An Orphan Gene Enhances Male Reproductive Success in Plutella xylostella

Abstract

Plutella xylostella exhibits exceptional reproduction ability, yet the genetic basis underlying the high reproductive capacity remains unknown. Here, we demonstrate that an orphan gene, lushu, which encodes a sperm protein, plays a crucial role in male reproductive success. Lushu is located on the Z chromosome and is prevalent across different P. xylostella populations worldwide. We subsequently generated lushu mutants using transgenic CRISPR/Cas9 system. Knockout of Lushu results in reduced male mating efficiency and accelerated death in adult males. Furthermore, our findings highlight that the deficiency of lushu reduced the transfer of sperms from males to females, potentially resulting in hindered sperm competition. Additionally, the knockout of Lushu results in disrupted gene expression in energy-related pathways and elevated insulin levels in adult males. Our findings reveal that male reproductive performance has evolved through the birth of a newly evolved, lineage-specific gene with enormous potentiality in fecundity success. These insights hold valuable implications for identifying the target for genetic control, particularly in relation to species-specific traits that are pivotal in determining high levels of fecundity.

Keywords: insulin; male reproductive fitness; orphan gene; sperm competition; sperm protein.

Graphical abstract

Fig. 1.

A sperm protein encoded by Lushu in P. xylostella. a) Lushu was highly expressed in male adult based on RNA-Seq data. RNA-Seq was conducted for newly laid egg, larvae (from first- to fourth-instar larvae, and the sample of fourth instar larvae is an equal mixture of males and females), pupa (>2 d, equal mixture of males and females), virgin male and female adults. b) Expression patterns of lushu at different developmental stages. Expression level represents the relative expression level using RIBP as the control gene for qRT-PCR. L1, L2 and L3, indicate first, second, and third instar larvae, respectively; L4F, fourth instar female larvae; L4M, fourth instar male larvae. Term “Male/Female pupa” refers to the developmental stages spanning from first- to fourth- day after pupation for males/females (samples of an equal mixture of each day). Male/Female adult includes the developmental stages from first- to fifth- day male/female adults (samples of an equal mixture of each day). The relative expression level is represented as the mean ± SD (n = 4). c) Lushu started to express at the later stage of male pupa and reached the highest expression level at male adult stage. P0–P3, first- to fourth- day male pupa; MA0–MA4, first- to fifth-day male adults. d) Expression patterns showed that lushu was highly expressed in male reproductive tract except testes including accessory gland, vas deferens and seminal vesicles. Expression level represents the relative expression level using RIBP as the control gene for qRT-PCR. e) Lushu was located in gonad, vas deferens, accessory gland, and seminal vesicles verified by immunofluorescence. ag, accessory gland; vd, vas deferens; sg, silkgland. Reproductive tissues were dissected from 1d-old male adults. (f) Lushu was located on sperm bundles dissected from testis. Ap, apyrene sperm bundles; ep, eupyrene sperm bundles.

Fig. 2.

Mutagenesis of Lushu using CRISPR/Cas9. a) Representative sequences from wild type (G88) strain and the lushu-null mutant strain showing a 2 bp- deletion (lushu-8), a 4 bp- deletion (lushu-4), and a 2 bp- insertion (lushu-2). b) Tissues of reproduction from male G88 and lushu-null DBM strains were incubated with Lushu polyclonal antibody and HRP-Goat Anti-Rabbit IgG. We cannot detect the Lushu (red) in lushu-null males, suggesting that Lushu was successfully knocked out in the mutant males. c) Comparison of mating rate between males of G88 and lushu-null. And mating within 30 min was indicated in darker color for each mating pair. Statistics were obtained from 10 biological replicates, with each replicate containing six mating pairs. The mating rate is represented as the mean ± SD (n = 10). Asterisks indicate significant difference: *P < 0.05, **P < 0.01 (Student's t-test). The mating rate of G88 males within 30 min was significantly higher than that of lushu-8 males (P = 0.0072), lushu-4 males (P = 0.0119), and lushu-2 males (P = 0.0197). Additionally, the overall mating rate of G88 males within 3 h was significantly higher than that of lushu-8 males (P = 0.0013), lushu-4 males (P = 0.0399), and lushu-2 males (P = 0.0304). d) Estimation and comparison of male fertility in terms of egg number between males of G88 and lushu-null under one-to-one mating. Totally 15 replicates were prepared for this analysis. The egg number is represented as the mean ± SD (n = 15). The statistical analysis was conducted using Student's t-test, and “ns” indicated nonsignificance. The number of eggs laid by females mating with G88 males was significantly higher than those mating with lushu-8 males (P = 0.042), lushu-4 males (P = 0.048), and lushu-2 males (P = 0.047).

