Cestodes are platyhelminths which are passively transmitted among several vertebrate species (Maule, 2006). Several members of this class, such as Echinococcus spp. and Taenia spp., represent a global public health issue as they are the etiological agents of human zoonotic diseases such as echinococcosis and taeniasis/cysticercosis, respectively (Moazeni et al., 2019, Wen et al., 2019). Often disregarded, these diseases are among the current 20 Neglected Tropical Diseases prioritised by the World Health Organization (WHO, 2020). Widespread around the world, these severe and potentially fatal infections mainly affect vulnerable populations in areas in which sanitation and hygiene are insufficient (Budke et al., 2006), poverty is rampant (Mitra and Mawson, 2017), and mainly where subsistence farming practices are performed (Cadavid Restrepo et al., 2016).

All cestodes are obligate endoparasites whose life cycles involve two or more hosts, undergoing morphological and physiological events that enable their survival and development in different species. Their notable phenotypic plasticity derives from a complex gene expression control system and the consequent activation/repression of different cellular pathways (Thompson et al., 2017). The subclass Eucestoda (true cestodes) presents increased fertility due to a key particularity in its biology which is the generation of serially repeated reproductive organs, known as proglottids (proglottization) (Olson et al., 2001). This developmental process is associated with proglottids’ external delimitation (segmentation) in medical and veterinary relevant species. It is called strobilation, when transition from the larval to adult stage occurs (Paludo et al., 2020). To date, the molecular basis of strobilation is not fully elucidated, and current knowledge is restricted to transcriptomic and proteomic works (Laschuk et al., 2011; Basika et al., 2016, Basika et al., 2019; Camargo de Lima et al., 2018) and identification of a few cellular signaling pathways (Olson et al., 2018; Montagne et al., 2019). The experimental study of the implicated mechanisms involved in strobilation represents a challenge due to the unfeasibility of using classic genetic and RNA interference methods for most species (Spiliotis et al., 2010).

An additional barrier associated with experimental work on cestode parasites is the limited availability of biological material due to the lack of in vitro and in vivo models for their maintenance, which makes it mandatory to rely on natural infections as the main supply of parasite specimens. To tackle this challenge, we used Mesocestoides vogae as a validated cestode laboratory model (Hemphill, 2010). The M. vogae larval developmental stage (tetrathyridium (TTy)) has a remarkable capacity for asexual reproduction in the peritoneal cavity of mice and some other mammalian hosts, allowing the continuous availability of large amounts of biological material. Also, it is easily cultured and is regarded as non-zoonotic, providing safety during manipulation of the parasites (Thompson et al., 1982, Hrčková et al., 1998). Furthermore, M. vogae strobilation can be induced in vitro under controlled laboratory conditions (Britos et al., 2000, Saldaña et al., 2001, Markoski et al., 2003). Thus, it was used to study the cestode parasite strobilation process (Saldaña et al., 2001, Lalanne et al., 2004, Koziol et al., 2010). In this cestode model, strobilation from the TTy larva to the mature strobilated adult (ST) encompasses evident modifications in external and internal structures, which is typical of cestodes (Camargo de Lima et al., 2020). Cestode larvae or pre-adult stages are commonly related to quiescent states, but in the case of TTy a highly metabolic activity was inferred according to gene expression and process enrichment analyses (Basika et al., 2019). Several genes are upregulated during the larval stage in M. vogae, which are supposed to be necessary for asexual reproduction, budding and carrying out specific behaviours of invasion of vertebrate host tissues and organs, such as the peritoneum and liver (Hrčkova et al., 2010). Identification of specific TTy genes related to binary fission and budding (Basika et al., 2019) provided insight into M. vogae development, although functional studies of the processes involved are still limited.

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of several biological processes, mainly through the repression of messenger RNAs (mRNAs) by typically binding to the 3’ untranslated region (3’UTR) of target genes (Bartel, 2018). As a result of this interaction, mRNA translation repression and/or exonucleolytic decay are induced (Bushati et al., 2007). The sequence located in the 5’ end of the miRNA, comprising nucleotides 2-7, is known as the ‘seed’ region, and its identity plays a key role in miRNA-target mRNA interaction (Bartel, 2018). The rising availability of helminth genomic sequences has enabled the bioinformatic and experimental identification of miRNAs in both nematodes and platyhelminths (Rosenzvit et al., 2013, Britton et al., 2014). In cestodes, these small RNAs have been described in zoonotic species such as Echinococcus granulosus sensu lato (Cucher et al., 2011, Bai et al., 2014, Macchiaroli et al., 2015), Echinococcus multilocularis (Cucher et al., 2015), Taenia solium (Pérez et al., 2017, Landa et al., 2019), Taenia saginata (Ai et al., 2012), Taenia asiatica (Liang et al., 2019) and Hymenolepis nana (Macchiaroli et al., 2019) as well as in the cestode model used in this work, M. vogae (Basika et al., 2016). Although studies on genome-wide miRNA identification, miRNA expression profile and miRNA target prediction are available for several species of cestodes, functional postgenomic studies and strategies are very limited to date. Among these, miRNA interference is a valuable tool for analyzing miRNA roles in cellular processes. It can be performed by the administration of inhibitors (generally miRNA complementary oligonucleotides, also called “anti-miRs”) or miRNA mimics, which can modify the activity of these small RNAs (Britton et al., 2014; Pérez et al., 2022).

miR-71-5p (miR-71 from here on) is one of the most expressed miRNAs in all cestode life cycle stages studied, including M. vogae (Cucher et al., 2015, Macchiaroli et al., 2015, Basika et al., 2016). This miRNA is absent in the genomes of vertebrate hosts and is known to target essential genes in Echinococcus spp. (Macchiaroli et al., 2017; Perez et al., 2019). However, target predictions for this miRNA have yet to be performed in M. vogae. In previous work, in vitro silencing of miR-71 in E. multilocularis cell cultures was successfully performed and shown to hamper early parasite development (Pérez et al., 2019). Thus, miR-71 represents a relevant miRNA that could aid in understanding the development and pathogenesis of parasitic cestodes, and could be considered a potential selective drug target to treat cestodiasis.

How miRNAs are involved in parasite biology and host interaction remains unclear. So far, successful whole worm knockdown of parasitic platyhelminth miRNAs has only been achieved in Schistosoma japonicum (Zhu et al., 2016). In this work, we aimed to analyze the function of miR-71 in M. vogae biology. We silenced miR-71 in whole larvae and assessed the effect of in vitro miR-71 knockdown on M. vogae larval development during strobilation induction and in experimental infection outcome. We also predicted and experimentally analyzed miR-71 target genes in this parasite model. The characterization and functional studies of miR-71 will contribute to a better understanding of the molecular bases of the development and survival of M. vogae. Further knowledge about cestode development biology could contribute to the search for, and design of, new therapies against Neglected Tropical Diseases caused by cestodes.