Haemonchus contortus (H. contortus) is a type of parasitic nematode that belongs to the Haemonchus spp. (Trichostrongylidae) and is a major threat to the economically important gastrointestinal nematode of small ruminants. It primarily infects the abomasum, and when 2,000 or more worms are present there they can cause severe harm. These worms can suck up to 30 mL of blood per day, in addition to the blood loss caused by their departure. This can result in severe infection in livestock and cause many issues, such as anemia, weight loss, and even death (1). It is believed that H. contortus lays more eggs than other nematodes of the Trichostrongylidae family, with females laying 5,000–10,000 eggs every day. Third-stage larvae (L3) are more resistant and can generally survive for 3 months in pastures, and they can remain dormant for up to 1 year in adverse environments, leading to the contamination of pastures and the environment. Recent epidemiological studies showed that the positivity rate of H. contortus infection in sheep in Iran, India, Egypt, Brazil, and Ethiopia was 84.6, 44.7, 38.8, 76.4, and 68.75%, respectively (2–6). These developing countries have experienced significant losses in their dominant animal product industries due to H. contortus infections.

The prevention and treatment of H. contortus can be accomplished through the use of anthelmintics, with the selection and breeding of livestock breeds that are resistant to parasitic infection, biological control methods, and improved feed management (7, 8). Among these methods, anthelmintics are the most effective and economical means of control. Since the invention of highly effective and broad-spectrum anthelmintics, their prolonged use has led to serious resistance problems. Researchers have made extensive efforts to address these problems (9–11). With the release of the complete genome of H. contortus in 2013 (chromosome-level assembly was completed in 2019) (12) and the advancement of post-genomics in the last 15 years (13, 14), there are numerous molecular targets that can be exploited for control. Although these molecular targets have the potential to control H. contortus, they still require the evaluation of some techniques before they can be used as drug screening or vaccine candidates (15).

RNA interference (RNAi) is an important tool for evaluating the gene function. It is based on the principle that exogenous genes, such as viral genes, artificially transferred genes, transposons, and so on, are randomly integrated into the host cell genome and often produce some double-stranded RNAs (dsRNAs), which are transcribed using the host cell. Dicer, a nucleic acid endonuclease in the host cell, cleaves the dsRNA into multiple small fragments of RNA, commonly known as small interfering RNA (siRNA), with a specific length and structure. These siRNA fragments are unwound into a righteous strand and an antisense strand by intracellular RNA decapping enzymes and are then bound by antisense siRNA to some enzymes in vivo. The RNA-induced silencing complex (RISC) specifically binds to the homologous region of exogenous mRNA and functions as a nuclease, cleaving the mRNA at the binding site, which is the area where the antisense strand of the siRNA is complementary to it. The cleaved and fragmented mRNA is then degraded, leading to a degradation response in the host cell against these mRNAs (Figure 1).

In recent years, the availability of genomic and transcriptomic data for H. contortus has highlighted the need for functional genomics techniques. Since there are limited methods to directly study gene function in nematodes, RNAi and its related technologies have been developed over time to successfully identify key genes, provide a rational method to develop new control strategies, and apply them in relevant gene function studies (16). Comparative genomic analyses, especially using data from free-living Caenorhabditis elegans (C. elegans), can help predict gene function (17). In this study, we reviewed recent studies on the RNAi technique applied to H. contortus development genes and drug target screening and discussed the problems and limitations of the technique. It provides a theoretical basis for further in-depth research on the application of RNAi technology in parasitic nematodes.

RNAi has been widely used in the functional identification genes of H. contortus. However, its problems are also evident and constantly arise in the following three respects.

