roberti (Rodentia: Echimyidae) collected during a survey of the fauna of Tapirapé-Aquirí National Forest (Brazil, Eastern Brazilian Amazon). The nematodes were fixed and processed for light microscopy and scanning electron microscopy (SEM). These nematodes were classified under the family Heligmonellidae and the subfamily Heligmonellinae. Although several species in the family Heligmonellidae exhibit discontinuous ridges, Squamasnema n. gen. and Trichotravassosia are the only genera with columns of scales along their entire body, as an apomorphy of the synlophe. Squamasnema n. gen. has columns of cuticular cells along its body, except for on the left flank, and exhibits a synlophe with no size gradient or inclination and does not present chitinized structures supporting the synlophe. Therefore, due to these morphological differences of Squamasnema n. gen., the creation of a new genus was necessary.

Proteomic profile of Ortleppascaris sp.: A helminth parasite of Rhinella marina in the Amazonian region.

Ortleppascaris sp. is a helminth that, in its larval stage, infects the liver parenchyma of the amphibian Rhinella marina, resulting in severe physiological and pathological changes. This study used a proteomic approach to determine the overall profile of proteins expressed in a somatic extract from the nematodes to investigate the relationship between the parasite and its host. A total of 60 abundant proteins were selected from the two-dimensional electrophoresis, identified by peptide mass fingerprinting, and grouped based on their Gene Ontology by the biological processes in which they are potentially involved. Important helminthic derivatives, such as the immunoreactive As37 antigen, guanylyl cyclases, proteolytic enzymes, and other proteins conserved among different parasites, were identified through homology. This study represents a new approach to helminth-related proteomic studies using an amphibian animal model. Furthermore, this study identified protein markers that are important to the host-parasite relationship and the viability, development, infectivity, and virulence of helminths.

Ortleppascaris sp. and your host Rhinella marina: A proteomic view into a nematode-amphibian relationship.

The success of the helminth-host relationship depends on a biochemical molecular arsenal. Perhaps the proteome is the largest and most important set of this weaponry, in which the proteins have a crucial role in vital processes to the parasite/host relationship, from basic metabolism and energy production to complex immune responses. Nowadays, the bioproducts expressed by the parasites are under the "spotlight" of immunoassays and biochemical analysis in helminthology, especially in proteomic analysis, which has provided valuable information about the physiology of the infecting agent. Looking into this point of view, why not turn to the infected agent as well? This study characterised the proteomic profile of fluid-filled fibrous cysts of encapsulated Ortleppascaris sp. larvae in the hepatic parenchyma of their intermediate host, the amphibian Rhinella marina. The proteins were separated by two-dimensional electrophoresis and identified by MS with the aid of Peptide Mass Fingerprint. A total of 54 molecules were analysed in this system, revealing a complex protein profile with molecules related to basic metabolic processes of the parasite, energy production, oxi-reduction and oxidative stress processes as well as molecules related to the host response. This study contributes to proteomic studies of protein markers of the development, infectivity, virulence and co-existence of helminths and their hosts.

Contracaecum larvae: morphological and morphometric retrospective analysis, biogeography and zoonotic risk in the amazon / Larva de Contracaecum: análise retrospectiva morfológica e morfométrica, biogeografia e risco zoonótico na amazônia

Abstract In Brazil there are several records of parasitic nematodes of fish with zoonotic potential, especially those belonging to the family Anisakidae. This study considers the morphology, morphometry and prevalence of Contracaecum in Astronotus ocellatus , fish consumed in the Amazon and sold as ornamental and it also performs a retrospective analysis of the diversity of fish with larvae of Contracaecum, in studies carried out in Brazil over a period of 90 years. 40 specimens of A. ocellatus were necropsied, and the nematodes were collected and fixed in 93 parts 70% ethyl alcohol, 5 parts formaldehyde, and 2 parts glacial acetic acid (AFA) for morphological analysis under light microscopy and scanning electron microscopy. Of the 40 fish collected during this work, 27 were parasitized by Contracaecum larvae with a total intensity of 150 larvae. Retrospective analysis of intermediate host diversity for Contracaecum larvae resulted in 16 orders, 49 families, 96 genera, 140 species and a hybrid morphotype. In the retrospective study, half of the fish were from freshwater, with the order Perciformes being the most representative, with 16 families, 30 genera and 37 species. In Brazil, the occurrence of larvae of Contracaecum in fish was reported in 15 of the 26 states, with Rio de Janeiro presenting the most information regarding fish harboring Contracaecum larvae.

Resumo No Brasil existem vários registros de nematóides parasitos de peixes com potencial zoonótico, especialmente aqueles pertencentes à família Anisakidae. Este estudo considera a morfologia, morfometria e prevalência de Contracaecum em Astronotus ocellatus, peixe consumido na Amazônia e vendido como ornamental, e também realiza uma análise retrospectiva da ictiofauna parasitada com larvas de Contracaecum, em estudos realizados no Brasil, durante um período de 90 anos. 40 espécimes de A. ocellatus foram necropsiados, e os nematóides foram coletados e fixados em 93 partes de álcool etílico 70%, 5 partes de formaldeído e 2 partes de ácido acético glacial (AFA) para análise morfológica em microscopia de luz e microscopia eletrônica de varredura. Dos 40 peixes coletados durante este trabalho, 27 estavam parasitados por Contracaecum, com intensidade total de 150 larvas. Análises retrospectivas da diversidade de hospedeiros intermediários para larvas de Contracaecum resultaram em 16 ordens, 49 famílias, 96 gêneros,140 espécies e um morfotipo híbrido. No estudo retrospectivo, metade dos peixes eram de água doce, sendo a ordem Perciformes a mais representativa, com 16 famílias, 30 gêneros e 37 espécies. No Brasil, a ocorrência de larvas de Contracaecum em peixes foi relatada em 15 dos 26 estados, com o Rio de Janeiro apresentando mais informações sobre peixes portadores de larvas de Contracaecum.