The Asian Citrus Psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the phytopathogens Candidatus Liberibacter americanus (Clam) (Coletta-Filho et al., 2005; Teixeira et al., 2005a; Teixeira et al., 2005b; Teixeira et al., 2005c) and Candidatus Liberibacter asiaticus (CLas) (Garnier and Bové, 1996; Bové, 2006). Both are phloem-restricted, belong to the alpha-proteobacteria group (Jagoueix et al., 1994), and are associated with the most serious citrus disease in the world, Huanglongbing (HLB) (McClean and Schwartz, 1970; Bové, 2006). The symptoms of HLB are well characterized, including reduced vigor (Etxeberria et al., 2009), a decline in fruit production, reduced fruit size and quality, aborted seeds, fruit asymmetry, an increase in juice acidity and, in more severe stages, death of the trees (Bové, 2006; Etxeberria et al., 2009) resulting in a significant economic loss. The life cycle of the D. citri vector includes an egg stage, 5 nymphal instars, and an adult stage; both immature and adult stages are classified as phloem feeding and sap-sucking insects and the duration of each developmental stage is dependent of the host-species and temperature (Tsai and Liu, 2000). D. citri population abundance is closely linked to the occurrence of young citrus leaf flush, which is the only suitable site for egg laying and nymph development (Hall and Albrigo, 2007). Given the high osmotic pressure and sugar content present in plant phloem-sap, hemipterans possess sucrose-transglucosidases in the alimentary canal that allow them to tolerate and transform the excessive sugars ingested into long chains of oligosaccharides that are subsequently excreted as honeydew (Douglas, 2006).

Currently, there is no known cure for HLB, and most measures associated with management of the disease focus on reducing D. citri populations. Over the past decade, most D. citri management programs have heavily relied on insecticide sprays (Qureshi et al., 2014; Boina and Bloomquist, 2011). However, this has resulted in significantly higher production costs, and unintended ecological consequences, such as decreased populations of beneficial insects and development of insecticide resistance among D. citri populations (Tiwari et al., 2011; Farnsworthn et al., 2014). Therefore, alternatives to insecticides must be developed and integrated into vector suppression.

One strategy that has received significant attention in recent years as a possible alternative to traditional insecticides is RNA interference (RNAi) (Huvenne and Smagghe, 2010; Mamta and Rajam, 2017; Yan et al., 2020). The purpose of RNAi is to silence specifically targeted genes involved in critical biological processes in the pest, but which are sufficiently specific to minimize non-target consequences. For example, knockdown of arginine kinase, abnormal wing disc, carboxylesterases, muscle protein 20, cathepsin D, chitin synthase and sucrose hydrolase genes cause various physical deformities and mortality in D. citri and could theoretically reduce D. citri populations (El-Shesheny et al., 2013; Hajeri et al., 2014; Andrade and Hunter, 2017; Galdeano et al., 2017; Kishk et al., 2017; Yu et al., 2017; Santos-Ortega and Killiny, 2018). The RNAi mechanism is triggered by a double-stranded RNA molecule (dsRNA) precursor, of variable length and origin, which is excised by RNase-III-like endonuclease (Dicer) into short ~20-30 nucleotides RNAs duplexes, called short interfering RNAs (siRNAs). The siRNAs are recognized by the RNA-induced silencing complex (RISC) and then this complex recognizes the expected target mRNA (single-strand), cleaving the mRNA molecule and thereby hindering the normal translational process (Elbashir et al., 2001; Hammond et al., 2001; Meister and Tuschl, 2004; Whyard et al., 2009). In agricultural systems, the potential implementation of RNAi delivery is based on transgenic and non-transgenic strategies. Among non-transgenic strategies to deliver RNAi, the use of Virus-induced gene silencing (VIGS) has been explored (Bachan and Dinesh-Kumar, 2012; Wuriyanghan and Falk, 2013; Hajeri et al., 2014; Khan et al., 2015). This strategy uses an engineered plant virus to deliver a targeted insect gene-sequence (dsRNA/siRNAs) during virus replication and/or transcription in the plant hosts (Bachan and Dinesh-Kumar, 2012). Upon feeding on a host colonized with the virus vector delivering the dsRNA/siRNA, the target pest ingests them leading to an inhibition of translation of the targeted genes within the insect (Hajeri et al., 2014; Khan et al., 2018).

