The α2δ Calcium Channel Subunit Accessorily and Independently Affects the Biological Function of Ditylenchus destructor

The α2δ subunit is a high-voltage activated (HVA) calcium channel (Cav1 and Cav2) auxiliary subunit that increases the density and function of HVA calcium channels in the plasma membrane of mammals. However, its function in plant parasitic nematodes remains unknown. In this study, we cloned the full-length cDNA sequence of the voltage-gated calcium channel (VGCC) α2δ subunit (named DdCavα2δ) in Ditylenchus destructor. We found that DdCavα2δ tends to be expressed in the egg stage, followed by the J3 stage. RNA-DIG in situ hybridization experiments showed that the DdCavα2δ subunit was expressed in the body wall, esophageal gland, uterus, post uterine, and spicules of D. destructor. The in vitro application of RNA interference (RNAi) affected the motility, reproduction, chemotaxis, stylet thrusting, and protein secretion of D. destructor to different degrees by targeting DdCα1D, DdCα1A, and DdCavα2δ in J3 stages, respectively. Based on the results of RNAi experiments, it was hypothesized that L-type VGCC may affect the motility, chemotaxis, and stylet thrusting of D. destructor. Non-L-type VGCC may affect the protein secretion and reproduction of D. destructor. The DdCavα2δ subunit gene also affected the motility, chemotaxis, and reproduction of D. destructor. These findings reveal the independent function of the VGCC α2δ subunit in D. destructor as well as give a theoretical foundation for future research on plant parasitic nematode VGCC.


Introduction
Intracellular Ca 2+ influx is primarily mediated by voltage-gated calcium channels (VGCC), which are widely distributed in biological cell membranes. Based on electrophysiological and pharmacological characteristics, VGCCs are classified as L-type, non-L-type, or T-type calcium channels [1]. Based on their activation properties, they are divided into high-voltage activated (HVA) calcium channels and low-voltage activated (LVA) calcium channels, with the HVA calcium channel being divided into L-type (Ca V 1) and non-L-type (Ca V 2) channels, whereas the LVA calcium channel only has T-type channels (Ca V 3) [2]. In mammals, calcium channels are found in a variety of cells, including neurons, neurosecretory cells, and muscle cells, and they play an important role in muscle contraction, hormone secretion, and neurotransmitter release. Ca V 1 and Ca V 2 channels are typically made up of a pore-forming α 1 subunit and β, α 2 δ, and γ auxiliary subunits, whereas only an α 1 subunit has been identified in Ca V 3 channels [3][4][5].
Although the major biophysical and pharmacological features of these channels are determined by the α 1 subunit, their expression is also influenced by auxiliary subunits β and α 2 δ. The β subunit is one of the major auxiliary subunits of HVA calcium channels, and four of its genes (β1-β4) have been cloned. The GK domain of the β subunit binds to the α-interacting domain (AID), thereby exerting its role in regulating the surface expression and gating properties of high-voltage activated calcium channels [6][7][8]. α 2 δ is another   There are 22 cysteine (Cys) residues in the DdCa v α 2 δ amino acid sequence and nine predicted N-glycosylation sites ( Figure 2). These cysteine residues may be associated with the formation of disulfide bonds. The amino acid sequence of DdCa v α 2 δ was compared with the unc-36 sequence from C. elegans and four α 2 δ sequences from humans. BLAST Protein analysis with the encoded amino acid sequences showed that DdCa v α 2 δ has features in common with other VGCC α 2 δ subunits, including two typical regions: the VWA domain and the Cache domain ( Figure 3). Moreover, according to Qin et al., we also predicted the breaking point between α 2 and δ subunits in the DdCa v α 2 δ sequence ( Figure 3) [12]. VWA domains are usually about 200 residues long, and there is a MIDAS motif in each VWA domain. We found that the VWA domains here all contained a "perfect" MIDAS motif, in which all five discontinuous and co-coordinating amino acids were present (DxSxS, A/T, D) [18]. The amino acid position of the VWA domain of the DdCa v α 2 δ sequence was present at 270-487 amino acids (Figure 1), and this domain was highly conserved when compared with the unc-36 domain, with a similarity of 81.3%. The Cache domains of DdCa v α 2 δ, unc-36, and tag-180 were all downstream of the VWA domains. The amino acid positions of the Cache domains in the DdCavα 2 δ sequence was 519-602 amino acids. The MIDAS motif in the DdCa v α 2 δ sequence was highly conserved in comparison to the unc-36 sequence. These sequences all contained five co-coordinating amino acids, including D, S, S, A, and D.     Other species' Cavα 2 δ amino acid sequences from NCBI database (https://blast.ncbi.nlm.nih.gov, accessed on 15 October 2022), with the following accession numbers: human-α 2 δ-1 (NM_000722), human-α 2 δ-2 (AF042793), human-α 2 δ-3 (AF516696), human-α 2 δ-4 (AF516695), unc-36 (NM_001047386.6).

