![Evolution of the Cucoid clade family in higher flies. Inferred gene duplications in the Cucoid clade based on data in this study and D. melanogaster gene ages reported elsewhere [55]. For details see text. Gene clusters (hexagons) and gene loci (triangles) are indicated. For color code see Fig 2.](https://www.researchgate.net/publication/366847081/figure/fig5/AS:11431281111431892@1672955239622/Evolution-of-the-Cucoid-clade-family-in-higher-flies-Inferred-gene-duplications-in-the_Q320.jpg)
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RESEARCH ARTICLE
Twenty-seven ZAD-ZNF genes of Drosophila
melanogaster are orthologous to the embryo
polarity determining mosquito gene cucoid
Muzi LiID, Koray Kasan, Zinnia Saha, Yoseop YoonID
¤
, Urs Schmidt-Ott*
Dept. of Organismal Biology and Anatomy, University of Chicago, Chicago, IL, United States of America
¤Current address: Department of Microbiology and Molecular Genetics, School of Medicine, University of
California, Irvine, CA, United States of America
*uschmid@uchicago.edu
Abstract
The C2H2 zinc finger gene cucoid establishes anterior-posterior (AP) polarity in the early
embryo of culicine mosquitoes. This gene is unrelated to genes that establish embryo polar-
ity in other fly species (Diptera), such as the homeobox gene bicoid, which serves this func-
tion in the traditional model organism Drosophila melanogaster. The cucoid gene is a
conserved single copy gene across lower dipterans but nothing is known about its function
in other species, and its evolution in higher dipterans, including Drosophila, is unresolved.
We found that cucoid is a member of the ZAD-containing C2H2 zinc finger (ZAD-ZNF) gene
family and is orthologous to 27 of the 91 members of this family in D.melanogaster, includ-
ing M1BP,ranshi,ouib,nom,zaf1,odj,Nnk,trem,Zif, and eighteen uncharacterized genes.
Available knowledge of the functions of cucoid orthologs in Drosophila melanogaster sug-
gest that the progenitor of this lineage specific expansion may have played a role in regulat-
ing chromatin. We also describe many aspects of the gene duplication history of cucoid in
the brachyceran lineage of D.melanogaster, thereby providing a framework for predicting
potential redundancies among these genes in D.melanogaster.
1. Introduction
Dipteran insects (true flies) begin embryogenesis with 12 or 13 synchronous nuclear division
cycles [1–4]. During this syncytial phase of embryonic development, a uniform blastoderm
forms in the cortical layer of the egg, activates the zygotic genome [5–7], and establishes axial
polarity [8,9]. Anterior determinants (ADs) establish the embryo’s head-to-tail polarity via
transcription factor gradients. In the fruit fly Drosophila melanogaster (D.melanogaster), the
AD is encoded by the homeobox gene bicoid [10], which has been studied extensively [11–17].
However, genes unrelated to bicoid are being used in species of different dipteran lineages for
the same developmental task [18,19]. This evolutionary plasticity, along with the simple anat-
omy of early dipteran embryos and their amenability to experimental perturbation in non-tra-
ditional model organisms, set the stage for an attractive experimental system to study the
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OPEN ACCESS
Citation: Li M, Kasan K, Saha Z, Yoon Y, Schmidt-
Ott U (2023) Twenty-seven ZAD-ZNF genes of
Drosophila melanogaster are orthologous to the
embryo polarity determining mosquito gene
cucoid. PLoS ONE 18(1): e0274716. https://doi.
org/10.1371/journal.pone.0274716
Editor: Rene
´Massimiliano Marsano, University of
Bari: Universita degli Studi di Bari Aldo Moro,
ITALY
Received: September 1, 2022
Accepted: December 16, 2022
Published: January 3, 2023
Copyright: ©2023 Li et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: ML was the recipient of a fellowship from
the Top Student Training Program of Peking
University, China, for an International Summer
Research Experience for Undergraduates (REU)
program of the Biological Sciences Division at the
University of Chicago. ZS was the recipient of a
Research Foundations in Genetics and Genomics
molecular and evolutionary basis of transcriptional network stability and co-option of new
central players in embryo development.
What are the molecular mechanisms that guide the co-option of new ADs? Bicoid has
many target genes [15,20–22], but it remains unclear how it adopted them. The bicoid gene
evolved from a gene duplication of the Hox3 ortholog of flies (also known as zerknüllt or zen),
more than 145 million years ago [23–25]. The diverged DNA-binding specificity of Bicoid,
compared to its closest paralogs, prompted detailed studies on the evolution of its DNA-bind-
ing homeodomain using ancestral sequence reconstruction, quantitative in vitro DNA binding
assays, and in vivo rescue experiments in Drosophila embryos [26–29]. These studies empha-
sized the importance of mutations that altered DNA-binding specificity of the Bicoid protein.
