Microfilariae develop into pre-sausage, sausage, and late sausage stages
Development of Dirofilaria immitis larvae was categorized as microfilaria, pre-sausage, sausage, or late sausage stage, based on their morphological differences (Fig. 1a-d). When first extracted from dog blood samples, parasites were in the microfilaria stage and appeared elongated and slender, with vigorous serpentine movements (Additional file 2: Vid 1). When microfilariae were incubated in vitro, the body of the parasite shortened and the posterior region began to enlarge after several days, making the tail more distinct; this stage was classified as the pre-sausage stage. Compared to the microfilaria stage, the pre-sausage stage was more granular in appearance, and parasite movement was slowed (Additional file 3: Vid 2). Over the next few days, the pre-sausage stage larvae grew shorter in length, resulting in a stumpy appearance with a fine tail at the tip, approaching the sausage stage. The sausage stage was more basophilic than the microfilaria, with heavy internal granulation, and movement was sluggish (Additional file 4: Vid 3). At the late sausage stage, movement of the parasite continued to decline (Additional file 5: Vid 4) and at the same time, the anterior region of the sausage stage larvae started to enlarge while the body became more elongated. The length and width of the parasites were measured to determine the mean body dimensions for the different stages (Fig. 1e).
Pre-sausage, sausage, and late sausage stage parasites are L1 larvae
To further study the morphological differences between the microfilaria, pre-sausage, sausage, and late sausage stage parasites and to observe possible signs of molting, SEM analysis and live-cell analysis were performed. Numerous transverse grooves in the cuticle were evident in all the developmental stages, giving the parasite a striated appearance (Fig. 2a-d). The grooves seemed to become more noticeable as the parasite became more developed. No visible difference was seen in the stoma region among the four stages; all the parasites had a circular tissue located at the tip of the head and layers of folded tissue that surrounded it. Results of the live-cell analysis with the IncuCyte ZOOM system also showed that development to the late sausage stage was not accompanied by any shedding (Additional file 6: Vid 5), although separation of the outer cuticle could be seen.
DMEM/Ham’s F-12 provides a relatively favorable condition for the in vitro cultivation of D. immitis microfilariae
To determine suitable conditions for the in vitro cultivation of D. immitis microfilariae, extracted microfilariae were incubated in various commercially available culture media at 26⁰C for 22 days. Results showed that both RPMI 1640 and Schneider’s insect medium were poor at sustaining the development and motility of the microfilariae (Fig. 3a-b). DMEM maintained microfilariae motility but was unable to initiate development into the pre-sausage stage (Fig. 3c), while Ham’s F-12 was beneficial to their development but motility was compromised (Fig. 3d). When mixed at a 1:1 ratio, DMEM/Ham’s F-12 provided a favorable condition for the microfilariae and supported larvae development to the pre-sausage and sausage stage (Fig. 3e).
When 10% heat-inactivated FBS was added to the microfilaria culture, development and motility of the parasite were both drastically lowered, and microfilariae appeared more lethargic and degenerated, with little development to the pre-sausage stage observed in most groups (Fig. 3a, b, d, e). An exception was seen in those that were incubated in DMEM and 10% FBS (Fig. 3c); although development was initiated in this group and some pre-sausages developed into sausages, most of the parasites appeared degenerated and motility was greatly reduced compared to the group without 10% FBS.
Anopheles gambiae cells and cell-secreted factors promote D. immitis microfilariae development
Anopheles gambiae cells were introduced to the culture of microfilariae incubated in DMEM/Ham’s F12 for 22 days to test the effect of insect cells on the development of the microfilariae. Consistent with results in Fig. 3, larvae incubated in DMEM/F-12 in the absence of mosquito cells developed into the pre-sausage stage at around day 4, with few that developed into the sausage stage throughout the 22-day incubation period. The presence of An. gambiae cells, however, initiated faster development and significantly higher levels of sausage stages were observed as early as day 8 (Fig. 4a-b). Also, some parasites were able to develop into the late sausage stage.