Phylogenetic Analysis of DdCa v α 2 δ
The phylogenetic analysis was based on the deduced amino acid sequences of the α 2 δ subunits of D. destructor and the corresponding subunits of C. elegans and other species. The amino acid sequences were obtained from the NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 15 October 2022) and WormBase (https://parasite.wormbase.org/index.html, accessed on 15 October 2022) databases ( Table 2). The phylogenetic tree shows the branches composed of vertebrate and invertebrate subunits. Particularly, the α 2 δ of D. destructor and the corresponding subunits of C. elegans were clustered in invertebrates, forming a different branch from vertebrates, and they were closely related to the evolutionary position of C. elegans (Figure 4). This implies that DdCa v α 2 δ may have the same evolutionary relationship and a similar physiological function as the unc-36 gene of C. elegans.

Developmental Expression Analysis of DdCa v α 2 δ
The expression level of DdCa v α 2 δ was assessed by RT-qPCR at different developmental stages (including egg, J2, J3, and J4). DdCa v α 2 δ was widely expressed at all developmental stages ( Figure 5). Results indicated that DdCa v α 2 δ plays an important role in all stages of D. destructor development. In particular, the expression of DdCa v α 2 δ was significantly higher in the egg stage than in other developmental stages. In addition, the mRNA expression level of DdCa v α 2 δ in the J3 and J4 were 3.4 and 2.7 times higher than in the J2 stage, respectively.

Tissue Localization of DdCa v α 2 δ
In situ hybridization was used to detect the site of DdCa v α 2 δ gene expression in the adult stages of D. destructor at the mRNA level ( Figure 6). The antisense-probe treated nematodes showed positive staining in the esophageal glands ( Figure 6E), body wall ( Figure 6F), uterus and post uterine area of females ( Figure 6G), and the spicules of males ( Figure 6H), whereas the sense-probe treated nematodes showed no hybridization signal ( Figure 6A-D).
accessed on 15 October 2022) and WormBase (https://parasite.wormbase.org/index.html, accessed on 15 October 2022) databases ( Table 2). The phylogenetic tree shows the branches composed of vertebrate and invertebrate subunits. Particularly, the α2δ of D. destructor and the corresponding subunits of C. elegans were clustered in invertebrates, forming a different branch from vertebrates, and they were closely related to the evolutionary position of C. elegans (Figure 4). This implies that DdCavα2δ may have the same evolutionary relationship and a similar physiological function as the unc-36 gene of C. elegans. Figure 4. Phylogenetic analysis of DdCavα2δ with α2δ genes from other species. The phylogenetic tree was constructed from the amino acid sequences of 20 VWA structural domain-containing α2δ genes. The tree was constructed using MEGA 6.0 based on the neighbor-joining method according to the amino acid sequences. The phylogenetic tree and sequences of other species were in the NCBI database; accession numbers are shown in Table 2.   Table 2.

Developmental Expression Analysis of DdCavα2δ
The expression level of DdCavα2δ was assessed by RT-qPCR at different developmental stages (including egg, J2, J3, and J4). DdCavα2δ was widely expressed at all developmental stages ( Figure 5). Results indicated that DdCavα2δ plays an important role in all stages of D. destructor development. In particular, the expression of DdCavα2δ was significantly higher in the egg stage than in other developmental stages. In addition, the mRNA expression level of DdCavα2δ in the J3 and J4 were 3.4 and 2.7 times higher than in the J2 stage, respectively.

Tissue Localization of DdCavα2δ
In situ hybridization was used to detect the site of DdCavα2δ gene expression in the adult stages of D. destructor at the mRNA level ( Figure 6). The antisense-probe treated

Silencing Efficiency of dsRNA Soaking
Following 24 h of dsRNA treatment, FITC was taken up by D. destructor. The expression levels of DdCα1D, DdCα1A, and DdCavα2δ mRNA in D. destructor were measured by qPCR. When dsCavα2δ was fed, the transcript level of DdCavα2δ was significantly reduced to 31.4%, whereas the transcript levels of DdCα1D and DdCα1A were unchanged relative to the control ( Figure 7A). When dsCα1D was fed, the transcript level of DdCα1D was significantly reduced to 67.8%, but the transcript levels of DdCα1A and DdCavα2δ were unchanged ( Figure 7B). When dsCavα2δ and dsCα1D were fed simultaneously, the transcript levels of DdCavα2δ and DdCα1D decreased to 45.7% and 67.4%, respectively, whereas the transcript level of DdCα1A was unchanged ( Figure 7D). When dsCα1A was fed, the transcript level of DdCα1A decreased to 44.7%, whereas there was no change in the transcript levels of DdCα1D and DdCavα2δ ( Figure 7C). When dsCavα2δ and dsCα1A were fed simultaneously, the transcript levels of DdCavα2δ and DdCα1A were decreased to 41.2% and 33.3%, respectively, and the transcript level DdCα1D was unchanged ( Figure  7E).