It was also shown that a feed-forward relay integrates certain regulatory activities of Bicoid
and Orthodenticle via shared DNA binding sites [28]. These homeodomain proteins have
qualitatively similar DNA affinity and Orthodenticle has a conserved zygotic function in head
development, which raised the question of whether Bicoid took over functions of Orthodenti-
cle [29]. However, comparative studies revealed ADs with distinct DNA binding domains and
DNA affinities and suggest that the AD of the last common ancestor of dipterans was encoded
by pangolin (Tcf) [18]. Therefore, the co-option of new ADs in different fly lineages may not
require shared target sites between the old and new ADs.
Why do specific genes adopt the AD function in addition to their other roles? The identifi-
cation of AD gene orthologs in Drosophila melanogaster provides a useful starting point
because many of its gene functions have been analyzed. For example, odd-paired, the only zic
(zinc finger of the cerebellum) gene family member of flies [30], opens specific chromatin
regions to advance the temporal progression of zygotic pattern formation in Drosophila
embryos [31,32]. This function appears to be conserved in moth flies, where odd-paired addi-
tionally adopted the AD function by acquiring a maternal transcription variant [18]. The abil-
ity of Odd-paired protein to drive the accessibility of specific chromatin regions, which is also
a property of Bicoid [22,33], could have facilitated their convergent co-option as ADs.
In culicine mosquitoes (e.g., Aedes and Culex), a previously uncharacterized C2H2 zinc fin-
ger gene, named cucoid, adopted the AD function. In these species, three cucoid transcript iso-
forms with alternative 3’ ends have been identified in embryos. The shortest isoform is
expressed maternally and is localized at the anterior pole of the egg. In culicine mosquitoes,
knockdown of cucoid by RNAi results in ectopic expression of posterior genes at the anterior
and the double abdomen phenotype [18]. However, the function of cucoid orthologs in other
species is unknown and obscured by a complex evolution of this gene in higher flies, including
Drosophila melanogaster [18]. Here we show that cucoid is a member of the ZAD-ZNF gene
family and is orthologous to at least 27 of D.melanogaster’s 91 ZAD-ZNF genes [34].
ZAD-ZNF gene family members encode C2H2 zinc finger proteins with an N-terminal Zinc-
finger-associated domain (ZAD) [34–39]. Most cucoid orthologs of D.melanogaster have not
yet been characterized but those that have been named and studied predominantly function in
early development and oogenesis and may affect chromatin states.
2. Materials and methods
2.1 Cucoid structure prediction and identification of cucoid orthologs
Protein structure was predicted using AlphaFold2_advanced with default settings [40]. Cucoid
orthologs were identified by reciprocal protein BLAST using default E value cut-off threshold
of 0.05 while setting maximum target sequences to 5000 to detect all potential orthologs in the
target species (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [41]. As queries we used Cucoid
sequences from Culex quinquefasciatus (C.quinquefasciatus; GenBank identifier QFQ59547.1)
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summer fellowship of the Biological Sciences
Collegiate Division at the University of Chicago. YY
was the recipient of an award of University of
Chicago Henry Hinds Funds for Graduate Student
Research in Evolutionary Biology. Research
reported in this publication was supported by the
National Institute of General Medical Sciences of
the National Institutes of Health under Award
Number R01GM127366. The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
and Aedes aegypti (A.aegypti; GenBank identifier XP_021704552.1). The Cucoid sequence of
C.quinquefasciatus is referred to as C-isoform in GenBank but corresponds to the non-trun-
cated A-isoform in [18]. The Cucoid sequence of A.aegypti is referred to as myoneurin in Gen-
Bank and correspond to the non-truncated A-isoform in [18]. The name myoneurin for cucoid
in A.aegypti appears to be a misnomer due to spurious similarity with non-orthologous myo-
neurin genes in other species. Therefore, cucoid and myoneurin are not synonyms and the
name myoneurin should not be used to designate cucoid orthologs. Candidate orthologs were
searched for conserved domains using NCBI’s Conserved Domain Database (CDD) with the
server’s default E value cut-off of �0.01 [42]. C2H2 zinc finger proteins with ZAD (also
known as zf-AD, smart00868, or pfam07776) were retained for reciprocal BLAST in A.aegypti
and C.quinquefasciatus, using server default E value cut-offs of �0.05.
Conservation of Cucoid clade genes of Drosophila melanogaster within the Brachycera was
assessed by reciprocal protein BLAST in Drosophila virilis,Lucilia cuprina,Bactrocera dorsalis,
and Hermetia illucens. Syntenies of Cucoid orthologs in these species were examined in Gen-
Bank and illustrated using the IBS server [43]. Accession numbers are provided as supplemen-
tary material (S1 Table).