When medium conditioned with An. gambiae cells was used instead of having physical cells in the culture, a similar effect in the development of microfilariae was also seen. In fact, there was a significant decrease in the percentage of microfilaria and an increase in the number of pre-sausage stage larvae during early incubation. In addition, more sausage stage larvae were observed when the cell-conditioned medium was used compared to when microfilariae were in physical contact with the insect cells (Fig. 4c-d).
In terms of motility, the presence of cells and cell-conditioned media seemed to improve and maintain parasite activity, whereas in the absence of cells, motility dropped at a faster rate and the parasites appeared more sluggish. The presence of physical cells in the culture also appeared to slow the drop in motility compared to parasites cultured in cell-conditioned media (Fig. 4e).
EcR, rxr-1 and downstream early regulatory genes are upregulated in developing microfilariae
As the steroid-nuclear hormone receptor signaling plays an important role in ecdysozoans development , microfilariae were collected every 3 days and the transcript levels of the DimEcR, Dim-rxr-1, and downstream early regulatory genes were measured to determine the transcriptional change in the ecdysteroid signaling pathway during microfilarial developmental changes. Dimnhr-7 and Dim-nhr-6, orthologues of E78 and E75, respectively, are both downstream early regulatory genes of the ecdysone receptor. Putative EcRE was found in the upstream promoter regions of both Dimnhr-7 and Dim-nhr-6, indicating that Dim-nhr-7 and Dimnhr-6 can potentially be regulated by EcR directly (Fig. 5). All primer pairs (Additional file 1: Table 1) were optimized for specificity and efficiency, and the cycle threshold (Ct) values from qPCR were used to estimate template concentrations for use on the ddPCR. After normalizing the genes of interest to three reference genes DimGAPDH, Dim-Actin, and Dim-β-tubulin, results in Fig. 6a-d show that Dim-EcR, Dim-rxr-1, Dim-nhr-7, and Dim-nhr-6 were all upregulated to different magnitudes in developing L1 larvae. In both no-cell and An. gambiae cell-conditioned media groups, transcript levels of all four genes consistently increased during the first few days of development. However, in no-cell groups, transcript levels incurred a drop at around day 13–16 for all four genes, after which an increase was seen again on day 19. In groups cultured in the cell-conditioned medium, a gradual increase throughout the 22-day incubation period was seen for Dim-EcR, Dim-rxr-1, and Dimnhr6, with a sharp rise on day 22. On the other hand, the transcript level of Dim-nhr-7 in the cell-conditioned medium group remained relatively constant during the early incubation period, but a sharp rise could be seen on day 22.
Overall, developing larvae in cell-conditioned medium groups expressed higher transcript levels than those that were not incubated in cell medium, an exception would be the transcript level of Dimnhr-7, where the no-cell group has a higher transcription level, but the transcript level of the cell-conditioned medium group quickly rose higher on day 22. In addition, the overall transcript level of Dim-nhr-6 in developing microfilariae was significantly higher than those of the other three genes. Transcript levels of female and male adults were also included as a comparison, and results showed that the transcript level of the ecdysteroid signaling system components was higher in females than in males for all four genes.
20-hydroxyecdysone promotes development to sausage and late sausage stage larvae
In view of the upregulation of genes involved in the ecdysone regulatory development pathway, the effects of 20-hydroxyecdysone (20E) were investigated on microfilariae cultured in vitro, without insect cells or insect cell-conditioned medium. When microfilariae were treated with 5 µM 20E every day, with (2-Hydroxypropyl)-β-cyclodextrin (HP-β-CD) as the carrier of the hydrophobic compound, there were significantly higher levels of sausage stage and late sausage stage larvae in the culture at the end of the 22-day incubation, compared to the DMSO + HP-βCD control group. This was accompanied by a drop in the level of the pre-sausage stage larvae (Fig. 7ab). In terms of the overall motility, there was no significant difference between the parasite activity of the 20E-treated group and the control group, except a slightly higher level of motility for the 20E-treated group on day 16 of the incubation (Fig. 7c).