Fig. 3.

Evidence for involvement of Lushu in sperm competition. a) Sperm competition conferred by lushu. The competition index (Ratio) calculated in this study is designated as P2 in the Materials and Methods. Given the ratios of offspring genotypes observed, successful paternity by G88 or lushu-null males can be calculated and illustrates the differences in sperm competition between genotypes. Ratio is represented as the mean ± SD (n = 3). Asterisks indicate significant differences: *P < 0.05, **P < 0.01, and ***P < 0.001 (Fisher's Exact Test). b) Expression patterns of lushu along different competition gradients. The relative expression level is represented as the mean ± SD (n = 4) for qPCR analysis. Asterisks indicate significant differences: *P < 0.05, **P < 0.01, and ***P < 0.001 and ns indicates no significant difference (Student's t-test).

Fig. 4.

Sperm numbers and testis size comparison. a) Comparison of sperm numbers in the Bursa after 30 min of mating with wild-type and mutant males. The sperm numbers were represented as the mean ± SD (n = 40). Student's t-test was utilized to conduct the statistical analysis. b) The comparison of sperm bundle numbers in the testes of 1-day-old wild-type and mutant males, before and after mating, is illustrated. The counts of sperm bundles are expressed as the mean ± SD (n = 25). Within each bar graph, the darker shade signifies the sperm bundle count before mating, while the lighter shade indicates the sperm bundle count after mating. The statistical analysis of sperm bundle counts in the testes of G88 versus lushu-null males was conducted using the Student's t-test. c) Testis size comparison between G88 males and mutant males. The testis size is represented as the mean ± SD (n = 15). Statistical analysis was conducted using Student's t-test. (d) Comparison of sperm bundle numbers in seminal vesicles of wild-type and mutant males at 1 d old. The sperm bundles numbers were represented as the mean ± SD (n = 25). Asterisks indicate significant differences: *P < 0.05, **P < 0.01, and ***P < 0.001 and ns indicate no significant difference.

Fig. 5.

Genetic basis of metabolic pathways in which lushu might function. a) A proposed model for an insulin signaling pathway in which lushu might function in P. xylostella. Genes with disordered expression patterns were marked as green in lushu-null males. The heatmap illustrated the relative expression levels of these genes with disordered expression patterns, assessed using RIBP as the control gene for qRT-PCR. Relative expression level is represented as the mean (n = 4) in heatmap. Genes that showed significant differential expression between G88 and all other three lushu-null strains were shown (Student's t-test). b) Key genes located on pathways controlling galactose metabolism, TCA cycle, and fatty acid metabolism are proved to be differently expressed between G88 and lushu-null males using qPCR. Genes that showed significant differential expression between G88 and all other 3 lushu-null strains were shown (Student's t-test). c) ILP levels in G88 male and lushu-null males. Significant differences were analyzed using Student's t-test (n = 6).

Fig. 6.

Evolutionary origination and model of DBM employing Lushu to achieve evolutionary success. a) Lushu is Z-linked in P. xylostella. b) Synteny analysis of lushu and its flanking genes. The lushu and its flanking genes have no obvious synteny blocks in M. sexta. c) Putative models for functions of lushu in P. xylostella.