Comparative transcriptome profiles of first-stage larvae and adult female Dracunculus medinensis (Guinea worm)

Dracunculus medinensis, also called the Guinea worm, is a nematode that causes dracunculiasis, a debilitating neglected tropical disease in humans. The parasite is currently targeted by the global Guinea Worm Eradication Program (GWEP). Historically, GWEP in endemic countries have focused on interrupting transmission of the disease through intervention such as isolation and management of patients, health education, provision of improved water sources and promotion of filtering drinking water from unimproved water sources to avoid ingestion of the copepod intermediate host (IH) that may contain infectious third-stage larvae, and treatment of water sources to kill copepods. The recent shift of Guinea worm infections in animals - particularly domestic dogs - has introduced an additional challenge to the eradication program, underscoring the urgent need for diagnostics and therapeutics. Understanding the parasite biology and survival strategies in the mammalian host, the copepod IH, and fresh water is pivotal to identifying new control measures. Comparative transcriptomic analysis provides a powerful tool to uncover the molecular mechanisms underlying parasite survival and adaptations. Here, we compared the transcriptome of adult gravid female and first-stage larvae (L1), the stage infective for the copepod IH. Comparative transcriptomic analysis of two adult females and their L1 revealed an upregulation of genes involved in translation, transcription, and DNA repair in L1, likely reflecting adaptations essential for survival in freshwater and subsequent infection of copepods. Additionally, genes involved in cuticle formation were upregulated in adult females highlighting the role of cuticle integrity in retaining millions of L1 until the gravid female worm emerges. We identified highly expressed genes in the adult female that may represent promising candidates for diagnostic markers.This study provides novel insights into the biology of the Guinea worm by examining the transcriptome of L1 and adult female stages. These findings could support the development of novel diagnostics and therapeutics to advance the ongoing eradication effort. Overall design: A ferret (Mustela furo) was experimentally infected at the University of Georgia (IACUC number: A2020 05-003) by feeding copepods infected with L3 stage of D. medinensis. Two subcutaneous female worms were recovered from the exposed ferret upon necropsy. Five pools of L1 stage were prepared from the two adult worms, counted, and stored at -80°C pending RNA extraction. Samples were thawed on ice and adult female tissues and L1 pools were homogenized with liquid N2 flash freezing and manual grinding using disposable pestles. Total RNA was extracted using the Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturer's instructions. RNA was quantified using QuantusTM Fluorometer (Promega, Madison, WI) and stored at -80°C until being shipped on dry ice to LC Sciences (LC Sciences, Houston, TX) for mRNA sequencing. mRNA sequencing was performed by LC Sciences Poly (A) RNA-seq Sequencing Service.