Background Current measures for the prevention of dirofilariasis, caused by the dog heartworm, Dirofilaria immitis, rely on macrocyclic lactones, but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, the aim of this study was to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development regulation. Methods Dirofilaria immitis microfilariae extracted from dog blood were incubated under various culture conditions to identify those most suitable for in vitro culture and development of the microfilariae, and to determine the effects of fetal bovine serum (FBS), mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript levels of the ecdysone signaling pathway components were measured with droplet digital PCR (ddPCR). Results In vitro conditions that best promote early development of D. immitis microfilariae to the “late sausage stage” have been identified, although shedding of the cuticle was not observed. FBS had inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to microfilarial growth. The transcript level study using ddPCR also showed that ecdysone signaling system components were upregulated in developing microfilariae and that 20-hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro. Conclusions The arthropod host environment provides cues required for the rapid development of D. immitis microfilariae, and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding of the developmental process of D. immitis microfilariae.

BackgroundCurrent Dirofilaria immitis prevention relies on macrocyclic lactones but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, this study aimed to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development. MethodsD. immitis microfilariae extracted from dog blood were incubated in various media conditions to identify suitable conditions for in vitro culture and development, and to determine the effects of FBS, mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript level of the ecdysone signaling pathway components were measured with droplet digital PCR. ResultsIn vitro conditions that best promote early development of D. immitis microfilariae to the “late sausage stage” have been identified, although shedding of the cuticle was not observed. FBS showed inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells was favorable to microfilarial growth. Transcript level study using droplet digital PCR also showed that developing microfilariae displayed upregulation of the ecdysone signaling system components, and 20‑hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro.ConclusionsThe arthropod host environment provides cues required for the rapid development of the D. immitis microfilariae and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding the developmental process of D. immitis microfilariae.

BackgroundCurrent Dirofilaria immitis prevention relies on macrocyclic lactones but evidence of drug-resistant isolates has called for the need for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo a rapid molt once inside the arthropod, this study aimed to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development.MethodsD. immitis microfilariae extracted from dog blood were incubated in various media conditions to identify suitable conditions for in vitro culture and development, and to determine the effects of FBS, mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript level of the ecdysone signaling pathway components were measured with droplet digital PCR.ResultsIn vitro conditions that best promote early development of D. immitis microfilariae to the “late sausage stage” have been identified. FBS showed inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells was favorable to microfilarial growth. Transcript level study using droplet digital PCR also showed that developing microfilariae displayed upregulation of the ecdysone signaling system components, and 20‑hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro.ConclusionsThe arthropod host environment provides cues required for the rapid development of the D. immitis microfilariae and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding the developmental process of D. immitis microfilariae.