Transovum Transmission of Trypanosomatid Cysts in the Milkweed Bug, Oncopeltus fasciatus

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.


Introduction
The family Trypanosomatidae is known by the severe human diseases caused by some of its species, which kill several thousand people yearly. Roughly 37 million people worldwide are infected with Trypanosoma brucei (African sleeping sickness), Trypanosoma cruzi (Chagas disease) and Leishmania species (different forms of leishmaniasis) [1]. Trypanosomatids are evolutionarily successful organisms, parasitizing a broad range of invertebrates, vertebrates, plants [2] and even other protozoans [3]. Sixty percent of trypanosomatid genera comprise of monoxenous insect parasites and the remainder forty percent comprise mostly of vertebrate parasites transmitted by insects [4]. Nevertheless, there are very few reports on the life cycles of insect trypanosomatids [5]. Oncopeltus fasciatus is a natural host of Leptomonas wallacei, which is a gut-restricted insect trypanosomatid [6][7][8]. L. wallacei develops free-swimming promastigotes, mainly in the midgut, and promastigotes attached to the intestine wall, mostly in the hindgut [6][7][8]. Promastigotes often display encysting stages (straphangers) adhered to the flagellum; these cystic resistant forms are frequently found in clusters of two to four. Free, mature cysts can also be found in the lumen of the intestinal tract, especially in the hindgut [6][7][8].
There are basically two forms of transmission of endoparasites among insects: vertical, when the parasite is transferred from the parent to its progeny, and horizontal, when the transfer occurs between two individuals, either host to host, or host to environment and then to host [20][21][22]. The vertical transmission is subdivided into two forms: transovarial and transovum, in which the parasites are present within the eggs or on the eggshells, respectively [20,23,24]. In the latter scenario, the parasites are acquired by the newly hatched nymphs or larvae by feeding on eggshell remnants [24]. Transmission and virulence of parasites are decisive factors that determine host-parasite relationships.
Vertical transmission demands precise integration of the parasite with the host biological functions and may be an exceptional predictor to ecological host specificity [20][21][22]. In migratory insects, such as O. fasciatus [11], vertical transmission may be crucial to the persistence of parasites in host populations [25].
The vertical transmission of trypanosomatids among invertebrate hosts has been proposed but so far not confirmed [26,27]. The main goal of the present study was to experimentally show the existence of transovum transmission of L. wallacei by O. fasciatus. For the first time, we demonstrated that trypanosomatid cysts may be vertically transmitted from insect females to their offspring through contamination of the egg surface with feces that contains cysts, and subsequent ingestion of these feces by newly hatched nymphs. Therefore, we believe that the differentiation of L. wallacei promastigotes to cystic forms and the vertical transmission of parasites to the host offspring may be connected and working together for the success of parasitism.

Oncopeltus fasciatus colony
A colony of O. fasciatus naturally infected with L. wallacei [7] was established and maintained in our laboratory under a 12 h 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 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 parental colony. In order to avoid recontamination of the parasitefree colony, these insects have been kept in a different, isolated, room from the parental colony. In order to validate the absence of L. wallacei in the parasite-free colony, samples of five insects Table 1. Infection rates of insects hatched from eggs laid by Leptomonas wallacei-infected Oncopeltus fasciatus females.  Leptomonas wallacei culture 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 28uC, supplemented with 10% fetal calf serum. In the logarithmic growth phase, the parasites were washed three times with phosphate-buffered saline, pH 7.2 (PBS) and harvested by centrifugation at 5.0006g for 10 min at 4uC for DNA extraction.

Analysis of L. wallacei infection during O. fasciatus life cycle
To establish the time course of O. fasciatus infection by L. wallacei, 450 O. fasciatus eggs were collected from the infected colony, separated in three groups of 150 eggs and kept in separated sterile plastic containers for eclosion. In the third-, fourth-and fifth instar nymphs and in adults, 30 insects were randomly collected from each one of the four groups and dissected in PBS. The guts were then extracted for analysis of infection by optical microscopy in a Zeiss Axioplan 2 light microscope (Oberkochen, Germany) equipped with a Color View XS digital video camera. Insects with at least one mobile flagellate in their alimentary tract were considered infected. Parasite detection was conducted through the analysis of optical micrographs taken from Giemsa-stained gut contents of the aforementioned insects. Promastigotes, encysting stages (straphangers) and free cystic forms were identified as described [6,28]. Some adult females were transferred to another plastic container for fresh feces and egg collection. The fresh and dried feces, the eggs and the gut of these adult insects were processed for scanning electron microscopy (SEM).

Transmission of L. wallacei via eggshells
Eggs collected from the infected colony were mechanically broken, mixed with sterile sunflower seeds and offered as the only source of food to 200 L. wallacei-free adult insects, in a sterile plastic pitcher. After one week, 20 insects were dissected in Petri dishes containing sterile PBS and the guts extracted for analysis of infection by SEM or to DNA extraction. DNA samples were PCRamplified for parasite detection.

