Fruit Fly Eggshell Gene Reveals Purifying Selection with Episodes of Positive Evolution

The defective chorion 1 gene, known as dec-1, has long been recognized in the fruit fly Drosophila melanogaster as a cornerstone of eggshell construction. Now, a study published in Development Genes and Evolution has traced this gene across two of the most economically important pest fruit flies in the Americas, Anastrepha fraterculus and Anastrepha obliqua, and found that while the gene is overwhelmingly governed by purifying selection, scattered sites show evidence of positive and episodic diversifying selection at multiple evolutionary scales.

The genus Anastrepha contains roughly 330 species arranged into 23 taxonomic groups, and its fraterculus species group includes 34 formally described species that are notoriously difficult to tell apart. A. fraterculus, the South American fruit fly, and A. obliqua, the West Indies fruit fly, are closely related, widely distributed, and infest a broad range of host fruits, though A. fraterculus tends to favor plants in the Myrtaceae family while A. obliqua prefers Anacardiaceae. Because eggshell morphology varies markedly among Anastrepha species, even among different morphotypes within A. fraterculus, the researchers reasoned that dec-1, which is crucial for chorion formation and female fertility, could yield markers informative for distinguishing these species and understanding their diversification.

To characterize the gene, the team mined reproductive and head transcriptomes previously produced in their laboratory, using the Drosophila melanogaster dec-1 sequence as a query. They also drew on a draft genome of Anastrepha ludens, generously shared before its publication, to validate exon structure, and consulted the recently released A. obliqua genome in GenBank. Comparative analysis revealed that dec-1 in these Anastrepha species contains six exons, one more than the five described for D. melanogaster, and produces three alternative transcripts, designated X1, X2 and X3, which correspond to the fc125, fc177 and fc106 proproteins respectively. In A. fraterculus, the inferred proteins measure 1472, 1192 and 955 amino acids, while in A. obliqua they measure 1526, 1181 and 950 amino acids. The first four exons are shared by all isoforms, and the differences arise in the arrangement of the last two exons, particularly exon 5, which shows alternative internal splicing sites.

Motif analysis added further texture. Two primary domains were identified in the putative DEC-1 proteins: an N-terminal region and a C-terminal region. Both motifs are highly conserved within the genus Anastrepha, with N-terminal identities close to or above 90 percent among Anastrepha species, but similarity drops sharply when compared with other flies, falling to roughly 27 percent for the N-terminal motif against Drosophila melanogaster and as low as 17 percent for the C-terminal motif against the diopsid Teleopsis dalmanni. Notably, unlike Drosophila, the Anastrepha proteins lack the nine-repeat region motif, and a novel RNAse P Rpr2/Rpp21/SNM1 subunit domain appeared on the longer X1 isoform of A. obliqua.

To probe selection at different hierarchical levels, the researchers built two datasets. The first, the Anastrepha dataset, comprised 31 sequences of a conserved portion of exon 3 amplified from natural populations of A. fraterculus and A. obliqua collected at eight sampling points across Brazil, plus sequences from A. ludens and the outgroup A. grandis. The second, the Cyclorrhapha dataset, assembled complete dec-1 sequences spanning Tephritidae genera such as Rhagoletis, Zeugodacus, Bactrocera and Ceratitis, several Drosophila species, and Teleopsis dalmanni as an outgroup. Using maximum likelihood phylogenies as frameworks, they applied codon-based models in PAML and a battery of HyPhy methods, including FEL, FUBAR, MEME, BUSTED and RELAX, to estimate the ratio of non-synonymous to synonymous substitutions, known as omega, across sites and branches.

The global picture was one of constraint. A significant contrast between the M0 and M3 models in both datasets indicated evolutionary rate heterogeneity along the gene, but tests comparing models that allow positive selection against those that do not found insufficient evidence of positive selection affecting the whole gene at the Anastrepha level. The BUSTED test suggested that 87.3 percent of branches in the Anastrepha dataset evolve under purifying selection, and the overall omega value of 0.1280 confirmed strong selective constraint. Yet the signal was not uniformly silent. In the Anastrepha dataset, the Bayes empirical Bayes analysis flagged seven candidate codons, with codon 523G reaching a significant 95 percent posterior probability at omega equal to 1.27. FUBAR independently identified codons 523, 560, 656, 743, 761 and 617 under positive selection, and MEME detected episodic diversifying selection at codons 617, 656 and 724, suggesting that positive selection has acted on these sites at specific evolutionary moments without sweeping the entire gene.

