Apoptosis of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae), which failed to migrate within its natural host

Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) naturally infects the mosquito Aedes albopictus (Chen, 1999). In general, infection by A. taiwanensis is not deleterious to its natural host (Walsh and Olson, 1976), although pathogenic effects have occasionally been seen among mosquitoes that did not allow the parasite to complete its developmental cycle (Rowton et al., 1987). Development of the parasite is initiated upon entry of sporozoites released from the ingested oocysts into epithelial cells in the midgut of mosquito larvae (Chen et al., 1997a). After a short intracellular stage, sporozoites subsequently leave the invaded epithelial cells to form extracellular trophozoites that attach to the epithelium by their epimerites (Chen et al., 1997b). Such trophozoites are mostly found in the posterior part of the midgut in which vacuolar-type H⁺-ATPase (V-ATPase) is actively expressed (Huang et al., 2006).

Trophozoites of A. taiwanensis in the midgut of mosquito larvae normally mature along with the development of the host (Chen and Yang, 1996). Extracellularly matured trophozoites then migrate from the midgut into the Malpighian tubules, by virtue of gliding motility (Wetzel et al., 2003), for sexual reproduction, which occurs only in the natural host (Chen, 1999). Migration from the midgut towards the Malpighian tubules is unidirectional and usually occurs among trophozoites that were liberated in the midgut of early pupae (usually <5 h after pupation) (Chen and Fan-Chiang, 2001).

Cells may die in response to stimuli or stresses, and the resulting death occurs mostly via necrosis or apoptosis (Kanduc et al., 2002). Extreme variance from physiological conditions, in association with infection, toxins or trauma, may cause necrosis of cells, resulting in direct damage to plasma membranes and fatal outcomes (Golstein and Kroemer, 2007). In contrast, apoptosis or programmed cell death is known to be the most common form of eukaryotic cell death, either for normal developmental processes or in response to extracellular stimuli, including infections (Kerr et al., 1994; James and Green, 2002). Although apoptosis is known to occur extensively in multicellular organisms (Bangs and White, 2000), it also occurs in unicellular eukaryotes (Jiménez-Ruiz et al., 2010), such as Trypanosoma cruzi (Ameisen et al., 1995; Welburn et al., 1997; Barcinski and DosReis, 1999) and Dictyostelium (Cornillon et al., 1994).

As mentioned, a low proportion of trophozoites of A. taiwanensis developing in the midgut of its mosquito host can successfully migrate into Malpighian tubules (Chen, 1999). In contrast, all residual trophozoites left in the midgut of mosquito pupae are ultimately lysed, hypothetically through apoptosis (Chen, 1999). The present study aimed to demonstrate the role of apoptosis in regulating the fate of these trophozoites; this unique feature is presumably involved in modulating a balanced state of the parasite load suitable for the survival of both the parasite and host.

A colony of Aedes albopictus (Skuse 1894) infected with Ascogregarina taiwanensis Lien and Levine 1980 has been maintained in the laboratory for a few years (Chen and Yang, 1996). To propagate the parasite, approximately 100–200 mosquito adults from the infected colony were ground in a 1.5 ml microfuge tube containing 1 ml of distilled water. The homogenate was passed through a nylon cloth (75 mesh) to remove tissue debris, and then fed to uninfected newly hatched larvae. Mosquitoes were reared at 28±1°C and 75% relative humidity, and under a 12 h:12 h light:dark photoperiod. Trophozoites of the parasite were collected ~8–10 days after eggs hatched from larvae or pupae of mosquitoes.

To collect trophozoites, larvae (fourth instar; no less than 24 h post third molt) or pupae (younger than 5 h or older than 5 h) of mosquitoes were dissected under a dissecting microscope to remove the midgut and/or Malpighian tubules on a glass slide. Food debris within the midgut lumen was first removed to provide an easier environment in which to operate on the tissues. Isolated trophozoites were subsequently transferred onto a 12-well Teflon-coated slide for observation under a light microscope or for running assays described below.

Trophozoites removed from the midgut of larvae (third or fourth instar) or pupae (<5 or >5 h) were distributed onto a 12-well Teflon-coated slide. Annexin V conjugated with fluorescein isothiocyanate (FITC) (BioVision, Mountain View, CA, USA) at 5 μl was added, incubated at room temperature for 5 min in the dark and then detected under an epifluorescence microscope (Olympus BX50, Tokyo, Japan).

