Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae)

Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were β-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and β-actin (for insecticide treatments), GAPDH, TATA, RPS18 (for starvation-induced stress), TATA, RPS6, and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions.

Correction to: Insights into wing dimorphism in worldwide agricultural pest and host-alternating aphid Aphis gossypii

An amendment to this paper has been published and can be accessed via the original article.

Insights into wing dimorphism in worldwide agricultural pest and host-alternating aphid Aphis gossypii

Background The worldwide pest Aphis gossypii has three-winged morphs in its life cycle, namely, winged parthenogenetic female (WPF), winged gynopara (GP), and winged male, which are all produced by a wingless parthenogenetic female (WLPF). Most studies on A. gossypii have focused on WPF, while few have investigated GP and male. The shared molecular mechanism underlying the wing differentiation in the three wing morphs of A. gossypii remains unknown. The wing differentiation of WPF was explored in a previous study. Herein, GP and male were induced indoors. The characters of the body, internal genitals, wing veins, and fecundity of GP and male were compared with those of WPF or WLPF. Compared with WLPF, the shared and separate differentially expressed genes (DEGs) were identified in these three-wing morphs. Results Newly-born nymphs reared in short photoperiod condition (8 L:16D, 18 °C) exclusively produced gynoparae (GPe) and males in adulthood successively, in which the sex ratio was GP biased. A total of 14 GPe and 9 males were produced by one mother aphid. Compared with WLPF, the three-wing morphs exhibited similar morphology and wing vein patterns but were obviously discriminated in the length of fore- and underwings, reproductive system, and fecundity. A total of 37 090 annotated unigenes were obtained from libraries constructed using the four morphs via RNA sequencing (RNA-Seq). In addition, 10 867 and 19 334 DEGs were identified in the pairwise comparison of GP versus WLPF and male versus WLPF, respectively. Compared with WLPF, the winged morphs demonstrated 2 335 shared DEGs (1 658 upregulated and 677 downregulated). The 1 658 shared upregulated DEGs were enriched in multiple signaling pathways, including insulin, FoxO, MAPK, starch and sucrose metabolism, fatty acid biosynthesis, and degradation, suggesting their key roles in the regulation of wing plasticity in the cotton aphid. Forty-four genes that spanned the range of differential expression were chosen to validate statistical analysis based on RNA-Seq through the reverse transcription quantitative real time polymerase chain reaction (RT-qPCR). The comparison concurred with the expression pattern (either up- or downregulated) and supported the accuracy and reliability of RNA-Seq. Finally, the potential roles of DEGs related to the insulin signaling pathway in wing dimorphism were discussed in the cotton aphid. Conclusions The present study established an efficiently standardized protocol for GP and male induction in cotton aphid by transferring newly-born nymphs to short photoperiod conditions (8 L:16D, 18 °C). The external morphological characters, especially wing vein patterns, were similar among WPFs, GPe, and males. However, their reproductive organs were strikingly different. Compared with WLPF, shared (2 235) and exclusively (1 470 in WLPF, 2 419 in GP, 10 774 male) expressed genes were identified in the three-wing morphs through RNA-Seq, and several signaling pathways that are potentially involved in their wing differentiation were obtained, including insulin signaling, starch and sucrose metabolism.

The miR-9b microRNA mediates dimorphism and development of wing in aphids

Wing dimorphism is a phenomenon of phenotypic plasticity in aphid dispersal. However, the signal transduction for perceiving environmental cues (e.g., crowding) and the regulation mechanism remain elusive. Here, we found that aci-miR-9b was the only down-regulated microRNA (miRNA) in both crowding-induced wing dimorphism and during wing development in the brown citrus aphid Aphis citricidus. We determined a targeted regulatory relationship between aci-miR-9b and an ABC transporter (AcABCG4). Inhibition of aci-miR-9b increased the proportion of winged offspring under normal conditions. Overexpression of aci-miR-9b resulted in decline of the proportion of winged offspring under crowding conditions. In addition, overexpression of aci-miR-9b also resulted in malformed wings during wing development. This role of aci-miR-9b mediating wing dimorphism and development was also confirmed in the pea aphid Acyrthosiphon pisum. The downstream action of aci-miR-9b-AcABCG4 was based on the interaction with the insulin and insulin-like signaling pathway. A model for aphid wing dimorphism and development was demonstrated as the following: maternal aphids experience crowding, which results in the decrease of aci-miR-9b. This is followed by the increase of ABCG4, which then activates the insulin and insulin-like signaling pathway, thereby causing a high proportion of winged offspring. Later, the same cascade, “miR-9b-ABCG4-insulin signaling,” is again involved in wing development. Taken together, our results reveal that a signal transduction cascade mediates both wing dimorphism and development in aphids via miRNA. These findings would be useful in developing potential strategies for blocking the aphid dispersal and reducing viral transmission.