Silencing Efficiency of dsRNA Soaking
Following 24 h of dsRNA treatment, FITC was taken up by D. destructor. The expression levels of DdCα1D, DdCα1A, and DdCa v α 2 δ mRNA in D. destructor were measured by qPCR. When dsCa v α 2 δ was fed, the transcript level of DdCa v α 2 δ was significantly reduced to 31.4%, whereas the transcript levels of DdCα1D and DdCα1A were unchanged relative to the control ( Figure 7A). When dsCα1D was fed, the transcript level of DdCα1D was significantly reduced to 67.8%, but the transcript levels of DdCα1A and DdCa v α 2 δ were unchanged ( Figure 7B). When dsCa v α 2 δ and dsCα1D were fed simultaneously, the transcript levels of DdCa v α 2 δ and DdCα1D decreased to 45.7% and 67.4%, respectively, whereas the transcript level of DdCα1A was unchanged ( Figure 7D). When dsCα1A was fed, the transcript level of DdCα1A decreased to 44.7%, whereas there was no change in the transcript levels of DdCα1D and DdCa v α 2 δ ( Figure 7C). When dsCa v α 2 δ and dsCα1A were fed simultaneously, the transcript levels of DdCa v α 2 δ and DdCα1A were decreased to 41.2% and 33.3%, respectively, and the transcript level DdCα1D was unchanged ( Figure 7E).
The above results showed that, when the dsRNA for each gene was fed, this led to a significant specific partial knock down of the target gene, indicating that our RNAi approach works. Furthermore, silencing of the DdCa v α 2 δ subunit gene did not lead to the downregulation of the transcript levels of the DdCα1D and DdCα1A subunit genes. Silencing of the DdCα1D or DdCα1A subunits also did not lead to the downregulation of the DdCa v α 2 δ subunit gene at the transcript level, indicating that the expression of DdCa v α 2 δ does not affect the transcription of the DdCα1D and DdCα1A subunits. Thus, it is presumed that the α 2 δ and HVA α 1 subunits do not appear to influence each other's mRNA expression levels.
proach works. Furthermore, silencing of the DdCavα2δ subunit gene did not lead to downregulation of the transcript levels of the DdCα1D and DdCα1A subunit genes. Sile ing of the DdCα1D or DdCα1A subunits also did not lead to the downregulation of DdCavα2δ subunit gene at the transcript level, indicating that the expression of DdCa does not affect the transcription of the DdCα1D and DdCα1A subunits. Thus, it is p sumed that the α2δ and HVA α1 subunits do not appear to influence each other's mR expression levels.   Results of the motility assay are shown in Figure 8. After washing dsRNA for 6 h, when dsGFP was fed, its passage rate was 33.7%. The sand column passage rates of other target dsRNA genes were significantly downregulated compared with dsGFP treatment. When dsCa v α 2 δ was fed, its passage rate was 25.3%. When dsCa1D was fed, its passage rate was 23.7%. When dsCa v α 2 δ and dsCα1D were fed simultaneously, the passage rate was 19.7%. When dsCa1A was fed, its passage rate (28.3%) was higher than that of dsCa1D. When dsCa v α 2 δ and dsCα1A were fed simultaneously, the passage rate (25.7%) was higher than that of dsCa v α 2 δ + dsCα1D. After washing dsRNA for 24 h, the sand column passage rate of D. destructor in all treatments increased significantly, but there were still significant differences compared to that observed in the dsGFP treatment.
when dsGFP was fed, its passage rate was 33.7%. The sand column passage rates of other target dsRNA genes were significantly downregulated compared with dsGFP treatment. When dsCavα2δ was fed, its passage rate was 25.3%. When dsCa1D was fed, its passage rate was 23.7%. When dsCavα2δ and dsCα1D were fed simultaneously, the passage rate was 19.7%. When dsCa1A was fed, its passage rate (28.3%) was higher than that of dsCa1D. When dsCavα2δ and dsCα1A were fed simultaneously, the passage rate (25.7%) was higher than that of dsCavα2δ + dsCα1D. After washing dsRNA for 24 h, the sand column passage rate of D. destructor in all treatments increased significantly, but there were still significant differences compared to that observed in the dsGFP treatment.
The passage rate of dsGFP treatment was 54.3%. When dsCavα2δ was fed, the passage rate of D. destructor was 44.7%, and the passage rate was 42.0% when dsCa1D was fed. In addition, the nematode passage rate was 41.0% when dsCavα2δ and dsCa1D were fed simultaneously. The passage rate was 46.7% for the nematodes fed dsCa1A and 48% for nematodes fed both dsCavα2δ and dsCα1A. The above results indicated that the effect of RNA interference diminished the motility of nematodes, and this decrease in motility recovered with time. Additionally, results indicate that both L-type and non-L-type VGCC affect the motility of D. destructor, and the Cavα2δ subunit affects D. destructor locomotion. The Cavα2δ subunit might play an auxiliary role in the locomotion of D. destructor that is mediated by L-type and non-L-type VGCCs. Figure 8. Effect of dsRNA soaking on the motility of D. destructor. One hundred J2-J3 worms treated with dsRNA were added to the sand column, and the number of worms passing through the sand column was counted at 6 h and 24 h, respectively, to calculate the migration rating. Each column represents the mean ± standard error of three replicates. Different letters indicate significant differences at p < 0.05 by Duncan's multiple range test. The experiments were performed three times with similar results.