A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was carried out using an in situ cell death detection kit following the protocol provided by the manufacturer (Roche Applied Science, Mannheim, Baden-Württemberg, Germany), which was performed for nick-end labeling of internucleosomal cleavage in the parasites. Trophozoites removed from the fourth instar larvae (no less 24 h post third molt) or pupae (<5 or >5 h) were distributed onto a 12-well Teflon-coated slide and fixed with 4% formaldehyde at room temperature for 30 min. After washing, samples were permeabilized in 20 μl of a solution containing 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. The slide was rinsed twice with phosphate-buffered saline (PBS; pH 7.3) and then air-dried. Subsequently, samples were labeled by adding 20 μl of TUNEL mix (Roche Applied Science) followed by incubation for 60 min at 37°C in the dark. After washing, samples were air-dried, and 20 μl of converter-AP was added to the samples followed by an incubation of 30 min at 37°C in the dark. The slide was rinsed again with PBS, and 20 μl of a substrate solution containing 3,3′-diaminobenzidine was added and incubated at room temperature for 10 min. After another wash, samples were mounted and observed under a light microscope; DNA-containing materials that were undergoing apoptosis were stained dark brown.

At least 500 trophozoites were harvested from either larvae (fourth instar; no less than 24 h post third molt) or pupae (<5 or >5 h) of infected mosquitoes for each test of DNA fragmentation. An apoptotic DNA ladder kit (Roche Applied Science) was used to extract DNA from the collected trophozoites. Extracted DNA was then separated on a 1% agarose gel. The pattern of DNA ladders shown on the gel was visualized on a UV transilluminator and photographed with a digital camera.

The CaspaTag In Situ Pan-Caspase Assay Kit (APT400, Millipore, Billerica, MA, USA) was used to detect caspase-like activity in living trophozoites that may have been undergoing apoptosis. The protocol used in this study was determined from instructions provided by the manufacturer. All trophozoites for the test were removed from the midgut of either mosquito larvae (fourth instar; no less than 24 h post third molt) or pupae (<5 or >5 h), which were first incubated in a working solution of the stain for 1 h at 37°C under 5% CO₂, followed by a 5 min incubation in Hoechst stain under the same conditions. After washing with PBS, propidium iodide was added, and trophozoites were immediately visualized under a fluorescent microscope (with excitation at 490 nm and emission at 520 nm).

Preparation for electron microscopy followed a previously described method (Chen et al., 1997a; Chen et al., 1997b). In brief, dissected tissues (the midguts from both larvae and pupae) were fixed with 2% (v/v) glutaraldehyde in 0.1 mol l⁻¹ cacodylate buffer for 2 h at 4°C, washed with 0.2 mol l⁻¹ cacodylate buffer three times, and then post-fixed in 1% (w/v) osmium tetroxide in 0.1 mol l⁻¹ cacodylate buffer for 2 h at room temperature, finally washed three times with 0.2 mol l⁻¹ cacodylate buffer and dehydrated through an ascending graded series of ethanol. Tissues were embedded in Spurr's resin, and polymerized tissue blocks were sectioned with an ultramicrotome (Reichert Ultracut S, Leica, Vienna, Austria) using a diamond knife. Thin sections were stained with saturated uranyl acetate in 50% ethanol and 0.08% lead citrate before being observed under a JEOL-JEM-1200 electron microscope (JOEL, Tokyo, Japan) at 80 kV.

Around the time of pupation of mosquito larvae (presumably the stage of pharate pupae), elongate mature trophozoites of A. taiwanensis that had formed in the space between the peritrophic membrane and the epithelium of the midgut began moving to the Malpighian tubules (Fig. 1). Based on a previous report, approximately half of the trophozoites present may migrate successfully (Chen, 1999). The migration of such trophozoites, driven by gliding motility (supplementary material Movie 1), is essential for subsequent sexual reproduction of Ascogregarina in their natural mosquito hosts.

An ultrastructural study showed that trophozoites that failed to migrate and were thus retained in the midgut of mosquito pupae had a highly vacuolated cytoplasm, and chromatin aggregates appeared in the nucleus (Fig. 2A). Ridges on the surfaces of the trophozoites became blunt and deformed (Fig. 2A); they were usually rigid and sharp in live trophozoites in the midgut of fourth instar larvae of the mosquito (Fig. 2B).

Translocation of phosphatidylserine to the outer surface of plasma membranes was detected through the affinity of FITC-labeled Annexin V protein. Using this technique, trophozoites removed from the midgut of pupae showed a crease in morphology to a certain extent (Fig. 3A), in which green fluorescence was observed on the plasma surface of trophozoites undergoing apoptosis (Fig. 3B). A negative control was shown by a trophozoite that was also removed from the pupal midgut (Fig. 3C) but did not show green fluorescence (Fig. 3D). In contrast, those trophozoites removed from mosquito larvae usually appeared to have structural integrity, i.e. absence of the crease (Fig. 3E), and only pale fluorescence was shown on their surfaces (Fig. 3F).

Cells undergoing apoptosis were detected by TUNEL, revealing fragmented DNA in the nucleus. Using this technique, shrunken trophozoites or apoptotic body-like structures that were removed from the midgut of older pupae were obviously nick-end-labeled and stained deep brown (Fig. 4A). However, no obvious staining was found in trophozoites harvested from the midgut of mosquito larvae or younger pupae (Fig. 4B), and none was seen in those from Malpighian tubules of older pupae (Fig. 4C).