Since the assembly of the robber fly Proctacanthus coquilletti in GenBank (GenBank identi-
fier GCA_001932985.1) is not annotated, we identified candidate exons of P.coquilletti cucoid
orthologs (S2 Table), using tblastn with default settings [41] and Hermetia illucens (H.illucens)
Cucoid orthologs and D.melanogaster CG9215, CG4424, and CG14711 as queries. Protein
sequences of four candidate Cucoid orthologs from the robber fly were assembled manually
and used for reciprocal protein BLAST in H.illucens and D.melanogaster.
2.2 Protein alignment and phylogenetic analysis
The list of D.melanogaster ZAD-ZNF genes has been reported elsewhere [34]. The respective pro-
tein sequences were downloaded from GenBank. MAFFT alignments were generated by MAFFT
v7.471with the L-INS-i strategy (https://mafft.cbrc.jp/alignment/software/) [44]. Protein align-
ments were visualized using Geneious Prime 2021.2.2 (https://www.geneious.com/). For the pro-
tein tree with 91 ZAD-ZNF sequences, the raw alignments were trimmed using TrimAl v1.3
(http://trimal.cgenomics.org/) [45] with a conservation threshold of 20, a gap threshold of 0.8, and
a similarity threshold of 0.05 to remove highly variable positions (columns). The trimmed align-
ments were further divided into two partitions corresponding to ZAD and ZNF regions. The best
molecular substitution model for each partition was selected by partition merging strategy (MFP+-
MERGE) using ModelFinder [46] implemented in IQ-TREE v2.1.3 [47], based on Bayesian Infor-
mation Criterion (BIC). Maximum likelihood trees were then built based on the selected
substitution models, with branch support values generated by the implemented ultrafast bootstrap
approximation [48], setting replicates to 3000. A majority rule consensus tree was generated form
bootstrap trees and visualized by FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Trees
are unrooted unless otherwise stated. Accession numbers of all sequences used in protein trees (S1
Table)and full-length alignments of D.melanogaster ZAD-ZNF proteins (S1 File)and the Cucoid
orthologs from D.melanogaster and D.virilis (S2 File)are provided as supporting information.
3. Results and discussion
3.1 Cucoid is a ZAD-ZNF protein with 27 orthologs in Drosophila
melanogaster
Reciprocal protein BLAST identified single copy cucoid orthologs in all major branches of the
lower (non-brachyceran) Diptera, including Tipulomorpha, Culicomorpha, Psychodomorpha
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[18], and Bibionomorpha (this study), as well as in the insect orders Siphonaptera (fleas) and
Lepidoptera (butterflies and moths) (S1 Fig). No cucoid orthologs were found in other insect
orders, suggesting that cucoid evolved during the radiation of holometabolous insects.
The genome of Drosophila melanogaster encodes around 300 C2H2 zinc finger proteins
[37,39], including multiple candidate orthologs of cucoid. To aid in the identification of cucoid
orthologs in D.melanogaster, we searched for diagnostic domains and motifs of Cucoid using
protein alignments and protein folding software [49] (Figs 1and S1). The alignment was con-
structed with previously reported single-copy Cucoid orthologs from the mosquitoes Culex
quinquefasciatus,Aedes aegypti, and Anopheles gambiae (Culicidae), the harlequin fly Chirono-
mus riparius (Chironomidae), the moth fly Clogmia albipunctata (Psychodidae), and the crane
fly Nephrotoma suturalis (Tipulidae) [18], as well as newly identified single-copy Cucoid
orthologs from the gall midge Contarinia nasturtii (Cecidomyiidae), the cat flea Ctenocepha-
lides felis (Siphonaptera), and the silk moth Bombyx mori (Lepidoptera) that we retrieved from
sequences deposited in GenBank (S1 Fig and S1 Table). The gall midge belongs to the Bibio-
nomorpha, the putative sister taxon of the Brachycera [50], while the cat flea and the silk moth
represent close outgroups of the Diptera [51]. We focused on these lineage representatives
because of the advanced state of genome resources for these species and because they yielded
best matches in reciprocal protein BLAST with our query sequences.
Cucoid proteins typically contain five C2H2 zinc finger domains. However, the Cucoid
ortholog of Chironomus lacks zinc fingers 4 and 5, and culicine mosquitoes also express
shorter isoforms without the 5
th
(Culex) or 5
th
, 4
th
, and C-terminal half of the 3
rd
zinc finger
domains (Aedes). We found that all these Cucoid orthologs also contain a conserved N-termi-
nal domain, known as Zinc-finger-associated domain (ZAD; Figs 1and S1) [35,38]. The ZAD
Fig 1. Predicted structure of Cucoid. Structure of a Cucoid homodimer (maternal isoform from C.quinquefasciatus,
GenBank: QFQ59549.1) as predicted by AlphaFold2 (AlphaFold identifier for Cucoid structure: A0A5P8HWN4) is
shown with the two chains colored in red and blue above a simplified sketch of full-length Cucoid protein with the N-
terminal end to the left and the C-terminal end to the right, and ZAD and ZNFs marked by colored rectangles.
