Genomic Identification and Functional Analysis of JHAMTs in the Pond Wolf Spider, Pardosa pseudoannulata

Juvenile hormone (JH) plays a critical role in many physiological activities of Arthropoda. Juvenile hormone acid methyltransferase (JHAMT) is involved in the last steps of JH biosynthesis as an important rate-limiting enzyme. In recent studies, an increasing number of JHAMTs were identified in arthropods, but no JHAMT was reported in spiders. Herein, eight JHAMTs were identified in the pond wolf spider, Pardosa pseudoannulata, all containing the well conserved S-adenosyl-L-methionine binding motif. JHAMT-1 and the other seven JHAMTs were located at chromosome 13 and chromosome 1, respectively. Multiple alignment and phylogenetic analysis showed that JHAMT-1 was grouped together with insect JHAMTs independently and shared high similarities with insect JHAMTs compared to the other seven JHAMTs. In addition, JHAMT-1, JHAMT-2, and JHAMT-3 were highly expressed in the abdomen of spiderlings and could respond to the stimulation of exogenous farnesoic acid. Meanwhile, knockdown of these three JHAMTs caused the overweight and accelerated molting of spiderlings. These results demonstrated the cooperation of multi-JHAMTs in spider development and provided a new evolutionary perspective of the expansion of JHAMT in Arachnida.

Recently, with the developments of the deep sequenced genomes and transcriptomes, JHAMT orthologs were also identified in Arachnida, including mite Tetranychus urticae [16,17], scorpion Mesobuthus martensii [18], ticks Ixodes scapularis [19,20], Dermacentor variabilis [20], and Ornithodoros turicata [20]. However, no study related to the identification and function of JHAMT in JH biosynthesis was reported in spiders. In this study, we took advantage of the genomic and transcriptomic databases to identify and characterize JHAMTs in the pond wolf spider Pardosa pseudoannulata. In addition, the function of JHAMTs in spider development was demonstrated by RNA interference (RNAi).

Characterization of JHAMTs
Eight full-length JHAMTs were identified in P. pseudoannulata, namely, JHAMT-1, -2, -3, -4, -5, -6, -7, and -8, and submitted to GenBank (GenBank accession number: MZ321024, MZ321025, MZ321026, MZ321027, MZ321028, MZ321029, MZ321030, and MZ321031, respectively). Their open reading frames (ORFs) were 822 base pairs (bp), 816 bp, 825 bp, 810 bp, 819 bp, 855 bp, 822 bp, and 861 bp which encoded a protein of 273 amino acids (aa), 271 aa, 274 aa, 269 aa, 272 aa, 284 aa, 273 aa, and 286 aa, respectively. The accuracy of the complete sequence was confirmed by PCR ( Figure S1). Eight JHAMTs were located at two chromosomes with JHAMT-1 at chromosome 13 and the rests at chromosome 1 ( Figure 1). The predicted amino acid sequences of eight JHAMTs contained the well conserved SAMbinding motif (motif I) and their secondary structure incorporated the alternation of nine α-helices and six β-strands comparing with the typical core fold of SAM-MTs ( Figure 2). related to the identification and function of JHAMT in JH biosynthesis was reported in spiders. In this study, we took advantage of the genomic and transcriptomic databases to identify and characterize JHAMTs in the pond wolf spider Pardosa pseudoannulata. In addition, the function of JHAMTs in spider development was demonstrated by RNA interference (RNAi).

Spatiotemporal Expression Profile
There were different expression profiles of eight JHAMTs in four developmental stages, egg, the second-instar spiderling, adult female and adult male ( Figure 4A). JHAMT-1 was expressed higher in males and lower in eggs. JHAMT-2 was significantly These JHAMTs were retrieved from the previous reports [9][10][11]19,21,22] or identified in the present study (Table S2). The gene ID was represented by the abbreviation of the Latin name of the species & its accession number. The phylogenetic tree was constructed by IQ-TREE and processed in Figtree software. The black JHAMTs were insects. The JHAMTs from an arachnid species were marked with the same color. The numbers at base of nodes were the branch times. The heatmap of similarities was processed in GraphPad Prism 7. Bommo, Bombyx mori; Aedae, Aedes aegypti; Trica, Tribolium castaneum; Drome, Drosophila melanogaster; Acypi, Acyrthosiphon pisum; Ixosc, Ixodes scapularis; Parps, Pardosa pseudoannulata; Parte, Parasteatoda tepidariorum; Stemi, Stegodyphus mimosarum; Tricl, Trichonephila clavipes; Censc, Centruroides sculpturatus.

Effect of Farnesoic Acid Administration
To determine the function of eight JHAMTs in JH synthesis, the changes of JHAMT transcriptional level were detected in spiderlings treated by FA. The transcriptional level of JHAMT-1 was significantly downregulated after FA treatment, but both JHAMT-2 and JHAMT-3 were significantly stimulated ( Figure 5). In addition, the other 5 JHAMTs showed no difference between FA treatment and control group ( Figure 5).

