Microfilariae develop into pre-sausage, sausage, and late sausage stages
As Dirofilaria immitis microfilariae develop, they progress into morphologically distinct forms which can be categorized as microfilaria, pre-sausage, sausage, or late sausage stage (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).
Fig 1. Morphological development and body dimensions of D. immitis microfilariae. Light microscopy images of methylene blue-stained (a) Microfilaria, (b) Pre-sausage, (c) Sausage, (d) Late sausage parasite at 40X magnification. (e) Mean body dimensions of microfilariae developmental stages. Data reported as means with standard deviation (SD) from ten counts.
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 of the cuticle (Additional file 6: Vid 5), although separation of the outer cuticle could be seen.
Fig 2. SEM analysis of D. immitis microfilaria, pre-sausage, sausage, and late sausage stages. (a) Microfilaria mounted on a double-sided tape showing whole parasite (left), enlarged head region (middle), ventral anterior view (right). (b) Pre‑sausage stage larvae mounted on a 3.0 µm polycarbonate membrane filter showing whole parasites (left), enlarged head region (middle and right). (c) Sausage staged larvae mounted on a 3.0 µm polycarbonate membrane filter showing whole parasite (left) and enlarged head region (middle and right). (d) Late sausage staged larvae mounted on a 3.0 µm polycarbonate membrane filter, showing whole parasite (left and middle) and enlarged region (right). Red arrows show where the stoma opening would be.
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 to the pre-sausage stage (Fig 3c), while Ham’s F-12 was beneficial to their development. Reduced motility was associated with this development (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 reduced, and microfilariae appeared more lethargic and degenerate, 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.
Fig 3. Development of D. immitis microfilariae and proportion of motile larvae in commercially available media and FBS. (a) RPMI-1640 medium (b) Schneider’s insect medium (c) DMEM (d) Ham’s F‑12 (e) DMEM/Ham’s F-12. All data reported as mean percentages, with error bars corresponding to the SD from at least four individual replicates.
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 F‑12 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 7 (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).
While motility always decreased as the microfilariae developed to the pre-sausage and later stages, the presence of cells and cell-conditioned media seemed to better maintain parasite activity, whereas in the absence of cells, motility dropped at a faster rate and the parasites appeared more sluggish. The presence of cells in the culture also appeared to slow the drop in motility compared to parasites cultured in cell-conditioned media, although the motility results were not significantly different (Fig 4e).
Fig 4. Development of D. immitis microfilariae cultured with An. gambiae cells or cell-conditioned media. Results show percentages of larvae at the microfilaria, pre‑sausage, sausage, and late sausage stages cultured (a) in the absence of An. gambiae cells, (b) with An. gambiae cells, (c) with An. gambiae cell-conditioned media. All data reported as mean percentages, with error bars corresponding to the SD from at least three individual replicates that were performed three times. The significance of the effects of An. gambiae cells on D. immitis development was analyzed by Kruskal-Wallis H test with Dunn’s multiple comparisons test using Prism 6.0c (Graph Pad Software, Inc.). Asterisks denote the statistical difference between total (*) and healthy (*) parasites versus the no-cell group. *p < 0.05, **p < 0.01, ***p < 0.001. (d) Light microscopy of D. immitis microfilariae cultured with no cells, An. gambiae cells, or An. gambiae cell-conditioned media at day 4 and day 13. (e) Proportion of motile D. immitis larvae cultured in the absence of cells, with An. gambiae cells, or with An. gambiae cell-conditioned media. The significance of the effects of An. gambiae cells or cell-conditioned media on D. immitis motility was analyzed by Kruskal-Wallis H test with Dunn’s multiple comparisons test using Prism 6.0c (Graph Pad Software, Inc.).
