Functional Study of Opsin Genes in Pardosa astrigera (Araneae: Lycosidae)

Abstract

Spiders are important predatory natural enemies in agricultural and forestry ecosystems, yet the role of vision in their predatory behavior remains unclear. In this study, we screened three opsin genes-corresponding to ultraviolet-sensitive and medium-to-long wavelength-sensitive opsins-from the transcriptome sequencing database of Pardosa astrigera. All three genes possess seven transmembrane topological structures and a lysine residue on the second transmembrane domain, which are typical characteristics of opsins. Using quantitative real-time PCR (RT-qPCR), we analyzed the expression patterns of these opsin genes in different tissues, developmental stages, and under the induction of light at three wavelengths. The results showed that all three opsin genes were significantly expressed in the cephalothorax and expressed across developmental stages with no significant differences. Under light induction, their relative expression first increased and then decreased in both male and female adult spiders. Subsequently, RNA interference (RNAi) was used to individually knock down each opsin gene, confirming their involvement in color vision. These results suggest that the three opsin genes are involved in spider vision, laying the foundation for further elucidating the role of vision in spider predation, and offering a new perspective for reducing the unintended killing of natural enemies by insect traps.

Keywords: Pardosa astrigera; biological control; opsin; phototactic behavior; vision.

Figure 1

The adult male and female P. astrigera and their eye arrangement diagram. (A) Female and male; (B) Ocular quadrangle of female and male. PL: aposterior lateral, PM: aposterior median, AL: anterior lateral, AM: anterior median. Scale bars = 1 mm.

Figure 2

Experimental device of selection response of P. astrigera to light source.

Figure 3

(A) Opsin genes conservative structure domain analysis (B) Opsin genes phylogenetic analysis (Csal: Cupiennius salei: the GenBank accession numbers of rhodopsin1, rhodopsin2, and rhodopsin3 are CCO61973.1, CCO61974.1, and CCO61975.1, respectively. Dmel: Drosophila melanogaster: the GenBank accession numbers of NINAE, rhodopsin2, rhodopsin3, rhodopsin4, rhodopsin5, rhodopsin6, rhodopsin7 are NP_524407.1, NP_524398.1, NP_52441.1, NP_476701, NP_477096.1, NP_524368.5, and NP_524035.2, respectively. Amel: Apis mellifera: the GenBank accession numbers of lws opsin, bws opsin and uvs opsin are NP_001011639.2, AAC13417.1 and AAC47455.1, respectively. Hada: Hasarius adansoni: the GenBank accession numbers of kumopsin1, kumopsin2 and kumopsin3 are BAG14330.1, BAG14331.1 and BAG14332.1, respectively. Ppay: Plexippus paykulli: the GenBank accession numbers of kumopsin1, kumopsin2 and kumopsin3 are BAG14333.1, BAG14334.1, and BAG14335.1. Past: Pardosa astrigera: the GenBank accession numbers of rhodopsin1, rhodopsin2, and rhodopsin3 are PV524665.1, PV524666.1, PV524667.1. Dpul: Daphnia pulex: the GenBank accession numbers of bws opsin, uvs opsin are XP_046438769.1, EFX75461.1. Lpol: Limulus polyphemus: the GenBank accession numbers of ocellar opsin, uvs and lateral eye opsin are NP_001301089.1, AEL29244.1, NP_001301044.1. Pcla: Procambarus clarkia: the GenBank accession numbers of opsin is AAB25036.1. Hsan: Hemigrapsus sanguineus: the GenBank accession numbers of opsin 1 and opsin 2 are BAA09132.1, BAA09133.1. Hera: Heliconius erato: the GenBank accession numbers of lws opsin, bws opsin and uvs opsin are AAY16540.1, AAY16539.1, AAY16537.1. Lser: Lasioderma serricorne: the GenBank accession numbers of lws opsin, uvs opsin are QPF71148.1, QPF71149.1 (Table S3).

Figure 4

The relative expression levels of PastRH at different developmental stages and organizations of P. astrigera. (A) The relative expression levels of PastRH1 at different developmental stages. (B) The relative expression levels of PastRH2 at different developmental stages. (C) The relative expression levels of PastRH3 at different developmental stages. (D) The relative expression levels of PastRH1 at different organizations in male and female adult spiders of P. astrigera. (E) The relative expression levels of PastRH2 at different organizations in male and female adult spiders of P. astrigera. (F) The relative expression levels of PastRH3 at different organizations in male and female adult spiders of P. astrigera. FAD: adult female, MAD: adult male, Ab: abdomen, Le: leg, Ce: Cephalothorax, The relative expression level was calculated using the 2^−ΔΔCt method. Data are presented as mean ± standard error. (A–C) were calculated using single factor ANOVA test followed by Tukey’s HSD multiple comparisons, a value of p < 0.05 was considered statistically significant. (D–F) were calculated using single factor ANOVA test followed by Tamhane’s T2 multiple comparisons, a value of p < 0.05 was considered statistically significant, compared only within the male and female groups. Capital letters and lowercase letters represent differences within male and female groups.

Figure 5

The relative expression of PastRH at different wavelengths light of P. astrigera. (A) The relative expression levels of PastRH1 at the green light (520–525 nm). (B) The relative expression levels of PastRH2 at the green light (520–525 nm). (C) The relative expression levels of PastRH3 at the blue light (460–465 nm). (D) The relative expression levels of PastRH3 at the ultraviolet light (370–375 nm), The relative expression level was calculated using the 2^−ΔΔCt method. Data are presented as mean ± standard error. (A–D) was calculated using single factor ANOVA test followed by Tukey’s HSD multiple comparisons, a value of p < 0.05 was considered statistically significant. Capital letters and lowercase letters represent differences within male and female groups.

Figure 6

Effect of RNA interference of PastRH on the relative expression level of PastRH of P. astrigera adult females and males. (A) The relative expression level of PastRH1 of adult females. (B) The relative expression level of PastRH2 of adult females. (C) The relative expression level of PastRH3 of adult females. (D) The relative expression level of PastRH1 of adult males. (E) The relative expression level of PastRH2 of adult males. (F) The relative expression level of PastRH3 of adult males. Data are presented as mean ± standard error. Independent sample t-test p-value, Significant differences are indicated by asterisks (**: p < 0.01, *: p < 0.05, ns: not significant).

Figure 7

Effect of RNAi of PastRH on behavioral selectivity of P. astrigera. (A) RNAi of PastRH1 reduces the behavioral selectivity of adult females at the green light (520–525 nm). (B) RNAi of PastRH2 of adult females at the green light (520–525 nm). (C) RNAi of PastRH3 of adult females at the blue light (460–465 nm). (D) RNAi of PastRH3 of adult females at the purple light (370–375 nm). (E) RNAi of PastRH1 of adult males at the green light (520–525 nm). (F) RNAi of PastRH2 of adult males at the green light (520–525 nm). (G) RNAi of PastRH3 of adult males at the blue light (460–465 nm). (H) RNAi of PastRH3 of adult males at the purple light (370–375 nm). The control was red light at 625–635 nm. Significant differences are indicated by Chi-square test (***: p < 0.001, **: p < 0.01, ns: not significant).