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is stabilized by zinc coordination via four invariant cysteine residues and can drive dimeriza-
tion [37,52] and nuclear localization [53].
Holometabolous insects evolved many ZAD-ZNF genes through lineage-specific gene
duplications [35,36,39], especially in dipterans. For example, 147 ZAD-ZNF proteins have
been found in Anopheles gambiae [36] and 91 in Drosophila melanogaster [34]. To identify the
ZAD-ZNF proteins in D.melanogaster most similar to Cucoid, we conducted protein BLAST
with all 91 ZAD-ZNF proteins of D.melanogaster in A.aegypti and C.quinquefasciatus. The
same seventeen Drosophila sequences retrieved cucoid in Aedes and Culex (54.1% sequence
conservation). The corresponding genes were therefore considered candidate orthologs of
cucoid (Table 1).
Next, we generated a Maximum Likelihood protein tree with all 91 ZAD-ZNF proteins of
D.melanogaster and examined the distribution of the candidate Cucoid orthologs on this pro-
tein tree (Fig 2). All seventeen candidate orthologs (marked by red triangles in Fig 2) mapped
to a monophyletic clade of 27 ZAD-ZNF proteins (henceforth Cucoid clade). The Cucoid
clade can be subdivided into two subclades with 9 members (subclade A) and 18 members
(subclade B), respectively. All 9 members of subclade A were included in the original list of
seventeen candidate Cucoid orthologs (Table 1). The top hit of that list, CG9215, also belongs
to subclade A. The 18 members of subclade B experienced on average an elevated substitution
Table 1. D.melanogaster ZAD-ZNF genes in the Cucoid clade and their relationship with mosquito Cucoid in BLAST.
GenBank protein accession number Gene Chromosome E value in
Aedes aegypti
E value in Culex quinquefasciatus %indentiy
Aedes/Culex
Rank of Cucoid
Aedes/Culex
NP_573045.1 CG9215 X 2.00E-50 3.00E-53 17.8/18.7 1st
NP_650094.1 CG14711 3R 1.00E-42 3.00E-45 20.2/19.7 1st
NP_650859.3 CG4424 3R 4.00E-41 5.00E-45 18.8/20.3 1st
NP_649825.1 M1BP 3R 7.00E-39 3.00E-41 19.3/18.1 1st
NP_649824.1 ranshi 3R 9.00E-38 9.00E-39 17.0/17.6 1st
NP_650860.1 CG4854 3R 9.00E-38 9.00E-38 17.4/17.8 1st
NP_650862.1 CG4936 3R 9.00E-37 3.00E-33 20.5/21.4 1st
NP_650861.1 trem 3R 7.00E-37 1.00E-36 19.8/19.6 1st
NP_649822.2 ouib 3R 2.00E-37 4.00E-39 15.7/16.0 1st
NP_001189188.1 Zif 3R 2.00E-34 2.00E-33 16.3/16.1 1st
NP_649823.2 CG8159 3R 3.00E-34 5.00E-33 12.5/12.7 1st
NP_001262384.1 nom 3R 1.00E-31 3.00E-30 15.2/15.3 1st
NP_001247055.1 Zaf1 3R 6.00E-33 2.00E-32 16.8/17.3 1st
NP_650092.4 CG14710 3R 1.00E-31 4.00E-34 18.9/19.7 1st
NP_731558.1 CG31441 3R 5.00E-31 1.00E-30 16.5/17.4 1st
NP_652712.2 CG18764 3R 2.00E-27 1.00E-27 18.0/17.8 1st
NP_651878.1 CG1792 3R 6.00E-27 2.00E-26 13.2/13.0 1st
NP_650658.1 CG17806 3R 2.00E-22 7.00E-21 14.9/14.0 2nd/19th
NP_650660.2 CG17801 3R 4.00E-21 1.00E-20 15.0/15.7 2nd/6th
NP_650060.1 CG31388 3R 4.00E-19 2.00E-18 13.7/15.0 13th/28th
NP_650659.2 Nnk 3R 7.00E-19 2.00E-17 14.6/15.6 >50th
NP_001163590. CG6813 3R 1.00E-18 2.00E-18 13.2/13.8 >50th/22nd
NP_650051.1 CG6689 3R 2.00E-17 5.00E-18 10.7/10.8 >50th
NP_650657.2 CG17803 3R 3.00E-15 5.00E-15 10.4/11.5 >50th
NP_001097748.1 CG4820 3R 3.00E-13 3.00E-13 12.7/13.6 5th/4th
NP_650661.1 odj 3R 2.00E-11 8.00E-13 12.4/13.8 >50th
NP_001014607.1 CG14667 3R 8.00E-11 2.00E-12 12.0/13.7 >50th
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Fig 2. Manually rooted maximum likelihood protein tree of ZAD-ZNF family members in D.melanogaster.