EcR, rxr-1 and downstream early regulatory genes are upregulated in developing microfilariae
As steroid-nuclear hormone receptor signaling plays an important role in ecdysozoans development , microfilariae were collected every 3 days and the transcript levels of the Dim‑EcR, Dim-rxr-1, and downstream early regulatory genes were measured to determine the transcriptional change in the ecdysteroid signaling pathway during microfilarial developmental changes. Dim‑nhr-7 and Dim-nhr-6, orthologues of E78 and E75, respectively, are both downstream early regulatory genes of the ecdysone receptor. The putative EcRE was found in the upstream promoter regions of both Dim‑nhr-7 and Dim-nhr-6, indicating that Dim-nhr-7 and Dim‑nhr-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 Dim‑GAPDH, 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 Dim‑nhr‑6, 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 Dim‑nhr-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.
Fig 5. Putative EcRE in the promoter region upstream of Dim-nhr-7 and Dim-nhr-6 start codon. Upstream promoter sequence of Dim-nhr-7 nDi.2.2.2.g02617 and Dim-nhr-6 nDi.2.2.2.g04428  were screened for EcRE. Start codons are marked with the blue box, putative EcREs are highlighted in yellow, and nucleotides that differ from the canonical EcRE are highlighted in red.
Fig 6. Transcript level of the ecdysone signaling system in D. immitis using ddPCR. Fold changes for both no-cell and cell-conditioned media groups were calculated relative to day 1 of each medium. Fold changes for adults were calculated relative to day 1 of the cell-conditioned media group. Insert figures show transcript level at day 1 for both media. All transcript levels were normalized to three reference genes: Dim‑GAPDH, Dim-Actin, and Dim-β-tubulin. (a) Dim-EcR (b) Dim-rxr‑1 (c) Dim-nhr-6 (d) Dim-nhr-7. All data reported as mean fold change, with error bars corresponding to the standard deviation from at least two individual replicates that were performed three times. The significance of the transcript level fold changes compared to day 1 baseline level was analyzed by unpaired t-test with Welch’s correction, two-tailed p-value, and 95% confidence interval using Prism 6.0c (Graph Pad Software, Inc.). Asterisks denote the statistical difference between no-cell microfilariae (*), cell-conditioned media microfilariae (*), adults (*) versus Day 1 groups (‑--). *p < 0.05, **p < 0.01, ***p < 0.001.
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 level of total pre-sausage stage larvae at day 19, and a significantly higher level of sausage stage larvae in the culture at the end of the 22-day incubation, compared to the DMSO + HP-β‑CD control group. With the increase in development to the sausage stage larvae in the 20E-treated parasites compared to the control group at day 22, as might be expected, there was a drop in the level of the pre-sausage stage larvae (Fig 7a‑b) at this time. There were no significant differences between the motility of parasites in the 20E-treated group and the control group, except a slightly higher proportion that were observed to be motile for the 20E-treated group on day 16 of the incubation (Fig 7c).
Fig 7. Development of D. immitis microfilariae treated with 20‑hydroxyecdysone. Results show percentages of larvae at the microfilaria, pre‑sausage, sausage, and late sausage stages cultured in (a) 0.05% DMSO + 0.004% HP-β-CD, (b) 5 µM 20E + 0.05% DMSO + 0.004% HP-β-CD. All data reported as mean percentages, with error bars corresponding to the SD from at least three individual replicates. The significance of the effects of 20E on D. immitis development was analyzed by Mann-Whitney U test, two-tailed p-value, and 95% confidence interval using Prism 6.0c (Graph Pad Software, Inc.). Asterisks denote the statistical difference between total (*) and healthy (*) parasites versus DMSO + HP-β-CD group. *p < 0.05, **p < 0.01, ***p < 0.001. (c) Proportion of motile D. immitis larvae cultured in 0.05% DMSO + 0.004% HP‑β‑CD or 5 µM 20E + 0.05% DMSO + 0.004% HP-β-CD. The significance of the effects of 20E on D. immitis the proportion of larvae that were motile was analyzed by Mann-Whitney U test, two-tailed p-value, and 95% confidence interval using Prism 6.0c (Graph Pad Software, Inc.). Asterisks denote the statistical difference between 5 µM 20E + DMSO + HP-β-CD versus DMSO + HP-β-CD group. *p < 0.05, **p < 0.01, ***p < 0.001.