The transcriptome of Icerya aegyptiaca (Hemiptera: Monophlebidae) and comparison with neococcoids reveal genetic clues of evolution in the scale insects

**Background:** Scale insects (Coccoidea) are sap-sucking parasites divided into neococcoids and non-neococcoids. Neococcoids are monophyletic and possess paternal genome elimination (PGE), while non-neococcoids like Iceryini have abdominal spiracles, compound eyes in males, abundant wax, a unique hermaphrodite system, and specific symbionts. Genetic studies have focused on neococcoids, leaving non-neococcoids poorly understood. This study sequenced the first transcriptome of Icerya aegyptiaca (Douglas), a globally invasive pest, to provide genetic resources and compare it with six neococcoid species to explore the evolution of structures, reproductive systems, and symbiosis.

**Methods:** RNA from four adult female I. aegyptiaca was sequenced on Illumina HiSeq 2500 (2×125 bp reads, 7.01–7.68 Gb per sample). Transcriptome assembly used Trinity v2.8.4, with foreign sequences removed by BLAST against Coccoidea and NCBI NT databases. Protein sets were constructed via EvidentialGene and annotated with InterPro, Eggnog, and GO. Orthologous groups (OGs) were assigned by OrthoFinder among I. aegyptiaca and six neococcoid species (Aclerda sp., Ericerus pela, Dactylopius confusus, Acutaspis umbonifera, Paratachardina pseudolobata, Maconellicoccus hirsutus) plus outgroup aphid Acyrthosiphon pisum. Phylogeny was inferred using IQ-TREE on a 434,622-aa supermatrix (848 OGs) and ASTRAL-Pro, with divergence times estimated by MCMCTREE using four fossils. Positive selection was detected via branch-site models in CODEML and BUSTED (HyPhy); selection intensification/relaxation via branch models and RELAX. Sex-specific DEGs in M. hirsutus and E. pela were identified using edgeR. Horizontal gene transfer (HGT) was detected using Alienness (AI > 15, % identity to non-Metazoa < 70) with DIAMOND searches against NCBI NR.

**Key Results:** The I. aegyptiaca transcriptome assembled into 235,635 contigs (N50=2,076 bp, GC=33.88%), yielding 72,003 proteins (92.7% complete BUSCO). A total of 12,995 OGs were identified across eight species. Phylogeny placed I. aegyptiaca diverging from neococcoids at ~247.14 Ma (95% CI: 221.87–266.95 Ma) in the Triassic; neococcoids originated in the Early Cretaceous (~137.39 Ma, 95% CI: 123.39–149.24 Ma). Positive selection at the I. aegyptiaca node identified 35 OGs enriched in neurogenesis, organ morphogenesis, and transcription, including Ets DNA-binding protein pokkuri (eye development), Casein kinase I isoform epsilon (DNA repair), and Tau-tubulin kinase homolog Asator (spindle/mitosis). At the neococcoid ancestor, 47 positively selected OGs were enriched in chromatin remodeling and embryo development, including Gustavus (reproduction), ASH1L (H3K36 methylation), and CHD7 (chromatin remodeling). At the whole neococcoid clade, 43 OGs were positively selected, including Mediator subunit 13 (eye-antennal disc development). Selection intensification in neococcoids (49 OGs) included Homeodomain-interacting protein kinase 2 (eye development), RAD54-like (DNA repair), and SWI/SNF-related chromatin remodeler. Selection relaxation in neococcoids (71 OGs) included Bicaudal-C (oogenesis) and Diaphanous (cytokinesis). A total of 216 genes were uniquely highly expressed (TPM ≥ 100) in I. aegyptiaca, with top 20 including 3 fatty acid desaturation/elongation genes. Common sex-specific DEGs in neococcoids showed male-biased genes under selection relaxation and female-biased genes under selection intensification (P < 0.05). A total of 791 candidate HTGs were identified (36.28% bacterial, 39.82% fungal origin). Ribosome recycling factor was present in all eight species. bioD (biotin synthesis) was found in scale insects but not aphids; bioB was exclusive to neococcoids and not detected in I. aegyptiaca.

**Clinical Implications:** Not applicable (basic entomological research). The findings have implications for pest control of I. aegyptiaca and related scale insects by identifying potential genetic targets (e.g., fatty acid biosynthesis genes for wax production, reproductive genes for hermaphroditism) and elucidating evolutionary mechanisms underlying invasive success and insecticide resistance.