Effect of Farnesoic Acid Administration
To determine the function of eight JHAMTs in JH synthesis, the changes of JHAMT transcriptional level were detected in spiderlings treated by FA. The transcriptional level of JHAMT-1 was significantly downregulated after FA treatment, but both JHAMT-2 and JHAMT-3 were significantly stimulated ( Figure 5). In addition, the other 5 JHAMTs showed no difference between FA treatment and control group ( Figure 5).

Discussion
In the present study, eight JHAMTs were identified in the whole genome of P. pseudoannulata. It was the first detailed characterization of JHMAT in spiders. Similarly, multicopies of JHAMT were also found in several arachnids, including three spiders P. tepidariorum, S. mimosarum, and T. clavipes, and a scorpion C. sculpturatus. Moreover, the numbers of 3 and 44 JHAMT genes have been reported in the genomic works of scorpion M. martensii [18] and tick I. scapularis [19], respectively. However, the same with T. urticae [16,17], only one JHAMT presented in two mite species, V. destructor and G. occidentalis. Therefore, there was the striking expansion of JHAMT in spiders, scorpions, and ticks, but not in mites. The duplication of JHAMT also occurred in insects, including T. castaneum [10], A. pisum [12], and B. mori [21], although only one JHAMT in most insect species. Interesting results from the phylogenetic tree showed that there was always a JHAMT in arachnids that was independent of the other copies and grouped together with that of insects. Further, JHAMT-1 from P. pseudoannulata showed high similarities of amino acid sequences with insect JHAMTs than the other seven JHAMTs. In the chromosomal distribution of eight JHAMT genes, JHAMT-1 was located at chromosome 13 and the remaining JHAMTs were located together at chromosome 1. From the above results, we speculated that an ancestral JHAMT gene was presented in both Arachnida and Insecta, and the new JHAMTs were developed and duplicated in arachnids after the separation of insects. In the future, more evidence related genomic analyses of arachnid species are needed to confirm this hypothesis.
JHAMT, as the key rate-limiting enzyme in regulation of JH titer, is involved in the last steps of the active JH product biosynthesis pathway in insects by transferring the methyl group of SAM to the carboxyl group of FA or JHA III [7]. Differing from corpora allata as the main biosynthetic site of JH III in insects [23], these JH biosynthesis-related genes were highly expressed in abdomen of P. pseudoannulata [24]. It indicated that JHAMT-1,

Discussion
In the present study, eight JHAMTs were identified in the whole genome of P. pseudoannulata. It was the first detailed characterization of JHMAT in spiders. Similarly, multi-copies of JHAMT were also found in several arachnids, including three spiders P. tepidariorum, S. mimosarum, and T. clavipes, and a scorpion C. sculpturatus. Moreover, the numbers of 3 and 44 JHAMT genes have been reported in the genomic works of scorpion M. martensii [18] and tick I. scapularis [19], respectively. However, the same with T. urticae [16,17], only one JHAMT presented in two mite species, V. destructor and G. occidentalis. Therefore, there was the striking expansion of JHAMT in spiders, scorpions, and ticks, but not in mites. The duplication of JHAMT also occurred in insects, including T. castaneum [10], A. pisum [12], and B. mori [21], although only one JHAMT in most insect species. Interesting results from the phylogenetic tree showed that there was always a JHAMT in arachnids that was independent of the other copies and grouped together with that of insects. Further, JHAMT-1 from P. pseudoannulata showed high similarities of amino acid sequences with insect JHAMTs than the other seven JHAMTs. In the chromosomal distribution of eight JHAMT genes, JHAMT-1 was located at chromosome 13 and the remaining JHAMTs were located together at chromosome 1. From the above results, we speculated that an ancestral JHAMT gene was presented in both Arachnida and Insecta, and the new JHAMTs were developed and duplicated in arachnids after the separation of insects. In the future, more evidence related genomic analyses of arachnid species are needed to confirm this hypothesis.
JHAMT, as the key rate-limiting enzyme in regulation of JH titer, is involved in the last steps of the active JH product biosynthesis pathway in insects by transferring the methyl group of SAM to the carboxyl group of FA or JHA III [7]. Differing from corpora allata as the main biosynthetic site of JH III in insects [23], these JH biosynthesis-related genes were highly expressed in abdomen of P. pseudoannulata [24]. It indicated that JHAMT-1, JHAMT-2, JHAMT-3, and JAHMT-6 were involved in JH biosynthesis in P. pseudoannulata due to their specific expressions in abdomen. Just as predicted, the gene expressions of JHAMT-1, JHAMT-2, and JAHMT-3 were significantly changed by exogenous FA application, but not JHAMT-6. JHAMT-2 and JHAMT-3 were stimulated by FA, but opposite in JHAMT-1. It might be that there was a cooperative mechanism between the JHAMTs to respond to the changes of FA. In addition, some JHAMTs might have functional differentiation, such as JHAMT-7 and JHAMT-8 involved in toxin production in the spider because of their high expressions in venom glands.
The development of spiderlings was affected by JHAMT silencing. JHAMT downregulation could increase the weight of spiderlings. Meanwhile, the accelerated molting was embodied in the higher molting rate of dsJHAMT treatments than that of control group at both 96 h and 120 h. However, JHAMT-3 had no effects in spiderling's development although there was a statistical difference in the weight of spiderlings, possibly due to the functional absence of JHAMT-3, which was rescued by the increased expression of JHAMT-1 in dsJHAMT-3-treated spiderlings. To sum up, JHAMTs regulate spider development in a coordinated way.