Orthologs that were identified based on reciprocal protein BLAST (see Table 1) are marked by red arrow heads.
Colored lines correspond to chromosomal gene clusters (see Fig 3).
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rate. This subclade includes 10 genes that we did not recover using reciprocal protein BLAST.
These 10 genes do not form a monophyletic clade but are nested within the Cucoid clade and
are therefore probably true Cucoid orthologs. We therefore conclude that at least 27 of D.mel-
anogaster’s 91 ZAD-ZNF genes are orthologous to cucoid.
3.2 Genomic organization and relationship of genes of the Cucoid clade
All proteins of the Cucoid clade, except CG9215, are encoded by genes on the right arm of
chromosome 3 and are organized in five gene complexes of 4–5 genes and three isolated sin-
gletons (Fig 3). These 26 genes share intron positions among each other and with the cucoid
orthologs from lower dipterans (Fig 4), consistent with an evolutionary origin by DNA-based
tandem gene duplications. CG9215 is an intron-less gene on the X-chromosome that may have
evolved by retro-transposition from the singleton zif, its most likely parent gene (Fig 2). We
denoted each gene cluster in D.melanogaster by the member that gave the smallest E value in
reciprocal BLAST with Cucoid (Table 1), that is: M1BP cluster (orange), CG4424 cluster
(blue), CG14711 cluster (green), and CG31441 cluster (purple). One cluster (light blue) did
not include any of the genes that we identified by reciprocal BLAST and was named after a pre-
viously described gene, oddjob (odj) [54].
The M1BP cluster genes form a monophyletic clade that can be traced to a single M1BP-
like precursor gene, preserved in other schizophoran flies such as blow flies and tephritid fruit
flies (see below). The first duplication of the M1BP-like precursor gave birth to M1BP/ranshi
and nom/ouib/CG8159. The precursor of nom/ouib/CG8159 duplicated twice, first generating
nom/ouib and CG8159 and then generating nom and ouib (Fig 2). These duplications occurred
before the split of the D.virilis and D.melanogaster lineages [55]. M1PB/ranshi duplicated
after the split of D.melanogaster and Drosophila ananassae [55].
The other cucoid-related gene clusters of D.melanogaster do not form monophyletic clades.
These incongruences between our protein tree and clustering in the D.melanogaster genome
Fig 3. Synteny of genes in the Cucoid clade. Genes of the Cucoid clade are distributed in clusters on the right arm of chromosome 3, except
CG9215 (not shown) which is located on the X chromosome. M1BP cluster (orange), CG14711 cluster (green), CG4424 cluster (blue), Odj cluster
(light blue), CG31441 cluster (purple), Zif (black), other dispersed genes (outlined).
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could have resulted from limitations of the phylogenetic inference methods that we used to build
the protein tree, such as model choice and long-branch attraction [56], or non-allelic recombina-
tion (gene conversion) within the ZAD-ZNF family [57,58]. However, in D.virilis, genes related
to members of the CG14711 cluster (green), the CG31441 cluster (purple), and the Odj cluster
(light blue) form a single gene complex with a different gene order, and this gene order is consis-
tent with the inferred close relationship of neighboring Cucoid clade genes (Fig 5A). Based on
synteny in D.virilis and phylogenetic inference with D.virilis and D.melanogaster orthologs (Fig
5B), CG18764 of the CG14711 cluster (green) is paralogous to all genes of the Odj cluster (light
blue) as well as CG6689 of the CG31441 cluster (purple). Additionally, we infer that CG6689 is a
paralog of CG17803 of the Odj cluster, even though a gene-specific N-terminal THAP (Thanatos
Associated Proteins) domain [59–61] that CG6689 inherited from its precursor, CG6689/
CG17803, is not preserved in CG17803 (S2 Fig). Finally, we infer that D.virilis lost the Nnk/
Fig 4. Conservation of Cucoid introns. Multiple sequence alignment of Cucoid orthologs from lower dipterans and Cucoid clade members of D.melanogaster.
Conserved intron positions are boxed, and the red zigzags represent the splicing points. Similarity of aligned amino acids was assessed using the Blosum62 matrix with
black representing 100% similarity, dark grey 80–100% similarity, light grey 60–80% similarity, and white less than 60% similarity.