Chemotaxis Assay
In order to detect changes in D. destructor chemotaxis to sweet potato after silencing the DdCavα2δ subunit gene, the number of nematodes attracted to sweet potato blocks placed on 1% agar for 36 h was counted separately. The results are shown in Figure 9A. One hundred J2-J3 worms treated with dsRNA were added to the sand column, and the number of worms passing through the sand column was counted at 6 h and 24 h, respectively, to calculate the migration rating. Each column represents the mean ± standard error of three replicates. Different letters indicate significant differences at p < 0.05 by Duncan's multiple range test. The experiments were performed three times with similar results.
The passage rate of dsGFP treatment was 54.3%. When dsCa v α 2 δ was fed, the passage rate of D. destructor was 44.7%, and the passage rate was 42.0% when dsCa1D was fed. In addition, the nematode passage rate was 41.0% when dsCa v α 2 δ and dsCa1D were fed simultaneously. The passage rate was 46.7% for the nematodes fed dsCa1A and 48% for nematodes fed both dsCa v α 2 δ and dsCα1A. The above results indicated that the effect of RNA interference diminished the motility of nematodes, and this decrease in motility recovered with time. Additionally, results indicate that both L-type and non-L-type VGCC affect the motility of D. destructor, and the Ca v α 2 δ subunit affects D. destructor locomotion. The Ca v α 2 δ subunit might play an auxiliary role in the locomotion of D. destructor that is mediated by L-type and non-L-type VGCCs.

Chemotaxis Assay
In order to detect changes in D. destructor chemotaxis to sweet potato after silencing the DdCa v α 2 δ subunit gene, the number of nematodes attracted to sweet potato blocks placed on 1% agar for 36 h was counted separately. The results are shown in Figure 9A. The attraction rate of the dsGFP treatment was 22.5%. Compared with the dsGFP treatment, the nematode attraction rates of the other target gene dsRNA treatments were decreased to various degrees. When dsCa1D was fed, the attraction rate of D. destructor was 7.3%. When dsCa v α 2 δ was fed, the attraction rate was 12.0%. When dsCa v α 2 δ and dsCa1D were fed simultaneously, the attraction rate was only 5.7%. The attraction rate was 12.7% when dsCa1A was fed and 9.0% for nematodes fed both dsCa v α 2 δ and dsCα1A. The co-silencing of DdCα1D or DdCα1A and DdCa v α 2 δ subunit genes also affected nematode chemotaxis, with the effect of dsCa v α 2 δ + dsCα1D treatment being greater. This indicates that both L-type and non-L-type VGCC affect the chemotaxis of D. destructor, and the Ca v α 2 δ subunit has an important auxiliary role in L-type and non-L-type VGCC-mediated nematode chemotaxis. The Ca v α 2 δ subunit also had an effect on the chemotaxis of D. destructor. The attraction rate of the dsGFP treatment was 22.5%. Compared with the dsGFP treatment, the nematode attraction rates of the other target gene dsRNA treatments were decreased to various degrees. When dsCa1D was fed, the attraction rate of D. destructor was 7.3%. When dsCavα2δ was fed, the attraction rate was 12.0%. When dsCavα2δ and dsCa1D were fed simultaneously, the attraction rate was only 5.7%. The attraction rate was 12.7% when dsCa1A was fed and 9.0% for nematodes fed both dsCavα2δ and dsCα1A. The cosilencing of DdCα1D or DdCα1A and DdCavα2δ subunit genes also affected nematode chemotaxis, with the effect of dsCavα2δ + dsCα1D treatment being greater. This indicates that both L-type and non-L-type VGCC affect the chemotaxis of D. destructor, and the Cavα2δ subunit has an important auxiliary role in L-type and non-L-type VGCC-mediated nematode chemotaxis. The Cavα2δ subunit also had an effect on the chemotaxis of D. destructor. Different letters indicate significant differences at p < 0.05 by Duncan's multiple range test. The experiments were performed three times with similar results.

Stylet Thrusting Assay
To assess the effect of HVA α1 and α2δ subunits on nematode stylet thrusting, nematodes were stimulated with serotonin, and the numb of stylets thrusting was counted over a one-minute period. It is evident from Figure 9B that the number of stylets thrusting following dsGFP treatment was 51.7 times/min. The number of stylets thrusting in dsCavα2δ, dsCa1A, and dsCavα2δ + dsCα1A treatments were not significantly different from that of dsGFP treatment: dsCavα2δ treatment nematodes had 49.7 stylets thrusting in one minute, dsCa1A treatment nematodes had 42.0 stylets thrusting, and dsCavα2δ + dsCα1A treatment nematodes had 41.0 stylets thrusting. The dsCa1D and dsCavα2δ + dsCα1D treatment nematodes showed significant differences in the number of stylets thrusting compared to that observed in the dsGFP treatment. The number of nematode stylets thrusting was 33.0