Degradation of chromosomal DNA is one of the events among cells undergoing apoptosis that usually presents DNA ladders as visualized on the agarose gel after electrophoresis. The present results showed that DNAs extracted from trophozoites retained in the midgut of older pupae, i.e. failed to migrate, revealed a ‘ladder’ pattern on the agarose gel (Fig. 5). Nevertheless, such ladders did not appear in DNA extracted from trophozoites living in mosquito larvae of the fourth instar (no less than 24 h post third molt) (Fig. 5).

Trophozoites harvested 3, 4, 7 and 21 h after pupation were tested for caspase-like activity (Fig. 6). The activity of fluorescent-labeled inhibitors of caspase, which is irreversibly bound to active caspases, allowed apoptotic cells to be identified by their bright-green fluorescence. According to the present results, stronger caspase-like activity can be detected in trophozoites removed from the midgut of older pupae. Apparently, the caspase-like activity within trophozoites increased along with the age of the pupae.

Apoptosis generally occurs with characteristic morphological and biochemical features, including shrinkage of cells, degradation of membrane integrity, chromatin aggregation, chromatin condensation and nuclear fragmentation (Cohen, 1993; Vaux and Strasser, 1996; Lüder et al., 2001). In many cases, cytoplasmic vacuolization has also been found (Kerr et al., 1972). Apoptosis is a tightly regulated physiological process in metazoans (Pridgeon et al., 2008); however, it is apparently not just occurring in multicellular organisms (Barcinski, 1997). A variety of parasitic protozoans such as Trypanosoma, Leishmania, Plasmodium, Toxoplasma, Trichomonas, Blastocystis, Entomoeba and Giardia were reported to be associated with apoptosis (Jiménez-Ruiz et al., 2010; Lüder et al., 2010). Similarly, some ookinetes of Plasmodium also die by a mechanism resembling apoptosis (Arambage et al., 2009).

As previously reported, only 50% (or fewer) of A. taiwanensis trophozoites can successfully migrate (Chen, 1999); others usually end up dead, likely through apoptosis according to the present observations of different markers. It seems that there are advantages of leaving from the midgut, which starts remodeling through histolysis during pupation (Parthasarathy and Palli, 2007). Morphologically, trophozoites undergoing apoptosis may become shrunken, and exhibit cytoplasmic vacuolation and chromatin condensation. Positive staining of Annexin V on the outer surface of plasma membranes indicates the initial stage of apoptosis among trophozoites (Jiménez-Ruiz et al., 2010). DNA fragmentation also appeared in extracted genomic materials, implying that apoptosis has occurred in most, if not all, trophozoites that did not successfully migrate. In some cases, shrunken or apoptotic body-like structures were seen in trophozoites removed from the midgut of older pupae that had failed to migrate (Elmore, 2007). In addition to features of programmed cell death in metazoans (Ch'ng et al., 2010), it seems to be unique for this parasite to have blunt or even deformed ridges on the surfaces of the trophozoites.

Caspase-like activity was detected in trophozoites undergoing apoptosis, indicating alteration of mitochondria. Activated caspases generally cause cell death by cleaving a number of cellular proteins such as poly-ADP-ribose-polymerase, a DNA repair enzyme (Lazebnik et al., 1994). The event reflects that apoptosis of A. taiwanensis trophozoites is actually triggered by internal signals through an intrinsic or mitochondrial pathway (Jiang and Wang, 2004; Mohamad et al., 2005).

Although apoptosis occurs extensively in multicellular organisms, its occurrence in protozoans provides an opportunity to elucidate the molecular and cellular biology of this process in single-celled organisms (Kaczanowski et al., 2011). In terms of parasite ecology, the occurrence of apoptosis is particularly mysterious, as cell death is not supposed to be beneficial for the species' continuation (Pollitt et al., 2010). However, apoptosis may be beneficial to the parasite because cell death in response to limited resources may help promote the survival of genetically related individuals (James and Green, 2002). In addition, the Malpighian tubule lumen may not be large enough to endure the entry of all those trophozoites from which the numerous oocysts contained in large-sized gametocysts would ultimately form (Chen et al., 1997b). As a result, differential support of the survival of individual trophozoites may help optimize the parasite load within the host, indicating a form of social control of cell survival and cell death (Ameisen, 2002). This may be a way for parasitic protozoans to sustain a balanced state between themselves and the host (Lüder et al., 2010), leading to maximal survival of both parasites and hosts. In turn, the differential fate among trophozoites may indicate the existence of altruistic behavior in unicellular parasites such as A. taiwanensis (James and Green, 2002).

The authors would like to thank Shi-Chien Su for her technical assistance.