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Fig 5. Cucoid clade protein tree is consistent with gene synteny in D.virilis.(A) Synteny of cucoid orthologs in D.virilis. Genes are color
coded to indicate their relationship to gene clusters in D.melanogaster (see Fig 3). (B) Manually rooted maximum likelihood protein tree of
Cucoid orthologs from D.melanogaster and D.virilis. Note that D.virilis has two CG17801 orthologs.
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CG17806 precursor because a CG17806-like precursor gene existed before the split of D.melano-
gaster and D.virilis but was not found in D.virilis. Duplication of the Nnk/CG17806 precursor
occurred after the split of the D.willistoni lineage from D.melanogaster lineage [55].
3.3 The Cucoid clade in Drosophila outgroups
Lineage-specific gene family expansions may reflect innovations or adaptations [38], but it is
unknown why the number of ZAD-ZNF genes independently increased so much in multiple
lineages of the Holometabola. To better understand when and how the Cucoid clade
expanded, we searched for orthologs of the Cucoid clade members in representatives of other
schizophoran fly species, including a blow fly (Lucilia cuprina) and a tephritid fruit fly (Bactro-
cera dorsalis), and in two representatives of the lower Brachycera, including a soldier fly (Her-
metia illucens) and a robber fly (Proctacanthus coquilleti). While non-brachyceran dipterans
have single cucoid orthologs (see section 3.1), we identified multiple cucoid orthologs in all the
brachyceran species, albeit in lower numbers than in Drosophila.
In Lucilia and Bactrocera, we identified a single M1BP-like gene (orange), and several genes
related to the CG4424 cluster (dark blue) and the CG14711 cluster (green), respectively, as
well as putative orthologs of zif and CG9215 (Fig 6A and 6B). The presence of a M1BP-like
gene in these unrelated species (they represent paraphyletic/parallel lineages of the Schizo-
phora [62]) suggests that the M1BP cluster expanded during, rather than before the radiation
of the Schizophora in the Tertiary epoch [63]. Whether the expansion of the M1BP cluster
within the Schizophora resulted in subfunctionalization or the acquisition of new gene func-
tions or a mix of both remains unknown, due to the lack of functional comparisons of the
M1BP-like gene in lower Schizophora with their multiple orthologs in Drosophila.
Two additional features of the genomic organization of Cucoid clade genes in Bactrocera
dorsalis deserve attention. First, the M1BP-like gene of this species is in the immediate vicinity
of the CG14711 cluster (green, Fig 6A). This finding may suggest that the founder gene of the
M1BP cluster originated as an offshoot of the CG14711 cluster, even though this is not apparent
in the protein tree (Fig 2). Second, CG14711 and CG14710 of B.dorsalis have merged; the pre-
dicted protein has two linear ZAD-ZNFs structures that correspond to CG14710 and CG14711,
respectively. Whether these genes resulted from the same duplication is unclear. Phylogenetic
analysis suggests that CG14711 is more closely related to CG4424 than to CG14710. However,
since CG14711 and CG4424 are more similar to Cucoid than CG14710 and other members of
the CG14711 and CG4424 clusters, the inferred close relationship of CG14711 and CG4424
might reflect their less diverged status rather than their duplication history.
In the genomes of lower Brachycera, we identified five cucoid loci on chromosome 6 of the
soldier fly Hermetia illucens (Hil_cucoid_1–5, GenBank accession numbers: XP_037925088.1,
XP_037925165.1, XP_037924715.1, CAD7093451.1, XP_037922166.1) [64] (Fig 6C) and four
cucoid loci in the robber fly Proctacanthus coquilleti (Pco_cucoid_1–4) [65] (S2 Table), which
seem to be orthologous to Hermetia cucoid orthologs 1, 2, 3, and 4/5, respectively (S3 Fig).
The lower brachyceran Cucoid proteins 1 and 2 are closely related to CG9215 judged by pro-
tein BLAST E value but retain conserved introns and are therefore potentially orthologous to
CG9215/Zif, whereas the lower brachyceran Cucoid orthologs 3 and 4 are closely related to
CG4424 and CG14711, respectively. Therefore, the last common ancestor of soldier flies, rob-
ber flies, and Drosophila may have had at least three cucoid orthologs, including a CG4424-like
member, CG14711-like member, and a CG9215-like ortholog of the Zif/CG9215 precursor.
No Hil_5 ortholog was found in D.melanogaster and the robber fly Proctacanthus coquilletti.
Its location within an intron of Hil_4 suggests that it was born by lineage-specific duplication
of Hil_4. Thus, Hil_5 may also be orthologous to CG14711.