At the deeper Cyclorrhapha level, the contrast between the M7 and M8 models was highly significant, indicating sites under positive selection at that level of divergence. Codons 1309G and 1314V showed posterior probabilities of 96 percent and 98.2 percent respectively in the PAML analysis, with FUBAR corroborating codon 1314V and FEL identifying seven additional sites under diversifying selection. MEME revealed 13 sites under episodic diversifying selection, affecting regions coding for the s25 or s60 proproteins but not s20. Branch-site tests added lineage-specific stories: the branch separating the genus Anastrepha showed codon 526N under positive selection with 96.8 percent posterior probability, the Tephritidae branch showed evidence of positive selection in roughly 20 percent of tested codons, and within Anastrepha, the A. obliqua branch showed a significant branch-site result with codon 724 at 95 percent posterior probability. The RELAX test indicated intensified selection on the A. fraterculus Clade VII branch and relaxed selection on the Multispecies branch.

The study also connected evolutionary rates to protein architecture. DEC-1 is cleaved into derivative proproteins that are deposited in the eggshell at different developmental times: the fc106 product yields s25 and later s60 and s20, while s25, s60 and s85 become structural components of the egg layers. The analyses showed that regions associated with proproteins incorporated earlier in egg formation, particularly s25 and the s20-coding region, are generally more conserved than regions tied to later-deposited products such as s60. TreeSAAP analysis of radical physicochemical amino acid changes reinforced this pattern, finding in the Anastrepha dataset that radical mutations in the s20 region occur more frequently on interior branches than on tips, a distribution consistent with purifying selection, whereas the s60 region showed no radical mutations on internal branches. In the broader Cyclorrhapha comparison, nearly 60 percent of codons in the s60 region showed significant radical physicochemical alterations.

The phylogenetic structure within A. fraterculus itself proved striking. Samples divided into three distinct lineages, one from central Brazil termed Clade VII, one from the Brazilian coast termed Clade VI, and a multispecies branch, while all A. obliqua samples clustered together. The authors note that A. fraterculus appeared paraphyletic in their analysis, corroborating earlier work suggesting it is a species complex and possibly not monophyletic. This topology mirrors patterns recovered from transcriptomic and genomic data by other researchers, and it matters practically: dec-1 appears to distinguish among A. fraterculus lineages for which only a handful of informative genetic markers exist, despite extensive prior investigation.

The researchers are careful about limits. Attempts to model the three-dimensional structure of DEC-1 yielded low confidence scores, and the protein appears to be largely intrinsically disordered, which prevented firm conclusions about how positively selected sites alter protein structure, although those sites seem to sit in disordered regions positioned externally on the protein. The authors also caution that dN/dS-based tests are designed for divergent lineages and can be influenced by demographic forces in recently split species, and that gene flow among A. fraterculus lineages may complicate within-species inference. Still, the combination of strong purifying selection, episodic positive selection, and exon-level rate variation makes dec-1 a promising marker for studying differentiation in Anastrepha and, potentially, for supporting the management of pest species whose eggs, quite literally, bear the imprint of this gene’s evolution.

Subject of Research: Molecular evolution and selection patterns of the defective chorion 1 eggshell gene in Anastrepha fruit flies

Article Title: Molecular evolution and genetic diversity of defective chorion 1 in Anastrepha fraterculus and Anastrepha obliqua (Diptera, Tephritidae)

Article References: de Cássia Bisio, M., dos Santos, E. M., Santos, C. A., Chahad-Ehlers, S., & de Brito, R. A. (2024). Molecular evolution and genetic diversity of defective chorion 1 in Anastrepha fraterculus and Anastrepha obliqua (Diptera, Tephritidae). Development Genes and Evolution, 234(2), 153-171. https://doi.org/10.1007/s00427-024-00723-3

Image Credits: AI Generated

DOI: 10.1007/s00427-024-00723-3

Keywords: Anastrepha fraterculus, Anastrepha obliqua, dec-1 gene, chorion, purifying selection, positive selection, Tephritidae, molecular evolution, alternative splicing, phylogenetics, fruit flies, dN/dS