Developmental regulation of Dirofilaria immitis microfilariae and evaluation of ecdysone signaling pathway transcript le
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RESEARCH
Developmental regulation of Dirofilaria immitis microfilariae and evaluation of ecdysone signaling pathway transcript level using droplet digital PCR Tsai‑Chi Shang Kuan* and Roger K. Prichard*
Abstract 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 develop‑ ment and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to micro‑ filarial 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 micro‑ filariae, 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. Keywords: Dirofilaria immitis, Microfilaria, Anopheles gambiae, Ecdysone signaling pathway, 20-Hydroxyecdysone Background Dirofilaria immitis, also called the dog heartworm, is the causative agent of cardiovascular dirofilariasis, a serious and potentially fatal disease in pets that is spread *Correspondence: tsai‑[email protected]; [email protected] Institute of Parasitology, McGill University, Sainte‑Anne‑de‑Bellevue, Montreal H9X3V9, Canada
by mosquitoes. All canids, regardless of age or sex, are highly susceptible to heartworm infections [1]. According to the 2019 American Heartworm Society (AHS) incidence survey, the total number of cases of dogs with heartworm disease reported in the USA has risen by about 12% since the o
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