Developmental regulation of Dirofilaria immitis microfilariae and evaluation of ecdysone signaling pathway transcript level using droplet digital PCR

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 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.

Current Applications of Digital PCR in Veterinary Parasitology: An Overview

“…The ddPCR was employed during the transcript-level study and provided data for the absolute quantification of the target nucleic acids present in the sample. This study displayed that the ecdysone signaling system might play an important role in filarial nematode developmental transitions, and gave another example of the potential applications of ddPCR in veterinary parasitology [8].…”

“…infections, the detection of mutations indicative of anthelmintic resistance, as well identification of new targets for drug development [5,[7][8][9].…”

“…Several studies also compare the sensitivity of dPCR with traditional parasitological techniques that are considered gold standard methods [3][4][5][6]. Other researchers described dPCR assays for surveillance purposes, species identification, and quantification in mixed infections, the detection of mutations indicative of anthelmintic resistance, as well as the identification of new targets for drug development [5,[7][8][9].…”

Digital PCR: modern solution to parasite diagnostics and population trait genetics

“…Other less conventional applications of the duplex assay approach in parasitology include, for example, the study of gene expression changes associated with in vitro development of the heartworm Dirofilaria immitis microfilariae [ 34 ]. The authors in this study were able to demonstrate that morphologically different larvae have different expression levels of target genes involved in the ecdysone signalling cascade.…”