In citrus, virus-based vectors have been created through the manipulation of Citrus tristeza virus (CTV) for heterologous gene expression (Folimonov et al., 2007; El-Mohtar and Dawson, 2014). CTV is a member of the Closterovirus genus within the family Closteroviridae; it possesses a single stranded RNA genome of ~19.3 kb (Bar-Joseph et al., 1979; Karasev et al., 1995), and it is considered the largest and most complex plant RNA virus with phloem limited tissue tropism where ACP feeds. The CTV-RNAi vectors were successfully used to target citrus and ACP genes (Hajeri et al., 2014). For example, engineering the CTV virus vector to deliver a 400 base, truncated RNA has been successfully demonstrated in citrus, by targeting the phytoene desaturase (PDS) gene, which resulted in photo-bleaching of citrus leaves upon infection with the CTV vector. Also, a CTV-RNAi vector delivering a truncated version of the Awd gene of D. citri resulted in reduced expression of the Awd gene upon ACP feeding on infected plants causing malformation of D. citri wings and high mortality of adults (Hajeri et al., 2014). This validates the use of CTV vectors for delivery of dsRNAs/siRNAs via citrus to target D. citri genes.

In our previous studies, two cathepsin genes were identified in D. citri, cathepsin B-like (DcCathB) (Ferrara et al., 2015) and cathepsin L-like (DcCathL) (Ferrara et al., 2020). Cathepsins are proteolytic cysteine-peptidase type enzymes (Storer and Ménard, 1994; Polgár, 2013; Rawlings et al., 2016), belonging to the family C1 and subfamily papain; cathepsins are proteases that occur broadly among animals and play important roles in immunity, metabolism, and development (Sajid and McKerrow, 2002; Wiederanders, 2003). In insects, cathepsins B and L play a key role in digestion and are involved in embryogenesis, immune defense, and ecdysis (Terra and Ferreira, 1994). In D. citri, DcCathB and DcCathL were highly expressed in the alimentary canal suggesting a role in digestion and were expressed during the egg stage suggesting involvement in embryonic development (Ferrara et al., 2020). We hypothesized that blocking activity of these enzymes using the recombinant CTV-RNAi vectors should disturb critical biological processes in D. citri. In this study, we evaluated the effects of RNAi targeting DcCathB and DcCathL genes in D. citri. The RNAi were constructed and delivered via modified CTV vectors. Our results showed a significant downregulation of both Cathepsin genes in ACP adults. Additionally, we examined the effects of these RNAi on ACP female reproduction, and our results indicated coincident downregulation of the Vitellogenin A1-like gene, which was associated with significant reduction in ovarial development among D. citri females feeding on RNAi-treated plants infected with modified CTV vectors.

Cathepsin B and cathepsin L enzymes in insects are associated with various biological processes including protein turnover, embryogenesis, mobilization, degradation of yolk proteins (Medina et al., 1988; Izumi et al., 1994; Cho et al., 1999; Zhao et al., 2002), metamorphosis (Takahashi et al., 1993; Shiba et al., 2001; Lee et al., 2009), and programmed cell death (Yang et al., 2020). We hypothesized that cathepsin enzymes should be useful targets for RNAi given their broad importance for D. citri development. Given the current lack of therapeutic treatments targeting the CLas pathogen, emphasis has been placed on reducing populations of the D. citri vector (Hajeri et al., 2014).

RNAi has been demonstrated on target insects for cathepsin genes using transgenic plants (Huang et al., 2006; Galdeano et al., 2017). In the present investigation, we utilized CTV-RNAi to target the cathepsin genes in D. citri. Delivery of dsRNA in citrus plants against a phloem feeding insect, such as D. citri, using CTV-based vectors offers advantages over the traditional transgenic approach in citrus. Engineering the virus is relatively easier and faster than producing transgenic plants. The engineered virus could colonize the mother plants from which budwood is used to generate citrus trees for establishing new grooves. Further, the CTV virus vector could be graft transmitted to current plants at any stage, including fully grown plants in the ground, obviating the need to remove the current generation of plants. However, despite the stability of CTV RNAi vectors for many years, at some stage, the engineered virus will recombine back into the wild type of the virus (El-Mohtar and Dawson, 2014). During this time, we expect that ongoing research may provide the ultimate solution for HLB via engineering a resistant/tolerant plant via biotechnology or traditional breeding.

DcCathB gene expression occurs in the D. citri gut of both nymph and adult stages, suggesting a possible role in digestion (Ferrara et al., 2015). Additionally, downregulation of DcCathB occurs in the presence of the CLas bacterium of both nymphs and adults, suggesting a possible role in immune response (Vyas et al., 2015). A different pattern of DcCathL expression has been reported compared to DcCathB, with expression observed in the head, thorax, and gut tissues of D. citri, suggesting function in more processes than digestion. Greater expression of the DcCathL gene in eggs than nymph and adult stages also suggests a role of in embryonic development (Ferrara et al., 2020). Additionally, DcCathL has been implicated in the innate immune response, like DcCathB (Yu et al., 2019). The preponderance of evidence collected to date would suggest that both DcCathB and DcCathL play roles associated with development in D. citri.