Stylet Thrusting Assay
To assess the effect of HVA α 1 and α 2 δ subunits on nematode stylet thrusting, nematodes were stimulated with serotonin, and the numb of stylets thrusting was counted over a one-minute period. It is evident from Figure 9B that the number of stylets thrusting following dsGFP treatment was 51.7 times/min. The number of stylets thrusting in dsCa v α 2 δ, dsCa1A, and dsCa v α 2 δ + dsCα1A treatments were not significantly different from that of dsGFP treatment: dsCa v α 2 δ treatment nematodes had 49.7 stylets thrusting in one minute, dsCa1A treatment nematodes had 42.0 stylets thrusting, and dsCa v α 2 δ + dsCα1A treatment nematodes had 41.0 stylets thrusting. The dsCa1D and dsCa v α 2 δ + dsCα1D treatment nematodes showed significant differences in the number of stylets thrusting compared to that observed in the dsGFP treatment. The number of nematode stylets thrusting was 33.0 times/min in the dsCa1D treatment, and 27.3 times/min in the dsCa v α 2 δ + dsCα1D treatment. These results indicate that the silencing of the DdCα1D subunit gene alone affects the stylets thrusting of D. destructor, and the co-silencing of the DdCα1D and DdCa v α 2 δ subunit genes increases the effect of L-type VGCCs on the function of stylets thrusting. However, the difference between dsCα1D and dsCa v α 2 δ + dsCα1D treatments was not significant. This indicates that L-type VGCCs affect the stylet thrusting function of the potato decay stem nematode, and non-L-type VGCCs have no effect on the stylet thrusting function of the potato decay stem nematode. Consequently, the auxiliary subunit Ca v α 2 δ has an important auxiliary effect on L-type VGCC-mediated stylet thrusting in D. destructor, but non-L-type VGCCs have no direct effect on the stylet thrusting function of D. destructor.

Protein Secretion Assay
Secreted proteins from nematodes were detected after 16 h of 0.1% resorcinol treatment. As shown in Figure 9C, the nematode secretory protein content in the dsGFP treatment was 20,222.22 µg/mL. When dsCa v α 2 δ and dsCα1D were fed, respectively, the protein content of D. destructor was not significantly different compared to that of the dsGFP treatment, which were 20,042.22 µg/mL and 20,004.44 µg/mL, respectively. Similarly, the protein content of D. destructor was 20.102 mg/mL and was not significantly different compared to that in the dsGFP treatment when dsCa v α 2 δ and dsCα1D were fed simultaneously. However, the protein content was significantly downregulated when dsCα1A or dsCa v α 2 δ + dsCα1A were fed, resulting in a protein secretion content of 19,740.00 µg/mL and 19,664.44 µg/mL, respectively. These results indicate that silencing the DdCα1A subunit gene alone affects protein secretion in potato rot stem nematodes and that co-silencing the DdCα1A and DdCa v α 2 δ subunits increases the effect of non-L-type VGCCs on the protein secretion function of D. destructor. This indicates that L-type VGCCs have no effect on protein secretion function, but non-L-type VGCCs affect the protein secretion function of D. destructor. The Ca v α 2 δ auxiliary subunit has an important auxiliary effect on nematode protein secretion mediated by non-L-type VGCCs, but it has no effect on the protein secretion function of D. destructor.

Reproduction Assay
The reproduction rate was significantly reduced 25 d after DdCα1D, DdCα1A, and DdCa v α 2 δ were silenced. As shown in Figure 9D, the reproduction coefficient of D. destructor was 62.3 in the dsGFP treatment. The reproduction coefficients were significantly reduced 25 d after DdCα1D, DdCα1A, and DdCa v α 2 δ were silenced. The reproduction coefficient was 35.2 for the dsCa v α 2 δ treatment, 32.4 for the dsCa1D treatment, and 31.5 for the dsCa v α 2 δ + dsCα1D treatment. When dsCa1A was fed, the reproduction coefficient was 22.2, and the reproduction coefficient was 17.2 when dsCa v α 2 δ and dsCα1A were both fed. The above results indicate that the silencing of DdCα1D, DdCα1A, and DdCa v α 2 δ subunit genes, respectively, all affected the reproduction coefficient of potato rot stem nematodes, and the silencing of the DdCα1A subunit gene had the greatest effect on the reproduction coefficient of nematodes. Co-silencing DdCα1D or DdCα1A with DdCa v α 2 δ subunit genes further reduced the reproduction coefficient of D. destructor, which was even lower in the dsCa v α 2 δ + dsCα1A treatment. These data indicate that both L-type and non-L-type VGCCs affect the reproduction of D. destructor, and the Ca v α 2 δ auxiliary subunit has an important auxiliary role in non-L-type VGCC-mediated nematode reproduction. The Ca v α 2 δ subunit also has an effect on the reproduction of nematodes. Among the Ca v α 2 δ subunits, non-L type VGCCs had the greatest effect on the reproduction of D. destructor.