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Orthologs of the mosquito gene cucoid in D.melanogaster
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4. Conclusions
D.melanogaster contains at least 27 cucoid orthologs, that is, almost one third of the 91 ZAD
ZNF genes of this species. Reciprocal BLAST, phylogenetic inference, and genomic
Fig 6. Syntenies of cucoid orthologs in Lucilia, Bactrocera, and Hermetia. Genes are color coded to indicate their relationship to gene clusters in D.
melanogaster. (A) Synteny of cucoid orthologs in Lucilia cuprina. (B) Synteny of cucoid orthologs in Bactrocera dorsalis. (C) Synteny of cucoid orthologs
in Hermetia illucens.
https://doi.org/10.1371/journal.pone.0274716.g006
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organization suggest that the Cucoid clade of D.melanogaster expanded gradually in the bra-
chyceran lineage (Fig 7), while its founder gene was already present in the last common ances-
tor of butterflies, fleas, and flies. The last common ancestor of the brachyceran species that we
analyzed may have had three cucoid orthologs that encoded proteins similar to CG9215/zif,
CG4424, and CG14711. We infer this because in protein BLAST against Drosophila proteins,
H.illucens and P.coquilletti Cucoid proteins 1 and 2 recovered CG9215 as the best hit, and
their Cucoid orthologs 3 and 4 recovered CG4424 and CG14711 as the best hits, respectively.
The founder of the monophyletic M1BP-cluster originated before the radiation of the Schizo-
phora. All other clusters of D.melanogaster may not have monophyletic origins.
Our study was motivated by the question of what is known about cucoid orthologs in
Drosophila melanogaster. Most of the 27 cucoid orthologs of D.melanogaster that we identi-
fied in this study did not affect viability when downregulated in previous large-scale screens
(Table 2) [34,66–68]. However, several orthologs have been characterized in greater depth
and display diverse, essential functions. For example, M1BP binds core promoters of thou-
sands of genes and functions during transcription activation and polymerase Ⅱpausing
while promoting chromatin accessibility surrounding the transcription start sites [69,70].
Other genes in the M1BP cluster show more specialized functions: ranshi regulates oocyte
differentiation [71], nom functions in muscle development, and ouib is necessary during
ecdysteroid synthesis by regulating spookier [72,73]. The closely related genes odj and Nnk
Fig 7. Evolution of the Cucoid clade family in higher flies. Inferred gene duplications in the Cucoid clade based on data in this study and D.
melanogaster gene ages reported elsewhere [55]. For details see text. Gene clusters (hexagons) and gene loci (triangles) are indicated. For color code
see Fig 2.
https://doi.org/10.1371/journal.pone.0274716.g007
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Orthologs of the mosquito gene cucoid in D.melanogaster
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have essential functions in heterochromatin regulation [34], zaf1 is a chromosome architec-
ture protein that serves as insulator in Drosophila melanogaster [74], trem is required for
binding Mei-P22 on meiotic chromosomes to initiate double strand breaks for homologous
recombination [75], and Zif is required for the expression and asymmetric localization of
aPKC in neuroblast cells to regulate their polarity and self-renewal [76,77]. All other genes
of the Cucoid clade remain uncharacterized. Taken together, our study suggests that many
cucoid orthologs of D.melanogaster function in oogenesis and embryogenesis and several of
them modify chromatin states. It will be interesting to find out whether single copy Cucoid
orthologs from lower dipterans function in similar ways to some D.melanogaster orthologs
and what structural and/or regulatory features enable Cucoid in culicine mosquitoes to reg-
ulate early zygotic segmentation genes.
Supporting information
S1 Table. Accession numbers.
(XLSX)
S2 Table. Locations of cucoid orthologs in P.coquiletti inferred from tblastn.
(XLSX)
Table 2. Functions of D.melanogaster cucoid orthologs.
Gene Functions Loss-of-function References
M1BP activates transcription; orchestrates RNA polymerase II pausing viable [69,70]
Zaf1 architectural protein, chromosome insulator viable [66–68,74,78]
Nnk heterochromatin binding lethal [34,66,79]
odj heterochromatin binding lethal [34,80]
ranshi involves in oocyte differentiation sterile [71]
ouib regulates ecdysteroid synthesis; stimulates transcription of spookier lethal [72,73]
nom regulates embryonic muscle morphogenesis viable [66–68,81]
Zif regulates neuroblast differentiation with aPKC lethal [66,67,76,77]
trem required for DSB formation in meiosis sterile [75]
CG14667 uncharacterized viable [68]
CG14710 uncharacterized viable [34]
CG14711 uncharacterized unknown
CG17801 uncharacterized viable [34,68]
CG17803 uncharacterized viable [34,66–68]
CG17806 uncharacterized viable [34,67]
CG1792 uncharacterized viable [34,67,68]
CG18764 uncharacterized viable [66–68]
CG31388 uncharacterized viable [66,67]
CG31441 uncharacterized lethal [66]
CG4424 uncharacterized viable [66–68]
CG4820 uncharacterized viable [66–68]
CG4854 uncharacterized viable [66–68]
CG4936 uncharacterized lethal [66,67,82]
CG6689 uncharacterized lethal [66]
CG6813 uncharacterized viable [34]
CG8159 uncharacterized viable [66]
CG9215 uncharacterized viable [66–68]
https://doi.org/10.1371/journal.pone.0274716.t002
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Orthologs of the mosquito gene cucoid in D.melanogaster
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S1 File. Full length alignment of 91 D. melanogaster ZAD-ZNF protiens.