Detection of parasite infection by PCR
L. wallacei promastigotes grown in axenic culture medium, insect guts or eggs were homogenized in 500 ml lysis buffer (Tris-HCl (pH 7.6), 0.1 M NaCl, 10 mM EDTA, 0.5% SDS and 300 mg/ml proteinase K), incubated at 52uC for 1 h and submitted twice to extraction with phenol:chloroform:isoamyl alcohol (25:24:1,v/v). The samples were centrifuged at 5,0006g for 10 min. After extraction, total DNA was precipitated from 200 ml-aliquots of the aqueous phase by centrifugation at 5.0006g for 10 min at room temperature, after the addition of 25 ml 3 M sodium acetate and 475 ml absolute ethanol. The pellet of DNA was dried and resuspended in distilled water. Primers specific for O. fasciatus were designed by our group using as target the sequence of O. fasciatus 16S rRNA gene (GenBank accession number AY252660.1). Since there are no L. wallacei gene

Interruption of L. wallacei transmission by egg surface asepsis
To verify a possible role for cystic forms found on O. fasciatus eggshells as infective forms, 450 eggs were collected from the infected colony and submitted to surface asepsis as described above in the topic Establishment of a parasite-free colony. Then the eggs were washed in distilled sterile water and dried on sterile filter paper. After asepsis, the eggs were kept in sterile plastic pitchers and the hatched insects maintained in the conditions described for the parental colony, being fed with sterilized water and sunflower seeds until the adult stage. Three pools of five adult insects were randomly collected, dissected in Petri dishes containing sterile PBS and their guts extracted for ruling out infection with L. wallacei, by SEM and PCR.

Scanning electron microscopy
Insect guts were vertically opened in PBS at 4uC before the fixation. Fresh feces were transferred to glass coverslips and immediately fixed or kept at room temperature to dry. The samples were fixed with a solution containing 2.5% glutaraldehyde, 4.0% formaldehyde, 3.7% sucrose and 5 mM CaCl 2 in 0.

