Milkweed Bug, Oncopeltus Fasciatus

Transovum Transmission of Trypanosomatid Cysts in the Milkweed Bug, Oncopeltus fasciatus Felipe de Almeida Dias1, Luiz Ricardo da Costa Vasconcellos2, Alexandre Romeiro3, Marcia Attias3, Thais Cristina Souto-Padro´ n2, Angela Hampshire Lopes2* 1 Instituto de Bioquı´mica Me´dica Leopoldo de Meis, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, 2 Instituto de Microbiologia Paulo de Goés, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, 3 Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Abstract Leptomonas wallacei is a trypanosomatid that develops promastigotes and cystic forms in the gut of the hemipteran insect Oncopeltus fasciatus. Insect trypanosomatids are thought to be solely transmitted from one host to another through the ingestion of parasite-contaminated feces. However, here we show that L. wallacei cysts present on the eggshells of eggs laid by O. fasciatus can also act as infective forms that are transmitted to the insect offspring. Newly hatched O. faciatus nymphs are parasite-free, but some of them become contaminated with L. wallacei after feeding on eggshell remnants. The present study is the first report of transovum transmission of a trypanosomatid, a process that may have a relevant role in parasite’s within-host population dynamics. Citation: Dias FdA, Vasconcellos LRdC, Romeiro A, Attias M, Souto-Padroń TC, et al. (2014) Transovum Transmission of Trypanosomatid Cysts in the Milkweed Bug, Oncopeltus fasciatus. PLoS ONE 9(9): e108746. doi:10.1371/journal.pone.0108746 Editor: Kostas Bourtzis, International Atomic Energy Agency, Austria Received May 24, 2014; Accepted August 25, 2014; Published September 26, 2014 Copyright: ß 2014 Dias et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundaçaõ Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Nacional de Cieˆncia e Tecnologia em Entomologia Molecular (INCTEM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction of these insects with their natural [7,8] or experimental trypanosomatid parasites [16–19]. The family Trypanosomatidae is known by the severe human There are basically two forms of transmission of endoparasites diseases caused by some of its species, which kill several thousand among insects: vertical, when the parasite is transferred from the people yearly. Roughly 37 million people worldwide are infected parent to its progeny, and horizontal, when the transfer occurs with Trypanosoma brucei (African sleeping sickness), Trypanosoma between two individuals, either host to host, or host to cruzi (Chagas disease) and Leishmania species (different forms of environment and then to host [20–22]. The vertical transmission leishmaniasis) [1]. Trypanosomatids are evolutionarily successful is subdivided into two forms: transovarial and transovum, in which organisms, parasitizing a broad range of invertebrates, vertebrates, the parasites are present within the eggs or on the eggshells, plants [2] and even other protozoans [3]. Sixty percent of respectively [20,23,24]. In the latter scenario, the parasites are trypanosomatid genera comprise of monoxenous insect parasites acquired by the newly hatched nymphs or larvae by feeding on and the remainder forty percent comprise mostly of vertebrate eggshell remnants [24]. Transmission and virulence of parasites parasites transmitted by insects [4]. Nevertheless, there are very are decisive factors that determine host-parasite relationships. few reports on the life cycles of insect trypanosomatids [5]. Vertical transmission demands precise integration of the parasite Oncopeltus fasciatus is a natural host of Leptomonas wallacei, with the host biological functions and may be an exceptional which is a gut-restricted insect trypanosomatid [6–8]. L. wallacei predictor to ecological host specificity [20–22]. In migratory develops free-swimming promastigotes, mainly in the midgut, and insects, such as O. fasciatus [11], vertical transmission may be promastigotes attached to the intestine wall, mostly in the hindgut crucial to the persistence of parasites in host populations [25]. [6–8]. Promastigotes often display encysting stages (straphangers) The vertical transmission of trypanosomatids among inverte- adhered to the flagellum; these cystic resistant forms are frequently brate hosts has been proposed but so far not confirmed [26,27]. found in clusters of two to four. Free, mature cysts can also be The main goal of the present study was to experimentally show the found in the lumen of the intestinal tract, especially in the hindgut existence of transovum transmission of L. wallacei by O. fasciatus. [6–8]. For