Noronha , 1970 ( Nematoda , Diplotriaenidae ) in Pitangus sulphuratus ( Linnaeus , 1766 ) ( Passeriformes , Tyrannidae ) from southern Brazil

Diplotriaena delirae Pinto & Noronha, 1970 is known to parasitize Pitangus sulphuratus (Linnaeus, 1766) in Peru and in the Midwestern and Southeastern regions of Brazil. Here, specimens of P. sulphuratus were collected in the southern state of Rio Grande do Sul, Brazil, and necropsied. Nematodes (n = 6) found in these specimens were identified as D. delirae based on their morphological traits. This is the first report of D. delirae from southern Brazil, expanding the knowledge of the helminth fauna of P. sulphuratus in the Neotropical region.

Pitangus sulphuratus is endemic to the New World, where it is distributed from southern Texas, USA, south-ward as far as Argentina (Sick 1997).This tyrannid has an omnivorous diet, feeding on fruit, invertebrate prey such as arachnids, coleopterans, dipterans, lepidopterans, hymenopterans, and small crustaceans (Argel- de-Oliveira et al. 1998), as well as vertebrates, including amphibians and fish (Andrade 1997), and processed human foods.

Methods
In 2015 and 2016, a research team from the Wild Animal Rescue Center (CETAS) of the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) retrieved 4 specimens of P. sulphuratus from the margins of the RS-040 state highway between the towns of Viamão (30°08ʹ08.0ʺS, 050°51ʹ27.5ʺW) and Capivari do Sul (30°09ʹ11.5ʺS, 050°26ʹ45.9ʺW), in the state of Rio Grande do Sul, Brazil.The bird specimens were taken to the laboratory of invertebrate zoology at the Brazilian Lutheran University (ULBRA) in Canoas for necropsy.The nematodes found in the specimens were fixed in A.F.A. (70° GL ethanol; formalin 37%; glacial acetic acid) at 65 °C for 48 h and then stored in 70° GL ethanol (Amato and Amato 2010).The internal structures were visualized in specimens mounted temporarily and cleared using Amann's lactophenol (Humason 1979).
The species was identified based on the morphological traits and morphometry of the specimens (Pinto and Noronha 1970, Vicente et al. 1983b, 1995).Nematode systematics followed De Ley and Blaxter (2002).The measurements were obtained under a light microscope with an ocular micrometer and are given in micrometers (µm), unless otherwise indicated.In the text, these measurements are presented as the range followed (between parentheses) by the mean, and standard deviation.The ecological terminology (prevalence, mean intensity, and mean abundance of infections) follows Bush et al. (1997).Photomicrographs were taken using a Nikon Coolpix S3300 camera attached to the microscope.Voucher specimens of the nematode were deposited in the Helminthological Collection (CHMU) in the ULBRA Natural Sciences Museum in Canoas, Rio Grande do Sul, Brazil.New records.Brazil: Rio Grande do Sul state: Municipality of Capivari do Sul (30°09ʹ11.5ʺS, 050°26ʹ45.9ʺW) (Fig. 2).Host: Pitangus sulphuratus (Linnaeus, 1766).Site of infection: coelomic cavity.Collection of parasites: Identification.Nematodes with slender body and transversely striated cuticle.Rudimentary buccal capsule.Anterior extremity with two chitinous structures in trident shape with little apparent roughness, tapering finely toward the anterior extremity.Esophagus divided into 2 portions, glandular portion longer than the muscular portion.No excretory pore observed.
The identification of the species of the genus Diplotriaena Railliet & Henry, 1909 is based primarily on the shape and size of the tridents and spicules (Vicente et al. 1983b(Vicente et al. , 1995)).Diplotriaena delirae is closely related to Diplotriaena attenuata-verrucosa (Molina, 1858) Henry & Ozoux, 1909, Diplotriaena henryi Blanc, 1919, and Diplotriaena zederi Pinto, Vicente & Noronha, 1981 due to its rough trident.However, D. delirae is closest to D. henryi due to its less apparent roughness, but can be distinguished from this species by the presence of tridents of more than 0.21 mm in length.

Discussion
The specimens examined here presented morphological measurements generally similar to those reported for D. delirae in previous studies (Pinto and Noronha 1970, Vicente et al. 1983b, Hon et al. 2013).While the measurements of our specimens were closest to those recorded by Hon et al. (2013), they are generally smaller than those reported by Pinto and Noronha (1970) and Vicente et al. (1983b) (Table 1).
Previous reports of D. delirae include the following hosts and localities (Fig. 2): Myiarchus tyrannulus (Statius Müller, 1776) from Jaén province, Peru (Hon et al. 2013), and P. sulphuratus from the Brazilian towns of Angra dos Reis, in Rio de Janeiro state (Pinto and Noronha 1970), Linhares in Espírito Santo, and Salobra in Mato Grosso do Sul (Vicente et al. 1983a).This is the first report of D. delirae parasitizing P. sulphuratus in Rio Grande do Sul, extending the distribution of D. delirae to the southernmost state of Brazil.
During its life cycle, the female D. delirae produces eggs that enter the air sacs of the bird host, and subsequently migrate to the throat and then the feces, where they reach the environment (Chabaud 1955, Anderson 2000).The eggs in the feces are ingested by insects, possibly the intermediate hosts, which are the prey of bird hosts in which the parasite completes its development (Anderson 2000).Given these features of the parasite's life cycle, the presence of D. delirae in P. sulphuratus reflects its omnivorous diet under natural conditions, which includes insects (Argel-de-Oliveira et al. 1998).
The microhabitat of the adult D. delirae is the air sacs of its birds hosts, although most studies have identified erroneously the coelomic cavity as the site of infection (Anderson 2000).The presence of D. delirae in the coelomic cavity of the specimens of P. sulphuratus examined by us is likely due to the impact of the vehicles that collided with the birds on the highway.The prevalence of D. delirae was 25%, with a mean abundance of 1.5 helminths per host.The study of the helminths collected from animals killed by traffic can provide important insights into the composition of the parasitic fauna of a given region, as well as aspects of the relationship with hosts and the life cycle of the parasite in specific environments.Despite this potential, the analysis of the helminth fauna of roadkill specimens is limited by the typically small numbers of specimens collected from this source.
Animals killed by traffic may provide a good source of specimens for the analysis of the helminth fauna of wild vertebrates, including endangered species (Gallas et al. 2014), and the systematic retrieval of carcasses should be stimulated as an alternative strategy for the collection of specimens without the need to sacrifice the hosts.Parasitological research provides important insights into the quality of the environment, which contributes to the development of effective conservation measures for wild species and the environments they inhabit (Lafferty 1997).

Figure 2 .
Figure 2. Map showing known geographic distribution (A-D) and the new record (E) of Diplotriaena delirae Pinto & Noronha, 1970. A. Province of Jaén, Peru.B. Linhares, state of Espírito Santo, Brazil.C. Salobra, state of Mato Grosso do Sul, Brazil.D. Angra dos Reis, state of Rio de Janeiro, Brazil.E. Capivari do Sul, state of Rio Grande do Sul, Brazil.