First, the sensitivity of H. contortus to RNAi varies with dsRNA delivery methods and target genes and is less reproducible. The results of many studies have shown that commonly used immersion, feeding, and injection methods can produce RNAi phenomena in H. contortus, but the immersion method is less efficient and more costly, and the storage time of larvae and batch effects have a significant impact on the results (40). The heterogeneous RNAi makes it difficult to accurately deliver dsRNA to specific sites and can easily lead to cellular damage. The feeding method is still the most widely used and is more likely to produce phenotypic effects and be easy to observe (41). When dsRNA or siRNA is delivered using electroporation, the phenotypic effects cannot be examined due to the high mortality of the treated larvae (42, 43). RNAi may inhibit gene expression under certain conditions. However, this only works on a limited number of genes, and in some cases, the effect is negligible, which may reflect differences in dsRNA uptake and transport to and from cells in H. contortus. The efficiency of gene interference in H. contortus has no particularly clear correlation with the gene itself or the delivery method; however, existing research found that gene interference may only occur in certain cell types. For example, the C. elegans rde-4 null mutants are amenable to RNAi with siRNA but not dsRNA (44). If parasitic nematodes lack rde-4 or a functional equivalent, RNAi should still be possible with siRNA delivery. They can still be delivered by siRNA; that is, siRNA is more effective than dsRNA. It is very likely that the ortholog of rde-4 or some undefined mechanism is still in operation. These examples can show that the efficiency of gene interference will become more controllable with further information and a more detailed search of genome sequencing projects. The insights gained from studies on other worms may also help optimize dsRNA delivery and determine which developmental stages may be best targeted.

Second, a single developmental stage for RNAi will make it difficult to assess phenotypes that may only be expressed at later developmental stages, as reliable culture systems have not been established. Further research is needed to develop reliable phenotyping systems. In vitro, reagents such as hypochlorite are widely used for sterilization and unsheathing of L3, which can affect larvae and subsequent stages of development even at very low concentrations (45). High concentrations of dsRNA delivery may be cytotoxic, so it may be necessary for us to determine the optimal concentration for each gene tested. It has also been reported that some of the phenotypes generated by RNAi can be replicated by chemical inhibitor target activity, providing proof of the principle that RNAi can replicate chemo-suppressive effects and confirming that RNAi screening is a potentially powerful and effective method for identifying novel drug targets. Based on RNAi, we need to further investigate the mechanism of these genes and improve the culture system of H. contortus to detect the long-term effects of RNAi; only then can RNAi become a reliable method for gene function detection.

In addition, there is also a large body of data indicating limitations in identifying potential H. contortus genes as a predictive model based on C. elegans data, with RNAi-mediated silencing being reliable for some genes but not others. H. contortus-specific genes are mainly located on autosomes, which have a higher recombination rate, and evolutionary mutations mainly occur in these regions, possibly being promoted by higher recombination. When these genes are silenced by RNAi, only a few genes can alter the phenotype, and in C. elegans, it is more difficult for us to observe the occurrence and alteration of these phenotypes. When conditions allow, we can further perform protein analyses to examine the decrease in mRNA and protein levels. Therefore, we need to be extra cautious and careful while using C. elegans as a model organism (46, 47). The RNAi technology will be more specific and targeted if these challenges are successfully overcome.

The current database has a large amount of genomic and transcriptomic data, and many studies have identified certain important genes that can successfully eliminate H. contortus. We currently need to move forward to the next level; RNAi technology has significantly improved its efficacy and ease of use and can be used to develop a more reliable and functional genomics platform. This can also be combined with technologies such as CRISPR/Cas and will provide a more rational approach to gene function identification. In vivo experiments have currently demonstrated that site-specific targeting of genes can improve their success rate, making in vivo RNAi a feasible option for identifying essential gene functions during infection, which is particularly important for elucidating non-conserved genes and speculating on the genes that play a role in influencing host immune responses. However, greater efforts are still needed to improve our understanding of H. contortus biology and to identify new approaches for parasitic control and prevention. The conclusion that can be drawn from the outlined studies is that RNAi is possible in parasitic nematodes and, if effectively utilized, can be a valuable tool for screening and validating new gene and vaccine targets.

SH: conceptualization. BH: writing—original draft preparation and writing—review and editing. YH: visualization. YH, BB, and SH: supervision, project administration, and funding acquisition. All authors read and agreed to the published version of the manuscript.