the first time, we demonstrated that trypanosomatid cysts may The milkweed bug O. fasciatus is a hemipteran insect, which be vertically transmitted from insect females to their offspring has been an important model for classical studies on embryogen- through contamination of the egg surface with feces that contains esis [9–11]. More recently, O. fasciatus has become a laboratory cysts, and subsequent ingestion of these feces by newly hatched model for reports on molecular development of insects [12,13], nymphs. Therefore, we believe that the differentiation of L. transcriptomes [14,15], as well as several aspects of the interaction wallacei promastigotes to cystic forms and the vertical transmission PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e108746 Transovum Transmission of Trypanosomatid Cysts in Oncopeltus fasciatus of parasites to the host offspring may be connected and working together for the success of parasitism. Materials and Methods SEM Oncopeltus fasciatus colony 8.67 ± A colony of naturally infected with [7] 5.77 2.2 O. fasciatus L. wallacei 0 6 6 6 6 was established and maintained in our laboratory under a 12 h mean of infection rate light/dark cycle at 28uC with 70–80% relative humidity, as previously described [6,18]. Establishment of a parasite-free colony In order to obtain a parasite-free colony, eggs collected from the infected colony were submitted to surface asepsis. The asepsis was infection rate (%) performed by treatment of the eggs with 2% sodium hypochlorite for 5 min, which was followed by washing the eggs in distilled sterile water and drying on sterile filter paper. After asepsis, the eggs were kept in sterile plastic containers and the newly-hatched insects maintained in the same conditions described for the not infected parental colony. In order to avoid recontamination of the parasite- free colony, these insects have been kept in a different, isolated, room from the parental colony. In order to validate the absence of females. L. wallacei in the parasite-free colony, samples of five insects Oncopeltus fasciatus infection rate (%) infected -infected not infected Leptomonas wallacei infection rate (%) infected not infected Groups (30 insects analyzed per group) Group 1* Group 2* Group 3* Figure 1. Scanning electron microscopy of Oncopeltus fasciatus guts infected with Leptomonas wallacei. (A) Midgut of L. wallacei- infected O. fasciatus. The arrows indicate large numbers of parasites near the midgut wall. (B) Hindgut of L. wallacei-infected O. fasciatus. The Infection rates of insects hatched from eggs laid by image shows a massive presence of flagellates. Most of those are attached to the intestinal wall of the hindgut by their flagella, so only their slender bodies can be seen. One of the short-sized flagellates can be seen in the lumen (arrow). Bars = 10 mm. Third instarFourth instar 17 15 13 15 56.7 50.0 8 9 22 21 26.7 30.0 13 12 17 18 43.3 40.0 42.23 40.0 Stage infected Table 1. Fifth instar 11 19 36.7 11 19 36.7 13 17 43.3 38.9 Adult*Groups 1–3 are biologicaldoi:10.1371/journal.pone.0108746.t001 replicates. 30 0doi:10.1371/journal.pone.0108746.g001 100.0 30 0 100.0 30 0 100.0 100 PLOS ONE | www.plosone.org 2 September 2014 | Volume 9 | Issue 9 | e108746 Transovum Transmission of Trypanosomatid Cysts in Oncopeltus fasciatus Figure 2. Scanning electron micrograph of feces and eggs of Oncopeltus fasciatus infected with Leptomonas wallacei. (A) Promastigote forms of L. wallacei in fresh feces showing typical characteristics of live cells and cystic forms (arrow). Bar = 5 mm. (B) High magnification of a promastigote form showing one cystic form (arrow) near to its flagella. Bar = 1 mm. (C) Promastigote form present in the naturally dried feces showing extensive membrane damage and three cystic forms. Note that the cystic forms (arrows) do not show any surface damage. Bar = 1 mm. (D) Low magnification of the surface of the eggs. Bar = 500 mm. (E) High magnification of egg surface showing typical cystic forms of L. wallacei (arrows). Bar = 1 mm. doi:10.1371/journal.pone.0108746.g002 collected at random have been weekly checked for the presence of transferred to another plastic container for fresh feces and egg L. wallacei in their guts by optical microscopy and PCR. collection. The fresh and dried feces, the eggs and the gut of these adult insects were processed for scanning electron microscopy Leptomonas wallacei culture (SEM). L. wallacei was grown in Warren modified medium (37.0 g/l brain infusion hearth, 10.0 mg/l folic acid and 1.0 mg/l hemin) at Transmission of L.

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