(AFA)
S2 File. Full length alignment of D.melanogaster and D.virilis Cucoid orthologs.
(AFA)
S1 Fig. Cucoid protein alignment and prediction of Cucoid homodimer. (A) Multiple
sequence alignment of Cucoid orthologs from Aedes aegypti (Aae), Anopheles gambiae (Aga),
Bombyx mori (Bmo), Chironomus riparius (Cri), Clogmia albipunctata (Cal), Contarinia nasturtii
(Cna), Ctenocephalides felis (Cfe), Culex quinquefasciatus (Cqu), and Nephrotoma suturalis (Nsu).
(B) A plot of the predicted alignment error of the best model acquired from AlphaFold2 output
which estimates the distance error for every pair of residues. Both axes represent the positions on
the dimer of Cucoid maternal isoform (499 aa) from C.quinquefasciatus. The color key is mea-
sured in angstrom. Very low position errors are found for the overlapping of residues in the ZAD
dimer as well as between zinc fingers on the same strand, indicating true packing of these
domains. (C) The plot of predicted local distance difference test (pLDDT) per position gives a
confidence level between 0–100 for each residue. All models predict ZAD and ZNF domain with
very high confidence, whereas the highly variable linker regions get deficient support.
(TIF)
S2 Fig. CG6689 acquired a DNA-binding THAP domain. The THAP domains from 9
THAP-containing proteins in D.melanogaster and a THAP-like fragment from CG17803 are
shown in alignment here. The color code for each column is based on similarity of aligned
amino acids, with black representing high similarity and white representing no similarity. The
N-terminal THAP domain of CG6689 is absent in all other Cucoid orthologs including its most
recent paralog, CG17803, which has incomplete THAP features. THAP is a zinc-coordinating
DNA binding domain with a conserved C2CH structure and shares features with the DNA bind-
ing domain of the P element transposase [59,60]. THAP-domain-containing proteins have been
found in human, D.melanogaster, and C.elegans [59]. In the nine D.melanogaster proteins that
have this domain, only CG6689 and CG10431 belong to the ZAD-ZNF family. CG10431 is only
distantly related to CG6689 and located on a different chromosome (2L), suggesting that even
within the ZAD-ZNF family THAP domains evolved de novo. The THAP domain of CG6689 is
encoded by the first two exons of this gene, which are only conserved in CG17803 (Fig 4).
(PDF)
S3 Fig. Phylogeny of Cucoid orthologs in H.illucnes and P.coquilletti.A phylogenetic tree
with Cucoid orthologs in H.illucnes,P.coquilletti and lower flies was constructed based on an
untrimmed alignment using 3 partitions inlucing ZAD, ZNF, and the other regions. Regions
outside the ZAD and ZNF domains include diagnostic features useful for inferring orthology.
This tree suggests that Hil_cucoid_1 to Hil_cucoid_4 are orthologous to Pco_cucoid_1 to Pco_-
cucoid_4, respectively.
(TIF)
Acknowledgments
We thank our colleagues Dr. Phoebe Rice and Dr. Manyuan Long for helpful discussions, and
Dr. Shengqian Xia and Dylan Sosa in the Long lab for technical assistance.
Author Contributions
Conceptualization: Muzi Li, Koray Kasan, Urs Schmidt-Ott.
PLOS ONE
Orthologs of the mosquito gene cucoid in D.melanogaster
PLOS ONE | https://doi.org/10.1371/journal.pone.0274716 January 3, 2023 14 / 19
Formal analysis: Muzi Li, Koray Kasan, Zinnia Saha.
Funding acquisition: Muzi Li, Zinnia Saha, Yoseop Yoon, Urs Schmidt-Ott.
Investigation: Muzi Li, Koray Kasan, Zinnia Saha, Yoseop Yoon, Urs Schmidt-Ott.
Supervision: Urs Schmidt-Ott.
Visualization: Muzi Li.
Writing – original draft: Muzi Li, Koray Kasan, Urs Schmidt-Ott.
Writing – review & editing: Muzi Li, Koray Kasan, Yoseop Yoon, Urs Schmidt-Ott.
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