A large genomic insertion containing a duplicated follistatin gene is linked to the pea aphid male wing dimorphism

Wing dimorphisms have long served as models for examining the ecological and evolutionary tradeoffs associated with alternative phenotypes. Here, we investigated the genetic cause of the pea aphid (Acyrthosiphon pisum) male wing dimorphism, wherein males exhibit one of two morphologies that differ in correlated traits that include the presence or absence of wings. We mapped this trait difference to a single genomic region and, using third generation, long-read sequencing, we identified a 120 kb insertion in the wingless allele. This insertion includes a duplicated follistatin gene, which is a strong candidate gene in the minimal mapped interval to cause the dimorphism. We found that both alleles were present prior to pea aphid biotype lineage diversification, we estimated that the insertion occurred millions of years ago, and we propose that both alleles have been maintained in the species, likely due to balancing selection.

Insights into wing dimorphism in the worldwide agricultural pest Aphis gossypii, the host-alternating aphid

Background: Three wing morphs exists in the life cycle of the worldwide pest Aphis gossypii, i.e., wing parthenogenetic female (WPF), gynopara (GP) and male, which were produced mostly by crowding and host quality, photoperiod, loss of X chromosome, respectively. However, the shared molecular mechanism underlying their wing differentiation remains an enigma. Here we firstly induced gynoparae and males indoors and compared the characters of these wing morphs in body, internal genitals and fecundity. Then we identified the shared and separate differentially expressed genes (DEGs) and signaling pathways potentially involved in the wing morphs regulation in WPF, GP and male compared to wingless parthenogenetic female (WLPF). Results: Newly-born nymphs reared in short photoperiod condition exclusively produce gynoparae and males in adulthood successively, in which the sex ratio is gynoparae biased. Compared with WLPF, three wing morphs have similar morphology in bodies but is obviously discriminated in the reproductive system and fecundity. Built upon our previous study, 37 090 annotated unigenes were obtained from libraries constructed by the four morphs above through RNA-sequencing, in which 10 867, 19 334 DEGs were identified in pairwise comparison of GP vs. WLPF, Male vs. WLPF, respectively. Furthermore, 2 335 shared DEGs including 1 658 up- and 677 downregulated were obtained in these wing morphs compared to WLPF. The 1 658 shared up-regulated DEGs were enriched in multiple signaling pathways including insulin, FoxO, MAPK, strarch and sucrose metabolism, fatty acid biosynthesis and degradation which hint their key roles in the regulation of wing plasticity in cotton aphid. Gene expression levels were validated by using Pearson’s correlation (r) and potential roles of 15 DEGs related to the insulin signaling pathway in cotton wing dimorphism were discussed. Conclusions: The results of this study establish a solid foundation for deciphering molecular mechanisms underlying the switch between wingless and wing morphs in the cotton aphid and provide valuable resources for future research on the host-alternating aphid species.

Comparative Insight into the Bacterial Communities in Alate and Apterous Morphs of Brown Citrus Aphid (Hemiptera: Aphididae)

Wing polyphenism (alate and apterous morphs) in aphids is a trade-off between dispersal and reproduction. How bacterial communities are associated with wing polyphenism in aphids is still not clearly understood. This study used 16S rRNA sequencing to examine the differences in diversity of the bacterial community between alate and apterous morphs in Aphis citricidus, the main vector of the Citrus tristeza virus. Eighty-one operational taxonomic units (OTUs) belonging to 37 orders, 34 classes, and 13 phyla were identified from all samples. Among these OTUs, Wolbachia (79.17%), Buchnera (17.64%), and Pseudomonas (2.99%) were the dominant bacterial genera. The diversity of symbionts varied between the two morphs; apterous morphs had more bacterial diversity (69 OTUs belonging to 45 families, 21 classes, and 12 phyla) than alate morphs (45 OTUs belonging to 36 families, 15 classes, and 10 phyla). In addition, the abundance of five OTUs was significantly different between two morphs. Among these OTUs, two Pseudomonas species (Pseudomonas_brenneri [OTU21] and unclassified_Pseudomonas [OTU13]) represented a high proportion (3.93% and 2.06%) in alate morphs but were present in low abundance (0.006% and 0.002%) in apterous morphs. RT-qPCR showed consistent results with high-throughput DNA sequencing. The preliminary survey showed the difference in composition and frequency of bacteria between alate and apterous morphs. Thus, the results contribute to anew insight of microorganisms that may be involved in wing dimorphism and helpful for controlling the dispersal of this pest through artificial elimination or reinfection of bacterial symbionts or targeting symbiosis-related host genes by RNA interference in future.