Our results provide evidence that DcCathB and DcCathL are associated with female reproductive development in D. citri ( Figure 3 ). Following knockdown of either DcCathB ( Figure 1 ) or DcCathL ( Figure 2 ), oocyte development was significantly compromised ( Figure 3B ) and resulted in an abnormal ovary phenotype ( Figure 3C ). Furthermore, maturation of ovaries was delayed, which appeared related to interference with vitellogenesis ( Figure 3 ). It is possible cathepsin gene regulation plays a role in vitellogenesis at some stage given the observed downregulation of the Vitellogenin A1-like gene coincident with downregulation of DcCathB or DcCathL ( Figure 3 ).

Vitellogenesis is crucial for insect embryonic development and egg production (Wu et al., 2021), and alterations in Vitellogenin (Vg) and associated genes can significantly interfere with normal development. For example, interference with Vg and the Vitellogenin receptor (VgR) hinder vitellogenin accumulation and resultant egg maturation (Ibanez et al., 2017; Peng et al., 2020). Similarly, in locusts, reduced expression of Vg gene resulted in arrested oocyte maturation and impaired egg production (Guo et al., 2014; Wu et al., 2016; Wu et al., 2018). In D. citri, knockdown of Vitellogenin-4 like (Vg-4 like) and VgR genes decreases the number of mature eggs in the ovary and causes phenotypic abnormalities in eggs and nymphs (Li et al., 2022). Also, RNAi knockdown of the VgR gene in Cadra cautella resulted in low fecundity and fertility (Husain et al., 2020). Cathepsin L (Mn-CTS L1) has been reported to play an important role in the ovarian development of the oriental river prawn, Macrobrachium nipponense. Interestingly, it was hypothesized that Mn-CTS L1 silencing can effectively inhibit Vg hydrolysis, thereby reducing the availability of energy required for ovarian development (Zhu et al., 2021). Cathepsin B has been shown to play a role in embryogenesis and egg development in the hard tick, Haemaphysalis longicornis (Zhang et al., 2019). Accordingly, we could observe a downregulation of Vg A1-like in those insects that fed on plants expressing dsRNA-DcCaths ( Figure 4 ). In addition, Cathepsins have been related to embryogenesis in insects as well the Vg hydrolysis (Moura et al., 2015; Zhang et al., 2019), for instance resulting in a reduction in developing oocytes and compromising ovarian maturation ( Figure 3 ).

However, how cathepsin and vitellogenin genes interact and why knockdown of DcCathB or DcCathL both appeared to coincidentally reduce vitellogenesis remain open questions.

Novel methodologies such as RNAi must be considered to mitigate economic and environmental damage of pathogen vectors (Zhang et al., 2013). We demonstrate here the silencing of cathepsin genes in D. citri by delivery of RNAi inducers via an engineered CTV vector colonizing citrus phloem tissue. Given that CTV accumulates and produces large amounts of dsRNA in the phloem, which is the sole source of nutrition for developing D. citri (Hajeri et al., 2014), use of CTV-based RNAi vectors is being explored as possible management tools for this phytopathogen vector. Our results indicate that reducing cathepsin gene expression causes visible phenotypic changes in D. citri, delaying and reducing ovarial development which appears related to a downregulation of vitellogenin expression. It will be important to determine whether reduced ovarial development is associated with changes to the fecundity and fertility of D citri (Bueno et al., 2020; Koji et al., 2020). We speculate that decreased ovarial development caused by silencing of cathepsin genes may result in reduced population growth of D. citri. This hypothesis will require further testing. The remarkable stability of the CTV vector, its effectiveness in delivering dsRNA into the plant phloem, and relatively fast activity further support its use for validation of biological targets and development into practical therapeutic tools. Our findings also indicate that Cathepsin B and L-like are promising target sites that should be further explored as a means of managing the D. citri vector. This research represents an initial step that will require scaling up to population-wide treatment of psyllids under authentic field conditions. Further biological testing of the consequences of cathepsin silencing in D. citri are needed to determine potential practical utility for population suppression of this phytopathogen vector, including effects on female fecundity and fertility.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

AS-C, FI-C, CEM, CR, WD and LS contributed to conception and design of the study. AS-C, SVR and FI-C wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.