Discussion
In mammals and insects, voltage-gated sodium channels (VGSCs) play an important role in maintaining cellular excitability and normal physiological functions, making them important targets for a variety of neurotoxins [36]. However, VGSCs have not been found in nematodes [37], whose neuronal activity is thought to be related to VGCCs. VGCCs regulate a number of physiological functions, including neuronal excitability, transmitter release, and muscle contraction and are mainly composed of several subunits, such as α 1 , β, α 2 δ with γ [38]. In our study, we cloned and characterized a VGCC α 2 δ subunit from D. destructor, named DdCa v α 2 δ. α 2 δ is a highly glycosylated extracellular protein containing a VWA domain that is normally found in extracellular matrix proteins and integrin receptors [39]. Therefore, it is highly likely that the interaction between α 2 δ and α 1 occurs extracellularly. The amino acid sequence deduced from the DdCa v α 2 δ cDNA sequence has a VWA domain and a MIDAS motif, and the domain and motif are also present in the unc-36 gene of C. elegans [19]. Thus, we tentatively defined DdCa v α 2 δ as the VGCC auxiliary subunit of D. destructor. The analysis of the protein amino acid sequence also revealed that there was no GPI anchor site, "CGG" or "GAS", as found in mammals, but a similar "GCS" sequence was found at the 903-905 amino acid position (underlined part of Figure 2). Therefore, further validation of the DdCa v α 2 δ structure that spans the membrane remains to be conducted.
Four α 2 δ subunits (α 2 δ-1, α 2 δ-2, α 2 δ-3, and α 2 δ-4) have been identified in mammals, and Dolphin et al. showed that they are expressed in skeletal muscle, neurons, the brain, and the testis [9,20]. Two VGCC α 2 δ subunits (UNC-36 and TAG-180) were identified in C. elegans [25]. It was found that UNC-36 was expressed in the body wall, vulva, and pharynx muscles of C. elegans [27]. In a further study, UNC-36 was also shown to be expressed in muscle and motor neurons and co-regulated with EGL-19 (L-type α 1 ) in C. elegans body muscles [26,27]. Ye et al. cloned three VGCC α 1 subunit genes in D. destructor, DdCα1D, DdCα1A, and DdCa1G, and found that they play a role in modulating locomotion, feeding, and reproduction, respectively [33]. Recently, we identified the DdCa v β subunit of D. destructor and showed that it has a complementary role in the biological functions of the DdCα1D and DdCα1A subunits [40].
In the present study, DdCa v α 2 δ was found to be expressed in the esophageal glands, body wall, uterus, and post uterine tissue of D. destructor, along with the spicules. This is highly consistent with studies on α 2 δ subunits in mammals and in C. elegans. Furthermore, we also showed that silencing the DdCα1D, DdCα1A, and DdCa v α 2 δ subunit genes, respectively, significantly reduced the nematode passage rate in sand columns and the attraction rate of D. destructor, with lower passage and attraction rates when the DdCα1D subunit gene was silenced alone. In addition, the passage and attraction rates were lowest when DdCa v α 2 δ was co-silenced with the DdCα1D subunit, but the rates were not significantly different from those observed with DdCα1D subunit gene silencing alone. The results showed that silencing of the DdCα1D subunit gene affected the stylet thrusting of D. destructor, and the co-silencing of the DdCα1D and DdCa v α 2 δ subunit genes increased the effect of L-type VGCCs on the stylet thrusting of nematodes, but the difference was not significant.
By examining the protein content in the supernatant of D. destructor, we found that silencing the DdCα1A subunit gene reduced the nematode's secretory protein content, and co-silencing of DdCa v α 2 δ with the DdCα1A subunit gene increased the effect of non-Ltype VGCCs on the nematode's secretory protein function. Interestingly, we found some differences in secretory protein content when DdCa v α 2 δ or DdCa v β were co-silenced with the DdCα1A subunit gene [40]. This may be due to the fact that the Ca v β and Ca v α 2 δ auxiliary subunits have different affinities for the HVA Ca v α 1 subunit, thus causing a difference in the auxiliary effect on the α 1 subunit. The binding of the Ca v β to Ca v α 1 subunits occurs at a high affinity action site [41], while the affinities between Ca v α 1 and Ca v α 2 δ, however, appear rather weak [42]. We also found that the silencing of DdCα1D, DdCα1A, and DdCa v α 2 δ subunit genes, respectively, all affected the reproduction coefficient of D. destructor, and the silencing of the DdCα1A subunit gene had the greatest effect on the reproduction coefficient. Co-silencing of DdCα1D or DdCα1A and DdCa v α 2 δ subunit genes further reduced the reproduction coefficient, whereas the reproduction coefficient of dsCa v α 2 δ + dsCα1A treated nematodes was even lower.
In this study, we found that DdCa v α 2 δ was expressed in the uterus, post uterine tissue, and spicules. DdCα1A was also expressed in the vulva and vas deferens [33]. Therefore, we conclude that DdCα1A plays a key role in the reproduction of D. destructor along with DdCa v α 2 δ. In the future, additional studies on the phenotypic effects of transgenic fungi on D. destructor should be conducted. This will provide a theoretical basis for control strategies against D. destructor.

Nematode
Ditylenchus destructor was isolated from infested sweet potatoes in Hebei, China, and was preserved in the storage roots of sweet potatoes. Sweet potatoes were washed with clean water, sterilized with 1% NaClO for 10 min, dried, and treated with UV light for 30 min. Approximately 1000 mixed stage D. destructor were inoculated into sweet potatoes by digging holes with a sterile hole punch, after which D. destructor were sealed in the sweet potato with paraffin. Inoculated sweet potatoes were incubated at 25 • C for 25-30 d in the dark, and nematodes in the mixed stage were collected using the modified Baermann method [43]. Nematode eggs were screened by density-gradient centrifugation with a 1500-mesh sieve [44]. Nematodes at different developmental stages were obtained at 1-week intervals.