Results and Discussion
The relationship between human trypanosomatids and their insect vectors have been thoroughly documented [29][30][31][32][33][34]. On the other hand, the studies on interactions of insect trypanosomatids with their hosts are still incipient [7,[16][17][18][19]35,36]. The occurrence of horizontal transmission of L. wallacei by O. fasciatus was evident in the present study, corroborating previous data [6]. Here, we collected in our colony at least 50 specimens of the third, fourth and fifth instars, as well as adult insects so as to evaluate the  (Table 1). Such different rates between nymphs and adults are due to cumulative infection and have been observed both in ticks and insects [37,38]. Therefore, we concluded that during the insect development, parasites were transmitted between contaminated individuals to parasite-free individuals, or to the environment and then to parasite-free insects.
In order to detect the infective forms of L. wallacei, we analyzed the opened guts of adult insects, feces and eggs collected at the insect colony by means of scanning electron microscopy (SEM). We observed many promastigote forms in the lumen of the midgut (Fig. 1A, arrows). In the hindgut, a massive presence of promastigotes was also evident, most of which were attached to the intestinal wall of the hindgut by the flagella, so that only the slender bodies could be seen (Fig. 1B). The arrow in figure 1B indicates a short-sized flagellate that can be seen in the lumen (Fig. 1B, arrow). We also observed promastigote forms in fresh feces ( Fig. 2A), some of which showed cystic forms in association with their flagella, near to the cell body (Fig. 2B). In dried feces there were promastigotes showing extensive cell membrane damage, while the cystic forms attached to them seemed intact, without any visible surface damage (Fig. 2C). Coprophagy among phytophagous and wood-feeding insects seems likely to have been positively selected throughout evolution, since this habit greatly facilitates the transmission of cellulose degrading gut microbiota between these insects [39]. Horizontal transmission by ingestion of parasites present in the insect feces, including cystic forms, has been extensively studied in the interaction between the trypanosomatid Blastocrithidia triatomae and the hematofagous insect Triatoma infestans, as well as other triatomines [27,35,36]. Along with cystic forms, live promastigote forms were observed in fresh feces, so we speculated that horizontal transmission takes place mostly by ingestion of these contaminated feces, which has already been observed for the trypanosomatid Crithidia bombi within a population of its natural host, bumblebees of the genus Bombus [21].
The characteristic cystic forms of L. wallacei were observed on the surface of eggs, while promastigotes were never observed there [6]. These cysts showed an ovoid ellipse shape with the expected body size (approximately 3 mm in length), slight body torsion and a small invagination in only one side of each cyst (Fig. 2D and 2E). These findings showed that the cystic forms of the parasites, which most likely contaminate the eggshells during oviposition, may act as infective forms if ingested by the newly hatched nymphs. Similar results have been described for moth eggs contaminated with microsporidian spores [20]. Considering that Porter [26] had suggested transovarial transmission of the trypanosomatid Crithidia guerridis in a waterbug Gerris paludum population, we searched for the presence of parasite DNA in the eggs collected from the infected colony that were submitted or not to surface asepsis. Total DNA was extracted from whole egg homogenates and PCR-amplified using primers specific for parasite DNA detection. No parasite DNA was found in eggs that were previously treated with sodium hypochlorite, which discarded the possibility of transovarial transmission in our system (Fig. 3A).
The transovum transmission strategy depends on the parasite ability to persist in the environment until egg hatching. Some invertebrate trypanosomatids, including other species of the genus Leptomonas, in addition to L. wallacei, develop resistant cysts that enable them to persist in the environment for a long time [5,6,40,41]. In order to investigate the remaining possibility of transovum vertical transmission of L. wallacei, we isolated eggs from the infected colony and observed newborn nymphs probing and feeding on egg remnants (data not shown). This nymph behavior led us to hypothesize that the cysts of L. wallacei that were observed on the eggshells of O. fasciatus (Fig. 2E) could be the resistant and infective parasite forms. In addition, we speculated that egg surface contamination occurred during the oviposition because of the contact of eggs with female feces, due to the proximity of the ovipositor with the anus.
Some phytophagous insects eat food sources other than plants, due to the need of a high-protein diet. A newly-hatched nymphs often consume the remainder of their eggshell because the sucking mouthparts of first to third instar nymphs are usually still too small to perforate plants [42,43]. To investigate the possibility that nymphs naturally acquire infection through the ingestion of cystic forms present on the eggshells after the oviposition, we performed the asepsis of the surface of the insect eggs using sodium hypochlorite [44]. When the insects that hatched from the treated eggs turned into adults, their guts were analyzed by PCR and SEM for the presence of parasites. All the DNA samples extracted from guts of insects developed from eggs treated with sodium hypochlorite were negative for the presence of L. wallacei, when tested by PCR (Fig. 3A). In addition, in contrast to the abundant presence of flagellates in the guts of naturally infected insects, only bacteria were observed in the guts of insects that hatched from treated eggs (Fig. 3B and 3C). Therefore, we conclude that transmission of L. wallacei from female O. fasciatus to the offspring was interrupted by the elimination of L. wallacei cystic forms, which usually contaminate the surface of the eggs. Similarly, hypochlorite-treated eggs from the European pine moth, Rhyacionia buoliana, gave rise to parasite-free larvae, without any significant decrease in egg hatch. Those larvae could also be aseptically maintained in sterile plastic containers [44].
To confirm our hypothesis that nymphs acquire infection by ingesting parasite forms present in the shell of hatched eggs, we added mechanically broken eggs, laid by infected females, to previously sterilized sunflower seeds. These seeds were then offered as the only source of food to parasite-free adult insects. After two weeks, the alimentary tract of these insects were dissected and the parasite infection investigated by optical microscopy and PCR, using primers specific for the detection of parasite DNA. All DNA samples that were extracted from intact guts of these insects were positive for the presence of L. wallacei DNA (Fig. 4A). In addition, mobile promastigote forms were found in the digestive tube of these insects (Fig. 4B).
Along the course of evolution parasites have adapted in order to persist within the host populations [25,26,45,46]. Trypanosomatids are amazingly successful parasites that can be found in all classes of vertebrates, several invertebrates, plants and other protozoa [2,3]. Also, trypanosomatids can be themselves hosts of endosymbiotic bacteria [47], viruses [48][49][50] or both [50]. Not surprisingly, trypanosomatids harbor ''foreign'' genetic material, probably originated from plants, bacteria and virus, integrated in their genomes [49,51,52] or solely within virus-like particles [50,52]. Intriguingly, these viruses play a major role in virulence and metastasis of the South American subgenus of the Leishmania parasite, L. (Viannia) [53]. Studying the relationships of trypanosomatids with their hosts is of upmost relevance for better understanding the uniqueness of these parasites. This is particularly true for monoxenous trypanosomatids, which are still poorly understood [54]. Transovum transmission is a strategy shown by different parasites, supposedly with low cost to the host fitness [22,55,56]. Even though vertical transmission has been speculated [27,35], trypanosomatids have only been described as horizontally transmitted by insects. The present study is the first experimental demonstration that vertical (transovum) transmission takes place in the interaction of O. fasciatus with L. wallacei, a process driven by the contamination of eggshells with resistant cystic parasite forms, which are ingested by the nymphs after hatching. The data described in this study are summarized in a cartoon that shows the horizontal and vertical modes of transmission of L. wallacei in O. fasciatus (Fig. 5). Based on our findings we hypothesize that, in addition to horizontal transmission, vertical transmission is a feature of the O. fasciatus-L. wallacei relationship may play an important role in the maintenance of the parasite within its host population.