Spiders
P. pseudoannulata adults were collected from a rice field in Nanjing (Jiangsu province, China) in May 2020 and housed in 500 mL plastic cups individually at 28 ± 1 • C and 16/8 h light/dark and fed with Nilaparvata lugens. The spiders were reared in laboratory conditions for at least one month before experiments began. Four developmental samples, egg, the second-instar spiderling, adult female, and adult male, were collected individually and 5-10 eggsacs or spiders were pooled as one sample. Two parts, cephalothorax and abdomen, were dissected from 10 the second-instar spiderlings. Six tissular samples, brain, venom gland, fat body, intestine, ovary, and testes, were dissected from 20 adult females and males. Each sample was carried out with three biological replicates.

Farnesoic Acid Treatment
FA was purchased from Echelon (Salt Lake City, UT, USA) and dissolved in absolute ethanol to get the stock solution of 10 mg/mL. The stock solution was diluted using sterilized water to get the working concentration of 0.05 mg/mL. Ethanol diluted 200 times was set as negative control. The day 1 second-instar spiderlings were starved in petri dishes (3.5 cm in diameter) individually for 12 h before bioassay. The working solution soaked in absorbent cotton was supplied to each spiderling and solution-cotton was refreshed every 24 h. The spiderlings were fed with a few N. lugens after exposure to working solution for 36 h. Ten spiderlings were harvested at 72 h to pool as one sample and each sample was carried out with three biological replicates.

RNA Interference
The gene specific primers with extended T7 RNA polymerase promoter sequence on the 5 end were designed using Beacon Designer (Table S1) and synthesized by Genscript. The gene fragment was amplified using Phanta ® Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) and then purified using GeneJET Gel Extraction Kit (Thermo Scientific, Carlsbad, CA, USA) according to the manufacturer's instructions. dsRNA was synthesized using T7 RiboMAX™ Express RNAi System (Promega, Madison, WI, USA) according to the manufacturer's instructions. The integrity and quantity of dsRNA were verified by 1.5% agarose gel electrophoresis and NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA) respectively. dsRNA against enhanced green fluorescent protein (eGFP, GenBank accession number: KC896843) was used as the negative control. Delivery of dsRNA by injection have been described in the previous report [33]. This method was used to investigate the biological function of JHMATs in P. pseudoannulata in the present study. Briefly, after anaesthetization by carbon dioxide, the day 1 second-instar spiderlings were kept in an agar gel plate and microinjected with 10 nL of 50 ng dsRNA individually from the injection site of ventral abdomen. The injected-spiderlings were transferred into petri dishes individually and fed with N. lugens. Individuals died of mechanical injury within 12 h were removed. Injected spiderlings were divided into two groups. Group I was used for quantitative PCR (qPCR). The number of 10 spiderlings of each dsRNA treatment were harvested at 48 h to pool as one sample. Group II was used for phenotypic responses. The number of 15-20 spiderlings of each dsRNA treatment were used to record the phenotypes. The spiderlings were weighted at 72 h and the counts of molts were recorded at both 96 h and 120 h. The experiment was conducted three times independently.

Real-Time Quantitative PCR
Total RNA was extracted using Trizol™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The integrity and quantity of RNA were verified by 1.5% agarose gel electrophoresis and NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA) respectively. cDNA was synthesized using PrimeScript RT Reagent Kit (TaKaRa, Kyoto, Japan) according to the manufacturer's instructions. Two reference genes of elongation factor-1 alpha (EF-1α, GenBank accession number: KJ888948) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH, GenBank accession number: KJ888949) were selected based on previous description [33]. Primers for qPCR were designed using Beacon Designer (Table S1) and synthesized by Genscript. The specificity and efficiency of the primers were verified via melting curve and standard curve assay respectively. The components of qPCR reaction were made using TB Green Premix Ex Taq II Kit (TaKaRa, Kyoto, Japan) according to the manufacturer's instructions and performed on QuantStudio Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each reaction was carried out with two technical replicates.

Data Analysis
The relative gene expression was related to the geometric mean of two reference genes by the 2 −∆CT method [34,35]. Gene expression, weight, and molting rate of spiderlings were presented as mean ± SEM from three independent biological replicates. Significant differences were analyzed with t-tests for gene expressions between cephalothorax and abdomen, FA treatments, and dsRNA treatments, and with one-way ANOVA followed by Tukey test for gene expressions in four developmental samples and six tissular samples, and weights and molting rates of spiderlings in dsRNA treatments using GraphPad Prism (version 7, GraphPad Software, San Diego, CA, USA) [36].