Cloning of the DdCa v α 2 δ Subunit
Total RNA was extracted from D. destructor using Trizol Reagent (Sangon Biotech Co., Ltd., Shanghai, China). RNA quality and concentration were determined with an ultra-micro spectrophotometer (Thermo, Shanghai, China), and RNA integrity was assessed using 1% gel electrophoresis. The first strand of cDNA was synthesized using the PrimeScript™III First-Strand Synthesis System (Invitrogen, Carlsbad, USA) for RT-PCR. The DdCa v α 2 δ gene putative sequence was obtained from the WormBase database (https://parasite.wormbase.org/index.html, accessed on 15 October 2022) by comparing WormBase database genes with the C. elegans unc-36 gene obtained from the National Center for Biotechnology Information (NCBI) database. First, to obtain the full length DdCavα 2 δ subunit coding region sequence, PCR was performed using gene-specific primers (D-F, D-R) ( Table 1). PCR was conducted using a standard procedure in a 25-µL volume that included 2.5 µL 10 × Ex Taq Buffer (TaKaRa, Japan), 2 µL dNTP Mixture (TaKaRa), 1 µL cDNA template, 1 µL of each primer, 0.5 µL of EX Taq polymerase (TaKaRa), and 17 µL of ddH 2 O. PCR conditions included an initial denaturation step at 94 • C for 5 min, followed by 35 cycles at 94 • C for 30 s, 55 • C for 30 s, 72 • C for 4 min, and a final step for 7 min at 72 • C. PCR products were purified using an agarose gel recovery kit (Trans, Beijing, China). The purified fragments were cloned into pMD™ 19-T vectors (TaKaRa,) and transformed into E. coli DH5α competent cells (Tiangen, Beijing, China). Three positive clones were randomly selected for bidirectional sequencing through the commercial service of the Sangon Biotech Co., Ltd. To obtain the length of the DdCa v α 2 δ subunit with its 5 -noncoding region sequence, PCR was performed using a spliced leader sequence, SL1, and a gene-specific primer (5 -R1, 5 -R2) ( Table 1). Finally, the first strand of cDNA with the 3 -noncoding region was synthesized using the 3 full RACE Core Set with PrimerScript TM RTase (TaKaRa) with the primers listed in Table 1.

Gene Characterization and Phylogenetic Analysis
Three overlapping fragments were spliced using DNAMAN software (DNAMAN 9.1; Lynnon BioSoft, Canada) to generate the full-length cDNA for the DdCa v α 2 δ subunit, and the amino acid sequence was deduced. The DdCa v α 2 δ conserved structural domains were predicted by the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 October 2022) and were mapped using IBS 1.0. A phylogenetic tree of the DdCa v α 2 δ subunit was constructed using the mega 6.0 neighbor joining method [45].

Stage-Specific Expression of DdCa v α 2 δ
To analyze the expression of the DdCa v α 2 δ subunit at each developmental stage in D. destructor, mRNA was extracted from approximately 1000 worms using the Dynabeads TM mRNA DIRECT TM Kit (Invitrogen). Additionally, cDNA was synthesized from mRNA using reverse transcription kits, according to the manufacturer's instructions. Primers were designed (Table 1), and SYBR Green Real-time PCR was performed using the 18S rRNA as an internal reference. The qPCR data were analyzed using the CFX Manager software and the Bio-Rad 2 −∆∆Ct method to calculate relative gene expression [46]. All experiments were repeated three times, and averages were calculated.

Tissue Localization of DdCa v α 2 δ
In situ hybridization was performed following a previously published method with modifications [47]. Fragments used as probes were amplified from the full-length cDNA from D. destructor using HindIIIF and EcoRI R primers (Table 1). DIG-labeled forward and antisense probes were synthesized using the DIG RNA Labeling Kit (Roche, Basel, Germany). Approximately 10,000 mixed-stage D. destructor specimens were fixed in 4% Paraformaldehyde Fix Solution (Sangon Biotech Co., Ltd.) for 18 h at 4 • C, followed by fixation for 4 h at room temperature. Ditylenchus destructor were then cut into 2-5 segments and treated with 0.5 mg/mL proteinase K at room temperature for 1 h. Hybridization was performed at 50 • C for 22 h, and they were treated with an antibody solution at 37 • C for 2 h. Samples were treated with chromogenic solution (Sangon Biotech Co., Ltd.) overnight and then observed and photographed under a microscope (Motic, Xiamen, China).

