Mealybugs are highly aggressive to a diversity of plants. The waxy layer covering the outermost part of the integument is an important protective defense of these pests. However, the molecular mechanisms underlying wax biosynthesis in mealybugs remain largely unknown. Here, we analyzed multi-omics data on wax biosynthesis by the cotton mealybug, Phenacoccus solenopsis Tinsley, and found that a fatty acyl-CoA reductase (PsFAR) gene, which was highly expressed in the fat bodies of female mealybugs, contributed to wax biosynthesis by regulating the production of the dominant chemical components of wax, cuticular hydrocarbons (CHCs). RNA interference (RNAi) against PsFAR by dsRNA microinjection and allowing mealybugs to feed on transgenic tobacco expressing target dsRNA resulted in a reduction of CHC contents in the waxy layer, and an increase in mealybug mortality under desiccation and deltamethrin treatments. In conclusion, PsFAR plays crucial roles in the wax biosynthesis of mealybugs, thereby contributing to their adaptation to water loss and insecticide stress.

The cotton mealybug, Phenacoccus solenopsis, is an invasive pest that can cause massive damage to many host plants of agricultural importance. P. solenopsis is highly polyphagous, and shows extreme sexual dimorphism between males and females. The functions of DNA methyltransferase (DNMT) enzymes in the cotton mealybug have not been well studied. Here, we carried out an investigation of DNMTs in cotton mealybug to study their roles in sexual dimorphism. We found that the cotton mealybug has two copies of PsDnmt1, but Dnmt3 is absent. We then amplified the full-length cDNAs of PsDnmt1A (2225 bp) and PsDnmt1B (2862 bp) using rapid amplification cDNA ends (RACE). Quantitative reverse transcriptase PCR shows that both PsDnmt1A and PsDnmt1B are highly expressed in adult males, while the expression of PsDnmt1B is 30-fold higher in gravid females than in virgin females. We knocked down PsDnmt1A and PsDnmt1B with small interfering RNAs (siRNAs), and both genes were successfully down-regulated after 24 h or 72 h in adult females and pupa (t-test, p < 0.05). Down-regulating the expression of these two DNMT genes led to offspring lethality and abnormal body color in adult females. Furthermore, the silencing of PsDnmt1B induced abnormal wing development in emerged adult males. Our results provide evidence that PsDnmt1 plays a crucial role in regulating sexual dimorphism in the cotton mealybug.

For insects, female density is closely related to reproductive output. However, little is known about the effects of female density on male mating and female postmating performances. Here, we explored the effects of female density in cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), an invasive, rapidly spreading pest in Asia damaging multiple crops and horticultural plants. Using USB digital microscopes, we investigated the frequency, duration, and intervals of mating for males that were individually supplied with 1, 5, 10, and 15 females. We also evaluated the reproduction of mated females and the sex ratio of their offspring. As the female density increased, males mated with more females while substantially shortening mating intervals. Mating occurred actively at the densities of 10 and 15 females, where males mated four times on average, and some mated 6–9 times. However, mating duration and the observed reproductive parameters of females (preoviposition period, overall period from formation of ovisacs to female death, fecundity, and offspring sex ratio) did not differ significantly with female density. A weak trade-off existed between males’ mating frequency and longevity, but there was no relationship between females’ fecundity and longevity. In conclusion, despite their short lifespan, P. solenopsis males have a high mating capacity, and their mating frequency and intervals can be significantly affected by female density. In contrast, female density has little influence on females’ postmating performance. Our findings indicate the significance of the reproductive biology and life history strategies for rapid establishment and population development of mealybugs in newly invaded regions.

Many insects are capable of developing two types of wings (i.e., wing polyphenism) to adapt to various environments. Though the roles of microRNAs (miRNAs) in regulating animal growth and development have been well studied, their potential roles in modulating wing polyphenism remain largely elusive. To identify wing polyphenism-related miRNAs, we isolated small RNAs from 1st to 5th instar nymphs of long-wing (LW) and short-wing (SW) strains of the brown planthopper (BPH), Nilaparvata lugens. Small RNA libraries were then constructed and sequenced, yielding 158 conserved and 96 novel miRNAs. Among these, 122 miRNAs were differentially expressed between the two BPH strains. Specifically, 47, 2, 27 and 41 miRNAs were more highly expressed in the 1st, 3rd, 4th and 5th instars, respectively, of the LW strain compared with the SW strain. In contrast, 47, 3, 29 and 25 miRNAs were more highly expressed in the 1st, 3rd, 4th and 5th instars, respectively, of the SW strain compared with the LW strain. Next, we predicted the targets of these miRNAs and carried out Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. We found that a number of pathways might be involved in wing form determination, such as the insulin, MAPK, mTOR, FoxO and thyroid hormone signaling pathways and the thyroid hormone synthesis pathway. Thirty and 45 differentially expressed miRNAs targeted genes in the insulin signaling and insect hormone biosynthesis pathways, respectively, which are related to wing dimorphism. Among these miRNAs, Nlu-miR-14-3p, Nlu-miR-9a-5p and Nlu-miR-315-5p, were confirmed to interact with insulin receptors (NlInRs) in dual luciferase reporter assays. These discoveries are helpful for understanding the miRNA-mediated regulatory mechanism of wing polyphenism in BPHs and shed new light on how insects respond to environmental cues through developmental plasticity.