dsRNA Synthesis and Soaking of D. destructor
Total RNA was extracted from D. destructor using the Trizol method, first strand cDNA was synthesized, and dsRNA was synthesized using gene-specific primers containing the T7 polymerase promoter sequence (Table 1) and a MEGAscript TM RNAi Kit (Invitrogen). The quality and concentration of dsRNA were assessed using 1% agarose gel electrophoresis and a Thermo ultra-micro spectrophotometer, respectively. Based on the findings of P. E. Urwin, 1 mg/mL FITC solution was used as an indicator of dsRNA entry into D. destructor [48]. About 10,000 J3 worms were collected and immersed in a solution containing dsRNA and 3 mg/mL spermidine (Sigma, Shanghai, China), 50 mM octopamine (Sigma), and 5% gelatin. The worms were incubated in the solution with shaking at 120 rpm for 24 h at 25 • C in the dark. In addition, J3 worms that were incubated in green fluorescent protein (GFP) dsRNA served as the control. After 24 h of soaking in the solution, D. destructor specimens were washed three times with DEPC-water and immediately stored at −80 • C for later detection of gene expression or were collected into 1.5-mL centrifuge tubes for later detection of motility, chemotaxis, stylet thrusting, protein secretion, and reproduction coefficient.

Gene Expression Analysis after Gene Knockdown
To determine levels of DdCα1D, DdCα1A, and DdCa v α 2 δ gene transcription following gene knockdown, approximately 1000 nematodes were collected after soaking in dsRNA for 24 h. mRNA was extracted, and cDNA was synthesized as described above. Specific qPCR primers were designed using NCBI, and 18S rRNA was used as the internal control for all qPCR assays ( Table 1). The qPCR reaction solution was a 20-µL mixture that included 1 µL cDNA template, 10 µL SYBR Green Premix Pro Taq HS qPCR mix (Accurate Biology, Changsha, China), 1 µL of each primer (0.2 mM), and 7 µL ddH 2 O. Quantitative analysis was performed using the 2 −∆∆CT method, as described above.

Phenotypic Analysis after Gene Knockout
Post RNAi phenotype analysis was performed by assessing mobility, chemotaxis, stylet thrusting, protein secretion, and reproduction.
Ditylenchus destructor mobility after the knockdown of DdCα1D, DdCα1A and DdCa v α 2 δ was assayed according to the procedure used by Kimber et al. [49]. One hundred treated J2s and control J2s were transferred into PVC tubes filled with moist sand, and the bottom of the PVC tubes were wrapped with 150-mesh nylon yarn. PVC tubes were transferred to a petri dish (50 mm) filled with 20 mL of ddH 2 O to cover the bottom of the sand column. Columns and petri dishes were kept in the dark at 25 • C. The number of worms passing through the sand column into the petri dish was counted at 6 h and 24 h, and the migration rating of the sand column was calculated. Migration rate (Mr) = Pp (passing population of nematodes)/100.
To determine whether chemotaxis was affected, we conducted experiments with 1-cm sweet potato blocks. Briefly, the blocks were placed on 1% agar in 90 mm petri dishes, 3 cm away from 200 J2s. Petri dishes were sealed with cling film and placed at 25 • C for 36 h. After incubation, the number of nematodes within 2 cm of the blocks was counted.
Worms were analyzed for stylet thrusting as previously reported by McClure et al., with slight modifications [50]. The concentrated suspension of aliquots (2 µL), containing approximately 50 J2 D. destructor specimens, was treated with 20 µL of 5 mM serotonin creatinine sulfate (Sigma) for 20 min, and 10 randomly selected J2s were observed for the frequency of stylet thrusting over a 1 min period. The number of twitches for each J2 in 60 s was calculated.
An assay using 0.1% resorcinol was developed to assess protein secretion in nematodes. Two thousand J2 nematodes from each treatment and control group were concentrated to 100 µL, an equal volume of 0.2% resorcinol was added, and specimens were incubated for 16 h at 25 • C. Lastly, the supernatant was aspirated, and the protein content was determined using a Modified BCA Protein Assay Kit (Sangon).
To assess whether the reproduction of nematodes treated with dsRNA was affected, 100 nematodes from each treatment group were inoculated into PDA medium filled with Botrytis cinerea. Then, 25 d after incubation at 25 • C, the total number of nematodes was counted, and the reproduction coefficient was calculated as the ratio of the final number of nematodes to the initial number of nematodes. Each assay experiment had three biological replicates and three technical replicates. Data were analyzed by one-way analysis of variance (ANOVA) in SPSS. Differences between treatments were tested using Duncan's multiple range test (DMRT) with F test (DMRT) with p < 0.05 [51]. The significant difference letter marking method first ranked all the means from largest to smallest and then marked the largest mean with a; the average was compared with the following averages. If the difference was not significant (p > 0.05), the letter a was marked, and if the difference was significant (p < 0.05), the letter b was marked. If the mean was significantly (p < 0.05) less than the mean of the group marked by letter b, it was marked by letter c.

Conclusions
This study identified the VGCC α 2 δ subunit in D. destructor and analyzed its transcriptional levels at different developmental stages and its tissue localization. In this study, silencing of the HVA α 1 and α 2 δ subunits in D. destructor was achieved simultaneously using multi-targeted dsRNA soaking. The function of DdCα1D, as well as DdCα1A, was further validated, and the auxiliary role of DdCa v α 2 δ was demonstrated, enhancing our understanding of VGCCs in plant parasitic nematodes.